Anti-gii.2 norovirus antibodies or antigen-binding fragments thereof, and methods of making and using the same

Abstract

The application belongs to the technical field of biology and specifically relates to an anti-GII.2 norovirus antibody or antigen-binding fragment thereof, a preparation method and application thereof. The antibody or antigen-binding fragment thereof can specifically recognize and bind to GII.2 norovirus or VP1 protein thereof, and has good affinity therewith, and has no cross reaction with GI.1, GII.3, GII.4, GII.6 and GII.17 norovirus or VP1 protein thereof. Meanwhile, the antibody or antigen-binding fragment thereof has good blocking activity on GII.2 norovirus VLP. The antibody or antigen-binding fragment thereof can be used for preparing products for diagnosing, preventing and / or treating GII.2 norovirus infection or diseases caused by the GII.2 norovirus, or detecting the presence or level of GII.2 norovirus VP1 protein in a sample.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies against GII.2 norovirus or their antigen-binding fragments, their preparation methods, and applications. Background Technology

[0002] Norovirus (NoV) is a single-stranded positive-strand RNA virus belonging to the Caliciviridae family. It has become a major cause of acute nonbacterial gastroenteritis worldwide. It is highly variable, and noroviruses that cause human infection can be divided into 6 genogroups (GI-GVI), each of which can be further divided into multiple genotypes.

[0003] Human norovirus comprises three open reading frames (ORFs), among which ORF2 encodes the major structural protein VP1, a key determinant of NoV antigens and a focus of NoV research. The major structural protein VP1 encoded by ORF2 includes an N-terminal shell domain (S domain) and a C-terminal protruding domain (P domain), connected by a hinge region. Because the P domain is located on the outside of the viral particle and contains the receptor-binding region and the major antigenic site, the VP1 protein can be packaged into virus-like particles (VLPs) in vitro.

[0004] Currently, there is a lack of specific monoclonal antibodies with blocking activity for the quantitative detection of antigen content in vaccines for various genotypes of human norovirus, especially new genotypes. The common antigen detection method is enzyme-linked immunosorbent assay (ELISA), which requires high antibody specificity and the ability to sensitively identify cross-reactivity with different genotype antigens. Therefore, there is an urgent need to develop antibodies against GII.2 norovirus or their antigen-binding fragments. Summary of the Invention

[0005] The first aspect of the present invention is to provide an antibody against GII.2 type norovirus 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 a multispecific antibody or an antigen-binding fragment thereof.

[0008] The fourth aspect of the present invention aims to provide biomaterials related to the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect.

[0009] The fifth aspect of this invention aims to provide a method for preparing an antibody or antigen-binding fragment thereof according to the first aspect of this invention, a chimeric antigen receptor according to the second aspect, or a multispecific antibody or antigen-binding fragment thereof according to the third aspect.

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

[0011] A seventh aspect of the present invention is to provide a pharmaceutical composition.

[0012] An eighth aspect of the present invention aims to provide a diagnostic or therapeutic reagent kit.

[0013] The object of the ninth 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 multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] A first aspect of the present invention provides an antibody against GII.2 norovirus or an antigen-binding fragment thereof, said antibody against GII.2 norovirus or an antigen-binding fragment thereof comprising:

[0016] 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: 3; or

[0017] 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).

[0018] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0019] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0020] b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 4, HCDR2 having the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 6; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 7, LCDR2 having the amino acid sequence GAT, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 8; or

[0021] 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);

[0022] The CDR is defined according to the IMGT numbering system.

[0023] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0024] c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 9, HCDR2 having the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 11; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 8; or

[0025] 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);

[0026] The CDR is defined according to the Kabat numbering system.

[0027] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0028] d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 14, HCDR2 having the amino acid sequence shown in SEQ ID NO: 15, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 11; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 8; or

[0029] 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);

[0030] The CDR is defined according to the Chothia numbering system.

[0031] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0032] e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 16, HCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 18; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 19, LCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 21; or

[0033] 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);

[0034] The CDR is defined according to the Contact numbering system.

[0035] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0036] In some embodiments, the heavy chain variable region of the anti-GII.2 norovirus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region.

[0037] In some embodiments, 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; further, it includes the framework region of the heavy chain variable region of mouse immunoglobulins, or a mutant thereof.

[0038] In some embodiments, the light chain variable region of the anti-GII.2 norovirus antibody or its antigen-binding fragment further includes the framework region of the light chain variable region.

