Anti-gii.6 norovirus antibodies or antigen-binding fragments thereof and methods of making and using the same

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to an anti-GII.6 norovirus antibody or antigen-binding fragment thereof, and a preparation method and application thereof. BACKGROUND

[0002] Human norovirus (NoV) is a single-stranded positive-strand RNA virus of the Caliciviridae family, which has become the main cause of acute non-bacterial gastroenteritis worldwide. Human norovirus is highly variable and can be divided into six genogroups (GI-GVI), and each genogroup can be further divided into multiple genotypes.

[0003] Human norovirus includes three open reading frames (ORF), of which the major structural protein VP1 encoded by ORF2 is the main determinant of NoV antigen and has been the focus of NoV research. The major structural protein VP1 encoded by ORF2 includes an N-terminal shell domain S region and a C-terminal protruding domain P region, which are connected by a hinge region. Since the P domain is located on the outside of the virus particle, it contains a receptor binding region and a major antigenic site, and 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 quantification of antigen content in the development of vaccines for each genotype of human norovirus, especially new genotypes. The common antigen detection method is enzyme-linked immunosorbent assay (ELISA), which requires high specificity of the antibody used in the detection and can sensitively make cross-reactivity of different genotypes of antigens, i.e., can specifically identify different genotypes of antigens. Therefore, it is urgent to develop an anti-GII.6 norovirus antibody or antigen-binding fragment thereof. SUMMARY

[0005] The first aspect of the present application aims to provide an anti-GII.6 norovirus antibody or antigen-binding fragment thereof.

[0006] The second aspect of the present application aims to provide a chimeric antigen receptor.

[0007] The third aspect of the present application aims to provide a multispecific antibody or antigen-binding fragment thereof.

[0008] The fourth aspect of the present application aims to provide a biological material 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.

[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.6 norovirus or an antigen-binding fragment thereof, said antibody against GII.6 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.6 norovirus antibody or its antigen-binding fragment comprises:

[0020] b1) a VH comprising 3 CDRs: a HCDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a HCDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a HCDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and / or, a VL comprising 3 CDRs: a LCDR1 having the amino acid sequence set forth in SEQ ID NO: 7, a LCDR2 having the amino acid sequence WAS, and a LCDR3 having the amino acid sequence set forth in SEQ ID NO: 8; or

[0021] b2) a VH comprising 3 CDRs: a HCDR1, a HCDR2, and a HCDR3 having one or more amino acid substitutions, deletions, or additions compared to the HCDR1, HCDR2, and HCDR3 as set forth in b1); and / or, a VL comprising 3 CDRs: a LCDR1, a LCDR2, and a LCDR3 having one or more amino acid substitutions, deletions, or additions compared to the LCDR1, LCDR2, and LCDR3 as set forth in b1);

[0022] wherein the CDRs are defined according to the IMGT numbering system.

[0023] In some embodiments, the anti-GII.6 norovirus antibody, or antigen binding fragment thereof, comprises:

[0024] c1) a VH comprising 3 CDRs: a HCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, a HCDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and a HCDR3 having the amino acid sequence set forth in SEQ ID NO: 11; and / or, a VL comprising 3 CDRs: a LCDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a LCDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a LCDR3 having the amino acid sequence set forth in SEQ ID NO: 8; or

[0025] c2) a VH comprising 3 CDRs: a HCDR1, a HCDR2, and a HCDR3 having one or more amino acid substitutions, deletions, or additions compared to the HCDR1, HCDR2, and HCDR3 as set forth in c1); and / or, a VL comprising 3 CDRs: a LCDR1, a LCDR2, and a LCDR3 having one or more amino acid substitutions, deletions, or additions compared to the LCDR1, LCDR2, and LCDR3 as set forth in c1);

[0026] wherein the CDRs are defined according to the Kabat numbering system.

[0027] In some embodiments, the anti-GII.6 norovirus antibody, or antigen-binding fragment thereof, comprises:

[0028] d1) a VH comprising a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 14, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 15, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 11; and / or, a VL comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 8; or

[0029] d2) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having one or more amino acid

[0030] wherein the CDRs are defined according to the Chothia numbering system.

