Novel coronavirus XBB.1.5 and related variant broad-spectrum neutralizing antibody and application

Abstract

The invention provides a broad-spectrum neutralizing antibody of novel coronavirus XBB.1.5 and related variants and application of the broad-spectrum neutralizing antibody. Specifically, a series of broad-spectrum neutralizing antibodies capable of efficiently neutralizing the novel coronavirus XBB.1. 5 and related variants are obtained by applying technologies in many fields of immunology, molecular biology, virology, protein structure analysis and the like, and the broad-spectrum neutralizing antibodies can be used for diagnosing, preventing and treating various diseases caused by infection of the novel coronavirus Ommitron mutant strain.

Description

Broad-spectrum neutralizing antibodies against novel coronavirus XBB.1.5 and related variants and their applications Technical Field

[0001] The present invention relates to the field of immunology, in particular to broadly neutralizing antibodies against the novel coronavirus (XBB.1.5) and related variants, and their applications. Specifically, the present invention utilizes techniques from a variety of fields, including immunology, molecular biology, virology, and protein structure analysis, to produce a series of broadly neutralizing antibodies that are highly effective against the novel coronavirus (XBB.1.5) and related variants. These antibodies can be used to diagnose, prevent, and treat various diseases caused by infection with the novel coronavirus (Omicron) mutant strain. Background Art

[0002] Since the end of 2019, SARS-CoV-2 has produced numerous variants, including five designated by the World Health Organization as variants of concern (VOC): Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Since the emergence of the Omicron strain in November 2021, it has gradually evolved into five subclades: BA.1, BA.2, BA.3, BA.4, and BA.5. Each subclade has further differentiated into a varying number of subclades, with BA.2 having the largest number of subclades. Regarding the Omicron variant, the WHO has designated seven subclades for monitoring based on the speed of transmission and potential impact of the variants, five of which are BA.2. Furthermore, BA.1 and BA.2 have also produced 28 new strains through recombination, including strains xe and xf that became prevalent in the UK earlier this year, xh in Denmark, and xj in Finland. On December 7, 2022, scientists from the University of Tokyo, including Masaki Imai, published an article in the New England Journal of Medicine titled "Efficacy of Antiviral Agents against Omicron Subvariants BQ.1.1 and XBB" (hereinafter referred to as the "Article"), stating that the Omicron subvariants BQ.1.1 and XBB have greater immune evasion capabilities than earlier Omicron variants.

[0003] Since the end of 2022, the proportion of infections caused by SARS-CoV-2 lineage XBB.1.5 has gradually increased in the United States, making XBB.1.5 one of the major lineages of SARS-CoV-2. XBB.1.5 is a subclade derived from XBB and was first detected in the United States on October 22, 2022. The World Health Organization has designated XBB.1.5 as a variant of interest (VOI).

[0004] The mortality rate of the Omicron mutant strain is generally lower than that of the early new coronavirus strains, but its transmission speed is also much higher than the original strain. It is estimated that in the absence of an immune basis, the R0 of XBB.1.5 may be as high as 25-30, which means that the XBB.1.5 pandemic will lead to more infections and pose greater challenges to the public health system.

[0005] The series of broadly neutralizing antibodies developed by this invention against the novel coronavirus (XBB.1.5) and related variants are derived from memory B cells of patients who have recovered from SARS or the novel coronavirus. These antibodies possess the broad-spectrum, high-neutralizing properties of XBB.1.5 and are distributed across multiple epitope clusters. Combining these antibodies can minimize the escape of XBB.1.5 mutants. Currently, no antibody combinations with the same or similar broad-spectrum, high-neutralizing activity against XBB.1.5 mutants and targeting multiple epitopes have been reported.

[0006] SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise complementarity determining regions of specific amino acid sequences.

[0008] In some embodiments, the heavy chain variable region and the light chain variable region comprise specific amino acid sequences.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin, for example, a heavy chain constant region and a light chain constant region comprising a specific amino acid sequence.

[0010] In some embodiments, the antigen binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, single domain antibody (sdAb), chimeric antibody, bispecific antibody or multispecific antibody.

[0011] In a second aspect, the present invention also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention, or the heavy chain variable region and / or light chain variable region thereof.

[0012] In some embodiments, the nucleic acid molecule is an mRNA molecule.

[0013] In some embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

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

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

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

[0017] (1) culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector of the present invention, and

[0018] (2) Isolating and purifying the antibody or antigen-binding fragment thereof expressed by the nucleic acid molecule or expression vector.

[0019] In a sixth aspect, the present invention further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0020] In a seventh aspect, the present invention also provides a pharmaceutical composition comprising the mRNA molecule of the present invention.

[0021] In an eighth aspect, the present invention also provides lipid nanoparticles comprising the mRNA molecules of the present invention.

[0022] In a ninth aspect, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention.

[0023] In the tenth aspect, the present invention also provides a method for preventing and / or treating diseases caused by infection with the novel coronavirus Omicron mutant strain, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof or any combination thereof, or the pharmaceutical composition of the present invention; the subject is preferably a mammal, more preferably a human.

[0024] In the eleventh aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof or any combination thereof of the present invention for preparing a drug for preventing and / or treating diseases caused by infection with the novel coronavirus Omicron mutant strain.

[0025] In some embodiments, the novel coronavirus Omicron mutant strain includes one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5 related mutant proteins.

[0026] In some embodiments, the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.5.

[0027] In some embodiments, the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

[0028] In some embodiments, the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

[0029] In a twelfth aspect, the present invention further provides a conjugate comprising the antibody or antigen-binding fragment thereof or any combination thereof of the present invention, and a detectable label linked to the antibody or antigen-binding fragment thereof or any combination thereof.

[0030] In some embodiments, 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.

[0031] In a thirteenth aspect, the present invention further provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, or any combination thereof, or the conjugate of the present invention.

[0032] In some embodiments, the kit comprises a conjugate of the invention.

[0033] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof, or any combination thereof, of the present invention, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof, or any combination thereof.

[0034] In some embodiments, 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.

[0035] In a fourteenth aspect, the present invention also provides a method for detecting the presence or level of a novel coronavirus Omicron mutant in a sample, comprising:

[0036] (1) contacting the sample with the antibody or antigen-binding fragment thereof of the present invention, or any combination thereof, or the conjugate of the present invention;

[0037] (2) detecting the binding of the antibody or antigen-binding fragment thereof or any combination or conjugate thereof to the target antigen in the sample.

[0038] In some embodiments, detection of the binding represents the presence of the novel coronavirus Omicron mutant in the sample.

[0039] In some embodiments, the strength of the binding is detected to represent the level of the novel coronavirus Omicron mutant strain in the sample.

[0040] In some embodiments, the sample is a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from a subject.

[0041] In some embodiments, the subject is a mammal, such as a human.