[0039] In some embodiments, the framework region of the light chain variable region includes the framework region of the light 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; further, it includes the framework region of the light chain variable region of mouse immunoglobulins, or a mutant thereof.

[0040] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0041] 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: 3, 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.

[0042] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment may further include a heavy chain constant region and / or a light chain constant region.

[0043] In some embodiments, the heavy chain constant region may include 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; and may further include at least a portion of the heavy chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0044] In some embodiments, the light chain constant region may include 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; and may further include the light chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0045] In some embodiments, the heavy chain constant region may include a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin.

[0046] In some embodiments, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:25, 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.

[0047] In some embodiments, the light chain constant region may include light chain constant regions derived from κ and λ immunoglobulins.

[0048] In some embodiments, the light chain constant region may include the amino acid sequence shown in SEQ ID NO: 29, 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.

[0049] In some embodiments, the anti-GII.2 norovirus 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.

[0050] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment may include, but is not limited to, monoclonal antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

[0051] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment comprises:

[0052] The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24, 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 comprises the amino acid sequence shown in SEQ ID NO: 28, 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.

[0053] In some embodiments, the N-terminus of the heavy chain further comprises a signal peptide; and / or

[0054] The N-terminus of the light chain also contains a signal peptide.

[0055] In some embodiments, the signal peptide of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23, 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.

[0056] In some embodiments, the signal peptide of the light chain comprises the amino acid sequence shown in SEQ ID NO: 27, 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.

[0057] In some embodiments, the anti-GII.2 norovirus antibody or its antigen-binding fragment specifically binds to the VP1 protein (i.e., the VP1 protein of GII.2 norovirus).

[0058] 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.

[0059] A third aspect of the invention provides a multispecific antibody or an antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the antibody or an antigen-binding fragment thereof of the first aspect of the invention.

[0060] A fourth aspect of the invention provides biological materials related to the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, or the multispecific antibody or antigen-binding fragment thereof of the third aspect, said biological materials comprising any one of n1)-n9):

[0061] n1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, or a multispecific antibody or antigen-binding fragment thereof of the third aspect;

[0062] n2) An expression cassette containing the nucleic acid molecule described in n1);

[0063] n3) A carrier containing the nucleic acid molecule described in n1);

[0064] n4) A carrier containing the expression box described in n2);

[0065] n5) A cell containing the nucleic acid molecules described in n1);

[0066] n6) Cells containing the expression cassette described in n2);

[0067] n7) Cells containing the carrier described in n3);

[0068] n8) Cells containing the carrier described in n4);

[0069] n9) Cells comprising an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, or a multispecific antibody or antigen-binding fragment thereof of the third aspect;

[0070] None of the cells described in n5)-n9) contain reproductive material.

[0071] 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.

[0072] 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 with a heavy chain encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule with a light chain encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0073] In some embodiments, the nucleic acid molecule of the heavy chain encoding the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 30, SEQ ID NO: 31, 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.

[0074] In some embodiments, the nucleic acid molecule of the light chain encoding the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 32, SEQ ID NO: 33, 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.

[0075] 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.

[0076] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0077] 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, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment 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.

[0078] 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.

[0079] A fifth 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, a chimeric antigen receptor from the second aspect, or a multispecific antibody or antigen-binding fragment thereof from the third aspect, obtained by culturing cells from the fourth aspect of the present invention.

[0080] A sixth 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.

[0081] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0082] 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.

[0083] 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.

[0084] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of GII.2 norovirus infection or diseases caused by it.

[0085] 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.

[0086] A seventh 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 multispecific antibody or antigen-binding fragment thereof of the third aspect, a biological material of the fourth aspect, or a conjugate of the sixth aspect; and a pharmaceutically acceptable carrier.

[0087] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0088] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating GII.2 norovirus infection or diseases caused by it.

[0089] 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.

[0090] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0091] 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.

[0092] An eighth 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 multispecific antibody or antigen-binding fragment thereof of the third aspect, a biomaterial of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0093] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0094] In some embodiments, the kit can be used to diagnose GII.2 norovirus infection or the disease it causes, and / or to detect the presence or level of GII.2 norovirus or its VP1 protein in a sample.

[0095] In some embodiments, the kit can be used to prevent and / or treat GII.2 norovirus infection or diseases caused by it.

