Anti-Omicro BA.4 / 5 type novel coronavirus antibody or antigen-binding fragment thereof and application of anti-Omicro BA.4 / 5 type novel coronavirus antibody or antigen-binding fragment thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-Omicro BA.4 / 5 type novel coronavirus antibody or an antigen binding fragment thereof and application of the anti-Omicro BA.4 / 5 type novel coronavirus antibody or the antigen binding fragment. The antibody or the antigen binding fragment of the antibody can specifically recognize and bind to Omicro BA.4 / 5 type novel coronavirus or RBD protein of the Omicro BA.4 / 5 type novel coronavirus, has good affinity with the Omicro BA.4 / 5 type novel coronavirus or the RBD protein of the Omicro BA.4 / 5 type novel coronavirus, and has no cross reaction with wild type SARS-CoV-2 or RBD protein of the wild type SARS-CoV-2; meanwhile, the compound has relatively good neutralizing activity on the Omicro BA.4 / 5 type new coronavirus; the kit can be used for diagnosing, preventing and / or treating Omicro BA.4 / 5 type novel coronavirus infection or diseases caused by Omicro BA.4 / 5 type novel coronavirus infection, detecting the existence or level of Omicro BA.4 / 5 type novel coronavirus or RBD protein thereof in a sample or developing or screening drugs for preventing and / or treating Omicro BA.4 / 5 type novel coronavirus infection or diseases caused by Omicro BA.4 / 5 type novel coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies against Omicron BA.4 / 5 novel coronavirus or their antigen-binding fragments and their applications. Background Technology

[0002] On March 11, 2020, the World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) a global pandemic. COVID-19 is a highly contagious infectious disease caused by a severe acute respirator syndrome coronavirus 2 (SARS-CoV-2), which has had a catastrophic impact on the world. As of March 11, 2023, more than 759 million cases of COVID-19 have been confirmed globally, and nearly 6.9 million deaths have been reported.

[0003] SARS-CoV-2 is an enveloped RNA virus that is prone to mutation. Mutation is an inherent characteristic of RNA viruses. 2021 was the period of outbreak of variants of concern (VOCs), with the original strain mutating into VOCs such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). 2022 marked the beginning of the Omicron era, with the emergence of Omicron BA.1 and its subtypes. From 2023 to 2024, the Omicron XBB lineage continued to evolve, resulting in mutants such as Omicron XBB.1.5, Omicron EG.5, and Omicron JN.1. By 2025, the current mainstream strain is FLiRT (KP.2 / KP.3). To address the challenge of the continuous mutation of the novel coronavirus, a "multi-layered synergistic defense" strategy is needed, with the development of iterative vaccines being a core defense measure against the continuous mutation of the novel coronavirus. Specific antibodies against variant strains play an important role in iterative vaccine development and the platformization of iterative vaccine development.

[0004] Currently, no commercially available monoclonal antibody can strictly meet the requirement of "specifically recognizing Omicron BA.4 / 5 RBD while showing no cross-reactivity with wild-type SARS-CoV-2 RBD protein." Therefore, there is an urgent need to develop antibodies against the Omicron BA.4 / 5 novel coronavirus or their antigen-binding fragments. Summary of the Invention

[0005] The first aspect of the present invention aims to provide an antibody against Omicron BA.4 / 5 novel coronavirus or an antigen-binding fragment thereof.

[0006] A second aspect of the present invention is to provide a chimeric antigen receptor.

[0007] A third aspect of the present invention aims to provide biomaterials.

[0008] The fourth aspect of this invention aims to provide a method for preparing the antibody or its antigen-binding fragment of the first aspect of this invention or the chimeric antigen receptor of the second aspect.

[0009] The fifth aspect of this invention aims to provide a coupling.

[0010] The sixth aspect of this invention aims to provide a pharmaceutical composition.

[0011] The seventh aspect of this invention aims to provide a diagnostic or therapeutic reagent kit.

[0012] The object of the eighth aspect of the present invention is to provide the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the biomaterial of the third aspect, the conjugate of the fifth aspect, or the pharmaceutical composition of the sixth aspect.

[0013] The object of the ninth aspect of this invention is to provide a method for preventing and / or treating infection with Omicron BA.4 / 5 novel coronavirus or the disease caused therefrom.

[0014] The object of the tenth aspect of this invention is to provide a method.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an antibody against Omicron BA.4 / 5 novel coronavirus or an antigen-binding fragment thereof, said antibody against Omicron BA.4 / 5 novel coronavirus or an antigen-binding fragment thereof comprising: a1) having HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or having LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 4; or a2) Having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1).

[0016] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0017] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, HCDR2 having the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 7; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 8, LCDR2 having the amino acid sequence YTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or b2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1); The CDR is defined according to the IMGT numbering system.

[0018] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1); The CDR is defined according to the Kabat numbering system.

[0019] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1); The CDR is defined according to the Chothia numbering system.

[0020] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 19; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 20, LCDR2 having the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 22; or e2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1); The CDR is defined according to the Contact numbering system.