[0031] In some embodiments, the anti-GII.6 norovirus antibody, or antigen-binding fragment thereof, comprises:

[0032] e1) a VH comprising a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and / or, a VL comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 20, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 21; or

[0033] e2) a VH comprising a HCDR1, a HCDR2, and a HCDR3 having one or more amino acid

[0034] wherein the CDRs are defined according to the Contact numbering system.

[0035] It will be understood by those skilled in the art that the above amino acid substitutions are conservative substitutions.

[0036] In some embodiments, the heavy chain variable region of the anti-GII.6 norovirus antibody or antigen-binding fragment thereof further comprises a framework region of the heavy chain variable region.

[0037] In some embodiments, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof; further comprising a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, or a mutant thereof.

[0038] In some embodiments, the light chain variable region of the anti-GII.6 norovirus antibody or antigen-binding fragment thereof further comprises a framework region of the light chain variable region.

[0039] In some embodiments, the framework region of the light chain variable region comprises a framework region of a light chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof; further comprising a framework region of a light chain variable region of an immunoglobulin derived from a murine, or a mutant thereof.

[0040] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises:

[0041] a heavy chain variable region (VH) comprising the amino acid sequence set forth 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 thereto; and / or, a light chain variable region (VL) comprising the amino acid sequence set forth 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 thereto.

[0042] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof can further comprise 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.6 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.6 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.6 norovirus antibody or antigen-binding fragment thereof comprises:

[0052] a heavy chain comprising an amino acid sequence set forth 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 thereto; and / or, a light chain comprising an amino acid sequence set forth 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 thereto.

[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 further comprises a signal peptide.

[0055] In some embodiments, the signal peptide of the heavy chain comprises an amino acid sequence set forth 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 thereto.

[0056] In some embodiments, the signal peptide of the light chain comprises an amino acid sequence set forth 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 thereto.

[0057] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof specifically binds to a VP1 protein (i.e., a VP1 protein of GII.6 norovirus).

[0058] In a second aspect of the present application, there is provided a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the antigen binding domain comprising the antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0059] In a third aspect of the application, there is provided a multispecific antibody or antigen binding fragment thereof comprising two or more (e.g. three or four) antigen binding domains, wherein one antigen binding domain comprises an antibody or antigen binding fragment thereof of the first aspect of the application.

[0060] In a fourth aspect of the application, there is provided a biological material associated with an antibody or antigen binding fragment thereof of the first aspect of the application, a chimeric antigen receptor of the second aspect of the application, or a multispecific antibody or antigen binding fragment thereof of the third aspect of the application, the biological material 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 application, a chimeric antigen receptor of the second aspect of the application, or a multispecific antibody or antigen binding fragment thereof of the third aspect of the application;

[0062] n2) an expression cassette comprising the nucleic acid molecule of n1 );

[0063] n3) a vector comprising the nucleic acid molecule of n1 );

[0064] n4) a vector comprising the expression cassette of n2);

[0065] n5) a cell comprising the nucleic acid molecule of n1 );

[0066] n6) a cell comprising the expression cassette of n2);

[0067] n7) a cell comprising the vector of n3);

[0068] n8) a cell comprising the vector of n4);

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

[0070] Any cell of n5)-n9) does not comprise reproductive material.

[0071] It will be understood by a person skilled in the art that nucleotides in a nucleic acid molecule can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimised.

[0072] In some embodiments, the nucleic acid molecule encoding an antibody or antigen binding fragment thereof of the first aspect of the application comprises a nucleic acid molecule encoding a heavy chain of an antibody or antigen binding fragment thereof of the first aspect of the application and a nucleic acid molecule encoding a light chain of an antibody or antigen binding fragment thereof of the first aspect of the application.

[0073] In some embodiments, the nucleic acid molecule encoding the heavy chain of the antibody or antigen-binding fragment thereof of the first aspect of the application 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 thereto.

[0074] In some embodiments, the nucleic acid molecule encoding the light chain of the antibody or antigen-binding fragment thereof of the first aspect of the application 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 thereto.

