Anti-GII.6 norovirus antibody or antigen binding fragment thereof, and preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-GII.6 type norovirus antibody or an antigen binding fragment thereof as well as a preparation method and application thereof. The antibody or the antigen binding fragment thereof can specifically recognize and bind GII.6 type norovirus or VP1 protein thereof, has good affinity with the GII.6 type norovirus or VP1 protein thereof, and has no cross reaction with GI.1, GII.2, GII.3, GII.4 and GII.17 type norovirus or VP1 protein thereof; meanwhile, the GII.6 type norovirus VLP has relatively good blocking activity on the GII.6 type norovirus VLP; the GII.6 type norovirus can be used for preparing products for diagnosing, preventing and / or treating GII.6 type norovirus infection or diseases caused by GII.6 type norovirus infection, or detecting the existence or level of GII.6 type 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 the present invention aims to provide a method for preparing 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.

[0010] The sixth aspect of the present invention aims to provide a conjugate.

[0011] The seventh aspect of the present invention aims to provide a pharmaceutical composition.

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

[0013] The purpose of the ninth aspect of the present invention is to provide an application 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.

[0014] In order to achieve the above object, the technical solution adopted by the present invention is: In a first aspect of the present invention, an anti-GII.6 norovirus antibody or an antigen-binding fragment thereof is provided, wherein the anti-GII.6 norovirus antibody or an antigen-binding fragment thereof comprises: a1) HCDR1, HCDR2 and HCDR3 comprised in a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or, LCDR1, LCDR2 and LCDR3 comprised in a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 3; or a2) HCDR1, HCDR2 and HCDR3 having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or, LCDR1, LCDR2 and LCDR3 having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1).

[0015] In some embodiments, the CDRs are defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering systems.

[0016] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: b1) a VH comprising 3 CDRs: a HCDR1 having an amino acid sequence of SEQ ID NO: 4, a HCDR2 having an amino acid sequence of SEQ ID NO: 5, and a HCDR3 having an amino acid sequence of SEQ ID NO: 6; and / or, a VL comprising 3 CDRs: a LCDR1 having an amino acid sequence of SEQ ID NO: 7, a LCDR2 having an amino acid sequence of WAS, and a LCDR3 having an amino acid sequence of SEQ ID NO: 8; or 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); wherein the CDRs are defined according to the IMGT numbering system.

[0017] In some embodiments, the anti-GII.6 norovirus antibody, or antigen binding fragment thereof, comprises: c1) a VH comprising 3 CDRs: a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, and a HCDR3 having an amino acid sequence of SEQ ID NO: 11; and / or, a VL comprising 3 CDRs: a LCDR1 having an amino acid sequence of SEQ ID NO: 12, a LCDR2 having an amino acid sequence of SEQ ID NO: 13, and a LCDR3 having an amino acid sequence of SEQ ID NO: 8; or 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); wherein the CDRs are defined according to the Kabat numbering system.

[0018] In some embodiments, the anti-GII.6 norovirus antibody, or antigen binding fragment thereof, comprises: d1) a VHcomprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 14, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 15, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 11; and / or, a VLcomprising LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 8; or d2) a VHcomprising HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to the HCDR1, HCDR2, and HCDR3 as set forth in d1); and / or, a VLcomprising LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to the LCDR1, LCDR2, and LCDR3 as set forth in d1); wherein the CDRs are defined according to the Chothia numbering system.

[0019] In some embodiments, the anti-GII.6 norovirus antibody, or antigen binding fragment thereof, comprises: e1) a VHcomprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; and / or, a VLcomprising LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 20, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 21; or e2) a VHcomprising HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to the HCDR1, HCDR2, and HCDR3 as set forth in e1); and / or, a VLcomprising LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to the LCDR1, LCDR2, and LCDR3 as set forth in e1); wherein the CDRs are defined according to the Contact numbering system.

[0020] It will be understood by one of skill in the art that the above amino acid substitutions are conservative substitutions.

[0021] 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 a heavy chain variable region.

[0022] 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, musteline, 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.

[0023] 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 a light chain variable region.

[0024] 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, musteline, 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.

[0025] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising an 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 an 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.

[0026] 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.

[0027] In some embodiments, the heavy chain constant region can comprise at least a portion of a heavy chain constant region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, musteline, chicken, duck, or goose, or a mutant thereof; further comprising at least a portion of a heavy chain constant region of an immunoglobulin derived from a murine, or a mutant thereof.