[0042] In some embodiments, the sample is not a sample from a subject, eg, the sample is from a vaccine sample.

[0043] In the fifteenth aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof of the present invention or any combination thereof or the conjugate of the present invention in the preparation of a kit for detecting the presence or level of a novel coronavirus Omicron mutant in a sample.

[0044] It should be understood that the antibodies or antigen-binding fragments thereof in the present invention can be more than one, for example, a combination of 2, 3, 4, 5, 6, 7 or 8 types.

[0045] For example, the combination can be a combination of any two of the antibodies or antigen-binding fragments thereof of the present invention.

[0046] The mRNA molecules in the present invention should be understood to include more than one type, for example, a combination of 2, 3, 4, 5, 6, 7 or 8 types.

[0047] The mRNA molecules of the present invention may encode more than one, for example, two, three, four, five, six, seven or eight antibodies of the present invention or their antigen-binding fragments, or their heavy chain variable regions and / or light chain variable regions.

[0048] Furthermore, the antibodies or antigen-binding fragments thereof of the present invention can also be combined with other antibodies or antigen-binding fragments thereof.

[0049] The novel coronavirus Omicron mutant strains described in the present invention include one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5 related mutant proteins.

[0050] In some embodiments, the XBB.1.5-related mutant protein has a mutation in the RBD region.

[0051] In some embodiments, the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

[0052] In some embodiments, the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

[0053] Currently, there are no reports on antibody combinations targeting multiple epitopes of XBB.1.5 and its related mutants with broad spectrum, high neutralizing activity levels.

[0054] The present invention isolated memory B cells specific for the SARS-CoV-2 antigen from recovered SARS and COVID-19 patients. Antibody sequences were obtained through 10xGenomics single-cell sequencing. After bioinformatics analysis and comparison, target antibody sequences were selected for synthesis to produce antibody proteins. The neutralizing activity of the antibody proteins against pseudoviruses of various mutant strains of Omicoron was tested. Escape epitopes were detected through high-throughput screening of SARS-CoV-2 antigens using yeast display, and the antibody proteins were subjected to epitope grouping.

[0055] The series of broadly neutralizing antibodies against XBB.1.5 and its variants developed by this invention are derived from memory B cells of patients who have recovered from SARS or COVID-19 infection. These antibodies possess the broad-spectrum, high-neutralizing properties of XBB and its variants, and are distributed across multiple epitope clusters. Combining these antibodies can minimize the escape of XBB.1.5 mutants. Currently, no antibody combinations with the same or similar broad-spectrum, high-neutralizing activity against XBB.1.5 mutants and targeting multiple epitopes have been reported.

[0056] Detailed Description of the Invention

[0057] 1. Definition

[0058] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0059] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a," "one," "one or more," and "at least one" are used interchangeably herein. In some aspects, the term "a" means "single." In other aspects, the term "a" includes "two or more" or "a plurality."

[0060] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or completely synthetically (e.g., recombinantly), including any fragment thereof that retains the binding specificity of a full-length immunoglobulin comprising at least a portion of the variable region of an immunoglobulin molecule. 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, intrabodies, 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-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The 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 IgA2), or subclass (e.g., IgG2a and IgG2b).

[0061] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the 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, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, 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). The fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 -10 8 M -1 Antibodies to the Ka) antigen.

[0062] 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 other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies, which comprises antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0063] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by the 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 continuously produce a specific monoclonal antibody. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0064] As used herein, "conventional antibodies" refers to antibodies comprising two heavy chains (which may be designated as H and H') and two light chains (which may be designated as L and L') and two antigen-binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include but are not limited to VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region thereof (e.g., light chains include but are not limited to VL chains and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

[0065] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies with the same domains produced synthetically.

[0066] As used herein, dsFv refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

[0067] As used herein, Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments having the same structure, for example, produced synthetically by recombinant methods. Fab fragments comprise a light chain (comprising VL and CL) and another chain comprising the variable domain (VH) of the heavy chain and one constant region domain (CH1) of the heavy chain.

[0068] As used herein, a F(ab')2 fragment is an antibody fragment resulting from pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, for example, by recombinant methods. A F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain portion of which contains several additional amino acids, including cysteine, which forms a disulfide bond linking the two fragments.

[0069] As used herein, a Fab' fragment is a fragment comprising half (one heavy chain and one light chain) of a F(ab')2 fragment.

[0070] As used herein, a scFv fragment refers to a fragment comprising variable light chains (V L ) and variable heavy chain (V H The length of the linker is such that the two variable domains are bridged without substantial interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues interspersed to increase solubility. n residue.

[0071] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0072] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0073] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to thereby improve one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their impact on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. In addition, the mutations in the CDRs are typically no more than one or two. Therefore, the humanized antibodies of the present invention also encompass antibodies comprising one or two amino acid mutations in the CDRs.

[0074] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0075] As used herein, variable domain or variable region is the specific Ig domain of antibody heavy chain or light chain, and it is included in the aminoacid sequence that changes between different antibodies.Each light chain and each heavy chain has a variable region domain VL and VH respectively.Variable domain provides antigen specificity, and is therefore responsible for antigen recognition.Each variable region comprises CDR and framework region (FR), and CDR is the part of antigen binding site domain.

[0076] As used herein, "antigen binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A natural, conventional full-length antibody molecule has two conventional antigen-binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. A conventional antigen-binding site comprises loops connecting antiparallel beta chains within the variable region domain. An antigen-binding site may comprise other portions of the variable region domain. Each conventional antigen-binding site comprises three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. Hypervariable regions are also referred to as complementarity determining regions (CDRs).

[0077] As used herein, "hypervariable region," "HV," "complementarity determining region," "CDR," and "antibody CDR" are used interchangeably to refer to one of the multiple portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain comprises three CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises three CDRs, designated VL CDR1, VL CDR2, and VL CDR3; and the heavy chain variable region domain comprises three CDRs, designated VH CDR1, VH CDR2, and VH CDR3. The three CDRs in a variable region are discontinuous along the linear amino acid sequence, but are proximal in the folded polypeptide. The CDRs are located within loops connecting the parallel strands of the beta sheet of the variable domain. As described herein, those skilled in the art will recognize and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). CDRs are identified herein based on Chothia numbering.

[0078] As used herein, framework regions (FRs) are domains within the antibody variable region domains that are located within the beta sheets; in terms of amino acid sequence, FR regions are relatively more conserved than hypervariable regions.

[0079] 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 conservative than the amino acid sequence of the 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 a hinge region, while IgE and IgM comprise CH1, CH2, CH3, and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thereby contributing to interaction with the antigen and rotation of the antibody arm. The antibody constant region can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins to which the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.

[0080] As used herein, a functional region of an antibody is an antibody portion comprising at least the VH, VL, CH (eg, CH1, CH2, or CH3), CL, or hinge region domains of the antibody, or at least a functional region thereof.

