Anti-novel coronavirus antibody or antigen binding fragment thereof as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-novel coronavirus antibody or an antigen binding fragment thereof as well as a preparation method and application thereof. The antibody or the antigen binding fragment of the antibody can specifically recognize and bind 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 on wild type SARS-CoV-2, Delta (B.1.617.2), Omicro BA.1 or the RBD protein of the Omicro BA.1; the recombinant plasmid provided by the invention has good neutralizing activity on Omicro BA.4 / 5 type new coronavirus, Omicro XBB.1 type new coronavirus and / or Omicro BQ.1.1 type new coronavirus; the compound can be used for diagnosing, preventing and / or treating novel coronavirus infection or diseases caused by the novel coronavirus infection, detecting existence or level of the novel coronavirus or RBD protein of the novel coronavirus in a sample or developing or screening drugs for preventing and / or treating the novel coronavirus infection or the diseases caused by the novel coronavirus infection.

Description

Technical Field

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

[0002] The novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) is an RNA virus that is prone to mutation during replication. Most mutations do not significantly affect viral function, but occasionally, mutant strains with survival advantages emerge, such as those with stronger transmissibility, greater immune evasion, or altered pathogenicity. When a mutant strain accumulates a series of specific, advantageous mutations and exhibits a clear transmissibility advantage or immune evasion in the population, it is identified as a "variant of concern," such as Delta, Omicron, and its numerous subtypes (BA.1, BA.2, BA.4, BA.5, XBB, JN.1, KP.2, KP.3, etc.). Since the emergence of Omicron, the mutation has shown significant immune evasion, exhibiting a strong ability to evade previous infection and vaccination-induced immunity, leading to an increase in reinfection and breakthrough infections. Omicron has evolved a large number of subtypes with different mutation combinations. The competition among them is fierce, and the replacement of dominant subtypes is accelerated (e.g., from BA.1 to BA.4 / 5, and then to XBB, JN.1, KP.2 / KP.3, etc.). The rapid replacement of subtypes / branches also brings challenges to vaccine development.

[0003] For the development of vaccines against novel coronavirus variants, the quantification of the target antigen is one of the indicators for evaluating its effectiveness. A common method for quantitative detection of the target antigen is enzyme-linked immunosorbent assay (ELISA), which requires high antibody specificity to specifically identify the target antigen. Therefore, there is an urgent need to develop antibodies against the novel coronavirus or their antigen-binding fragments. Summary of the Invention

[0004] The first aspect of the present invention is to provide an antibody against the novel coronavirus or an antigen-binding fragment thereof.

[0005] A second aspect of the present invention is to provide a chimeric antigen receptor.

[0006] A third aspect of the present invention aims to provide biomaterials.

[0007] 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.

[0008] The fifth aspect of this invention aims to provide a coupling.

[0009] The sixth aspect of this invention aims to provide a pharmaceutical composition.

[0010] The seventh aspect of this invention aims to provide a diagnostic or therapeutic reagent kit.

[0011] 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.

[0012] The object of the ninth aspect of this invention is to provide a method for preventing and / or treating novel coronavirus infection or diseases caused by it.

[0013] The object of the tenth aspect of this invention is to provide a method.

[0014] 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 the novel coronavirus or an antigen-binding fragment thereof, said antibody against the 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).

[0015] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0016] In some embodiments, the anti-novel coronavirus antibody 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 RAS, 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.

[0017] In some embodiments, the anti-novel coronavirus antibody 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); The CDR is defined according to the Kabat numbering system.

[0018] In some embodiments, the anti-novel coronavirus antibody 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); The CDR is defined according to the Chothia numbering system.

[0019] In some embodiments, the anti-novel coronavirus antibody 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); The CDR is defined according to the Contact numbering system.

[0020] In some embodiments, the anti-novel coronavirus antibody 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.

[0021] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0022] In some embodiments, the heavy chain variable region of the anti-novel coronavirus antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region.

[0023] 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.

[0024] In some embodiments, the light chain variable region of the anti-novel coronavirus antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0025] 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.

[0026] In some embodiments, the anti-novel coronavirus antibody 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.

[0027] In some embodiments, the anti-novel coronavirus antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0032] In some embodiments, the anti-novel coronavirus antibody 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.