[0028] In some embodiments, the heavy chain constant region can comprise at least a portion of a heavy chain constant region of an immunoglobulin derived from a mouse, a primate, a bovine, a horse, a pig, a sheep, a goat, a dog, a cat, a rabbit, a camel, an ass, a deer, a mink, a chicken, a duck, or a goose, or a mutant thereof; further comprising a heavy chain constant region of an immunoglobulin derived from a mouse, or a mutant thereof.

[0029] In some embodiments, the heavy chain constant region can comprise a heavy chain constant region derived from an IgAl, an IgA2, an IgD, an IgE, an IgGl, an IgG2, an IgG3, an IgG4, or an IgM immunoglobulin.

[0030] In some embodiments, the heavy chain constant region can comprise an amino acid sequence set forth 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 thereto.

[0031] In some embodiments, the heavy chain constant region can comprise a heavy chain constant region derived from an IgAl, an IgA2, an IgD, an IgE, an IgGl, an IgG2, an IgG3, an IgG4, or an IgM immunoglobulin.

[0032] In some embodiments, the heavy chain constant region can comprise an amino acid sequence set forth 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 thereto.

[0033] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof can be a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; further a mouse-derived antibody.

[0034] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof can comprise, but is not limited to, a monoclonal antibody, a nanobody, a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, a F(ab’)2 fragment, an Fv fragment, a single-chain Fv (scFv), a dsFv, or a Fd fragment.

[0035] In some embodiments, the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: a heavy chain comprising an amino acid sequence as 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 as 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.

[0036] In some embodiments, the N-terminus of the heavy chain further comprises a signal peptide; and / or the N-terminus of the light chain further comprises a signal peptide.

[0037] In some embodiments, the signal peptide of the heavy chain comprises an amino acid sequence as 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.

[0038] In some embodiments, the signal peptide of the light chain comprises an amino acid sequence as 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.

[0039] 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).

[0040] 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.

[0041] In a third aspect of the present 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 of the antigen binding domains comprises the antibody or antigen binding fragment thereof of the first aspect of the present application.

[0042] In a fourth aspect of the application, there is provided a biological material associated with the antibody or antigen-binding fragment thereof of the first aspect of the application, the chimeric antigen receptor of the second aspect of the application, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the application, the biological material comprising any one of n1 )-n9): n1 ) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the application, the chimeric antigen receptor of the second aspect of the application, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the application; n2) an expression cassette comprising the nucleic acid molecule of n1 ); n3) a vector comprising the nucleic acid molecule of n1 ); n4) a vector comprising the expression cassette of n2); n5) a cell comprising the nucleic acid molecule of n1 ); n6) a cell comprising the expression cassette of n2); n7) a cell comprising the vector of n3); n8) a cell comprising the vector of n4); n9) a cell comprising the antibody or antigen-binding fragment thereof of the first aspect of the application, the chimeric antigen receptor of the second aspect of the application, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the application; n5)- n9) do not comprise reproductive material.

[0043] 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.

[0044] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the application comprises a nucleic acid molecule encoding the heavy chain of the antibody or antigen-binding fragment thereof of the first aspect of the application and a nucleic acid molecule encoding the light chain of the antibody or antigen-binding fragment thereof of the first aspect of the application.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In some embodiments, the vector can be selected from a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0049] 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 by the expression vector 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, a NS0 cell, a 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.

[0050] In some embodiments, any of the cells of n5) - n9) can be an immune cell. In some embodiments, the immune cell can comprise, but not limited to, a T cell, a NK cell, a DC cell, and a macrophage. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the application (i.e., a modified immune cell).

[0051] In a fifth aspect of the application, there is provided a method of preparing the antibody or antigen-binding fragment thereof of the first aspect of the application, the chimeric antigen receptor of the second aspect of the application, or the multispecific antibody or antigen-binding fragment thereof of the third aspect of the application, by culturing the cell of the fourth aspect of the application.

[0052] The sixth aspect of the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and a coupling moiety.

[0053] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label or a therapeutic agent.

[0054] In some embodiments, the detectable label can be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical, chemical, or other means. Such labels 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)), acridinium ester compounds, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding to modified avidins (e.g., streptavidin) of the above labels. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label described above can be linked to the antibody or antigen-binding fragment thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0055] In some embodiments, the detectable label may include, but is not limited to, an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), a colored substance, biotin, and the like.

[0056] In some embodiments, the therapeutic agent may include, for example, but not limited to, a drug for preventing and / or treating GII.6 norovirus infection or a disease caused by it.