[0096] A ninth aspect of the present 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 multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in any one of c1)-c4):

[0097] c1) Prepare products for the diagnosis of GII.2 norovirus infection or diseases caused by it;

[0098] c2) To prepare products for the prevention and / or treatment of norovirus type G12.2 infection or diseases caused by it;

[0099] c3) Prepare products for detecting the presence or level of GII.2 norovirus or its VP1 protein in samples;

[0100] c4) Non-diagnostic target detection of the presence or level of GII.2 norovirus or its VP1 protein.

[0101] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0102] In some embodiments, the body fluid includes at least one of blood and lymph.

[0103] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0104] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0105] 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).

[0106] In some implementations, the subject of the test includes humans.

[0107] In this invention, the diseases caused by GII.2 type norovirus infection include acute gastroenteritis.

[0108] The beneficial effects of this invention are:

[0109] This invention provides an antibody against GII.2 norovirus or its antigen-binding fragment, which specifically recognizes and binds to GII.2 norovirus or its VP1 protein, exhibiting good affinity for it and showing no cross-reactivity with GII.1, GII.3, GII.4, GII.6, and GII.17 noroviruses or their VP1 proteins. Simultaneously, it demonstrates good blocking activity against GII.2 norovirus VLP. It can be used to prepare products for the diagnosis, prevention, and / or treatment of GII.2 norovirus infection or diseases caused by it, or for detecting the presence or level of GII.2 norovirus VP1 protein in samples.

[0110] Specifically, the antibody against GII.2 norovirus or its antigen-binding fragment has high specificity and no cross-reactivity with GII.1, GII.3, GII.4, GII.6, GII.17 norovirus or their VP1 protein. It can quantitatively and specifically detect GII.2 from antigens of multiple valences, making it suitable as a quality control method in the preparation of norovirus multivalent vaccines.

[0111] This antibody against GII.2 norovirus or its antigen-binding fragment has blocking activity, BT 50 With a value of 609, it has the potential advantage of preventing and / or treating GII.2 norovirus infection or diseases caused by it, and can be used to prepare related preventive and / or therapeutic drugs;

[0112] This anti-GII.2 norovirus antibody or its antigen-binding fragment has the ability to recognize target antigen conformational epitopes. This characteristic indicates that it can act as a neutralizing antibody and play a key role in the prevention and / or treatment of viral infections. At the same time, in detection methods based on natural antigens (such as ELISA, immunofluorescence, or flow cytometry), conformational epitope antibodies 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

[0113] Figure 1 The results of Western Blot (WB) detection of 15 antibody-binding epitopes are shown.

[0114] Figure 2 The results of purity detection of three antibodies by reduced SDS-PAGE are shown: in the reduced SDS-PAGE, lane 1 is the marker, lane 2 is 22LKM4-1-M001, lane 3 is 22LKM4-1-M150, lane 4 is the marker, and lane 5 is 22LKM4-1-M181. Detailed Implementation

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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's BLAST 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.

[0121] 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).

[0122] 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.

[0123] 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.

[0124] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0125] Example 1. Establishment and screening of a phage library

[0126] The purpose of this embodiment is to screen for phage libraries enriched with antibodies against GII.2 VP1 protein (i.e., VP1 protein of GII.2 norovirus), select monoclonal phages from the enriched libraries for expression, and construct antibodies.

[0127] 1. Animal immunization and serum antibody titer detection

[0128] Five female mice (6-8 weeks old, Balb / C, designated SBI220324A, SBI220324B, SBI220324C, SBI220324D, and SBI220324E) were immunized with GII.2 VP1 protein (purchased from SPAF (Beijing) Biotechnology Co., Ltd.). Each mouse was immunized three times, with each immunization spaced 14 days apart. The dose for each immunization was 50 μg based on the immunogen (GII.2 VP1 protein). For the first immunization, the immunogen was emulsified with an equal volume of complete Fusarium adjuvant (Sigma, catalog number F5881) and administered via multiple subcutaneous injections into the abdomen. Every two weeks, the same dose of immunogen and an equal volume of incomplete Fischer adjuvant (BD Biosciences, catalog number 263910) were mixed to form an emulsion, which was then injected subcutaneously at multiple sites in the abdomen. One week after the third immunization, blood was collected from the orbital venous plexus of mice, incubated overnight at 4°C, and centrifuged at 4000 rpm for 15 min. The supernatant serum was then used for titer analysis. Before immunization, blood was collected from one randomly selected mouse as negative serum.