[0081] As used herein, a functional region of a VH domain is at least a portion of an intact VH domain that retains at least some of the binding specificity of the intact VH domain (e.g., by retaining one or more CDRs of the intact VH domain), such that the functional region of the VH domain binds to the antigen alone or in combination with another antibody domain (e.g., a VL domain) or a region thereof. An exemplary functional region of a VH domain is a region comprising CDR1, CDR2, and / or CDR3 of the VH domain.

[0082] As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least some of the binding specificity of the complete VL domain (e.g., by retaining one or more CDRs of the complete VL domain), such that the functional region of the VL domain binds to the antigen alone or in combination with another antibody domain (e.g., a VH domain) or a region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of a VL domain.

[0083] As used herein, "specific binding" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present in a biological sample. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about 1 x 10 7 M -1 or 1x 10 8 M-1 or greater affinity constant Ka (or 1x 10 -7 M or 1×10 -8 M or lower dissociation constant (K d )) binds to the antigen. Affinity constants can be determined by standard kinetic methods for antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0084] As used herein, the term "competition" with respect to antibodies refers to a first antibody or its antigen-binding fragment binding to an epitope in a manner sufficiently similar to a second antibody or its antigen-binding fragment, whereby the binding result of the first antibody to its associated epitope is detectably reduced in the presence of the second antibody compared to the absence of the second antibody. Alternatively, 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 be the case, but not necessarily so. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, in the case where each antibody can detectably inhibit the binding of another antibody to its associated epitope or ligand, whether to the same, higher or lower degree, the antibodies are referred to as "cross-competing" with each other in binding to their respective epitopes. Competition and cross-competing antibodies are encompassed in 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 that such competition and / or cross-competing antibodies are encompassed in the present invention and can be used in the methods disclosed herein based on the teachings provided herein.

[0085] As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

[0086] An "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that: (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cells from which the polypeptide naturally originates will be "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, i.e., using protein purification techniques well known in the art.

[0087] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0088] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, electric charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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 amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0089] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0090] 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), usually linked together by phosphodiester bonds.

[0091] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in the natural origin of nucleic acid molecules. " Isolated " nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesis. The exemplary isolated nucleic acid molecules provided herein comprise the isolated nucleic acid molecules of the antibody or Fab provided by coding.

[0092] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0093] 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 can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

[0094] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. 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 that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0095] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0096] As used herein, "expression" refers to the process of producing a polypeptide through 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 for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0097] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0098] "Codon optimization" refers to a process 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 (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which is believed to be dependent on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See, Nakamura et al., 2001; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2029; 2030; 2040; 205 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0099] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0100] 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 the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0101] As used herein, the term "prevention" refers to a method implemented 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 implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0102] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0103] 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., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0104] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0105] As used herein, the term "subject" preferably refers to a mammal, such as a human. In certain embodiments, the subject (e.g., a human) has a SARS-CoV-2 infection or a disease associated with a SARS-CoV-2 infection (e.g., COVID-19), or is at risk of having the above-mentioned disease.

[0106] As used herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), formerly known as "novel coronavirus" or "2019-nCov," belongs to the genus Betacoronavirus and is an enveloped, single-stranded, positive-sense RNA virus. SARS-CoV-2 contains at least three membrane proteins, including the surface spike protein (S), the integral membrane protein (M), and the membrane protein (E). Like SARS-CoV, the receptor for SARS-CoV-2 specifically binds to angiotensin-converting enzyme 2 (ACE2) on the host cell through the receptor binding domain (RBD) on the S protein, which then specifically binds to the host cell's angiotensin-converting enzyme 2 (ACE2), leading to viral membrane fusion and entry into the cell. This plays a crucial role in viral infection of cells.

[0107] As used herein, the term "COVID-19" refers to pneumonia caused by infection with SARS-CoV-2.

[0108] The sequence of the Omicron clade strain involved in the present invention is as follows:

[0109] BA.1 is based on Wuhan-Hu-1 with the following mutations: A67V+HV69-70del+T95I+GVYY142-145D+NL211-212I+ins214EPE+G339D+S371L+S373P+S375F+K417N+N440K+G446S+S477N+T478K+E484A+Q493R+G496S+Q498R+N501Y+Y505H+T547K+D614G+H655Y+N679K+P681H+N764K+D796Y+N856K+Q954H+N969K+L981F;

[0110] BA.2 is based on Wuhan-Hu-1 with the following mutations: T19I+LPPA24-27S+G142D+V213G+G339D+S371F+S373P+S375F+T376A+D405N+R408S+K417N+N440K+S477N+T478K+E484A+Q493R+Q498R+N501Y+Y505H+D614G+H655Y+N679K+P681H+N764K+D796Y+Q954H+N969K;

[0111] BA.2.75 has the following mutations based on BA.2: K147E+W152R+F157L+I210V+G257S+D339H+N460K+G446S+R493Q;

[0112] BA.2.86 is based on BA.2 and has mutations 16MPLFins+R21T+S50L+H69del+V70del+V127F+Y144del+F157S+R158G+N211del+L212I+L216F+H245N+A264D+1332V+D339H+K356T+R403K+V445H+G446S+N450D+L452W+N460K+N481K+V483del+A484K+F486P+R493Q+E554K+A570V+P621S+H681R+S939F+P1143L;

[0113] BA.2.12.1 is based on BA.2 and has mutations L452Q+S704L;

[0114] BA.5 has the following mutations based on BA.2: HV69-70del-+L452R+F486V+R493Q;

[0115] XBB has the following mutations based on BA.2: V83A+Y144del+H146Q+Q183E+G213E+D339H+R346T+L368I+V445P+G446S+N460K+F486S+F490S+R493Q;

[0116] BF.7 is based on BA.5 and has mutation R346T;

[0117] BQ.1 is based on BA.5 and has the following mutations: K444T+N460K;

[0118] BQ.1.1 is based on BQ.1 with mutation R346T;

[0119] BQ.1.19 is based on BQ.1 and has the mutation S494P;

[0120] XBB.1 has a mutation G252V based on XBB;

[0121] XBB.1.5 is based on XBB.1 and has the mutation S486P;

[0122] XBB.1.5-related mutant proteins have mutations in the RBD region, wherein the mutations are selected from one or more of the following sites: R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505. For example, the XBB.1.5-related mutant proteins include XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3, and XBB.1.5-S13.