[0021] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: f1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 23, HCDR2 having the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or f2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in f1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in f1); The CDR is defined according to the AbM numbering system.

[0022] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0023] In some embodiments, the heavy chain variable region of the anti-Omicron BA.4 / 5 novel coronavirus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region.

[0024] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; more preferably, it includes the framework region of the heavy chain variable region of immunoglobulin derived from mouse or a mutant thereof.

[0025] In some embodiments, the light chain variable region of the anti-Omicron BA.4 / 5 novel coronavirus antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0026] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; more preferably, it includes the framework region of the light chain variable region of immunoglobulin derived from mouse, or a mutant thereof.

[0027] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid 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 with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid 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 with it.

[0028] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0029] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes at least a portion of the heavy chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0030] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes the light chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0031] In some embodiments, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin; more preferably, it includes a heavy chain constant region derived from IgG1 immunoglobulin.

[0032] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0033] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment may be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; more specifically, it may be a murine antibody.

[0034] In some embodiments, the anti-Omicron BA.4 / 5 novel coronavirus antibody or its antigen-binding fragment may include, but is not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

[0035] In some embodiments, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment specifically binds to the Omicron BA.4 / 5 novel coronavirus receptor-binding domain (RBD) protein.

[0036] A second aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof from the first aspect of the invention.

[0037] A third aspect of the invention provides biomaterials related to the antibody or antigen-binding fragment of the first aspect of the invention or the chimeric antigen receptor of the second aspect, said biomaterials comprising any one of n1)-n9): n1) A nucleic acid molecule encoding an antibody or its antigen-binding fragment for the first aspect of the present invention or a chimeric antigen receptor for the second aspect of the present invention; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) Cells containing an antibody or antigen-binding fragment thereof from the first aspect of the present invention or a chimeric antigen receptor from the second aspect of the present invention; None of the cells described in n5)-n9) contain reproductive material.

[0038] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0039] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0040] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 1, or a nucleotide sequence having at least 60%, at least 70%, 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 with it.

[0041] In some embodiments, the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 3, or a nucleotide sequence having at least 60%, at least 70%, 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 with it.

[0042] In some embodiments, any of the vectors n3)-n4) can be expression vectors. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0043] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0044] In some embodiments, any of the cells (n5)-n9) can be host cells conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the antibody or its antigen-binding fragment or chimeric antigen receptor of the present invention. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0045] In some embodiments, any of the cells (n5)-n9) can be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptor (i.e., modified immune cells) described above in this invention.

[0046] A fourth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof from the first aspect of the present invention or a chimeric antigen receptor from the second aspect of the present invention, obtained by culturing cells from the third aspect of the present invention.

[0047] A fifth aspect of the invention provides a conjugate comprising an antibody or an antigen-binding fragment thereof from the first aspect of the invention; and a conjugation portion.

[0048] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0049] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), 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)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention using linkers of different lengths to reduce potential steric hindrance.

[0050] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0051] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused thereby.

[0052] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0053] A sixth aspect of the invention provides a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof of the first aspect of the invention, a chimeric antigen receptor of the second aspect, a biological material of the third aspect, or a conjugate of the fifth aspect; and a pharmaceutically acceptable carrier.

[0054] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0055] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating Omicron BA.4 / 5 novel coronavirus infection or the disease it causes.

[0056] In some embodiments, the antibody or its antigen-binding fragment, chimeric antigen receptor, biomaterial, or conjugate is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0057] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0058] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0059] A seventh aspect of the present invention provides a diagnostic or therapeutic kit comprising: an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a biomaterial of the third aspect, a conjugate of the fifth aspect, or a pharmaceutical composition of the sixth aspect.

[0060] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0061] In some embodiments, the kit can be used to diagnose Omicron BA.4 / 5 SARS-CoV-2 infection or the disease it causes, to detect the presence or level of Omicron BA.4 / 5 SARS-CoV-2 or its RBD protein in a sample, and / or to develop or screen drugs for the prevention and / or treatment of Omicron BA.4 / 5 SARS-CoV-2 infection or the disease it causes.

[0062] In some embodiments, the kit can be used to prevent and / or treat Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom.

[0063] An eighth aspect of the invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, the biomaterial of the third aspect, the conjugate of the fifth aspect, or the pharmaceutical composition of the sixth aspect in any one of c1)-c6): c1) Prepare products for the diagnosis of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes; c2) To prepare products for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes; c3) Prepare products for detecting the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein in samples; c4) Detect the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein; c5) Prepare products for drug development or screening, said drug for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom; c6) Drug development or screening for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom.

[0064] In some implementations, the applications described in c4) and c6) do not involve the diagnosis or treatment of diseases.

[0065] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0066] In some embodiments, the body fluid includes at least one of blood and lymph.

[0067] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0068] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0069] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0070] In some implementations, the subject of the test includes humans.

[0071] In some embodiments, the product is a pharmaceutical product, a reagent, or a reagent kit.