[0129] The indirect ELISA method was used to detect mouse serum titers as follows: Immunogens (GII.2 VP1 protein) diluted to 5 μg / ml with coating buffer (1×PBS buffer) (10×PBS, Solarbio, catalog number P1022) or decross-free antigen proteins (GI.1 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.6 VP1 protein, GII.17 VP1 protein) were coated onto microplates. 100 μL was added to each well, and the plates were incubated overnight at 4°C. The plates were then washed with washing buffer (1×PBST buffer) (10×PBST, Solarbio, catalog number P1033), and 300 μL of blocking buffer (PBST + 3% skim milk powder) (skim milk powder, Shanghai Sangon Biotech, catalog number A600669-0250) was added to each well, and the plates were blocked at room temperature for 1 h. Wash the plate with washing buffer. Serially dilute the serum using sample dilution buffer (PBST + 1% skim milk powder). Add 100 μL of serially diluted serum sample, negative serum, or sample dilution buffer (Blank). Then add 100 μL of diluted horseradish peroxidase-labeled 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 200 μL / well of chromogenic buffer (Solarbio, catalog number PR1200) and incubate at room temperature in the dark for 12 minutes. Add 50 μL / well of stop solution (Solarbio, catalog number C1058), mix well, and read the OD values ​​using a microplate reader. 450 Value. When serum is diluted 16000 times and coated with GII.2VP1 protein as the antigen, OD 450 The mean value minus Blank > 1.0 was considered the acceptable titer criterion. Results showed that all five mice met the acceptable titer criterion. Among the animals with acceptable titers, the OD proteins of each type of cross-linking antigen were preferentially selected. 450 Mice with smaller mean-Blank differences were selected for phage library construction. The serum of mouse SBI220324D showed the weakest binding to various cross-linking antigen proteins (GI.1 VP1, GII.3 VP1, GII.4 VP1, GII.6 VP1, and GII.17 VP1) compared to other mice; therefore, mouse SBI220324D was chosen for phage library construction. The results of indirect ELISA detection of mouse serum titers are shown in Table 1.

[0130] Table 1. Results of indirect ELISA detection of mouse serum titers

[0131]

[0132]

[0133] 2. Preparation of phage antibody library

[0134] Spleen tissue from mice with serial number SBI220324D was used for phage library construction. RNA was extracted from the spleen tissue using TriPure Isolation Reagent reagent, and cDNA was obtained by reverse transcription using a reverse transcription kit. Using cDNA as a template, the light chain variable region and heavy chain variable region sequences of mouse antibodies were amplified by PCR. The nucleotide sequence encoding scFv was obtained by overlap extension splicing PCR (the linker sequence used in scFv is: SSGGGGSGGGGGGSSRSS, SEQ ID NO: 1). The sequence was then ligated into the phage vector pComb3x by restriction endonuclease Sfil, and the phage display scFv antibody library for immunizing mice was constructed by electroporation into XL1-Blue competent cells. The successfully constructed phage library had a volume of 3.29 × 10⁻⁶. 9 CFU.

[0135] 3. Phage antibody library screening

[0136] The primary antibody library (the phage library obtained in step 2) was used to prepare the original antibody library after infection with helper phages. Immunogen (GII.2 VP1 protein) was coated onto an ELISA plate, and the original antibody library was added for binding. Non-specific binding was removed by washing with 1×PBST. The antigen-bound phages were eluted with elution buffer, amplified and precipitated in *E. coli*, and then enriched for the next round. After 2-4 rounds of adsorption, elution, amplification, and screening, a phage library enriched with positive antibodies against GII.2 VP1 protein was obtained. Single phage clones were selected from the enriched library for expression. Indirect ELISA was used to detect the binding of single phage clones to immunogen (GII.2 VP1 protein) and cross-linked antigens (GII.1 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.6 VP1 protein, GII.17 ... The binding of VP1 protein was investigated to obtain phage library antibody monoclonals that specifically bind to the immunogenic protein (GII.2VP1 protein). Fifteen antibody clones with high specific binding affinity to the immunogenic protein were screened (monoclonal numbers 22LKM4-1-M001-M, 22LKM4-1-M010-M, 22LKM4-1-M015-M, 22LKM4-1-M023-M, 22LKM4-1-M040-M, 22LKM4-1-M082, etc.). The results of phage antibody monoclonal expression detection by indirect ELISA (22LKM4-1-M122-M, 22LKM4-1-M148-M, 22LKM4-1-M149-M, 22LKM4-1-M150-M, 22LKM4-1-M170-M, 22LKM4-1-M181-M, 22LKM4-1-M193-M, 22LKM4-1-M195-M, 22LKM4-1-M214-M) are shown in Table 2. Sequencing analysis was performed on 15 antibody monoclonals to obtain the heavy and light chain variable region sequences. The amino acid sequences of the variable region and CDR of antibody 22LKM4-1-M181 are shown in Table 3.