[0057] In some embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), methyl, ethyl, or sugar group.

[0058] In a seventh aspect of the present application, there is provided a pharmaceutical composition comprising: 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.

[0059] In some embodiments, the pharmaceutical composition can further comprise an additional pharmaceutically active agent.

[0060] 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.

[0061] 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.

[0062] In some embodiments, the pharmaceutical composition can be administered by, for example, parenteral, subcutaneous injection, sublingual, rectal, nasal, intravenous injection, intramuscular injection, oral, ocular, topical, etc.

[0063] 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, etc.

[0064] In an eighth aspect of the present application, there is provided a diagnostic or therapeutic kit comprising: 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.

[0065] In some embodiments, the kit can further comprise an instruction and / or a device for administration.

[0066] 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.

[0067] In some embodiments, the kit can be used for preventing and / or treating GII.6 norovirus infection or a disease caused thereby.

[0068] In a ninth aspect of the present application, there is provided 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) to c4): c1) preparing a product for diagnosing GII.6 norovirus infection or a disease caused thereby; c2) preparing a product for preventing and / or treating GII.6 norovirus infection or a disease caused thereby; c3) preparing a product for detecting the presence or level of GII.6 norovirus or VP1 protein thereof in a sample; c4) detecting the presence or level of GII.6 norovirus or VP1 protein thereof for non-diagnostic purposes.

[0069] 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.

[0070] In some embodiments, the bodily fluid comprises at least one of blood, lymph.

[0071] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spot, whole blood.

[0072] In some embodiments, the excretion comprises at least one of urine, feces, tears.

[0073] 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).

[0074] In some embodiments, the subject to be tested comprises a human.

[0075] In the present application, the disease caused by GII.6 norovirus infection comprises acute gastroenteritis.

[0076] The present application has the following advantages: The present invention provides an anti-GII.6 norovirus antibody or an antigen-binding fragment thereof, which can specifically recognize and bind to GII.6 norovirus or its VP1 protein, has good affinity thereto, and has no cross-reaction to GI.1, GII.2, GII.3, GII.4 and GII.17 norovirus or their VP1 proteins; at the same time, it has good blocking activity against GII.6 norovirus VLP; it can be used to prepare products for diagnosing, preventing and / or treating GII.6 norovirus infection or the disease caused by it, or detecting the presence or level of GII.6 norovirus or its VP1 protein in a sample.

[0077] Specifically, the anti-GII.6 norovirus antibody or its antigen-binding fragment has high specificity, has no cross-reaction to GII.1, GII.2, GII.3, GII.4 and GII.17 norovirus or their VP1 protein, can quantitatively and specifically detect GII.6 from multiple valence antigens, and is suitable as a quality control method for the preparation of norovirus multivalent vaccines; The anti-GII.6 norovirus antibody or its antigen-binding fragment has blocking activity, BT 50 A value greater than 50,000 indicates potential advantages in preventing and / or treating GII.6 norovirus infection or diseases caused by it, and can be used to prepare related preventive and / or therapeutic drugs; This anti-GII.6 norovirus antibody or its antigen-binding fragment has the ability to recognize the conformational epitope of the target antigen. This property indicates that it can become a neutralizing antibody and play a key role in preventing and / or treating 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, which is crucial for sensitivity and specificity in clinical diagnosis or epidemiological studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The results of Western Blot (WB) detection of the binding epitopes of 12 antibodies are shown.

[0079] Figure 2 The results of reducing SDS-PAGE detection of the purity of three antibodies are shown: Lane 1 in 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 DESCRIPTION

[0080] For the purposes of the present invention, the term "about" means ±20% of the value stated. In some embodiments, the term "about" means ±10% of the value stated. In some embodiments, the term "about" means ±5% of the value stated.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are applied to the descriptions of the present application and throughout this document, and apply equally to both the singular and plural form of the terms.

[0082] Unless the context clearly indicates otherwise, as used herein, the singular forms "a," "an" and "the" include plural referents. For example, a reference to "a cell" includes a plurality of such cells, as well as equivalents thereof known to those skilled in the art, and so forth.

[0083] As used herein, the term "about" means a range of ±20% of the value stated. In some embodiments, the term "about" means a range of ±10% of the value stated. In some embodiments, the term "about" means a range of ±5% of the value stated.

[0084] The "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences is the number of identical matches between two sequences in a comparison window, accounting for any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A match is any position in the target sequence and the reference sequence where the same nucleotide or amino acid is present. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Likewise, since only the nucleotides or amino acids from the target sequence are counted, and not those from the reference sequence, gaps present in the reference sequence are not counted.