[0072] A ninth aspect of the present invention provides a method for preventing and / or treating infection with Omicron BA.4 / 5 novel coronavirus or the disease caused therefrom, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a biomaterial of the third aspect, a conjugate of the fifth aspect, or a pharmaceutical composition of the sixth aspect.

[0073] In some implementations, the subjects include mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0074] In some implementations, the subjects include humans.

[0075] A tenth aspect of the present invention provides a method comprising, under conditions allowing an antibody or antigen-binding fragment thereof of the first aspect of the present invention to form a complex with the Omicron BA.4 / 5 novel coronavirus RBD protein, contacting a sample with an antibody or antigen-binding fragment thereof of the first aspect of the present invention, and detecting the formation of the complex; The method is used for any one of f1)-f3): f1) Diagnosis of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes; f2) Detect the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein in the sample; f3) Develop or screen drugs for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom.

[0076] In some embodiments, the sample is the sample of the eighth aspect of the present invention.

[0077] In this invention, the Omicron BA.4 / 5 novel coronavirus is the Omicron BA.4 / 5 strain.

[0078] In this invention, the disease caused by Omicron BA.4 / 5 novel coronavirus infection is selected from one or more of the following: respiratory infection, acute respiratory syndrome, lung tissue inflammation, gastroenteritis, cough, fever, chills, vomiting, headache, chills, shortness of breath, cytokine storm.

[0079] The beneficial effects of this invention are: This invention provides an antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment, which can specifically recognize and bind to Omicron BA.4 / 5 novel coronavirus or its RBD protein, and has good affinity for it, with no cross-reactivity to wild-type SARS-CoV-2 or its RBD protein; at the same time, it has good neutralizing activity against Omicron BA.4 / 5 novel coronavirus; it can be used for the diagnosis, prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes, for detecting the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein in samples, or for developing or screening drugs for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes.

[0080] Specifically, the antibody against Omicron BA.4 / 5 type novel coronavirus or its antigen-binding fragment has high specificity, no cross-reactivity with wild-type SARS-CoV-2, and can specifically detect Omicron BA.4 / 5 type from antigens of multiple valences, making it suitable as a quality monitoring method in the preparation of multivalent COVID-19 vaccines; This antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment exhibits strong Omicron BA.4 / 5 pseudovirus neutralizing activity, with a neutralizing antibody titer of 2774. In detection methods based on natural antigens (such as ELISA, immunofluorescence, or flow cytometry), antibodies with neutralizing activity can specifically bind to viral proteins in their natural state, avoiding false negative results caused by antigen denaturation. This is crucial for sensitivity and specificity in clinical diagnosis or epidemiological studies. Attached Figure Description

[0081] Figure 1 The molecular weight (reduced SDS-PAGE) results of the 10-H5-B5 antibody are shown: where MW is the marker and 1 is the 10-H5-B5 antibody.

[0082] Figure 2 The results of the 10-H5-B5 antibody purity assay (SEC-HPLC) are shown. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0084] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0085] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0086] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0087] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0088] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0089] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0090] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0091] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0092] Example 1. Establishment and screening of hybridoma cells The purpose of this embodiment is to screen hybridoma cells enriched with positive antibodies against Omicron BA.4 / 5 antigen, subclone them using the limiting dilution method to obtain monoclonal cell lines, and then establish cell lines after obtaining positive monoclonal cell lines that meet the requirements.

[0093] 1. Animal immunization and serum antibody titer detection Seven mice (purchased from Spifon Biotech (Beijing) Co., Ltd.) aged 6-8 weeks, female, Balb / C, numbered #1, #2, #3, #4, #5, #6, and #7, were immunized with Omicron BA.4 / 5 receptor-binding domain protein (Omicron BA.4 / 5 RBD protein). Each mouse was immunized four times, with each immunization spaced 14 days apart. The initial immunization dose was 50 μg (based on Omicron BA.4 / 5 RBD protein), and the doses for the second, third, and fourth immunizations were 25 μg. One week after the four immunizations, blood was collected from the orbital venous plexus of the mice, incubated overnight at 4°C, and centrifuged at 4000 rpm for 15 min. The supernatant serum was used for titer analysis.