[0137] Table 2. Results of indirect ELISA detection of phage monoclonal expression

[0138]

[0139] Table 3. Full-length, variable region, and CDR amino acid / nucleotide sequences of the 22LKM4-1-M181 antibody.

[0140]

[0141]

[0142]

[0143] Example 2. Antibody construction, expression, purification, and screening

[0144] The purpose of this embodiment is to construct 15 antibodies, transfect them into HEK293 cells, express and purify them, and then perform ELISA specificity, Western Blot (WB), concentration, purity (SDS-PAGE, SEC-HPLC), titer and blocking activity assays on the antibodies, in order to screen out antibodies with specificity and blocking activity that can recognize the target antigen conformational epitopes from the 15 antibody strains.

[0145] 1. Antibody construction: Based on the variable region amino acid sequences of 15 antibody clones, the heavy and light chain variable regions of the antibodies were constructed into the constant regions of the heavy and light chains of mouse IgG using conventional methods (sequences are shown as SEQ ID NO: 25 and SEQ ID NO: 29, respectively), thus constructing 15 antibodies.

[0146] 2. HEK293 cells were passaged in serum-free CD medium (SMM 293-TI, Beijing Sinocare Medical Technology Co., Ltd.) and the 15 antibody plasmid DNAs to be expressed (taking 22LKM4-1-M181 as an example): the full-length heavy chain nucleotide sequence containing the signal peptide and the full-length light chain nucleotide sequence containing the signal peptide of 22LKM4-1-M181 from Table 3 were inserted into the pcDNA3.1 expression vector (Thermo). In fish (e.g., plasmid DNA expressing the heavy chain of 22LKM4-1-M181 and plasmid DNA expressing the light chain of 22LKM4-1-M181), were obtained and mixed with transfection reagent TF2 (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number STF02) and added to HEK293 cells. Serum-free feed solution 293 (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number M293-SUPI-100) was added on days 1, 3, and 5 post-transfection. Shake-flask culture conditions: 5% CO2, temperature 37℃, shaker speed 175 rpm, cultured for 7 days, and cell slurry was collected.

[0147] 3. Antibody purification: Centrifuge the cell suspension (1000g, 20min) using a benchtop centrifuge (Beckman). Collect the cell supernatant and filter it through a 0.45μM filter membrane (Sartorius, catalog number 17598-K). Use Protein... Affinity chromatography column was used to purify the supernatant of 15 filtered cells, yielding 15 mouse monoclonal candidate antibodies (antibody numbers: 22LKM4-1-M001, 22LKM4-1-M010, 22LKM4-1-M015, 22LKM4-1-M023, 22LKM4-1-M040, 22LKM4-1-M082, 22LKM4-1-M122, 22LKM4-1-M148, 22LKM4-1-M149, 22LKM4-1-M150, 22LKM4-1-M170, 22LKM4-1-M181, 22LKM4-1-M193, 22LKM4-1-M195, 22LKM4-1-M214).

[0148] 4. Antibody screening

[0149] (1) ELISA Specificity Assay: The binding of the purified 15 antibodies to immunogenic proteins and decross-free antigen proteins was detected by indirect ELISA, as follows: Immunogen (GII.2 VP1 protein) and decross-free antigen proteins (GI.1 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.6 VP1 protein, GII.17 VP1 protein) were diluted to 1 μg / ml with coating buffer (1×PBS) and coated with 100 μL per well overnight at 4°C. The plates were washed with washing buffer (1×PBST), and blocking buffer (PBST + 5% skim milk powder) was added and incubated at room temperature for 1 h. The plates were washed with washing buffer, and 100 μL of the 15 monoclonal antibodies diluted to 1 μg / ml were added to each well and incubated at room temperature for 2 h. Wash the plate with washing buffer, add 100 μL of diluted horseradish peroxidase-labeled goat anti-mouse IgG Fc (IGL, catalog number GGFC-90P) to each well, and incubate at room temperature for 1 h. Wash the plate again with washing buffer, add 200 μL / well of chromogenic buffer, and incubate at room temperature in the dark for 20 min. Add 50 μL / well of stop solution, mix well, and read the OD values ​​on a microplate reader. 450 The results showed that all 15 antibodies had good specificity. Except for antibody number 22LKM4-1-M170, the other 14 antibodies had strong binding affinity. The detection data are shown in Table 4.