[0085] 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.

[0086] The term "conservative substitution" as used herein means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with residues having similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue has a side chain that is similar in size, charge, and / or polarity to the side chain of the original amino acid residue. Methods of identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0087] Examples and figures are provided below to help understand the present application. It should be understood, however, that these examples and figures are only intended to illustrate the present application, but do not constitute any limitation. The actual protection scope of the present application is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present application. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0088] It should be noted that, in the examples, the specific conditions not specified are carried out according to the conventional conditions, the manufacturer's recommendations or the publicly reported experimental conditions. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by commercial purchase. The reagents used are specified by the manufacturer, and similar products of other manufacturers have substitutability.

[0089] In the quantitative tests in the following examples, three repeated experiments were set up unless otherwise specified, and the results were averaged.

[0090] Example 1. Establishment and screening of phage library The purpose of this example is to screen a 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 obtained antibody.

[0091] 1. Animal immunization and detection of serum antibody titer 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.

[0092] 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 Biotech, A600669-0250) was added to each well for 1 hour 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 then 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 hours. 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 minutes in the dark. 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. When the coating antigen was GII.6 VP1 protein, 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, so mouse SBI220325D was selected for phage library construction. The results of indirect ELISA method for detecting mouse serum titer are shown in Table 1.

[0093] Table 1 Experimental results of indirect ELISA method for detecting mouse serum titer

[0094]

[0095] 2. Preparation of phage antibody library Phage display library construction was performed using spleen tissue from mouse SBI220325D. RNA was extracted from the spleen using TriPure Isolation Reagent and reverse transcribed using a reverse transcription kit to obtain cDNA. Using the cDNA as a template, PCR was performed to amplify the light and heavy chain variable region sequences of the mouse antibody. Overlap extension PCR was used to obtain the nucleotide sequence encoding the scFv (linker sequence used in the scFv is: SSGGGGSGGGGGGSSRSS, SEQ ID NO: 1). The nucleotide sequence was then digested with the restriction endonuclease Sfil and ligated into the phage vector pComb3x. The phage display scFv antibody library for immunized mice was then electroporated into XL1-Blue competent cells to construct the library. The successfully constructed phage library had a capacity of 3.53×10 9 cfu.

[0096] 3. Phage antibody library screening The primary antibody library (the phage library obtained in step 2) is infected with helper phage to prepare the original antibody library. The immunogen protein (GII.6 VP1 protein) is coated on the enzyme-labeled plate and added to the original antibody library for binding. The washing solution (1×PBST) is used to wash to remove non-specific binding. The phage bound to the antigen is eluted with the elution solution, amplified and precipitated in Escherichia coli, and then the next round of enrichment is carried out. After 2 to 4 rounds of adsorption, elution, and amplification screening, a phage library enriched with anti-GII.6 antigen positive antibodies is obtained. Monoclonal phages are selected from the enriched library for expression. The indirect ELISA method is used to detect the interaction of phage monoclonals with the immunogen protein (GII.6 VP1 protein) and the de-crossed antigen proteins (GI.1 VP1 protein, GII.2 VP1 protein, GII.3VP1 protein, GII.4 VP1 protein, GII.17 VP1 protein), and obtained phage library antibody monoclonal clones that specifically bound to the immunogen protein. Twelve antibody clones with high specific binding to the immunogen protein were screened and obtained (monoclonal numbers were 22LKM4-2-M002-M, 22LKM4-2-M003-M, 22LKM4-2-M008-M, 22LKM4-2-M038-M, 22LKM4-2-M0 Table 2 shows the results of indirect ELISA for phage antibody monoclonal expression. Sequencing analysis was performed on 12 antibody monoclonal clones to obtain the heavy and light chain variable region sequences. The amino acid sequences of the variable region and CDRs of antibody 22LKM4-2-M008 are shown in Table 3.

[0097] Table 2 Experimental results of indirect ELISA detection of phage monoclonal expression

[0098] Table 3 Amino acid / nucleotide sequences of full length, variable region and CDR of 22LKM4-2-M008 antibody

[0099]

[0100]

[0101] Example 2. Antibody construction, expression, purification and screening The purpose of this example is to construct 12 antibodies, transfect them into HEK293 cells, express and purify them, and detect the ELISA specificity, Western Blot (WB), concentration, purity (SDS-PAGE, SEC-HPLC), titer and blocking activity of the antibodies, in order to screen from the 12 antibodies the one with specificity and blocking activity and capable of recognizing the target antigenic conformational epitope.