[0094] Indirect ELISA was used to detect mouse serum titers. Immunogen (Omicron BA.4 / 5 RBD protein) and cross-linked antigen-free protein (NCP-RBD protein, which is the RBD protein of wild-type SARS-CoV-2) were diluted to 5 μg / ml in coating buffer (carbonate buffer, CBS) (AtaGenix) to each well of an ELISA plate (Corning, catalog number 42592). 100 μl of each well was added and incubated overnight at 4°C. The plate was washed with washing buffer (1×PBST buffer) (10×PBST, Solarbio, catalog number P1033), and 300 μl of blocking buffer (PBS + 3% BSA (Solarbio, catalog number A8020)) was added to each well and incubated overnight at 4°C. Wash the plate with washing buffer. Serially dilute the serum using sample dilution buffer (PBST + 1% BSA). Add 100 μl of serially diluted serum sample and PBS (Blank, negative control), then add 100 μl of diluted horseradish peroxidase-labeled goat anti-mouse IgG Fc (IGL, catalog number GGFC-90P). Incubate at 37°C for 1 h. Wash the plate again with washing buffer. Add 100 μl / well of chromogenic reagent (Solarbio, catalog number PR1200) and incubate at room temperature in the dark for 5–10 min. Add 100 μl / well of stop solution (Solarbio, catalog number C1058), mix well, and read the OD values ​​using a microplate reader. 450 Value. OD 450 ≥cut-off value (mean OD of negative control) 450 Value × 2.1, mean OD of negative control 450A value < 0.05 is considered positive (calculated as 0.05), and the corresponding dilution factor is the antibody titer. Results showed that all 7 mice achieved titers greater than 128,000; among the 7 mice, the preferred antibody titer was the one with the de-crosslinked antigen protein OD. 450 Cell fusion was performed on mice with lower average values. The serum of mouse #7 showed the weakest binding to the cross-linked antigen protein (NCP-RBD protein) compared to other mice. Therefore, mouse #7 was selected for cell fusion. The serum titer of the mice was detected by indirect ELISA and is shown in Table 1.

[0095] Table 1. Results of indirect ELISA detection of mouse serum titers

[0096] 2. Cell fusion (1) Balb / c mice #7 were fixed, their eyes were removed and blood was collected. They were then euthanized by cervical dislocation and placed in 75% alcohol for at least 30 seconds for disinfection. Whole blood was left to stand at room temperature for 1 hour, then stored overnight at 4°C. The next day, the blood was centrifuged at 3000 rpm for 15 min, and the supernatant serum was collected as a positive control for hybridoma screening. The serum was then aliquoted and stored at -20°C.

[0097] (2) Fix the mouse's limbs with pins, making it face up. Use tweezers and scissors to cut open the epidermis and abdominal muscle layer, separate and remove the spleen. Take three 10 cm diameter culture dishes and add 10 ml of 1640 basic culture medium (WISENT, catalog number 350-000-CL). Rinse the spleen in the culture dishes to remove residual connective tissue on the surface of the spleen (be careful not to tear the spleen capsule). Finally, gently grind the spleen with the ground surfaces of two glass slides to break the spleen capsule, and obtain spleen cells. After filtering the spleen cells through a cell sieve, transfer them to a 50 ml sterile centrifuge tube. Take 10 ml of 1640 basic culture medium and rinse the culture dish repeatedly 2-3 times. After filtering through a cell sieve, transfer the cells to the same centrifuge tube and centrifuge at 1500 rpm for 6 min.

[0098] (3) After centrifugation, discard the supernatant, add 40 ml of 1640 basic culture medium, mix thoroughly to ensure the spleen cell pellet is homogeneous, centrifuge at 1500 rpm for 6 min, and repeat once. Discard the supernatant, add 5 ml of 1640 basic culture medium, resuspend the spleen cell pellet, and mix thoroughly. Take 0.2 ml of the cell suspension, dilute it 20-40 times, and perform spleen cell counting. Place at room temperature before fusion.

[0099] (4) During the intercentrifugation phase of spleen cells, myeloma cells (sp2 / 0 cells) were collected in a 50 ml sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 40 ml of 1640 basal medium was added, the myeloma cell pellet was resuspended, mixed well, and centrifuged at 1000 rpm for 5 min. This operation was repeated once. The supernatant was discarded, and the myeloma cell pellet was resuspended by repeatedly pipetting 10-15 times with 5 ml of 1640 basal medium. Approximately 0.2 ml of cell suspension was taken, diluted 10-20 times, and cell counting was performed. The cells were placed at room temperature before fusion.

[0100] (5) Before fusion, preheat PEG (SIGMA, catalog number P7181) and 1640 basic culture medium in a 37°C water bath. Based on the cell count results, mix the required spleen cells and myeloma cells separately in a 5:1 ratio in a 50ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently tap the centrifuge tube wall to loosen the cell pellet. Place the centrifuge tube in a 37°C water bath and add the preheated PEG (1ml over 1 min) to the cell pellet at a uniform rate. While adding PEG, gently stir with the tip of a pipette while rotating the centrifuge tube, and let stand for 90 s. Add the preheated 1640 basic culture medium at a uniform rate in 3 additions: first add 1ml over 1 min, then add 2ml over 1 min, and finally add 9ml over 3 min, gently stirring while adding, until 40 ml is reached. Place the centrifuge tube in a 37°C water bath and let stand for 3 min. Centrifuge the fused cell suspension at 800 rpm for 5 min, remove the supernatant, add 5 ml of HAT medium, gently pipette the cell pellet 10 times, add an appropriate amount of HAT medium according to the number of spleen cells, pipette mix well, and seed into a 96-well cell culture plate.