[0033] In some embodiments, the anti-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.

[0034] In some embodiments, the anti-novel coronavirus antibody or its antigen-binding fragment specifically binds to the novel coronavirus receptor-binding domain (RBD) protein.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0048] 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.

[0049] 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.

[0050] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of novel coronavirus infection or the disease caused by it.

[0051] 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.

[0052] 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.

[0053] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0054] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating novel coronavirus infection or the disease it causes.

[0055] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0056] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0057] 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.

[0058] 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.

[0059] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0060] In some embodiments, the kit can be used to diagnose novel coronavirus infection or the disease it causes, detect the presence or level of novel coronavirus or its RBD protein in a sample, and / or develop or screen drugs for the prevention and / or treatment of novel coronavirus infection or the disease it causes.

[0061] In some embodiments, the kit can be used to prevent and / or treat novel coronavirus infection or the disease it causes.

[0062] 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 diagnosing novel coronavirus infection or the disease it causes; c2) Prepare products for the prevention and / or treatment of novel coronavirus infection or the disease it causes; c3) Prepare products for detecting the presence or level of the novel coronavirus or its RBD protein in samples; c4) Detect the presence or level of the novel coronavirus or its RBD protein; c5) Prepare products for drug development or screening, said drug for the prevention and / or treatment of novel coronavirus infection or the disease caused by it; c6) Drug development or screening for the prevention and / or treatment of novel coronavirus infection or the disease caused by it.

[0063] In some implementations, the applications described in c4) and c6) do not involve the diagnosis or treatment of diseases.

[0064] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0065] In some embodiments, the body fluid includes at least one of blood and lymph.

[0066] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0067] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0068] 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).

[0069] In some implementations, the subject of the test includes humans.

[0070] In some embodiments, the product is a pharmaceutical product, a reagent, or a reagent kit.

[0071] A ninth aspect of the present invention provides a method for preventing and / or treating novel coronavirus infection or diseases caused by it, 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.

[0072] 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).

[0073] In some implementations, the subjects include humans.

[0074] 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 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 novel coronavirus infection or the disease it causes; f2) Detect the presence or level of the novel coronavirus or its RBD protein in the sample; f3) Develop or screen drugs for the prevention and / or treatment of novel coronavirus infection or the disease caused by it.

[0075] In some embodiments, the sample is the sample of the eighth aspect of the present invention.

[0076] In this invention, the novel coronavirus includes Omicron BA.2 or its progeny variants; more preferably, it includes Omicron BA.4 / 5, Omicron XBB.1 and / or Omicron BQ.1.1; even more preferably, it includes Omicron BA.4 / 5.

[0077] In this invention, the disease caused by the novel coronavirus infection is selected from one or more of the following: respiratory system infection, acute respiratory syndrome, lung tissue inflammation, gastroenteritis, cough, fever, chills, vomiting, headache, chills, shortness of breath, and cytokine storm.

[0078] The beneficial effects of this invention are: This invention provides an antibody against the novel coronavirus or its antigen-binding fragment, which can specifically recognize and bind to Omicron BA.4 / 5 type novel coronavirus or its RBD protein, and has good affinity for it. It shows no cross-reactivity with wild-type SARS-CoV-2, Delta (B.1.617.2), Omicron BA.1 or its RBD protein; simultaneously, it exhibits good neutralizing activity against Omicron BA.4 / 5, Omicron XBB.1 and / or Omicron BQ.1.1 type novel coronavirus. It can be used for the diagnosis, prevention and / or treatment of novel coronavirus infection or the disease it causes, for detecting the presence or level of novel coronavirus or its RBD protein in samples, or for developing or screening drugs for the prevention and / or treatment of novel coronavirus infection or the disease it causes.