[0123] XBB.1.5-L455F is based on XBB.1.5 with mutation L455F;

[0124] XBB.1.5-L455F+F456L is based on XBB.1.5 with mutations L455F+F456L;

[0125] XBB.1.5-S6 is based on XBB.1.5 and has mutations R403K+F456L+H505Y+K444T+K440N+K478R;

[0126] XBB.1.5-S8 is based on XBB.1.5 and has mutations R403K+F456L+H505Y+K444T+K440N+A484P+K478R+Y453F;

[0127] XBB.1.5-S12 is based on XBB.1.5 and has mutations R403K+F456L+H505Y+N405K+K444T+K440N+Y449D+K478R;

[0128] XBB.1.5-S3 is based on XBB.1.5 with mutations R403K+L455S+K478R+H505Y;

[0129] XBB.1.5-S13 is based on XBB.1.5 and has mutations R403K+N405K+K440N+K444T+Y453F+F456L+K478R+A484P+H505Y;

[0130] HK.3.1 is based on XBB.1.5 with mutations Q52H+F157L+L455F+F456L;

[0131] JD.1.1 is based on XBB.1.5 and has mutations L455F+F456L+A475V;

[0132] GW.5 is based on XBB.1.5 with mutations L455F+F456L+E554K+K478I;

[0133] JF.1 is based on XBB.1.16 and has mutations L455F+F456L.

[0134] As used herein, the term "neutralizing activity" refers to the functional activity of antibodies or antibody fragments that bind to antigenic proteins on viruses, thereby preventing virus-infected cells and / or the maturation of viral progeny and / or the release of viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent the amplification of viruses, thereby inhibiting or eliminating viral infection.

[0135] 2. Antibodies or Antigen-Binding Fragments

[0136] The present invention provides an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a complementarity determining region (CDR) of a specific amino acid sequence, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to the specific amino acid sequence.

[0137] In some embodiments, the heavy chain variable region and the light chain variable region comprise a specific amino acid sequence or a variant thereof, wherein the variant has one or more 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.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin, for example, a heavy chain constant region and a light chain constant region comprising a specific amino acid sequence.

[0139] In some embodiments, the constant region is not naturally occurring.

[0140] The information of the partial sequences of the present invention is provided in Table 1 below, specifically involving 31 antibodies (numbered as BD55-1205, BD55-5483, BD57-0129, BD57-0702, BD57-1169, BD57-1479, BD57-1492, BD57-1501, BD57-1513, BD57-1519, BD57-1520, BD57-1531, BD57-1540, BD57-1551, 78, BD57-1689, BD57-1908, BD57-2050, BD56-1247, BD57-1476, BD57-1912, BD57-1497, BD58-0730, BD57-2073, BD57-2118, BD57-2128, BD57-2140, BD57-2222, BD57-2223, BD57-2225, BD57-2243, BD57-2294). These 31 antibodies have common CH (SEQ ID NO: 145) and CL (SEQ ID NO: 146) sequences.

[0141] Table 1: Description of sequences (using the Chothia numbering system to identify CDRs)

[0142] 3. Antibody Preparation

[0143] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0144] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0145] Therefore, in another aspect, the present invention provides a kind of isolated nucleic acid molecule, it comprises the nucleotide sequence of encoding antibody of the present invention or its Fab, or its heavy chain variable region and / or light chain variable region.In some embodiments, the isolated nucleic acid molecule encodes antibody of the present invention or its Fab, or its heavy chain variable region and / or light chain variable region.

[0146] 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 present 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 molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0147] In some embodiments, the nucleic acid molecule is an mRNA molecule.

[0148] In some embodiments, the mRNA molecule comprises a chemically modified nucleobase, such as a chemically modified uracil, such as pseudouracil, N1-methylpseudouracil, 5-methoxyuracil, etc. The mRNA molecules of the present invention can comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0149] In some embodiments, the mRNA molecule comprises a heterologous 5' and / or 3' untranslated region (UTR). A UTR is a nucleic acid segment of a polynucleotide that is before the untranslated start codon (5'UTR) and after the stop codon (3'UTR). UTRs may have features that provide regulatory effects, such as increased or decreased stability, localization, and / or translation efficiency. The 5'UTR is an mRNA segment immediately adjacent to the translation start codon and has features that play a role in translation initiation. The 3'-UTR is an mRNA segment immediately following the translation stop codon and typically contains regulatory regions that affect gene expression after transcription. The regulatory region within the 3'-UTR can affect polyadenylation, translation efficiency, localization, and stability of the mRNA.

[0150] In some embodiments, the mRNA molecule comprises a 5' terminal cap (e.g., Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methyl G-cap, or analogs thereof) and a poly A tail. Both the 5' terminal cap and the poly A tail can increase the stability of the mRNA molecule.

[0151] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) encoding an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or a light chain variable region thereof.

[0152] In some embodiments, the mRNA molecule comprises one ORF. In some embodiments, the mRNA molecule may comprise more than one, for example, two, three, four, or more ORFs. Thus, the mRNA molecule may encode more than one, for example, two, three, four, five, six, seven, or eight, antibodies or antigen-binding fragments thereof of the present invention, or heavy chain variable regions and / or light chain variable regions thereof.

[0153] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In some embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, or the like.

[0154] In some embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the 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.

[0155] In some embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention, and / or a second nucleotide sequence encoding the light chain variable region of an antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0156] In some embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof of the present invention, and / or a second nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0157] In another aspect, the present invention provides a host cell transformed by the nucleic acid molecule of the present invention or the expression vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0158] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector of the present invention, and isolating and purifying the antibody or antigen-binding fragment thereof expressed by the nucleic acid molecule or expression vector.

[0159] IV. Pharmaceutical Composition

[0160] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention or any combination thereof.

[0161] In another aspect, the present invention also provides a pharmaceutical composition comprising the mRNA molecule of the present invention.

[0162] In another aspect, the present invention also provides a lipid nanoparticle comprising the mRNA molecule of the present invention.

[0163] In some embodiments, the mRNA of the present invention is formulated in lipid nanoparticles (LNPs). Lipid nanoparticles generally comprise ionizable cationic lipids, non-cationic lipids, sterols, and PEG lipid components, as well as nucleic acid molecules of interest. Components, compositions, and methods generally known in the art can be used to produce lipid nanoparticles of the present invention, see, for example, PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US20 16 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[0164] In another aspect, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention.

[0165] As used herein, "pharmaceutically acceptable carriers and / or excipients" include 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 carriers and / or excipients are 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, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0166] The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. 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, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0167] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0168] Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. In many cases, it is preferred to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium hydroxide in the composition. Prolonged absorption of injectable drugs can be achieved by including agents that delay absorption, such as monostearate and gelatin in the composition.

[0169] 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 to the extent that they are incompatible with the active compound, may be included in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

[0170] Therapeutic compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained.

[0171] Sterile injections can be prepared by mixing the active compound in a suitable solvent in the required amount and adding one or a combination of the ingredients listed above as needed, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injections, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), from which a powder of the active ingredient plus any additional required ingredients is obtained from a previously sterile-filtered solution thereof.

[0172] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0173] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as required by the exigencies of the therapeutic 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 unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specific specification of the dosage unit form of the present invention is limited by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art for formulating such active compound for treating individual sensitivity.