[0101] (6) Screening of specific positive master clones: After fusion, the cell culture supernatant was aspirated for indirect ELISA detection (the method is the same as "1. Animal immunization and serum antibody titer detection"). Wells that showed positive binding to Omicron BA.4 / 5 RBD protein were recorded, and the medium was partially changed. The next day, the positive wells that had been changed were retested for Omicron BA.4 / 5 RBD protein and NCP RBD protein were screened again. The clone number of positive hybridoma cells with stable OD values ​​was recorded and subcloning was performed.

[0102] 3. Subcloning screening Positive hybridoma cells were subcloned using the limiting dilution method to obtain monoclonal cell lines. During each subcloning period, 2-3 rounds of indirect ELISA screening were performed (cell lines that specifically recognize the Omicron BA.4 / 5 RBD protein antigen but do not recognize the NCP-RBD protein). After obtaining the required positive monoclonal cell lines, cell lines were established. The specific experimental steps are shown below.

[0103] (1) Preparation of cell suspension: Under a microscope, select positive hybridoma cells with good growth status to prepare cell suspension; accurately count the number of cells in the cell suspension according to the cell counting method, which is generally about 10 cells / ml.

[0104] (2) Place a new 24-well culture plate in a clean bench. Add 900 μl of 15% HT selective medium (SIGMA, catalog number H0137) to wells A1, A2, and A3 (i.e., row 1 of column 3). Mix the hybridoma cells from the limited dilution 24-well culture plate and take 100 μl of the cell suspension. Add this suspension to well A1 of the new 24-well culture plate. Use a 1 ml pipette to repeatedly pipette about 10 times. Then, take 100 μl from well A1 using a single-channel pipette (20-100 μl) and transfer it to well A2. Repeat this process until well A3 is reached.

[0105] (3) Take 120 cells from well A3 and place them into a V-shaped groove. Use a 10ml pipette to aspirate 15% HT selective culture medium into the V-shaped groove twice, so that the total volume of the culture medium in the V-shaped groove is 16ml. Repeat the pipetting and agitation about 8 times. When seeding 96-well culture plates, add 200μl / well to columns 1-6, which is 1.5 cells per well. Add another 6.4ml of 15% HT selective culture medium (SIGMA, catalog number H0137) to the remaining 6.4ml of cell suspension, repeat the pipetting and agitation about 8 times, and add 200μl / well to columns 7-12, which is 0.75 cells per well.

[0106] (4) Place in a 37℃ 8% CO2 incubator and incubate for 5 days. On the seventh day, small cell clones can be seen under an inverted microscope. Mark "1" on the plate cover if there is a single cell clone cluster, and mark "√" on the plate cover if there are two or more cell clone clusters. Record the results and make statistics.

[0107] (5) The culture supernatant can be harvested around day 8-9 for antibody detection.

[0108] (6) Select single-clone growth wells with good positive results, transfer them to 24-well plates for clonal culture or expansion culture.

[0109] The results of indirect ELISA detection of the supernatant titers of the established cell lines are shown in Table 2. A total of 4 clones were successfully established, namely 10-H5-B5, 2-A1-C8, 24-H4-G5 and 32-F2-F7. The NCP-RBD titers of the four cell lines were very low. The 10-H5-B5 monoclonal cell line with the highest RBD titer of Omicron BA.4 / 5 was selected to prepare antibodies.

[0110] Table 2. Results of indirect ELISA assay for cell line supernatant.

[0111] Example 2: Antibody purification and detection The purpose of this embodiment is to produce 10-H5-B5 antibodies and to perform ELISA specificity, antibody subtype, SDS-PAGE, and pseudovirus neutralizing antibody titer detection on the antibodies to determine the specificity and pseudovirus neutralizing activity of the 10-H5-B5 antibodies.

[0112] 1. Preparation of ascites Select the 10-H5-B5 monoclonal cell line and obtain the cell supernatant after culture as the immunogen. Mice that have been injected with Freund's incomplete adjuvant need to be prepared 7-21 days before the cell line injection. The specific method is as follows: Draw Freund's incomplete adjuvant (Sigma, catalog number F5506) into a 2.5 ml or 5 ml syringe. Disinfect the mouse's abdomen with an alcohol swab. Hold the mouse's ears with your left thumb and forefinger, grasp as much of the skin on the back of the neck as possible with your middle and ring fingers, and hold the base of the tail with your little finger. Position the mouse head down, allowing the abdominal organs to slide towards the thoracic cavity to prevent organ damage. Hold the needle between your right index and middle fingers and insert it into the abdominal cavity. Only after puncturing the cavity should you push in the incomplete adjuvant. After injecting 0.5 ml, gently shake and slowly withdraw the needle to prevent leakage. Then adjust the hybridoma cell density to 2 × 10⁻⁶. 6 Cells were collected at a concentration of [number] cells / ml into a 1.5 ml EP tube. Approximately 0.5 ml of cell suspension was drawn using a 2.5 ml syringe. Mice that had previously received Freund's incomplete adjuvant (within 7-21 days) were selected. The mouse's ears were grasped with the thumb and forefinger of the left hand, while the middle and ring fingers grasped as much of the skin on the back of the neck as possible. The little finger held the base of the tail. The mouse was positioned head down, allowing the abdominal organs to slide towards the thoracic cavity, preventing organ damage. The needle was held between the index and middle fingers of the right hand and inserted into the abdominal cavity from one side. The cell suspension was injected only after the puncture site was pierced. After injection, the needle was gently trembled and slowly withdrawn to prevent leakage. It is important that the injection be completed within 30 minutes of cell collection. Ascites fluid can be collected 7-12 days after cell injection. The mice with ascites fluid were euthanized by cervical dislocation. The skin on the lower abdomen was lifted with curved forceps in the left hand, and a small incision was made. The left hand should not be released to prevent ascites fluid leakage. Using your right hand, slowly insert a plastic pipette along the abdominal wall to aspirate the ascites fluid. When you reach the intestines, gently blow on them; do not poke or puncture them to avoid damaging the intestines and abdominal wall, which could affect the ascites fluid. Place the aspirated ascites fluid into a 5ml or 15ml centrifuge tube.