[0079] Specifically, the antibody against the novel coronavirus or its antigen-binding fragment is highly specific and shows no cross-reactivity with SARS-CoV-2, Delta (B.1.617.2), and Omicron BA.1. It can specifically detect Omicron BA.4 / 5 from multiple valence antigens, making it suitable as a quality monitoring method in the preparation of multivalent COVID-19 vaccines. This anti-novel coronavirus antibody or its antigen-binding fragment exhibits strong neutralizing activity against Omicron BA.4 / 5 pseudoviruses, with a neutralizing antibody titer greater than 12150. It also shows some cross-neutralizing activity against Omicron XBB.1 and Omicron BQ.1.1 pseudoviruses. Furthermore, it recognizes the spatial conformational epitopes of the target protein. In detection methods based on natural antigens (such as ELISA, immunofluorescence, or flow cytometry), antibodies with neutralizing activity can specifically bind to the viral protein in its 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

[0080] Figure 1 The results of molecular weight (reduced SDS-PAGE) detection of 21B7H5-A5 antibody are shown: where M is: Marker; 1 is: 21B7H5-A5 antibody.

[0081] Figure 2 The results of the 21B7H5-A5 antibody purity assay (SEC-HPLC) are shown.

[0082] Figure 3AThe results of Western blotting (WB) of the positive control antibody (COVID-19 rabbit polyclonal antibody) are shown: where M is: Marker; 1 is: Omicron BA.4 / 5 RBD protein; 2 is: negative control (Loading Buffer).

[0083] Figure 3B The results of Western blotting (WB) of the 21B7H5-A5 antibody are shown: where M is the marker; 1 is the Omicron BA.4 / 5 RBD protein; and 2 is the negative control (Loading Buffer). Detailed Implementation

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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).

[0091] 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.

[0092] 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.

[0093] Example 1. Cell fusion, screening, and establishment of stable cell lines The purpose of this embodiment is to screen for positive master clones against Omicron BA.4 / 5 using hybridoma cell fusion technology, subclone them using limiting dilution to obtain specific positive monoclonal cell lines, and establish stable cell lines.

[0094] 1. Animal immunization and serum antibody titer detection Five 6-week-old female BALB / c mice (purchased from Vital Rivers) were immunized with Omicron BA.4 / 5 novel coronavirus receptor-binding domain protein (Omicron BA.4 / 5 RBD protein). The initial immunization consisted of 100 μg of protein per mouse, emulsified with an equal volume of Freund's complete adjuvant (Sigma, catalog number F5881), and administered via multiple subcutaneous injections. Two weeks later, a second immunization was performed, with each mouse receiving 50 μg of protein, emulsified with an equal volume of Freund's incomplete adjuvant (Sigma, catalog number F5506), and administered via multiple subcutaneous injections. One week later, a third immunization was performed, with each mouse receiving 50 μg of protein, emulsified with an equal volume of Freund's incomplete adjuvant, and administered via multiple subcutaneous injections. One week after the third immunization, blood samples were collected for serum titer determination using an indirect ELISA method.

[0095] Indirect ELISA was used to detect mouse serum titers. Omicron BA.4 / 5 RBD protein was diluted to 1 μg / ml with carbonate-buffered saline (CBS) and coated onto 96-well microplates. After incubation overnight at 2–8°C, the plates were blocked with 0.1% casein blocking buffer (casein purchased from Sigma, catalog number C8654) at 37°C for 1 h. After washing, mouse serum was serially diluted 1000-fold starting at 1000-fold and then serially diluted 2-fold. Pre-immunization serum was diluted 1000-fold as a pre-immunization serum control. The diluted buffer served as a negative control. The diluted buffer was added to the blocked microplates and incubated at 37°C for 40 min. After washing, HRP-labeled goat anti-mouse IgG (H+L) (Anhui Global Gene Co., Ltd., catalog number US2108) was diluted 1:10000 with antibody diluent and added to the microplates. The plates were incubated at 37°C for 40 min. After washing the plate, add TMB chromogenic buffer (Solarbio, catalog number PR1200), incubate at 37°C for 5 min, and then measure the OD using a microplate reader. 450 The results showed that the serum titers of all five mice were greater than 64,000 after three immunizations, and the OD values ​​of the five mice were all between 2.1 and 2.3 when diluted 64,000 times, with very little difference. Therefore, mice #1 and #2 were selected for cell fusion, and the serum titers of the mice were detected by indirect ELISA, as shown in Table 1.

[0096] Table 1 Results of mouse serum titer detection

[0097] 2. Cell fusion (1) Prepare mouse myeloma cells SP2 / 0 at least one week before fusion, adjust the cell state to the logarithmic growth phase, select mice #1 and #2, and give the mice a booster immunization 3 days before fusion, 50 μg / mouse.