[0174] For the administration of antibody molecules, dosage range is about 0.0001 to 100 mg / kg, more generally 0.01 to 20 mg / kg recipient body weight. For example, dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or within the range of 1-20 mg / kg. Exemplary treatment regimens require weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or initial dosing interval slightly shorter (such as once a week to once every three weeks) later dosing interval lengthening (such as once a month to once every 3-6 months).

[0175] Alternatively, the antibody molecule of the present invention can also be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. Generally, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are given at less frequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, higher doses are sometimes needed at shorter intervals until the progression of the disease is alleviated or stopped, preferably until the patient shows partial or complete improvement in symptoms of the disease. Afterwards, the patient can be administered with a preventive regimen.

[0176] Actual dosage levels of the active ingredients 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 for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention being employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds, and / or materials being used in combination with the particular composition being employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0177] The antibodies of the present invention or their antigen-binding fragments or pharmaceutical compositions of the present invention can be administered by one or more routes of administration utilizing one or more methods well known in the art. It will be understood by those skilled in the art that routes of administration and / or modes vary depending on the desired outcome. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0178] Alternatively, the antibodies or antigen-binding fragments thereof of the invention or the pharmaceutical compositions of the invention can also be administered non-parenterally, such as topically, epidermally or mucosally, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0179] The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0180] The therapeutic composition can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention can be administered using a needle-free subcutaneous injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of known implants and modules that can be used in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a medical infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0181] In some embodiments, the antibodies of the present invention can be formulated to ensure proper distribution in the body. For example, the blood-brain barrier (BBB) ​​blocks many highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the present invention can cross the BBB (if necessary), they can be formulated in, for example, liposomes. As for methods for preparing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548 and 5,399,331. Liposomes contain one or more targeting moieties that can be selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29: 685). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.

[0182] 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 liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0183] 5. Disease prevention and / or treatment

[0184] The antibodies or antigen-binding fragments thereof of the present invention can be used to neutralize the novel coronavirus Omicron mutant strain in vitro or in vivo in a subject, blocking or inhibiting the infection of the novel coronavirus Omicron mutant strain on cells, thereby preventing and / or treating various diseases caused by infection with the novel coronavirus Omicron mutant strain.

[0185] In another aspect, the present invention provides a method for preventing and / or treating diseases caused by infection with the novel coronavirus Omicron mutant strain, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention.

[0186] In some embodiments, the novel coronavirus Omicron mutant strain comprises one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5 related mutant proteins.

[0187] In some embodiments, the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.5.

[0188] In some embodiments, the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

[0189] In some embodiments, the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

[0190] In some embodiments, the subject is preferably a mammal, more preferably a human.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof, or any combination thereof, or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the invention and the other pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0192] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof or any combination thereof of the present invention for preparing a medicament for diagnosing, preventing or treating a disease caused by infection with a novel coronavirus Omicron mutant strain, wherein the medicament is used for one or more of the following:

[0193] (1) neutralizing the novel coronavirus Omicron mutant in vitro or in a subject (e.g., human);

[0194] (2) Block or inhibit the binding of the novel coronavirus Omicron mutant to the ACE2 receptor;

[0195] (3) blocking or inhibiting the infection of cells by the novel coronavirus Omicron mutant strain; and / or

[0196] (4) Used to prevent and / or treat infection with the novel coronavirus (COVID-19) mutant strain or diseases associated with the novel coronavirus (COVID-19) mutant strain in subjects.

[0197] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. 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 present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0198] Antibody of the present invention or its Fab or pharmaceutical composition of the present invention can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In some embodiments, antibody of the present invention or its Fab or pharmaceutical composition are given by intravenous injection or push injection.

[0199] In this context, the dosage regimen may be adjusted to obtain the optimal intended response (e.g., therapeutic or preventive response). For example, the dosage may be administered in a single dose, multiple doses may be administered over a period of time, or the dosage may be proportionally reduced or increased as the exigencies of the therapeutic situation warrant.

[0200] 6. Conjugates

[0201] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Typically, the derivatization (e.g., labeling) of an antibody or its antigen-binding fragment will not adversely affect its binding to the novel coronavirus Omicron mutant strain. Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that can mediate binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), a methyl or ethyl group, or a glycosyl group. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

[0202] Thus, in some embodiments, the antibodies or antigen-binding fragments thereof of the invention are detectably labeled.

[0203] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0204] In some embodiments, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable label can be selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, or β-galactosidase), chemiluminescent reagents (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins, such as FITC, TRITC, or PE), radionuclides, or biotin.

[0205] In some embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0206] VII. Kit and Test Purpose

[0207] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, or any combination thereof, or the conjugate of the present invention.

[0208] In some embodiments, the kit comprises a conjugate of the invention.

[0209] In other embodiments, the kit comprises an antibody or antigen-binding fragment thereof of the present invention or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In some embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0210] In some embodiments, the second antibody is specific for an antibody from the species (eg, human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the invention is derived.

[0211] 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.

[0212] In some embodiments, the kit of the present invention may further comprise a reagent for detecting 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), tetramethylbenzidine (TMB), ABTS, or a luminol compound for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0213] In another aspect, the present invention provides a method for detecting the presence or level of a novel coronavirus Omicron mutant in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof, or any combination or conjugate thereof, of the present invention; detecting the binding of the antibody or antigen-binding fragment thereof, or any combination or conjugate thereof, to a target antigen in the sample; wherein the detection of the binding represents the presence of a novel coronavirus Omicron mutant in the sample, or the detection of the strength of the binding represents the level of the novel coronavirus Omicron mutant in the sample.

[0214] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0215] In some embodiments, the methods comprise using a conjugate of the invention.

[0216] In other embodiments, the method comprises using an antibody or antigen-binding fragment thereof of the present invention or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof does not contain a detectable label. In some embodiments, the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).

[0217] In some embodiments, the second antibody is specific for an antibody from the species (eg, human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the invention is derived.

[0218] 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.

[0219] In some embodiments, the method can be used for diagnostic purposes, for example, to diagnose whether a subject is infected with a novel coronavirus Omicron mutant strain based on the presence or level of the novel coronavirus Omicron mutant strain in a sample. In such embodiments, the sample can be a blood sample (e.g., whole blood, plasma, or serum), feces, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

[0220] In some embodiments, the subject is a mammal, such as a human.

[0221] In some embodiments, the methods can be used for non-diagnostic purposes, eg, the sample is not a sample from a subject, such as a vaccine sample.

[0222] In some embodiments, the novel coronavirus Omicron mutant strain includes one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5 related mutant proteins.

[0223] In some embodiments, the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.5.

[0224] In some embodiments, the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

[0225] In some embodiments, the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

[0226] In another aspect, provided is the use of an antibody or antigen-binding fragment thereof of the present invention or any combination thereof or a conjugate of the present invention in the preparation of a kit for detecting the presence or level of a novel coronavirus Omicron mutant in a sample.