[0113] 2. Purification of 10-H5-B5 antibody Collect 5000g of ascites fluid, centrifuge for 5 min, and collect the supernatant. Add the supernatant to a Protein G (Tiandi Renhe Company, catalog number SA032005) column, and collect the flow-through into a 15 ml centrifuge tube. After loading, wash the column with 10 column volumes of PBS. Elute with 1 ml of 0.1M glycine solution (pH 2.5), and collect the eluent in a 1.5 ml EP tube. Immediately after collection, invert the EP tube to mix, and test the pH of the eluent with pH paper to ensure it is neutral. After elution, add 3 column volumes of 0.1M glycine solution (pH 2.5) to the Protein G column again to thoroughly elute the column, then wash the column with 5 column volumes of dihydrochloric acid, followed by 2 column volumes of 20% ethanol. After the flow-through is complete, tightly cap the bottom of the column, add 2 column volumes of 20% ethanol, and store the column at 4°C. The eluted antibody was centrifuged at 12,000 rpm for 2-3 minutes at room temperature, the supernatant was collected and the precipitate was discarded, and finally a mouse monoclonal antibody (antibody name 10-H5-B5) was obtained.

[0114] 3. Detection of 10-H5-B5 antibodies (1) ELISA Specificity Assay: The binding of purified antibodies to immunogenic proteins and decross-free antigen proteins was detected using an indirect ELISA method. Immunogenic proteins (Omicron BA.4 / 5 RBD protein) and decross-free antigen proteins (NCP-RBD protein) diluted to 5 μg / ml with coating buffer (CBS) (AtaGenix) were coated onto the microplates, 100 μl per well, and incubated overnight at 4°C. The plates were washed with washing buffer (1×PBST buffer) (10×PBST, Solarbio, catalog number P1033), and 300 μl of blocking buffer (PBS + 3% BSA) was added to each well, incubating overnight at 4°C. Wash the plate with washing buffer. Serially dilute the 10-H5-B5 antibody using sample dilution buffer (PBST + 1% BSA). Add 100 μl of the serially diluted 10-H5-B5 antibody sample and PBS (Blank, negative control). Then add 100 μl of the diluted secondary antibody horseradish peroxidase-conjugated goat anti-mouse IgG Fc (IGL, catalog number GGFC-90P). Incubate at room temperature for 2 hours. Wash the plate again with washing buffer. Add 100 μl / well of chromogenic buffer (Solarbio, catalog number PR1200) and incubate at room temperature in the dark for 5–10 minutes. Add 100 μl / well of stop solution (Solarbio, catalog number C1058), mix well, and read the OD values ​​using a microplate reader. 450 Value. OD 450 ≥cut-off value (mean OD of negative control) 450 Value × 2.1, mean OD of negative control 450A value < 0.05 is considered positive (calculated as 0.05), and the corresponding dilution factor is the antibody titer. Results showed that the 10-H5-B5 antibodies exhibited good specificity and strong binding affinity; the detection data are shown in Table 3.