[0098] (2) Fusion: Spleens from mice #1 and #2 were mechanically disrupted, and spleen cells were collected, filtered through a 200-mesh sieve, and washed three times with PBS. SP2 / 0 cells were collected and washed three times with PBS. After cell counting, the cells were mixed at a ratio of SP2 / 0 cells to spleen cells of 1:2.5. After centrifugation, the PBS was discarded, and cell fusion was performed using an electrofusion apparatus. After fusion, the cells were centrifuged and then added to DMEM complete medium containing HAT (HAT purchased from Gibco, catalog number 21060017; fetal bovine serum purchased from Kangyuan Biotechnology, catalog number KY-01000S; DMEM purchased from Gibco, catalog number C11965500BT; penicillin and streptomycin antibiotics purchased from Solarbio, catalog number P1400). The cells were resuspended, mixed, and plated in 96-well plates. The medium was changed every 5 days after fusion.

[0099] 3. Specific monoclonal screening, strain establishment, and antibody subtype identification Positive hybridoma cells were subcloned using the limiting dilution method to obtain monoclonal cell lines. Cell lines that specifically recognized Omicron BA.4 / 5 RBD protein but did not recognize NCP RBD protein and DO RBD protein were screened by indirect ELISA. (NCP RBD protein is the wild-type SARS-CoV-2 RBD protein, and DO RBD protein is a recombinant protein obtained by tandemly fusing the RBD regions of Delta (B.1.617.2) and Omicron BA.1.) After obtaining the specific monoclonal cell lines that meet the requirements, cell lines were established and antibody subtypes were identified. The specific experimental steps are shown below.

[0100] (1) Screening of specific positive master clones Seven days after fusion, the cell culture supernatant was aspirated for indirect ELISA detection (method as in "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 after medium change were retested for Omicron BA.4 / 5 RBD protein and NCP RBD protein and DO RBD protein were screened. Clones with stable OD values ​​were recorded and subcloning was performed.

[0101] (2) Establishment of stable cell lines 1) First Subcloning: Subcloning was performed on wells containing positive cells from the previous step using limiting dilution in DMEM complete medium containing HAT. Cells were seeded at one cell per well in 96-well plates. After 7 days, the cells were observed under a microscope, and wells with single clones were marked. Indirect ELISA was then performed (using the same method as in "1. Animal Immunization and Serum Antibody Titer Detection"). Clones that turned negative were discarded. For positive clones, wells with vigorous growth were selected for a one-half medium change, followed by re-examination and reverse screening the next day. The results showed three specific positive clones: 12G7, 18B11, and 21B7. The OD value of the supernatant from 18B11 cells coated with Omicron BA.4 / 5 RBD protein was lower than that of 12G7 and 21B7, therefore, the 18B11 cell line was not considered. From the 12G7 and 21B7 cell lines, well 21B7-H5, with better cell condition, was selected for secondary subcloning. The results are shown in Table 2.

[0102] Table 2 Results of the first subclone-specific monoclonal cell line screening experiment

[0103] 2) Secondary subcloning: Positive clones 21B7-H5 in good condition were selected from the previous step for secondary subcloning. Screening was performed using limiting dilution with DMEM complete medium containing HAT. Cells were seeded into 96-well plates at a density of one cell per well. After 7 days, the cells were observed under a microscope, and the wells with single clones were marked. Indirect ELISA was performed (using the same method as "1. Animal immunization and serum antibody titer detection"). Clones that turned negative were discarded. After secondary subcloning, the ELISA positivity rate of the specific single clone 21B7 was 100%, meaning that the single clones in the four wells (numbered 21B7H5-C1, 21B7H5-B3, 21B7H5-A5, and 21B7H5-C5) did not bind to NCP RBD and DO RBD proteins. The 21B7H5-A5 cell line in good condition was selected for expansion culture, cryopreservation, and antibody preparation. The screening results are detailed in Table 3.