[0102] 1. Antibody construction: according to the variable region amino acid sequences of the 12 antibodies, the heavy and light chain variable regions of the antibodies were respectively constructed into the constant regions of the murine IgG heavy and light chains (sequences shown in SEQ ID NO: 25, SEQ ID NO: 29) using conventional methods, and 12 antibodies were constructed.

[0103] 2. HEK293 cells were subcultured with 293 serum-free CD medium (Beijing Yiqiao Shenzhou Technology Co., Ltd., item number SMM 293-TI), and the plasmid DNA of the 12 antibodies to be expressed (take 22LKM4-2-M008 as an example: the full-length nucleotide sequence of the heavy chain of 22LKM4-2-M008 containing the signal peptide and the full-length nucleotide sequence of the light chain of 22LKM4-2-M008 containing the signal peptide in Table 3 were inserted into pcDNA3.1 expression vector (Thermo Fishe) to obtain plasmid DNA expressing the heavy chain of 22LKM4-2-M008 and plasmid DNA expressing the light chain of 22LKM4-2-M008) were mixed with transfection reagent TF2 (Beijing Yiqiao Shenzhou Technology Co., Ltd., item number STF02) and added to HEK293 cells. After transfection, 293 serum-free feeding solution (Beijing Yiqiao Shenzhou Technology Co., Ltd., item number M293-SUPI-100) was added on the 1st, 3rd and 5th day. The shake flask culture conditions were 5% CO2, temperature 37°C, shaker speed 175 rpm, culture for 7 days, and collection of cell liquid.

[0104] 3. Antibody purification: centrifuge (1000g, 20min) the cell liquid with a bench centrifuge (Beckman), collect the supernatant after centrifugation, and filter it with a 0.45μM filter membrane (Sartorius, item number 17598-K). Use a Protein A affinity chromatography column to purify the 12 filtered cell supernatants, respectively, and harvest 12 murine monoclonal candidate antibodies (antibody numbers are 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, 22LKM4-2-M163).

[0105] 4. Antibody screening (1) ELISA specific detection: use indirect ELISA method to detect the binding of the 12 purified antibodies to immunogen protein and cross-antigen protein, respectively, as follows: dilute the immunogen protein (GII.6 VP1 protein) and cross-antigen protein (GI.1 VP1 protein, GII.2 VP1 protein, GII.3 VP1 protein, GII.4 VP1 protein, GII.17 VP1 protein) to 1μg / ml with coating solution (1×PBS) for coating, 100μL per well, 4℃ overnight. Wash the plate with washing solution (1×PBST), add blocking solution (PBST+5% skimmed milk powder), and incubate at room temperature for 1h. Wash the plate with washing solution, add 12 monoclonal antibodies diluted to 1μg / ml, 100μL per well, and incubate at room temperature for 2h. Wash the plate with washing solution, add diluted enzyme-labeled secondary antibody goat anti-mouse IgG Fc (IGL, item number GGFC-90P) labeled with horseradish peroxidase, 100μL per well, and incubate at room temperature for 1h. Wash the plate with washing solution, add 200μL / well color developing solution, and incubate at room temperature for 20min in the dark, add 50μL / well stop solution, mix well, and read OD 450 values on the enzyme-labeled instrument. The results show that, except for No. 22LKM4-2-M116, the other 11 antibodies have good specificity and strong binding force, and the detection data results are shown in Table 4.

[0106] Table 4 Experimental results of indirect ELISA detection of antibodies

[0107] (2) Western Blot (WB) antibody binding epitope detection: WB detection of 12 antibodies binding to immunogen protein (GII.6 VP1 protein), as follows: GII.6 VP1 protein was denatured, 30 ng was loaded, constant voltage 100 V electrophoresis, wet transfer instrument (Bio-rad) constant voltage 110 V membrane transfer for 90 min. After membrane transfer, immerse in blocking solution (PBST + 5% skimmed milk powder) 4°C overnight, wash with washing buffer (1x PBST), then add 12 antibodies, incubate at room temperature for 2 h, wash again, then add goat anti-mouse IgG (H+L) / HRP (Jackson company), incubate at room temperature for 2 h, finally develop with chemiluminescence imaging system (ProteinSimple company). The results showed that the purified antibodies 22LKM4-2-M008, 22LKM4-2-M152 and 22LKM4-2-M163 could not recognize the denatured immunogen protein (GII.6 VP1 protein), i.e. the results were negative, suggesting that the GII.6 VP1 protein epitope recognized by antibodies 22LKM4-2-M008, 22LKM4-2-M152 and 22LKM4-2-M163 may be a spatial conformation epitope. The detection data are shown in Table 5 and Figure 1 .