[0150] Table 4. Experimental results of indirect ELISA detection of antibodies.

[0151]

[0152] (2) Western Blot (WB) detection of antibody binding epitopes: WB was used to detect the binding of 15 antibodies to the immunogenic protein (GII.2 VP1 protein), as follows: GII.2 VP1 protein was denatured and loaded at a sample volume of 30 ng. Electrophoresis was performed at a constant voltage of 100 V, and the membrane was transferred at a constant voltage of 110 V for 90 min using a wet transfer apparatus (Bio-rad). After the transfer, the membrane was immersed in blocking buffer (PBST + 5% skim milk powder) at 4°C overnight. After washing with washing buffer (1×PBST), 15 antibodies were added and incubated at room temperature for 2 h. After washing again, goat anti-mouse IgG (H+L) / HRP (Jackson) was added and incubated at room temperature for 2 h. Finally, the membrane was developed using a chemiluminescence imaging system (ProteinSimple). The results showed that, except for antibody number 22LKM4-1-M195, all purified antibodies failed to recognize the denatured immunogenic protein (GII.2 VP1 protein), i.e., the results were negative. This suggests that the GII.2 VP1 protein epitope recognized by the other 14 antibodies (excluding 22LKM4-1-M195) may be a spatial conformational epitope. The detection data are shown in Table 5. Figure 1 .

[0153] Table 5. Results of Western Blot (WB) Detection of Antibodies

[0154]

[0155] Based on the ELISA specificity and WB detection results, a total of 13 antibodies were screened and obtained (numbered 22LKM4-1-M001, 22LKM4-1-M010, 22LKM4-1-M015, 22LKM4-1-M023, 22LKM4-1-M040, 22LKM4-1-M082, 22LKM4-1-M122, 22LKM4-1-M148, 22LKM4-1-M149, ... Three antibodies (numbered 22LKM4-1-M001, 22LKM4-1-M150, and 22LKM4-1-M214) that met both ELISA specificity and WB negative were selected for concentration, purity, titer, and blocking activity assays. These antibodies (numbered 22LKM4-1-M001, 22LKM4-1-M150, and 22LKM4-1-M181) showed relatively high binding affinity to the immunogenic protein (GII.2VP1 protein).

[0156] (3) Concentration detection: Using a micro spectrophotometer (Thermo Scientific), first add the blank control (1×PBS) to the detection base, lower the sample arm, click Blank, and perform blank calibration. OD 280 and OD 320When the absorbance value is within ±0.015, it indicates that the instrument baseline is stable and the sample can be detected. Then, spot the three antibody samples separately and record the absorbance values. Antibody concentration (mg / ml) = (OD) 280 - OD 320 ) / AU, where AU is the extinction coefficient of IgG (1.414). The antibody concentration detection data are shown in Table 6.

[0157] (4) Purity detection: The purity of the three antibodies was detected by SDS-PAGE and SEC-HPLC, respectively. In the SDS-PAGE purity detection, the stacking gel of reducing electrophoresis was 3.9% and the separating gel was 13%. For sample preparation, 5 μg of sample was added to 5 μl of 4× loading buffer, heated in a water bath at 100℃ for 8 min, and then centrifuged at 10000 rpm. 1×SDS electrode buffer was added to the electrophoresis tank, and the sample solution and protein molecular weight standard (Beyotime, 14.4-116 kDa, non-prestained) were added to the sample well. 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 was performed using an Agilent high-performance liquid chromatograph (HPLC). 40 μg of sample and a marker (Self-Produced, catalog number 215000-0101) were added to the inner tube and placed in the sample tray. The ChemStation online software was opened, and the wash pump accessory was turned on to flush the 10% isopropanol wash solution at a rate of 2-3 drops per minute. The D line was replaced with the SEC mobile phase: 0.2M Na₂HPO₄ (Sinopharm Reagent Company), 0.1M Arginine (Sinopharm Reagent Company), 1% IPA (isopropanol) (Sinopharm Reagent Company), pH 6.5. After removing air bubbles from the tubing, the column was correctly packed. The pump flow rate was set to 0.5 mL / min, and the column was equilibrated with the SEC mobile phase for 40 min. After equilibration, the DAD was illuminated. Once the baseline stabilized, the analysis program was run. After the program finished, the HP-SEC.M method was used for integration, and the data were calculated using the area normalization method. The results showed that the purity of the three antibodies (SDS-PAGE and SEC-HPLC) was greater than 95.0%, indicating high purity. The heavy and light chain molecular weights of the three antibodies were approximately 50 kDa and 25 kDa, respectively, and their structures were intact. The detection data are shown in Table 6. Figure 2 .