[0104] Table 3 Results of secondary subcloning-specific monoclonal cell line screening experiments

[0105] (3) Antibody subtype identification Omicron BA.4 / 5 RBD protein was diluted to 1 μg / ml using CBS and coated onto 96-well microplates. After incubation overnight at 2–8°C, the plates were blocked with 0.1% casein blocking buffer (casein purchased from Sigma, catalog number C8654) at 37°C for 1 hour. After washing the plates, cell culture supernatant from a stable 21B7H5-A5 cell line was added, with 7 replicates per cell line, and the plates were incubated at 37°C for 1 hour. After washing the plate, add HRP-labeled goat anti-mouse heavy chain subtype specific secondary antibody (Jackson, IgG1 115-035-205, IgG2a 115-035-206, IgG2b 115-035-207, IgG2c 115-035-208, IgG3 115-035-209, IgM 115-035-075), and a negative control (antibody dilution). Incubate at 37°C for 30 min. After washing the plate again, add TMB chromogenic buffer and incubate at 37°C for 5 min. After incubation, measure the OD using a microplate reader. 450 Antibody subtype identification results showed that the antibody produced by the 21B7H5-A5 cell line was IgG1, as shown in Table 4.

[0106] Table 4 Antibody Subtype Identification

[0107] Example 2: Antibody purification and detection The purpose of this embodiment is to produce 21B7H5-A5 antibody and to test the antibody concentration, specificity, titer, molecular weight (SDS-PAGE), purity (SEC-HPLC), Western blot, pseudovirus neutralizing activity, and affinity to obtain qualified antibody.

[0108] 1. Preparation of ascites The 21B7H5-A5 monoclonal cell line was selected and used as an immunogen after culture, at a dose of 1×10⁻⁶. 6 One cell per mouse was injected intraperitoneally. Ten days later, the mice were sacrificed after significant peritoneal distension. Ascites fluid was collected and its titer was detected by indirect ELISA (method as in "1. Animal Immunization and Serum Antibody Titer Detection"). The results showed that the titer of Omicron BA.4 / 5 RBD protein was greater than 792000 and it did not bind to either NCP RBD protein or DO RBD protein. Specific results are shown in Table 5.

[0109] Table 5 Results of Ascites Potency Test

[0110] 2. Purification of 21B7H5-A5 antibody 1) Column equilibration: Wash the Protein G purification column with 10 column volumes of deionized water to remove the ethanol protective solution, and then rinse the purification column with 10 column volumes of 0.01M PBS (pH 7.4) to equilibrate the purification column.

[0111] 2) Column chromatography: After centrifugation of ascites fluid, the solution was filtered through a 0.45 μm pore size filter membrane and subjected to column chromatography. Elution was performed using 0.1 M glycine solution (pH 2.5) (glycine purchased from Sigma, catalog number V900144). The purified product was collected and immediately neutralized with 1 M Tris-HCl solution (pH 9.0) (Tris purchased from Sigma, catalog number V900483) to obtain 21B7H5-A5 antibody.

[0112] 3) Dialyze the 21B7H5-A5 antibody with 0.01M PBS (pH 7.4), concentrate it, and store it at ≤-20℃.

[0113] 3. Antibody testing (1) Antibody concentration detection: Using the Nano-500 instrument, click the "Protein A280" icon on the main interface to enter the initial protein detection interface, and select the protein type "IgG". Add 2 μl of blank solution to the lower base, lower the upper base and click "Blank Calibration". Wipe the blank solution off the base with clean, dust-free paper. Add 2 μl of 21B7H5-A5 antibody to the lower base, lower the upper base, and click "Sample Detection". The antibody concentration detected by the Nano-500 is 2 mg / ml.

[0114] (2) Antibody specificity and titer detection: Indirect ELISA was used to detect antibody specificity and titer (method as in "1. Animal immunization and serum antibody titer detection"). Omicron BA.4 / 5 RBD protein, NCP RBD protein and DORBD protein were diluted to 1 μg / ml with PBS and coated onto the ELISA plate overnight at 2-8℃. The next day, blocking buffer was added and the plate was blocked at 37℃ for 2 h. The 21B7H5-A5 antibody was initially diluted 50 times and then serially diluted 2 times to 655-3600 times. The diluted buffer was used as a negative control and added to the ELISA plate and incubated at 37℃ for 1 h. After washing the plate, goat anti-mouse HRP secondary antibody (Merck, catalog number AP308P) diluted 1:5000 was added and incubated at 37℃ for 1 h. After washing the plate, TMB chromogenic solution (Solarbio, catalog number PR1200) was added and incubated at 37℃ for 5 min. After termination, OD was measured using an ELISA reader. 450 Antibody titer is antibody OD 450 The value should not be less than the highest dilution factor corresponding to the cut-off. The results showed that the 21B7H5-A5 antibody had good specificity and did not bind to either NCP RBD or DO RBD, with a titer of 1,638,400. The detection results are shown in Table 6.