[0075] In some embodiments, any of the vectors of n3) - n4) can be an expression vector. In some embodiments, the expression vector can comprise a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector can comprise, for example, but not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector can comprise, but not limited to, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0076] In some embodiments, the vector can be selected from a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0077] In some embodiments, any of the cells of n5) - n9) can be a host cell routinely used in the art, as long as the expression vector can stably express the nucleic acid molecule carried thereby as the above-mentioned antibody or antigen-binding fragment thereof, chimeric antigen receptor, or multispecific antibody or antigen-binding fragment thereof of the application. In some embodiments, the host cell can be a prokaryotic cell, for example, which can comprise E. coli, and / or a eukaryotic cell, for example, which can comprise a CHO cell, a HEK293 cell, a BHK cell, an NSO cell, an SP2 / 0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell, a HeLa cell, a Vero cell, an Expi293 cell, a hybridoma cell, a yeast cell, and an insect cell.

[0078] In some embodiments, the cell of any one of n5) - n9) can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, a T cell, an NK cell, a DC cell, and a macrophage. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present application (i.e., is a modified immune cell).

[0079] In a fifth aspect of the present application, there is provided a method of preparing the antibody or antigen-binding fragment thereof of the first aspect of the present application, the chimeric antigen receptor of the second aspect of the present application, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the present application, by culturing the cell of the fourth aspect of the present application.

[0080] In a sixth aspect of the present application, there is provided a conjugate comprising the antibody or antigen-binding fragment thereof of the first aspect of the present application; and, a conjugating moiety.

[0081] In some embodiments, the conjugating moiety can include, but is not limited to, a detectable label or a therapeutic agent.

[0082] In some embodiments, the detectable label can be any substance that can be detected by, for example, fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin reagents (e.g., streptavidin) modified to bind the above labels. In some embodiments, such labels can be suitable for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In some embodiments, the detectable label is selected from the group consisting of a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present application via linkers of varying lengths to reduce potential steric hindrance.

[0083] In some embodiments, the detectable label can include, but is not limited to, an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), a colored substance, biotin, etc.

[0084] In some embodiments, the therapeutic agent can include, for example, but not limited to, a drug for preventing and / or treating GII.6 norovirus infection or a disease caused thereby.

[0085] In some embodiments, the conjugating moiety is selected from a substance capable of improving the biological properties of the antibody, for example, increasing the serum half-life, which can be, for example, a chemical group, such as polyethylene glycol (PEG), methyl, ethyl, or a sugar group.

[0086] In a seventh aspect of the present application, a pharmaceutical composition is provided, which comprises: 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, or the conjugate of the sixth aspect; and a pharmaceutically acceptable carrier.

[0087] In some embodiments, the pharmaceutical composition can further comprise an additional pharmaceutically active agent.

[0088] In some embodiments, the additional pharmaceutically active agent can be a drug having a biological activity, for example, a drug capable of preventing and / or treating GII.6 norovirus infection or a disease caused thereby.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as a mixture.

[0090] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ocularly, topically, and the like.

[0091] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, a suspension, a powder, a tablet, a capsule, a granule, a powder, a pill, a disintegrant, a syrup, a spray, a gel, an emulsion, an injection, an elixir, a lozenge, a suppository, and the like.

[0092] In an eighth aspect of the present application, a diagnostic or therapeutic kit is provided, which comprises: 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.

[0093] In some embodiments, the kit can further comprise an instruction and / or a device for administration.

[0094] In some embodiments, the kit can be used for diagnosing GII.6 norovirus infection or a disease caused thereby, and / or detecting the presence or level of GII.6 norovirus or VP1 protein thereof in a sample.

[0095] In some embodiments, the kit can be used for preventing and / or treating GII.6 norovirus infection or a disease caused thereby.