[0227] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0228] In some embodiments, the kit detects the presence or level of the novel coronavirus Omicron mutant strain, or cells infected with the novel coronavirus Omicron mutant strain in a sample by the detection method described above, and optionally diagnoses whether the subject is infected with the novel coronavirus Omicron mutant strain based on the detection results.

[0229] In some embodiments, the sample is a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from a subject (eg, a mammal, preferably a human).

[0230] In some embodiments, the novel coronavirus Omicron mutant strain includes one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5 related mutant proteins.

[0231] In some embodiments, the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.5.

[0232] In some embodiments, the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

[0233] In some embodiments, the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

[0234] In the above technical solutions of the present invention, such as pharmaceutical compositions, conjugates, kits, methods or uses for preventing and / or treating diseases, detection methods, and the preparation and use of drugs or kits, it should be understood that there can be more than one antibody or antigen-binding fragment thereof, for example, a combination of 2, 3, 4, 5, 6, 7, or 8. Taking a combination of 2 antibodies or antigen-binding fragments thereof as an example, it includes a combination of any two of the antibodies or antigen-binding fragments thereof of the present invention, and no further enumeration is given in this application. However, all such combinations should be understood to be described in this application and fall within the scope of protection of this application.

[0235] The antibodies or antigen-binding fragments thereof of the present invention can also be combined with other antibodies or antigen-binding fragments thereof. Preferably, the combination of antibodies or antigen-binding fragments thereof has a more excellent binding activity and neutralizing effect against the novel coronavirus Omicron mutant strain.

[0236] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

[0237] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0238] Example 1: Memory B cell isolation

[0239] Whole blood was collected from patients who had recovered from SARS-CoV infection and COVID-19 infection. The blood samples were first diluted with PBS (Invitrogen) containing 2% FBS (Gibco) and subjected to Ficoll (Cytiva) gradient centrifugation. After lysis and washing, the samples were resuspended in PBS (Invitrogen) containing 2% FBS (Gibco) for downstream B cell isolation or added to FBS containing 10% DMSO (Sigma-Aldrich) for long-term storage at -80°C. B cells were enriched by positive selection using a CD19+ B cell isolation kit (STEMCELLS). Enriched B cells were stained with the following human antigens and antibodies in FACS buffer (1×PBS, 2% FBS, 1mM EDTA): FITC anti-CD19 antibody (Biolegend), FITC anti-CD20 antibody (Biolegend), Brilliant Violet 421 anti-CD27 Antibody (Biolegend), PE / Cyanine7 anti-IgM and fluorophore-labeled RBD (SARS340 CoV-2 and SARS-CoV RBD, Sino Biological Inc.) and ovalbumin (Ova) on ice for 30 minutes. Cells were stained with 7-AAD for 10 minutes before sorting, and single CD19 or CD20+CD27+IgM-Ova-RBD-PE+RBD-APC+ B cells were sorted into PBS containing 30% FBS on an Astrios EQ (Beckman Coulter). After flow cytometry sorting, the obtained cells were subjected to 5'-mRNA and single-cell V(D)J library preparation, and further subjected to Illumina sequencing using a double-sided sequencing mode of 26 bp (Barcode) + 91 bp (insertion sequence) on the HiSeq 2500 platform.

[0240] Example 2: Acquisition and identification of antibody sequences

[0241] Raw FASTQ files were processed using Cell Ranger (version 6.1.1) software with reference to the GRCh38 human sequence database. Sequences were generated using "cellranger multi" or "cellranger vdj" with default parameters. Protein sequences were then extracted and processed using IMGT / DomainGapAlign (version 4.10.2) to obtain annotations of V(D)J and CDR regions and mutation frequencies. V gene amino acid mutation rate = mutation count / V gene peptide length.

[0242] Example 3: Antibody Preparation and Purification

[0243] Paired immunoglobulin heavy and light chain genes obtained from 10X Genomics V(D)J sequencing and analysis were submitted for recombinant monoclonal antibody synthesis. The heavy and light chain sequences were cloned into separate expression vectors using Gibson assembly, and both plasmids were co-transfected into HEK293F cells. The secreted monoclonal antibodies in the cell culture medium were then purified by Protein A affinity chromatography.

[0244] Example 4: Evaluation of broad-spectrum neutralizing activity of antibodies

[0245] Neutralization assays were performed using a pseudovirus method to screen broadly binding antibodies to assess the neutralizing capacity of antibodies and plasma. A series of two-fold dilutions of the antibodies were first incubated with VSV pseudoviruses expressing spike proteins of various Omicron lineages (labeled with luciferase) for 1 hour, and then the mixture was incubated with Huh-7 cells. After 24 hours of incubation in a 37°C incubator, the cells were harvested and lysed with luciferase substrate (PerkinElmer), and the luminescence intensity was measured using a microplate reader. IC50 and NT50 were determined by a four-parameter nonlinear regression model (see Table 2).

[0246] All 18 antibodies showed high neutralizing activity against the latest prevalent mutant strain XBB.1.5 and its mutants of Omicron, IC 50 The neutralization activity against the main epidemic mutant strains of BA.5 and BQ.1.1 was also high, IC 50 Less than 0.03 μg / mL. 13 antibodies still maintained a high neutralization level for D614G (based on the D614G mutation of the Wuhan-Hu-1 sequence, which is widely used as a control for the original strain of the new coronavirus), and their neutralization activity remained at IC 50 Levels below 0.2 μg / mL.

[0247] Example 5: High-throughput yeast display of predicted binding epitopes and escape maps

[0248] We used a site-directed mutagenesis PCR strategy to construct a yeast display library of SARS-nCoV2-RBD mutant proteins with an additional randomly matched 26-nucleotide barcode. The display protein contains a Myc tag, and a unique list of barcodes and mutant sequences was obtained through third-generation sequencing. Protein A magnetic beads were coupled to the antibody to be tested and then incubated with the yeast mutant library, and unbound yeast cells were collected. Non-specific yeast cells that did not express proteins were further removed using anti-Myc magnetic beads, the culture was expanded, and the plasmid was extracted for PCR amplification. The changes in the proportion of mutant sequences before and after screening were compared by second-generation sequencing, and the mutation sites that escaped the sample antibodies were inferred and the antibodies were classified. The specific steps are as follows:

[0249] 5.1 Construction of the SARS-CoV-2 RBD mutation library

[0250] The SARS-nCoV2-RBD yeast display mutant library was constructed using a site-directed mutagenesis PCR strategy based on the experimental method reported by J. Bloom's laboratory. The SARS-nCoV2-RBD sequence generated by gene synthesis was cloned into the pETcon vector and used as a template for site-directed mutagenesis PCR reactions. This generated a yeast library containing 200 amino acid point mutations in the SARS-nCoV2-RBD. A different barcode sequence was randomly inserted into each vector. The resulting yeast library was sequenced using third-generation sequencing, and a mapping table between barcodes and mutant sequences was constructed using the Python program provided by J. Bloom's laboratory.