[0115] Table 6. Results of antibody specificity and titer testing

[0116] (3) Detection of 21B7H5-A5 antibody molecular weight (SDS-PAGE): Take 21B7H5-A5 antibody and add 5×Loading buffer (Shanghai Chemical Company, catalog number 71008060) at a ratio of 21B7H5-A5 antibody: 5×Loading buffer = 4:1 (volume ratio). Mix well by pipetting, centrifuge, and heat at 100℃ for 5 min. Assemble the gel in the electrophoresis tank, pour in the electrophoresis buffer, add the treated 21B7H5-A5 antibody to the gel wells, and add marker as a control. Cover the electrophoresis tank, ensuring red is at the positive electrode and black is at the negative electrode. Turn on the power and start electrophoresis. Set the initial voltage to 130V, and change it to 160V after 15 minutes. Stop electrophoresis when the bromophenol blue indicator migrates to 0~0.5 cm from the bottom edge of the gel. Turn off the power, carefully remove the gel, and stain. After staining for 30 minutes, destaining solution was added until the gel was colorless and transparent. ImageJ software was then used for molecular weight analysis. Reduced SDS-PAGE results showed that the antibody contained heavy and light chain bands, with the heavy chain around 50 kDa and the light chain around 25 kDa, indicating that the 21B7H5-A5 antibody structure was intact and correctly assembled. The molecular weights of the heavy and light chains were consistent with theoretical values. (See attached image for details.) Figure 1 .

[0117] (4) Purity detection of 21B7H5-A5 antibody (SEC-HPLC): SEC-HPLC was performed using an Agilent Technologies 1260 Infinity II high-performance liquid chromatograph. The mobile phase was 0.2M NaH2PO4 + 0.1M 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. The purity was calculated using the area normalization method. The results showed that the SEC purity of the 21B7H5-A5 antibody was 91.5%. The detection results are shown in […]. Figure 2 .

[0118] (5) Western blotting (WB) detection of 21B7H5-A5 antibody: Omicron BA.4 / 5 RBD protein was denatured with reducing test sample buffer at a loading volume of 1 μg and an initial voltage of 80 V, which was adjusted to 180 V when entering the separating gel. The membrane was transferred at 300 mA for 30 min. After the transfer, the membrane was immersed in blocking buffer (PBST + 5% skim milk powder) at 2~8℃ overnight. After washing with washing buffer (1×PBST), 1 μg / ml of 21B7H5-A5 antibody was added and incubated at 37℃ for 1 h. Incubation with rabbit polyclonal antibody against COVID-19 was used as a positive control. The membrane was then developed using an HRP-DAB colorimetric kit (Tiangen Biotech, catalog number PA110) and photographed. The results showed that the 21B7H5-A5 antibody could not recognize the denatured Omicron BA.4 / 5 RBD protein, and no obvious band was observed. This indicates that the antibody binds to the spatial conformational epitope of the target protein. (See attached results). Figure 3A , 3B .

[0119] (6) Detection of pseudovirus neutralizing activity: The pseudovirus neutralizing activity of 21B7H5-A5 antibody was detected using Omicron BA.4 / 5 (Beijing Yunling Biotechnology, catalog number 80123), SARS-CoV-2 (Beijing Yunling Biotechnology, catalog number 80033), Delta (B.1.617.2) (Beijing Yunling Biotechnology, catalog number 80048), Omicron BA.1 (Beijing Yunling Biotechnology, catalog number 80099), Omicron XBB.1 (Beijing Yunling Biotechnology, catalog number 80136) and Omicron BQ.1.1 (Beijing Yunling Biotechnology, catalog number 80126). In 96-well plates, antibodies were serially diluted using 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 mixture, and a virus control was included. The plates were then incubated in a cell culture incubator (37℃, 5% CO2) for 1 h to neutralize. Huh-7 cells (80%–90% confluence) were removed from the incubator, digested with 0.25% trypsin-EDTA, and then diluted to 2 × 10⁶ cells / well using 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. 4Set 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 and 5% CO2 for 20–28 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 21B7H5-A5 antibody had neutralizing activity against Omicron BA.4 / 5 pseudoviruses, but no neutralizing activity against SARS-CoV-2, Delta (B.1.617.2), or Omicron BA.1 pseudoviruses. It also showed some cross-neutralizing activity against Omicron XBB.1 and Omicron BQ.1.1 pseudoviruses, and was a specific neutralizing antibody against Omicron BA.2 or its progeny variants. Specific results are shown in Table 7.