[0096] In a ninth aspect of the present application, there is provided a 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 of c1) - c4):

[0097] c1) manufacturing a product for diagnosing GII.6 norovirus infection or a disease caused thereby;

[0098] c2) manufacturing a product for preventing and / or treating GII.6 norovirus infection or a disease caused thereby;

[0099] c3) manufacturing a product for detecting the presence or level of GII.6 norovirus or VP1 protein thereof in a sample;

[0100] c4) detecting the presence or level of GII.6 norovirus or VP1 protein thereof for non-diagnostic purposes.

[0101] In some embodiments, the sample is selected from at least one of a bodily fluid, a tissue, a cell, an excretion of a subject to be tested.

[0102] In some embodiments, the bodily fluid comprises at least one of blood, lymph.

[0103] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spot, whole blood.

[0104] In some embodiments, the excretion comprises at least one of urine, feces, tears.

[0105] In some embodiments, the subject to be tested comprises a mammal, such as a human, a non-human primate (e.g., chimpanzee, ape), a rodent (e.g., rat, mouse, guinea pig), a pet (e.g., cat, dog), a livestock (e.g., horse, cow, sheep, pig, rabbit).

[0106] In some embodiments, the subject to be tested comprises a human.

[0107] In the present application, the disease caused by GII.6 norovirus infection comprises acute gastroenteritis.

[0108] The present application has the following beneficial effects:

[0109] The present application provides an anti-GII.6 norovirus antibody or antigen-binding fragment thereof, which can specifically recognize and bind to GII.6 norovirus or VP1 protein thereof, and has good affinity therewith, and has no cross-reaction with GI.1, GII.2, GII.3, GII.4 and GII.17 norovirus or VP1 protein thereof; at the same time, it has good blocking activity on GII.6 norovirus VLP; and it can be used for preparing a product for diagnosing, preventing and / or treating GII.6 norovirus infection or diseases caused thereby, or detecting the presence or level of GII.6 norovirus or VP1 protein thereof in a sample.

[0110] Specifically, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof has high specificity, has no cross-reaction with GI.1, GII.2, GII.3, GII.4 and GII.17 norovirus or VP1 protein thereof, can quantitatively and specifically detect GII.6 from various valence antigens, and is suitable as a quality control means for preparing a norovirus multivalent vaccine;

[0111] The anti-GII.6 norovirus antibody or antigen-binding fragment thereof has blocking activity, BT 50 The value is greater than 50000, which has potential advantages in preventing and / or treating GII.6 norovirus infection or diseases caused thereby, and can be applied to prepare related prevention and / or treatment drugs;

[0112] The anti-GII.6 norovirus antibody or antigen-binding fragment thereof has the ability to recognize the target antigen conformational epitope, which indicates that it can be a neutralizing antibody and play a key role in preventing and / or treating viral infection; at the same time, in a detection method based on natural antigens (such as ELISA, immunofluorescence or flow cytometry), the conformational epitope antibody can specifically bind to the virus protein in the natural state, avoiding false negative results caused by antigen denaturation, which is crucial for the sensitivity and specificity in clinical diagnosis or epidemiological research. BRIEF DESCRIPTION OF DRAWINGS

[0113] Figure 1 The results of Western Blot (WB) detection of 12 antibody binding epitopes are shown.

[0114] Figure 2 The results of reducing SDS-PAGE detection of the purity of 3 antibodies are shown: lane 1 in the reducing SDS-PAGE is Marker, lane 2 is 22LKM4-2-M008, lane 3 is Marker, lane 4 is 22LKM4-2-M152, and lane 5 is 22LKM4-2-M163. 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 percent sequence identity can be calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software programs commonly available to those skilled in the art for online use and download. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing 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 Bioinformatics Institute (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 phage library

[0126] The purpose of this example is to screen the positive antibody enriched phage library against GII.6 VP1 protein (i.e. VP1 protein of GII.6 type norovirus), to pick single clone phage from the enriched library for expression, and to construct the antibody.