[0108] Table 5 Western Blot (WB) detection of antibody experimental results

[0109] Based on the above ELISA specificity and WB detection results, 3 antibodies (numbered 22LKM4-2-M008, 22LKM4-2-M152 and 22LKM4-2-M163) were screened and met the ELISA specificity and WB negative at the same time. The concentration, purity, titer and blocking activity of the 3 antibodies were detected.

[0110] (3) Concentration detection: using microspectrophotometer (Thermo company), first add blank control (1x PBS) to the detection base, put down the sample arm, click Blank, calibrate the blank, OD 280 and OD 320 absorbance values between ±0.015, indicating that the instrument baseline is stable and the detection of the sample to be tested can be carried out. Then spot the 3 antibody samples, record the absorbance values, and the antibody concentration (mg / ml) = (OD 280 - OD 320AU, AU is IgG extinction coefficient (1.414), the concentration of the three antibodies 22LKM4-2-M008, 22LKM4-2-M152, 22LKM4-2-M163 is 0.92 mg / ml, 1.20 mg / ml, 0.68 mg / ml respectively.

[0111] (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 reducing electrophoresis concentrated gel was 3.9%, the separation gel was 13%, the sample preparation was to take 5 μg sample and add 5 μl 4x loading buffer, heat in 100°C water bath for 8 min, then centrifuge at 10000 rpm, add 1x SDS electrode buffer in the electrophoresis tank, add sample solution and protein molecular weight marker (Biyun Tian, 14.4-116 kDa, non-pre-dye) in the loading hole, connect the power supply for electrophoresis, after electrophoresis, carry out room temperature staining and room temperature decolorization in turn, until a clean background is obtained for gel imaging analysis; SEC-HPLC detection was performed using a high performance liquid chromatograph (Agilent Company), 40 μg sample and Marker (Sephadex Company, item number 215000-0101) were added to the interpolation tube and then placed in the sample disc, the instrument software ChemStation online was started, the washing pump accessory was opened, and the washing liquid 10% isopropyl alcohol was discharged at a rate of 2-3 drops per minute, the D pipeline was replaced with SEC mobile phase 0.2M Na2HPO4 (National Pharmaceutical Reagent Company), 0.1M Arginine (National Pharmaceutical Reagent Company), 1% IPA (National Pharmaceutical Reagent Company), pH 6.5, after the bubbles in the pipeline were discharged, the column was correctly installed, the pump flow rate was set to 0.5 ml / min, and the SEC mobile phase was used to equilibrate the column for 40 min. After equilibration, the DAD was turned on, and after the baseline was stable, the analysis program was run, after the program ended, the HP-SEC.M method was used for integration, and the data was calculated by area normalization method. The results showed that the molecular weight of the heavy chain and light chain of the three antibodies was about 50 kDa and 25 kDa, and the structure was complete. The purity (SDS-PAGE and SEC-HPLC) of the two antibodies numbered 22LKM4-2-M008 and 22LKM4-2-M152 was higher, both more than 95.0%, the purity (SEC-HPLC) of the antibody numbered 22LKM4-2-M008 was higher, the detection data results are shown in Table 6 and Figure 2 .

[0112] Table 6 Antibody concentration and purity detection experimental results

[0113] (5) Titer detection: the titer of the three antibodies was detected by indirect ELISA method, as follows: the immunogen protein (GII.6 VP1 protein) was diluted with coating solution (1xPBS) to 1 pg / ml for coating, 100 mI per well, 4°C overnight. The plate was washed with washing solution (1xPBST), blocking solution (PBST+5% skim milk) was added, and incubated at room temperature for 1 h. The plate was washed with washing solution, and each gradient of the three monoclonal antibodies was diluted to 64,000 times, 100 mI per well, and incubated at room temperature for 2 h. The plate was washed with washing solution for 3 times, Rabbit Anti-Mouse IgG F(ab)2 / HRP (Jackson company) was added after dilution, 100 mI per well, and incubated at room temperature for 1 h. The plate was washed with washing solution for 3 times, 200 mI / well of color developing solution was added, and incubated at room temperature for 20 min in the dark, 50 mI / well of stop solution was added, mixed well, and then the OD 450 value was read on the enzyme label instrument. The OD 450 value of blank was greater than the cut-off value (the average OD 450 value of negative control x 2.1) was positive, and the corresponding dilution multiple was the titer of the antibody; wherein, blank was the negative control (the antibody was replaced with sample diluent). The results showed that the titer of the three antibodies was high, and the detection data results were shown in Table 7.