[0251] 5.2 Magnetic Bead Sorting (MACS)

[0252] First, add antibodies to the Protein A magnetic beads for co-incubation: dilute the pre-washed Protein A magnetic beads to 1.8 mg / mL with PBST (PBS buffer containing 0.02% Tween-20) and dispense into a 96-well deep-well plate, 500 μL per well. Then, add 2 μg of neutralizing antibody and place on a 96-well plate mixer. Incubate at room temperature at 700 rpm for 30 minutes. The antibodies captured by the Protein A magnetic beads are adsorbed by the magnetic rack, and the Protein A magnetic bead-antibody complex is resuspended in PBST for later use.

[0253] The constructed RBD mutant library was thawed and inoculated into SD-CAA medium and cultured with shaking at 30°C for 16-18 hours. 120 OD units were diluted into SG-CAA medium and induced for RBD surface expression. First, 10 OD units of cells were subjected to two rounds of sequential negative selection using Protein A magnetic beads (Thermo Fisher) to isolate yeast cells that did not bind to the antibody adsorbed to Protein A. The Protein A magnetic beads (Thermo Fisher) were washed and resuspended in PBST (PBS containing 0.02% Tween-20). The beads were then incubated with neutralizing antibody at room temperature for 30 minutes. The antibody-bound beads were washed and resuspended in PBST. The induced yeast library was washed and incubated with the antibody-bound beads at room temperature for 30 minutes with stirring. The supernatant was separated and subjected to a second round of negative selection to ensure complete removal of antibody-bound yeast. MYC-tagged RBD positive selection was then performed: anti-c-Myc magnetic beads (Thermo Fisher) were first washed with 1X TBST and resuspended, and then the magnetic beads prepared after two rounds of negative selection were incubated with antibodies against escaped yeast at room temperature for 30 minutes. Yeast bound to the anti-c-Myc magnetic beads were collected, washed with 1X TBST, and grown overnight in SD-CAA to expand the yeast population.

[0254] 5.3 Yeast plasmid extraction and library construction

[0255] Overnight cultures of MACS-sorted antibody escape and ACE2 pre-selected yeast populations were subjected to plasmid extraction using a yeast plasmid extraction kit (ZymoResearch) and PCR was performed using specific primers to amplify the N26 barcode sequence. PCR products were purified using 0.9X Ampure XP beads (Beckman Coulter) and submitted to the Illumina Nextseq 500 platform for single-end 75bp sequencing.

[0256] 5.4 Antibody Cluster Analysis

[0257] The barcodes in the sequencing results are located within the first 16 bases of each single-end reaction. The dms_tools2 package developed by J. Bloom's laboratory, combined with custom scripts, was used to align the sequenced barcodes to a barcode-mutation sequence mapping table to identify escape mutation sequences detected in each sample. Antibody clustering and epitope group identification were performed based on an N×M escape score matrix, where N is the number of antibodies that passed the quality control filter and M is the number of informative sites on the SARS-CoV-2 RBD. Each entry in the matrix Anm represents the total escape score for each mutation at site m in the antibody. The difference between two antibodies was defined by the Pearson correlation coefficient of their escape score vectors, Dij = 1-Corr(Ai,Aj). Sites with at least six escape antibodies (site escape score > 1) were selected for dimensionality reduction and clustering. The cleaned escape matrix was converted to an N×6 feature matrix using multidimensional scaling (MDS) with the various metrics described above using the cmdscale R function. Unsupervised k-medoids clustering was then performed within this six-dimensional antibody feature space. Finally, the Rtsne package is used to generate two-dimensional tSNE embeddings for visualization.

[0258] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region comprises: VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence 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 comprising the amino acid sequence of SEQ ID NO: 3 or comprising 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 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 comprising 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 comprising the amino acid sequence of 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 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 comprising 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 comprises the amino acid sequence shown in SEQ ID NO: 78 or the amino acid sequence corresponding to SEQ ID NO: NO:78 has an amino acid sequence with 1 or 2 amino acid residues substituted, deleted or added; or (3) The heavy chain variable region comprises: VH CDR1 comprising the amino acid sequence of 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 comprising the amino acid sequence of 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 comprising 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 comprising 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 (4) 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 comprising 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 comprising the amino acid sequence of 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 (5) The heavy chain variable region comprises: VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 121 or the amino acid sequence corresponding to SEQ ID NO: NO:121 has an amino acid sequence with 1 or 2 amino acid residues substituted, deleted or added, VH CDR2 comprising the amino acid sequence of 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 comprising 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 comprising 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 comprising 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 (6) 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 comprising 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 comprising the amino acid sequence of 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 (7) The heavy chain variable region comprises: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 211 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 211, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 212, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 213, or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted, or added relative to SEQ ID NO: 213; The light chain variable region comprises: VL CDR1 comprising the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 214, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 215 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 215, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 216, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted, or added relative to SEQ ID NO: 216; or (8) The heavy chain variable region comprises: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 219 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 219, VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 220 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 220, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 221, or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted, or added relative to SEQ ID NO: 221; The light chain variable region comprises: VL CDR1 comprising the amino acid sequence of SEQ ID NO: 222 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 222, VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 223 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 223, and VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 224 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:

224.

2. The isolated antibody or antigen-binding fragment thereof of 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: 79 or a variant thereof; the light chain variable region comprises the sequence shown in SEQ ID NO: 80 or a variant thereof; or (3) 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 (4) 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 (5) 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 (6) 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 (7) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 217 or a variant thereof; the light chain variable region comprises the sequence shown in SEQ ID NO: 218 or a variant thereof; or (8) the heavy chain variable region comprises the sequence shown in SEQ ID NO: 225 or a variant thereof; the light chain variable region comprises the sequence shown in SEQ ID NO: 226 or a variant thereof; in, The variant has one or several amino acid substitutions, deletions or additions, for example 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.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a constant region derived from a 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: 145 and / or a light chain constant region as shown in SEQ ID NO:

146.

4. The isolated antibody or antigen-binding fragment thereof of claim 3, wherein the constant region is not naturally occurring.

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, 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.

6. An isolated nucleic acid molecule encoding the isolated 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 5.

7. The isolated nucleic acid molecule of claim 6, wherein the nucleic acid molecule is an mRNA molecule.

8. The isolated nucleic acid molecule of claim 7, wherein the mRNA molecule is chemically modified.

9. The isolated nucleic acid molecule of claim 7 or 8, wherein the mRNA molecule comprises a heterologous 5' and / or 3' UTR.

10. The isolated nucleic acid molecule of claim 6, wherein the nucleic acid molecule is operably linked to an expression control sequence.