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

[0128] Five mice (6-8 weeks old, female, Balb / C, SBI220325A, SBI220325B, SBI220325C, SBI220325D, SBI220325E, respectively) were immunized with GII.6 VP1 protein (purchased from SBI (Beijing) Biotech Co., Ltd.). Each mouse was immunized a total of 3 times, with an interval of 14 days. The single immunization dose was 50 μg of immunogen (GII.6 VP1 protein). The immunogen was emulsified with an equal volume of complete Freund's adjuvant (Sigma, F5881) for the first immunization, and was injected subcutaneously in multiple points on the abdomen. Every two weeks, the same dose of immunogen was emulsified with an equal volume of incomplete Freund's adjuvant (BD, 263910) for injection, and was injected subcutaneously in multiple points on the abdomen. One week after the third immunization, blood was collected from the orbital venous plexus of the mouse, and was allowed to stand at 4°C overnight. The upper serum was collected by centrifugation at 4000 rpm and 4°C for 15 min, and was subjected to titer detection. Before immunization, one mouse was optionally bled to serve as negative serum.

[0129] The mouse serum titer was detected by indirect ELISA method as follows: The enzyme-labeled plate was coated with 100 μL of immunogen protein (GII.6 VP1 protein) or cross-antigen protein (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.17 VP1 protein) diluted to 5 μg / mL with coating solution (1×PBS buffer) (10×PBS, Solarbio, P1022) at 4°C overnight. The plate was washed with washing solution (1×PBST buffer) (10×PBST, Solarbio, P1033), and 300 μL of blocking solution (PBST+3% skim milk) (skim milk, Shanghai Sangon, A600669-0250) was added to each well for 1 h at room temperature. The plate was washed with washing solution, and the serum was gradiently diluted with sample diluent (PBST+1% skim milk). 100 μL of gradiently diluted serum sample, negative serum, or sample diluent (Blank) was added, and 100 μL of diluted secondary antibody, horseradish peroxidase-labeled goat anti-mouse IgG Fc (IGL, GGFC-90P), was added. The mixture was incubated at room temperature for 2 h. The plate was washed with washing solution, 200 μL / well of color developing solution (Solarbio, PR1200) was added, and the mixture was incubated at room temperature for 12 min. 50 μL / well of stop solution (Solarbio, C1058) was added, and the mixture was mixed and then the OD 450 values were read on a microplate reader. The serum was diluted 16,000 times, and the OD 450 average-Blank>1.0 was the qualified standard for titer. The results showed that the titers of the five mice met the qualified standard. Among the animals with qualified titers, the mice with smaller differences in OD 450 average-Blank were selected for phage library construction. The serum of mouse SBI220325D had the weakest binding to each type of cross-antigen protein (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.17 VP1 protein) compared to other mice, and therefore, mouse SBI220325D was selected for phage library construction. The results of indirect ELISA method for detecting mouse serum titer are shown in Table 1.

[0130] Table 1 Experimental results of indirect ELISA method for detecting mouse serum titer

[0131]

[0132]

[0133] 2. Preparation of phage antibody library

[0134] The spleen tissue of the numbered SBI220325D mouse was taken for phage library construction. The TriPure Isolation Reagent reagent was used for RNA extraction of the spleen tissue, and a reverse transcription kit was used for reverse transcription to obtain cDNA. The cDNA was used as a template for PCR amplification of the light chain variable region sequence and the heavy chain variable region sequence of the mouse antibody. The nucleotide sequence encoding the scFv was obtained by overlap extension splicing PCR (the linker sequence used in the scFv was: SSGGGGSGGGGGGSSRSS, SEQ ID NO: 1). Then, the scFv was cleaved by the restriction enzyme Sfil and ligated into the phage vector pComb3x. The XL1-Blue competent cells were electrotransformed to construct the phage display scFv antibody library of the immunized mouse. The library capacity of the successfully constructed phage library was 3.53 x 1010 9 cfu.