[0158] Table 6. Results of antibody concentration and purity detection experiments

[0159]

[0160] (5) Titer assay: The titers of the three antibodies were detected by indirect ELISA as follows: Immunogen (GII.2 VP1 protein) was diluted to 1 μg / ml with coating buffer (1×PBS) and coated with 100 μL per well, incubated overnight at 4°C. The plate was washed with washing buffer (1×PBST), and blocking buffer (PBST + 5% skim milk powder) was added and incubated at room temperature for 1 h. The plate was washed with washing buffer, and 100 μL of each of the three monoclonal antibodies, serially diluted twofold to 64,000 times, was added to each well and incubated at room temperature for 2 h. The plate was washed three times with washing buffer, and 100 μL of diluted Rabbit Anti-Mouse IgG F(ab)2 / HRP (Jackson) was added to each well and incubated at room temperature for 1 h. The plate was washed three times with washing buffer, and 200 μL of chromogenic buffer was added and incubated at room temperature in the dark for 20 min. 50 μL of stop buffer was added to each well, mixed well, and the OD was read on a microplate reader. 450 Value, OD 450 -Blank ≥ cut-off value (mean OD of negative control) 450 A value multiplied by 2.1 indicates a positive result, and the corresponding dilution factor represents the antibody titer. Blank is the negative control (the antibody is replaced with the sample diluent). The results show that all three antibody strains have high titers, and the detection data are shown in Table 7.

[0161] Table 7 Results of Antibody Titer Detection Experiment

[0162]

[0163] (6) Blocking activity assay: The blocking activity of the three antibodies was detected by ELISA as follows: Porcine gastric mucin was diluted to 10 μg / ml with coating buffer (1×PBS), and 100 μl was added to each well of a 96-well microplate. The plate was incubated at 37°C for 2 hours. The plate was washed with washing buffer (1×PBST), and blocking buffer (PBST+1% BSA) was added. The plate was blocked overnight at 2–8°C. Three antibody strains were each diluted 50-fold as the initial dilution, and then serially diluted 2-fold to 6400-fold. Immunogen (GII.2 VP1 protein) and decross-crossing antigen proteins (GI.1 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.6 VP1 protein, and GII.17 VP1 protein) were diluted to working concentrations as VLPs (virus-like particles assembled from in vitro expressed norovirus VP1 protein). GI.1 VP1, GII.4 VP1, and GII.17 VP1 proteins were diluted to 200 ng / ml, GII.3 VP1 and GII.6 VP1 proteins to 500 ng / ml, and GII.2 VP1 protein to 5000 ng / ml. The VLP solutions diluted to working concentrations were added to the diluted antibody solutions at a 1:1 volume ratio and incubated at 37°C for 1 hour (sample group). Wash the ELISA plate with washing buffer. Transfer the antigen-antibody solution after co-incubation to the coated ELISA plate. Set each type of VP1 protein (VLP) as a positive control (using sample dilution to replace the antibody compared to the sample group), and the sample dilution as a blank control (using sample dilution to replace both the antibody and VLPs compared to the sample group). Incubate at 37°C for 1 hour. Wash the ELISA plate with washing buffer again, and add rabbit polyclonal antibodies for the corresponding types (GI.1, GII.2, GII.3, GII.4, GII.6, GII.17) at a 1:5000 dilution. Incubate at 37°C for 1 hour. Wash the ELISA plate with washing buffer again, and add goat anti-rabbit-HRP (Abmart) diluted 1:5000. Incubate at 37°C for 1 hour. Wash the ELISA plate with washing buffer again, and develop the color at room temperature in the dark. After stopping the reaction, read the absorbance value at 450 nm. Inhibition index (%) = (Positive control OD) / (VLP) * (VLP * (VLP) ...) * (VLP) * (VLP)) * (VLP)) * (VLP)) * (VLP)) * (VLP)) * (VLP)) * (VLP)) * (VLP))) * (VLP)) 450 -Sample OD 450 ) / (Positive control OD 450 - Blank control OD 450 ) × 100. After calculating the inhibition index corresponding to each serum dilution gradient, the BT of each antibody is calculated according to dose-response-inhibition statistical analysis. 50 The value represents the highest antibody dilution capable of blocking the binding of 50% of VLPs to the porcine gastric mucin HBGA receptor. Results showed that all three antibodies possessed blocking activity and exhibited no cross-blocking activity with other decross-linked proteins. Among them, 22LKM4-1-M181 showed slightly higher blocking activity, and BT...50 The value is 609. The test data results are shown in Table 8.