11. An expression vector comprising the isolated nucleic acid molecule of claim 6 or 10.

12. A host cell transformed with the isolated nucleic acid molecule according to any one of claims 6 to 10 or the expression vector according to claim 11.

13. A method for preparing an antibody or an antigen-binding fragment thereof, comprising: (1) culturing the host cell of claim 12 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.

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or any combination thereof.

15. The pharmaceutical composition of claim 14, wherein the antibodies or antigen-binding fragments thereof are more than one, such as two, three, four, five, six, seven or eight.

16. The pharmaceutical composition of claim 15, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

17. A pharmaceutical composition comprising at least one mRNA molecule according to any one of claims 7 to 9.

18. A lipid nanoparticle comprising at least one mRNA molecule according to any one of claims 7 to 9.

19. A pharmaceutical composition comprising the lipid nanoparticles of claim 18.

20. The pharmaceutical composition or lipid nanoparticle of any one of claims 17-19, wherein the mRNA molecule comprises an open reading frame encoding at least one isolated antibody or antigen-binding fragment thereof according to any one of claims 1-5, or a heavy chain variable region and / or a light chain variable region thereof.

21. The pharmaceutical composition or lipid nanoparticle of claim 20, wherein the mRNA molecule comprises an open reading frame encoding at least two isolated antibodies or antigen-binding fragments thereof according to any one of claims 1 to 5, or heavy chain variable regions and / or light chain variable regions thereof.

22. The pharmaceutical composition of any one of claims 14 to 17 or claims 19 to 21, further comprising a pharmaceutically acceptable carrier and / or excipient.

23. A method for preventing and / or treating a disease caused by infection with a novel coronavirus mutant strain, the method comprising administering to a subject an effective amount of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5, or any combination thereof, or the pharmaceutical composition of any one of claims 14-17 or claims 19-22.

24. The method of claim 23, wherein there are more than one, e.g., two, three, four, five, six, seven, or eight, isolated antibodies or antigen-binding fragments thereof of any one of claims 1-5, or mRNA molecules encoding more than one, e.g., two, three, four, five, six, seven, or eight, isolated antibodies or antigen-binding fragments thereof of any one of claims 1-5, or heavy chain variable regions and / or light chain variable regions thereof.

25. The method of claim 24, comprising a combination of any two of the isolated antibodies or antigen-binding fragments thereof according to any one of claims 1 to 5, or mRNA molecules encoding any two of the isolated antibodies or antigen-binding fragments thereof according to any one of claims 1 to 5, or heavy chain variable regions and / or light chain variable regions thereof.

26. Use of the isolated antibody or antigen-binding fragment thereof or any combination thereof according to any one of claims 1 to 5 for the preparation of a drug for diagnosing, preventing or treating diseases caused by infection with a novel coronavirus mutant strain.

27. The use of claim 26, wherein the number of the antibodies or antigen-binding fragments thereof is more than one, such as two, three, four, five, six, seven or eight.

28. The use according to claim 27, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

29. The method of any one of claims 23-25 ​​or the use of any one of claims 26-28, wherein the novel coronavirus Omicron mutant strain comprises one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5-related mutant proteins.

30. The method or use of claim 29, wherein the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.

5.

31. The method or use of claim 30, wherein the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

32. The method or use of claim 30, wherein the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.

33. The method of any one of claims 23-25 ​​or claims 29-32, wherein the subject is a mammal, such as a human.

34. A conjugate comprising the isolated antibody or antigen-binding fragment thereof, or any combination thereof, of any one of claims 1 to 5, and a detectable label attached to the isolated antibody or antigen-binding fragment thereof, or any combination thereof.

35. The conjugate of claim 34, wherein the antibodies or antigen-binding fragments thereof are more than one, such as two, three, four, five, six, seven or eight.

36. The conjugate of claim 35, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

37. The conjugate of any one of claims 34 to 36, 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.

38. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or any combination thereof, or the conjugate according to any one of claims 34 to 37.

39. The kit of claim 38, wherein the kit comprises the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or any combination thereof, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

40. The kit of claim 38 or 39, wherein the antibodies or antigen-binding fragments thereof are more than one, such as two, three, four, five, six, seven or eight.

41. The kit of claim 40, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

42. The kit of any one of claims 39-41, 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.

43. A method for detecting the presence or level of a novel coronavirus Omicron mutant in a sample, comprising: (1) contacting the sample with the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or any combination thereof, or the conjugate according to any one of claims 34 to 37; (2) detecting binding of the antibody or antigen-binding fragment thereof or any combination thereof or the conjugate to the target antigen in the sample; The detection of the binding represents the presence of the novel coronavirus Omicron mutant strain in the sample, or the detection of the strength of the binding represents the level of the novel coronavirus Omicron mutant strain in the sample.

44. The method of claim 43, wherein the antibodies or antigen-binding fragments thereof are more than one, such as two, three, four, five, six, seven or eight.

45. The method of claim 44, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

46. ​​The method of any one of claims 43-45, wherein the sample is a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from the subject.

47. The method of claim 46, wherein the subject is a mammal, such as a human.

48. The method of any one of claims 43-47, wherein the sample is not a sample from a subject, eg, the sample is from a vaccine sample.

49. Use of the isolated antibody or antigen-binding fragment thereof or any combination thereof according to any one of claims 1 to 5 or the conjugate according to any one of claims 34 to 37 in the preparation of a kit for detecting the presence or level of a novel coronavirus Omicron mutant in a sample.

50. The use of claim 49, wherein the number of the antibodies or antigen-binding fragments thereof is more than one, such as two, three, four, five, six, seven or eight.

51. The use according to claim 50, comprising a combination of any two of the antibodies or antigen-binding fragments thereof listed in numbers (1) to (8).

52. The method of any one of claims 43-48 or the use of any one of claims 49-51, wherein the novel coronavirus Omicron mutant strain comprises one or more of BA.1, BA.2, BA.2.75, BA.2.86, BA.2.12.1, BA.5, BQ.1.1, BQ.1.19, XBB, XBB.1.5, XBB.1.5.10, XBB.1.16, HK.3.1, JD.1.1, JF.1, GW.5, BF.7 and XBB.1.5-related mutant proteins.

53. The method or use of claim 52, wherein the XBB.1.5-related mutant protein has a mutation in the RBD region compared to XBB.1.

5.

54. The method or use of claim 53, wherein the mutation is selected from one or more of positions R403, N405, K440, K444, Y449, Y453, L455, F456, K478, A484, and H505.

55. The method or use of claim 54, wherein the XBB.1.5-related mutant protein is selected from one or more of XBB.1.5-L455F, XBB.1.5-L455F+F456L, XBB.1.5-S6, XBB.1.5-S8, XBB.1.5-S12, XBB.1.5-S3 and XBB.1.5-S13.