[0115] Table 3. Results of 10-H5-B5 antibody titer assay

[0116] (2) Detection of 10-H5-B5 antibody subtypes: The antibody subtypes of 10-H5-B5 antibodies were detected using an indirect ELISA method. 100 μl of 10-H5-B5 antibody was diluted 1:1000 and coated onto an ELISA plate at 37°C for 2 h. The plate was washed with washing buffer (1×PBST buffer) (10×PBST, Solarbio, catalog number P1033), and 300 μl of blocking buffer (PBS + 3% BSA) was added to each well. The plate was then blocked at 37°C for 1.5 h. Wash the plate with washing buffer, then add 100 μl of the following subtype secondary antibodies: Peroxidase AffiniPure Goat Anti-Mouse IgG, Subclass 1 Specific (Jackson, Catalog No. 115-005-205), Peroxidase AffiniPure Goat Anti-Mouse IgG, Subclass 2a Specific (Jackson, Catalog No. 115-005-206), Peroxidase AffiniPure Goat Anti-Mouse IgG, Subclass 2b Specific (Jackson, Catalog No. 115-005-207), Peroxidase AffiniPure Goat Anti-Mouse IgG, Subclass 2c Specific (Jackson, Catalog No. 115-005-208), and Peroxidase AffiniPure Goat Anti-Mouse IgG, Subclass 3. Specific (Jackson, catalog number 115-005-209), incubated at 37°C for 1 hour. Wash the plate three times with washing buffer, add 100 μl / well of chromogenic reagent (Solarbio, catalog number PR1200), incubate at 37°C in the dark for 10 minutes, add 100 μl / well of stop solution (Solarbio, catalog number C1058), mix well, and read the OD on a microplate reader. 450 The results showed that the 10-H5-B5 antibody subtype was IgG1, as detailed in Table 4.

[0117] Table 4. Results of 10-H5-B5 antibody subtype detection experiments

[0118] (3) Detection of 10-H5-B5 antibody purity: The purity of 10-H5-B5 antibody was detected by reduced SDS-PAGE and SEC-HPLC, respectively. 2 μg of sample was added to 5 μl of 4× loading buffer, heated in a water bath at 100℃, and then centrifuged at 10,000 rpm. 1× SDS electrode buffer was added to the electrophoresis tank, and sample solution and protein molecular weight standard (Thermo Fisher Scientific 26616, 10~180 KD) were added to the sample wells. The power supply was connected for electrophoresis. After electrophoresis, room temperature staining and destaining were performed sequentially until a clean background was obtained for gel imaging analysis. SEC-HPLC analysis was performed using an Agilent Technologies 1260 Infinity II high-performance liquid chromatograph (HPLC). The mobile phase was 0.2 M NaH₂PO₄ + 0.1 M Arginine + 1% IPA (isopropanol) solution (pH 6.5). The sample loading was 50 μg. Isocratic elution was used. The detection wavelength was 280 nm, the flow rate was 0.5 mL / min, the detection time was 30 min, and the column temperature was 25 °C. Purity was calculated using the area normalization method. Results showed that the reduced SDS-PAGE results indicated the presence of heavy and light chain bands, with the heavy chain around 56.8 kDa and the light chain around 23.7 kDa. This indicates that the 10-H₅-B₅ antibody structure was intact and correctly assembled. The molecular weights of the heavy and light chains were consistent with theoretical values. The purity of the 10-H₅-B₅ antibody (SDS-PAGE and SEC-HPLC) was high, both greater than 90.0%. The results are shown in Table 5. Figure 1-2 .

[0119] Table 5. Results of 10-H5-B5 antibody purity detection experiment

[0120] (4) Detection of pseudovirus neutralizing activity: The pseudovirus neutralizing activity of 10-H5-B5 antibody was detected using pseudoviruses from Omicron BA.4 / 5 (catalog number 80123, Beijing Yunling Biotechnology Co., Ltd.), SARS-CoV-2 (catalog number 80033, Beijing Yunling Biotechnology Co., Ltd.), Delta (B.1.617.2) (catalog number 80048, Beijing Yunling Biotechnology Co., Ltd.), Omicron BA.1 (catalog number 80099, Beijing Yunling Biotechnology Co., Ltd.), and Omicron XBB (catalog number 80125, Beijing Yunling Biotechnology Co., Ltd.). In 96-well plates, the antibody was serially diluted with DMEM complete medium (DMEM high glucose medium + 10% FBS + 1% penicillin antibody + 25mM HEPES solution) to a final antibody volume of 100 μl. 50 μl of pseudovirus was added to the antibody to be tested, and a virus control was set up. The plates were placed in a cell culture incubator (37℃, 5% CO2) for 1 h for neutralization. After incubation for 30 minutes, Huh-7 cells can be processed. Remove the pre-prepared Huh-7 cells (with a confluence of 80%–90%) from the incubator, digest the cells with 0.25% trypsin-EDTA, and then dilute the cells to 2 × 10⁶ cells / mL with DMEM complete medium. 5 Cells / ml. After neutralization, add 100 μl of cells to each well of the 96-well plate, making the cell density 2 × 10⁶ cells per well. 4 Set up a cell control. Gently shake the 96-well plate back and forth and side to side to disperse the cells evenly in the wells. Place the 96-well plate in a cell culture incubator and incubate at 37°C with 5% CO2 for 24 hours. After incubation, remove the 96-well plate and use a multichannel pipette to aspirate 150 μl of supernatant from each sample well. Then add 100 μl of luciferase assay reagent and react at room temperature in the dark for 2 minutes. After the reaction, use a multichannel pipette to repeatedly pipette and aspirate the liquid in the reaction wells 6-8 times to ensure complete cell lysis. Aspirate 150 μl of liquid from each well and add it to the corresponding 96-well chemiluminescence assay plate. Place the plate in a chemiluminescence assay device and read the luminescence value immediately after color development. Calculate the neutralizing antibody titer based on the chemiluminescence value. The results showed that the 10-H5-B5 antibody could only be detected with high neutralizing titers using Omicron BA.4 / 5 pseudoviruses, while no neutralizing titers were detected with SARS-CoV-2, Delta (B.1.617.2), Omicron BA.1, and Omicron XBB pseudoviruses at the initial concentrations. This indicates that the 10-H5-B5 antibody has good specificity. The specific results are shown in Table 6.