[0164] Table 8 Results of antibody blocking activity assay

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] 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 GII.2 norovirus or its antigen-binding fragment, wherein the antibody against GII.2 norovirus or its antigen-binding fragment comprises: The heavy chain variable region VH, which has the amino acid sequence shown in SEQ ID NO: 2, includes HCDR1, HCDR2 and HCDR3; and, having the light chain variable region VL including LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 3; The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-GII.2 norovirus antibody or its antigen-binding fragment includes: b1) A VH comprising the following three CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 4, HCDR2 with the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 6; and / or a VL comprising the following three CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 7, LCDR2 with the amino acid sequence GAT, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 8, wherein the CDRs are defined according to the IMGT numbering system; or c1) A VH comprising the following three CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 9, HCDR2 with the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 11; and / or a VL comprising the following three CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 12, LCDR2 with the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat numbering system; or d1) A VH comprising the following three CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 14, HCDR2 with the amino acid sequence shown in SEQ ID NO: 15, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 11; and / or a VL comprising the following three CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 12, LCDR2 with the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Chothia numbering system; or e1) A VH comprising the following three CDRs: HCDR1 with the amino acid sequence shown in SEQ ID NO: 16, HCDR2 with the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 18; and / or a VL comprising the following three CDRs: LCDR1 with the amino acid sequence shown in SEQ ID NO: 19, LCDR2 with the amino acid sequence shown in SEQ ID NO: 20, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 21, wherein the CDRs are defined according to the Contact numbering system.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain variable region of the anti-GII.2 norovirus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region; and / or The light chain variable region of the anti-GII.2 norovirus antibody or its antigen-binding fragment also includes the framework region of the light chain variable region.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and / or The framework region of the light chain variable region includes the framework region of the light chain variable region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The anti-GII.2 norovirus antibody or its antigen-binding fragment includes: The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 2; and / or, the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 3; or The anti-GII.2 norovirus antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region includes at least a portion of the heavy chain constant region derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; or The light chain constant region includes at least a portion of the light chain constant region derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; or The heavy chain constant region includes heavy chain constant regions derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM immunoglobulins; or The light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins; or The heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:25; or The light chain constant region includes the amino acid sequence shown in SEQ ID NO: 29; or The antibody against GII.2 norovirus or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; or The anti-GII.2 norovirus antibody or its antigen-binding fragment includes monoclonal antibodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv or dsFv; or The anti-GII.2 norovirus antibody or its antigen-binding fragment includes: The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24; And / or, a light chain comprising the amino acid sequence shown in SEQ ID NO:

28.

7. A biological material relating to the antibody or antigen-binding fragment thereof according to any one of claims 1-6, said biological material comprising 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 1-6; 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 comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-6; None of the cells described in n5)-n9) contain reproductive material.

8. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is obtained by culturing the cells described in claim 7.

9. A conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-6; and a conjugation portion; The coupling portion is a detectable marker.

10. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the biological material according to claim 7; And pharmaceutically acceptable carriers.

11. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-6, the biological material according to claim 7, the conjugate according to claim 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 1-6, the biomaterial according to claim 7, the conjugate according to claim 9, or the pharmaceutical composition according to claim 10 in any one of c1)-c4): c1) Prepare products for the diagnosis of GII.2 norovirus infection or diseases caused by it; c2) To prepare products for the prevention and / or treatment of norovirus type G12.2 infection or diseases caused by it; c3) Prepare products for detecting the presence or level of GII.2 norovirus or its VP1 protein in samples; c4) Non-diagnostic target detection of the presence or level of GII.2 norovirus or its VP1 protein.

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