[0135] 3. Screening of the phage antibody library

[0136] The primary antibody library (the phage library obtained in step 2) is prepared after being infected with helper phage. The immunogen protein (GII.6 VP1 protein) is coated on an enzyme-labeled plate, the primary antibody library is added for binding, and the non-specific binding is removed by washing with a washing solution (1x PBST). The phage bound to the antigen is eluted with an elution solution, amplified in E. coli, and precipitated before the next round of enrichment. After 2-4 rounds of adsorption, elution, and amplification screening, the phage library enriched with positive antibodies against GII.6 antigen is obtained, and a single clone of phage is selected for expression. The binding of the phage single clone to the immunogen protein (GII.6 VP1 protein) and the cross-antigen protein (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, and GII.17 VP1 protein) is detected by indirect ELISA to obtain the phage library antibody single clone that specifically binds to the immunogen protein. Twelve antibody clones (single clone numbers: 22LKM4-2-M002-M, 22LKM4-2-M003-M, 22LKM4-2-M008-M, 22LKM4-2-M038-M, 22LKM4-2-M083-M, 22LKM4-2-M109-M, 22LKM4-2-M110-M, 22LKM4-2-M116-M, 22LKM4-2-M117-M, 22LKM4-2-M119-M, 22LKM4-2-M152-M, and 22LKM4-2-M163-M) with high specific binding to the immunogen protein are screened, and the results of the expression of the phage antibody single clone are shown in Table 2 by indirect ELISA.

[0137] Table 2 Experimental results of indirect ELISA detection of phage single clone expression

[0138]

[0139] Table 3 Amino acid / nucleotide sequences of the full length, variable region, and CDR of 22LKM4-2-M008 antibody

[0140]

[0141]

[0142]

[0143] Example 2. Construction, expression, purification, and screening of antibodies

[0144] The purpose of this embodiment is to construct 12 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 target antigen conformational epitopes from the 12 antibody strains.

[0145] 1. Antibody construction: Based on the variable region amino acid sequences of 12 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 12 antibodies.

[0146] 2. HEK293 cells were passaged in serum-free CD medium (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number SMM 293-TI). The 12 antibody plasmid DNAs to be expressed (taking 22LKM4-2-M008 as an example) were inserted into the pcDNA3.1 expression vector (Thermomix) using the heavy chain full-length nucleotide sequence containing the signal peptide of 22LKM4-2-M008 and the light chain full-length nucleotide sequence containing the signal peptide of 22LKM4-2-M008, respectively. In fish (a transfection assay), plasmid DNA expressing the heavy chain of 22LKM4-2-M008 and plasmid DNA expressing the light chain of 22LKM4-2-M008 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 solution (1000g, 20min) using a benchtop centrifuge (Beckman), collect the cell supernatant after centrifugation, and filter it through a 0.45μM filter membrane (Sartorius, catalog number 17598-K). The supernatant from the filtered cells of the 12 strains was purified using a Protein A affinity chromatography column to obtain 12 mouse monoclonal candidate antibodies (antibody numbers: 22LKM4-2-M002, 22LKM4-2-M003, 22LKM4-2-M008, 22LKM4-2-M038, 22LKM4-2-M083, 22LKM4-2-M109, 22LKM4-2-M110, 22LKM4-2-M116, 22LKM4-2-M117, 22LKM4-2-M119, 22LKM4-2-M152, and 22LKM4-2-M163).

[0148] 4. Antibody screening

[0149] (1) ELISA Specificity Assay: The binding of the purified 12 antibodies to immunogenic proteins and decross-free antigen proteins was detected by indirect ELISA, as follows: Immunogen (GII.6 VP1 protein) and decross-free antigen proteins (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 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 12 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, except for antibody number 22LKM4-2-M116, the other 11 antibodies had good specificity and 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 12 antibodies to the immunogenic protein (GII.6 VP1 protein), as follows: GII.6 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), 12 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 purified antibodies 22LKM4-2-M008, 22LKM4-2-M152, and 22LKM4-2-M163 could not recognize the denatured immunogenic protein (GII.6 VP1 protein), i.e., the results were negative. This suggests that the GII.6 VP1 protein epitopes recognized by antibodies 22LKM4-2-M008, 22LKM4-2-M152, and 22LKM4-2-M163 may be spatial conformational epitopes. 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 above ELISA specificity and WB test results, a total of 3 antibodies (numbered 22LKM4-2-M008, 22LKM4-2-M152, and 22LKM4-2-M163) were screened and met both ELISA specificity and WB negative. The concentration, purity, titer, and blocking activity of these 3 antibodies were then tested.