[0121] Table 6. Results of the test for neutralizing activity of pseudoviruses

[0122] In summary, through pseudovirus neutralizing antibody detection, specificity and titer detection, antibody subtype detection, and purity (SDS-PAGE and SEC-HPLC) analysis, the 10-H5-B5 antibody showed high neutralizing titer and potency against the immunogen (Omicron BA.4 / 5 RBD), with high specificity. The antibody subtype detection result was IgG1. The SDS-PAGE and SEC-HPLC results showed that the 10-H5-B5 antibody had a complete structure and correct assembly, with no abnormal bands except for the target band. The molecular weights of the heavy and light chains were consistent with the theoretical values, and the purity was >90%. Therefore, it was selected as a candidate antibody.

[0123] (5) Determination of the amino acid / nucleotide sequence of the variable region and its complementarity-determining region (CDR) of the 10-H5-B5 antibody. The variable region of the 10-H5-B5 antibody was sequenced, and its CDR was determined, as shown in Table 7.

[0124] Table 7. Variable region and its CDR amino acid / nucleotide sequence of the 10-H5-B5 antibody.

[0125] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody against Omicron BA.4 / 5 novel coronavirus or an antigen-binding fragment thereof, wherein the antibody against Omicron BA.4 / 5 novel coronavirus or an antigen-binding fragment thereof comprises: a1) having HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or having LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 4; or a2) Having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1).

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, HCDR2 having the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 7; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 8, LCDR2 having the amino acid sequence YTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or b2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1); Wherein, the CDR is defined according to the IMGT numbering system; or The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1); Wherein, the CDR is defined according to the Kabat numbering system; or The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1); Wherein, the CDR is defined according to the Chothia numbering system; or The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 19; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 20, LCDR2 having the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 22; or e2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1); Wherein, the CDR is defined according to the Contact numbering system; or The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: f1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 23, HCDR2 having the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 9; or f2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in f1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in f1); The CDR is defined according to the AbM numbering system; Preferably, the heavy chain variable region of the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment further includes the framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; more preferably, it includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, or a mutant thereof. Preferably, the light chain variable region of the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment further includes the framework region of the light chain variable region; Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; more preferably, it includes the framework region of the light chain variable region of immunoglobulin derived from mouse or a mutant thereof.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid 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 with it; and / or, the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid 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 with it; Preferably, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes at least a portion of the heavy chain constant region or a mutant thereof derived from mouse immunoglobulins. Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes the light chain constant region or a mutant thereof derived from mouse immunoglobulins. Preferably, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin; more preferably, it includes a heavy chain constant region derived from IgG1 immunoglobulin. Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the antibody against Omicron BA.4 / 5 novel coronavirus or its antigen-binding fragment includes monoclonal antibodies, bispecific antibodies, multispecific antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

5. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the antibody or an antigen-binding fragment thereof as described in any one of claims 1-4.

6. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1-4 or the chimeric antigen receptor as described in claim 5, wherein the biomaterial comprises any one of n1)-n9): n1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-4 or the chimeric antigen receptor as described in claim 5; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) A cell comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4 or the chimeric antigen receptor as described in claim 5; None of the cells described in n5)-n9) contain reproductive material.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-4 or the chimeric antigen receptor according to claim 5, obtained by culturing the cells according to claim 6.

8. A conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; and a conjugation portion.

9. The coupling according to claim 8, characterized in that, The coupling portion includes a detectable marker or therapeutic agent; Preferably, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin.

10. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor as described in claim 5, the biomaterial as described in claim 6, or the conjugate as described in any one of claims 8-9; and a pharmaceutically acceptable carrier.

11. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to claim 5, the biomaterial according to claim 6, the conjugate according to any one of claims 8-9, or the pharmaceutical composition according to claim 10.

12. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to claim 5, the biomaterial according to claim 6, the conjugate according to any one of claims 8-9, or the pharmaceutical composition according to claim 10 in any one of c1)-c6): c1) Prepare products for the diagnosis of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes; c2) To prepare products for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease it causes; c3) Prepare products for detecting the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein in samples; c4) Detect the presence or level of Omicron BA.4 / 5 novel coronavirus or its RBD protein; c5) Prepare products for drug development or screening, said drug for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom; c6) Drug development or screening for the prevention and / or treatment of Omicron BA.4 / 5 novel coronavirus infection or the disease caused therefrom.

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