[0120] Table 7. Results of the pseudovirus neutralization activity assay

[0121] (7) 21B7H5-A5 antibody affinity assay: The 21B7H5-A5 antibody was diluted to 5 μg / ml using HEPES running buffer, and the three analytical proteins (Omicron BA.4 / 5 RBD, NCP RBD, DO RBD) were diluted to 300 nM (18 μg / ml). HEPES running buffer was used as a control. The antibody and analytical proteins were loaded into EP tubes and placed on the reagent rack. 10 mM glycine regeneration solution (pH 1.7) was loaded into EP tubes and placed on the reagent rack. The reagent rack was placed in the Biacore T200 sample compartment. The Biacore T200 control software was opened, the manual program was run, and the corresponding signal values ​​were recorded. The results showed that the 18 μg / ml Omicron BA.4 / 5 RBD protein had a very obvious binding signal compared to the control. When the 18 μg / ml NCP RBD and DO RBD proteins were injected, the binding signals were comparable to the control signals and were both negative (the negative signal was generated by slight dissociation after capture antibody). This indicates that the NCP RBD and DO RBD proteins do not have a specific binding signal with the 21B7H5-A5 antibody. The specific results are shown in Table 8.

[0122] Table 8. Results of Antibody Affinity Detection

[0123] (8) Determination of the amino acid / nucleotide sequence of the variable region and its complementarity-determining region (CDR) of the 21B7H5-A5 antibody. The variable region of the 21B7H5-A5 antibody was sequenced, and its CDR was determined, as shown in Table 9.

[0124] Table 9. Amino acid / nucleotide sequences of the variable region and its CDR of the 21B7H5-A5 antibody.

[0125] In summary, through antibody subtype identification, specificity and titer detection, molecular weight (SDS-PAGE), purity (SEC-HPLC), Western blot (WB), pseudovirus neutralizing antibody detection, and affinity assay, the 21B7H5-A5 antibody subtype was determined to be IgG1. It exhibits high neutralizing activity and titer with OmicronBA.4 / 5 RBD protein, and demonstrates good specificity. SDS-PAGE results showed that the 21B7H5-A5 antibody has a complete and correctly assembled structure, with both heavy and light chain molecular weights consistent with theoretical values. SEC-HPLC results showed an antibody purity of 91.5%. WB results revealed the spatial conformational epitopes of the target protein bound by the antibody. Affinity assays showed a very clear binding signal with Omicron BA.4 / 5 RBD protein, but no specific binding signal with NCP RBD and DO RBD proteins. Therefore, the 21B7H5-A5 antibody is a specific antibody for Omicron BA.4 / 5 types and does not recognize SARS-CoV-2, Delta (B.1.617.2), or Omicron BA.1 types, and can be used as a candidate antibody.

[0126] 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 the novel coronavirus or an antigen-binding fragment thereof, wherein the antibody against the 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 the 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 RAS, 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 the 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 the 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 the 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 the 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 anti-novel coronavirus antibody 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 anti-novel coronavirus antibody 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 the 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 anti-novel coronavirus antibody 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 the novel coronavirus or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the anti-novel coronavirus antibody 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 diagnosing novel coronavirus infection or the disease it causes; c2) Prepare products for the prevention and / or treatment of novel coronavirus infection or the disease it causes; c3) Prepare products for detecting the presence or level of the novel coronavirus or its RBD protein in samples; c4) Detect the presence or level of the novel coronavirus or its RBD protein; c5) Prepare products for drug development or screening, said drug for the prevention and / or treatment of novel coronavirus infection or the disease caused by it; c6) Drug development or screening for the prevention and / or treatment of novel coronavirus infection or the disease caused by it.

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