[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 320 When 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: the concentrations of the three antibodies 22LKM4-2-M008, 22LKM4-2-M152 and 22LKM4-2-M163 are 0.92 mg / ml, 1.20 mg / ml and 0.68 mg / ml, respectively.

[0157] (4) Purity detection: The purity of the three antibodies was detected by SDS-PAGE and SEC-HPLC respectively. In SDS-PAGE purity testing, the reducing electrophoresis stacking gel was 3.9% and the separating gel was 13%. Sample preparation involved adding 5 μg of sample to 5 μl of 4× loading buffer, heating in a 100℃ water bath for 8 min, followed by centrifugation at 10000 rpm. 1×SDS electrode buffer was added to the electrophoresis tank, and sample solution and protein molecular weight standards (Beyotime, 14.4-116 kDa, unstained) were added to the wells. Electrophoresis was then performed, followed by room temperature staining and destaining until a clean background was obtained for gel imaging analysis. SEC-HPLC detection was performed using a high-performance liquid chromatograph (Agilent Technologies). 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 instrument software ChemStation online was opened, and the wash pump accessory was activated to flush out 10% isopropanol at a rate of 2-3 drops per minute. The D line was then replaced with SEC. The mobile phase consisted of 0.2M Na₂HPO₄ (Sinopharm Reagent Company), 0.1M Arginine (Sinopharm Reagent Company), and 1% IPA (isopropanol) (Sinopharm Reagent Company), pH 6.5. After removing air bubbles from the piping, the column was correctly packed. The pump flow rate was set to 0.5 mL / min, and the column was equilibrated with SEC mobile phase for 40 min. After equilibration, the DAD was illuminated, and the analytical program was run after the baseline stabilized. After the program ended, the data were integrated using the HP-SEC.M method, and the data were calculated using the area normalization method. The results showed that the heavy chain and light chain molecular weights of the three antibodies were approximately 50 kDa and 25 kDa, respectively, and their structures were intact. The antibodies numbered 22LKM4-2-M008 and 22LKM4-2-M152 showed high purity (SDS-PAGE and SEC-HPLC), both greater than 95.0%. The antibody numbered 22LKM4-2-M008 showed even higher purity (SEC-HPLC). 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.6 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 antibodies were each diluted 50-fold as the initial dilution, and then serially diluted 2-fold to 6400-fold. Immunogen (GII.6 VP1 protein) and decross-crossing antigen proteins (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 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. 50The value is the highest dilution of antibody capable of blocking 50% of VLPs from binding to the porcine gastric mucin HBGA receptor. Results showed that antibodies 22LKM4-2-M008 and 22LKM4-2-M152 possessed blocking activity and showed no cross-blocking activity with other decross-linked proteins. Furthermore, both antibodies exhibited high blocking activity. 50 All values ​​are greater than 50,000. The test data results are shown in Table 8.

[0164] Table 8 Results of antibody blocking activity assay

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] Based on the results of ELISA specificity assays, Western Blot (WB) antibody binding epitope assays, concentration assays, purity assays, titer assays, and blocking activity assays, both antibodies numbered 22LKM4-2-M008 and 22LKM4-2-M152 are superior, but antibody number 22LKM4-2-M008 has higher purity (SEC-HPLC). Ultimately, antibody number 22LKM4-2-M008 was selected for application.

[0171] 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.6 norovirus or its antigen-binding fragment, wherein the antibody against GII.6 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.6 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 WAS, 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.6 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.6 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 of immunoglobulins derived 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.6 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.6 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.6 norovirus or its antigen-binding fragment is a murine antibody, chimeric antibody, humanized antibody, or fully human antibody; or The anti-GII.6 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.6 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.6 norovirus infection or diseases caused by it; c2) Prepare products for the prevention and / or treatment of norovirus infection type G1.6 or the diseases caused by it; c3) Prepare products for detecting the presence or level of GII.6 norovirus or its VP1 protein in samples; c4) Non-diagnostic target detection of the presence or level of GII.6 norovirus or its VP1 protein.

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