[0114] Table 7 Antibody titer detection experiment results

[0115] (6) Blocking activity detection: the blocking activity of the three antibodies was detected by ELISA method, as follows: porcine gastric mucin was diluted to 10 μg / ml with coating solution (1xPBS), 100 μl per well, and incubated at 37°C for 2 hours. The plate was washed with washing solution (1xPBST), and blocking solution (PBST+1%BSA) was added and incubated at 2-8°C overnight. The three antibodies were diluted 50 times as the starting dilution, and then diluted by two times to 6400 times. 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, GII.17 VP1 protein) were diluted to the working concentration as VLPs (VP1 protein of norovirus expressed in vitro self-assembled virus-like particles), GI.1 VP1 protein, GII.4 VP1 protein, GII.17 VP1 protein were diluted to 200 ng / ml, GII.3 VP1 protein, GII.6 VP1 protein were diluted to 500 ng / ml, and GII.2 VP1 protein was diluted to 5000 ng / ml. The VLP solution diluted to the working concentration was added to the diluted antibody solution in a volume ratio of 1:1, and incubated at 37°C for 1 hour (sample group). The enzyme-labeled plate was washed with washing solution, and the antigen-antibody solution after co-incubation was transferred to the coated enzyme-labeled plate. The VP1 protein (VLP) of each type was set as the positive control (compared with the sample group, the sample diluent was used to replace the antibody), and the sample diluent was set as the blank control (compared with the sample group, the sample diluent was used to replace the antibody and VLPs), and incubated at 37°C for 1 hour. The enzyme-labeled plate was washed with washing solution, and the corresponding type (GI.1, GII.2, GII.3, GII.4, GII.6, GII.17) rabbit polyclonal antibody was added, which was diluted at 1:5000, and incubated at 37°C for 1 hour. The enzyme-labeled plate was washed with washing solution, and goat anti-rabbit-HRP (Abmart Company) diluted at 1:5000 was added, and incubated at 37°C for 1 hour. The enzyme-labeled plate was washed with washing solution, and color development was carried out at room temperature in the dark. After stopping the reaction, the absorbance value was read at 450 nm wavelength. Inhibition index (%) = (positive control OD 450 - sample OD 450 ) / (positive control OD 450 - blank control OD 450 ) x 100. After calculating the inhibition index corresponding to each serum dilution gradient, the BT 50The value, i.e. the highest dilution of the antibody capable of blocking 50% of the VLPs from binding to the porcine gastric mucin HBGA receptor. The results show that the two antibodies numbered 22LKM4-2-M008 and 22LKM4-2-M152 have blocking activity and no cross-blocking activity with other de-crossed proteins, and the blocking activity of the two antibodies is relatively high, BT 50 The values are all greater than 50000, and the detection data results are shown in Table 8.

[0116] Table 8: Results of antibody blocking activity detection experiment

[0117]

[0118]

[0119]

[0120]

[0121] According to the above-mentioned ELISA specificity detection, Western Blot (WB) antibody binding epitope detection, concentration detection, purity detection, titer detection, and blocking activity detection results, it can be known that the two antibodies numbered 22LKM4-2-M008 and 22LKM4-2-M152 are relatively optimal, but the antibody numbered 22LKM4-2-M008 has higher purity (SEC-HPLC). Finally, one antibody numbered 22LKM4-2-M008 is selected for application.

[0122] The technical scheme of the present application is not limited to the above-mentioned specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. An anti-GII.6 norovirus antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 comprised in a heavy chain variable region VH having an amino acid sequence of SEQ ID NO: 2; and, LCDR1, LCDR2 and LCDR3 comprised in a light chain variable region VL having an amino acid sequence of SEQ ID NO:

3. 2.The antibody or antigen-binding fragment thereof of claim 1, wherein the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: b1) a VH comprising HCDR1 of an amino acid sequence of SEQ ID NO: 4, HCDR2 of an amino acid sequence of SEQ ID NO: 5 and HCDR3 of an amino acid sequence of SEQ ID NO: 6; and / or, a VL comprising LCDR1 of an amino acid sequence of SEQ ID NO: 7, LCDR2 of an amino acid sequence of WAS and LCDR3 of an amino acid sequence of SEQ ID NO: 8, the CDRs being defined according to the IMGT numbering system; or c1) a VH comprising HCDR1 of an amino acid sequence of SEQ ID NO: 9, HCDR2 of an amino acid sequence of SEQ ID NO: 10 and HCDR3 of an amino acid sequence of SEQ ID NO: 11; and / or, a VL comprising LCDR1 of an amino acid sequence of SEQ ID NO: 12, LCDR2 of an amino acid sequence of SEQ ID NO: 13 and LCDR3 of an amino acid sequence of SEQ ID NO: 8, the CDRs being defined according to the Kabat numbering system; or d1) a VH comprising HCDR1 of an amino acid sequence of SEQ ID NO: 14, HCDR2 of an amino acid sequence of SEQ ID NO: 15 and HCDR3 of an amino acid sequence of SEQ ID NO: 11; and / or, a VL comprising LCDR1 of an amino acid sequence of SEQ ID NO: 12, LCDR2 of an amino acid sequence of SEQ ID NO: 13 and LCDR3 of an amino acid sequence of SEQ ID NO: 8, the CDRs being defined according to the Chothia numbering system. ​ ​ e1) a VHcomprising HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 16, HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 17, and HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 18; and / or, a VLcomprising LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 19, LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 20, and LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 21, the CDRs defined according to the Contact numbering system.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein: the heavy chain variable region of the anti-GII.6 norovirus antibody or antigen-binding fragment thereof further comprises a framework region of a heavy chain variable region; and / or the light chain variable region of the anti-GII.6 norovirus antibody or antigen-binding fragment thereof further comprises a framework region of a light chain variable region.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein: the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region derived from an immunoglobulin of a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose; and / or the framework region of the light chain variable region comprises a framework region of a light chain variable region derived from an immunoglobulin of a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein: the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 2; and / or, a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 3; or the anti-GII.6 norovirus antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein: the heavy chain constant region comprises at least a portion of a heavy chain constant region derived from an immunoglobulin of a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose; or the light chain constant region comprises at least a portion of a light chain constant region derived from an immunoglobulin of a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose; or the heavy chain constant region comprises a heavy chain constant region derived from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM immunoglobulin; or the light chain constant region comprises a light chain constant region derived from a kappa and lambda immunoglobulin; or the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 25; or the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO:

26. the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 29; or the anti-GII.6 norovirus antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; or the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises a monoclonal antibody, a nanobody, a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab')2 fragment, an Fv fragment, a single chain Fv, a dsFv, or a Fd fragment; or the anti-GII.6 norovirus antibody or antigen-binding fragment thereof comprises: a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 24; and / or a light chain comprising an amino acid sequence as set forth in SEQ ID NO:

28.

7. A biological material related to the antibody or antigen-binding fragment thereof of any one of claims 1-6, the biological material comprising any one of n1)-n9): n1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6; n2) an expression cassette comprising the nucleic acid molecule of n1); n3) a vector comprising the nucleic acid molecule of n1); n4) a vector comprising the expression cassette of n2); n5) a cell comprising the nucleic acid molecule of n1); n6) a cell comprising the expression cassette of n2); n7) a cell comprising the vector of n3); n8) a cell comprising the vector of n4); n9) a cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6; the cell of any one of n5)-n9) does not comprise reproductive material.

8. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-6, by culturing the cell of claim 7.

9. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6; and, a conjugating moiety.

10. The conjugate of claim 9, wherein the conjugating moiety comprises a detectable label or a therapeutic agent.

11. A pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-6, the biological material of claim 7, or the conjugate of any one of claims 9-10; and, a pharmaceutically acceptable carrier.

12. A diagnostic or therapeutic kit comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-6, the biological material of claim 7, the conjugate of any one of claims 9-10, or the pharmaceutical composition of claim 11.

13. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the biological material of claim 7, the conjugate of any one of claims 9-10, or the pharmaceutical composition of claim 11, in any one of c1)-c4): c1) preparing a product for diagnosing a GII.6 norovirus infection or a disease caused thereby; c2) preparing a product for preventing and / or treating a GII.6 norovirus infection or a disease caused thereby; c3) preparing a product for detecting the presence or level of a GII.6 norovirus or a VP1 protein thereof in a sample; c4) detecting the presence or level of GII.6 norovirus or a VP1 protein thereof for non-diagnostic purposes.

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