A fully human monoclonal antibody binding to h3n2 ha protein and uses thereof

Abstract

The application discloses a full human monoclonal antibody binding to H3N2 HA protein and application. The antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of a certain amino acid sequence, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of a certain amino acid sequence. The antibody or antigen binding fragment thereof according to the embodiment of the application has at least the following beneficial effects: the antibody or antigen binding fragment thereof provided in the embodiment of the application can be combined with the influenza virus H3N2 HA protein with high affinity, and thus can be effectively applied to the diagnosis of the influenza virus.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and in particular to a fully human monoclonal antibody that binds to H3N2 HA protein and its application. Background Art

[0002] Influenza virus, also known as influenza virus, is a representative species of the Orthomyxoviridae family, mainly including four types: A, B, C, and D. Based on the differences in the two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza viruses can be divided into different subtypes. The influenza viruses closely related to human seasonal influenza infections mainly include two subtypes of influenza A virus, H1N1 and H3N2. Influenza vaccination remains the best way to prevent influenza virus infection. To this end, rapid and clear laboratory diagnosis in advance and accurate typing of influenza viruses are necessary measures to achieve targeted vaccination.

[0003] Current specific pathogenic testing for influenza A virus infection includes: positive influenza A antigen detection, positive nucleic acid detection, isolation and culture of influenza A virus, and significantly elevated influenza A-specific IgG antibody titers in acute and convalescent serum. Numerous studies have developed specific antibodies targeting different subtypes, but their clinical efficacy is limited. This is due to their low affinity. Therefore, there is a need to develop antibodies with higher affinity to provide a potential new tool for the diagnosis of H3N2 influenza virus. Summary of the Invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a fully human monoclonal antibody that binds to the H3N2 HA protein and its application, which has a high affinity for the H3N2 influenza virus.

[0005] In a first aspect of the present application, an antibody or antigen-binding fragment thereof that binds to H3N2 HA protein is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are any one of the following A1) or A2);

[0006] A1) The heavy chain variable region comprises:

[0007] The amino acid sequence is shown in SEQ ID NO.1 as HCDR1: GYNFNNYY,

[0008] HCDR2 with the amino acid sequence shown in SEQ ID NO. 2: INPDSGGT, and

[0009] HCDR3 with the amino acid sequence shown in SEQ ID NO. 3: ARGYNWNSDLWWLDP;

[0010] The light chain variable region includes:

[0011] The amino acid sequence is shown in SEQ ID NO.4 LCDR1: QAIRND,

[0012] LCDR2:TAS with an amino acid sequence as shown in SEQ ID NO.5, and

[0013] LCDR3 having an amino acid sequence as shown in SEQ ID NO. 6: LQDYNFPIT;

[0014] A2) The heavy chain variable region includes:

[0015] The amino acid sequence of HCDR1 is shown in SEQ ID NO.7: GFTVSSNY,

[0016] HCDR2 with the amino acid sequence shown in SEQ ID NO.8: IYSGGRT, and

[0017] HCDR3 with the amino acid sequence shown in SEQ ID NO. 9: ARMHSSGWYSPGWFDP;

[0018] The light chain variable region includes:

[0019] The amino acid sequence is shown in SEQ ID NO.10 LCDR1: QSLSSTY,

[0020] LCDR2:GAS with an amino acid sequence as shown in SEQ ID NO.11, and

[0021] The amino acid sequence of LCDR3 is shown in SEQ ID NO.12: QQYSSSPTWT.

[0022] The antibodies or antigen-binding fragments thereof according to the embodiments of the present application have at least the following beneficial effects:

[0023] The antibodies or antigen-binding fragments thereof provided in the examples of the present application can bind to the influenza virus H3N2 HA protein with high affinity and can therefore be effectively used in the diagnosis of influenza virus.

[0024] In some embodiments, the heavy chain variable region and the light chain variable region are any of the following A1) or A2);

[0025] A1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13:

[0026] QVQLVQSGAEVKKPGASVKVSCKAFGYNFNNYYLHWVRQAPGQGLEWMGWINPD SGGTNFAQKFQGRVTMTRDTSISTAYMELTRLTSDDAAVYFCARGYNWNSDLWWLDPWG QGTLVTVSS,

[0027] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.14:

[0028] AIQMTQSPSSSLSASVGDRVTITCRASQAIRNDLGWYQQKPGKAPKLLIYTASSLYSGV PSRFSGSGSGTYFTLTISSLQPEDFATYYCLQDYNFPITFGQGTRLEIK;

[0029] A2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.15:

[0030] EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSIIYSGGRT YYADSVRGRFTISRDNSKNTLHLQLNSLRAEDTAVYYCARMHSSGWYSPGWFDPWGQGT LVTVSS,

[0031] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.16:

[0032] EIVLTQSPGTLSLSPGERATLSCRASQSLSSTYLAWYQQKPGQAPRLLIYGASTRATGIP DRFSGSGSGPDFTLTISRLEPEDFAVYYCQQYSSSPTWTFGQGTKVEIK.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof is selected from at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

[0034] In some embodiments, the antibody or its antigen-binding fragment further comprises at least one of a heavy chain constant region and a light chain constant region. In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment further comprises a light chain constant region. In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region.

[0035] In some embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO. 17:

[0036] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0037] In some embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO. 18:

[0038] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

[0040] In a second aspect of the present application, there is provided a biological material, the biological material comprising any one of B1) to B4):

[0041] B1) a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof;

[0042] B2) an expression cassette containing the nucleic acid molecule of B1);

[0043] B3) a recombinant vector containing the nucleic acid molecule of B1) or the expression cassette of B2);

[0044] B4) a recombinant cell containing the nucleic acid molecule of B1) or the expression cassette of B2) or the recombinant vector of B3).

[0045] In a third aspect of the present application, there is provided a pharmaceutical composition comprising an effective dose of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned biological material.

[0046] In some embodiments, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier, a diluent, a buffer, and an excipient.

[0047] In a fourth aspect of the present application, a kit is provided, comprising the aforementioned antibody or antigen-binding fragment thereof.

[0048] The fifth aspect of the present application provides a use of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition, or the aforementioned kit in the preparation of a diagnostic, preventive or therapeutic product for H3N2 influenza virus.

[0049] In a sixth aspect of the present application, a method for detecting H3N2 influenza virus in a sample for non-diagnostic purposes is provided, comprising contacting the sample with the aforementioned antibody or antigen-binding fragment thereof.

[0050] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is an SDS-PAGE image of the H3N2 HA protein in Example 1. The lanes from left to right are the Marker, the results under +DTT reducing conditions, and the results under -DTT non-reducing conditions.

[0052] Figure 2 This is the evaluation of the antibody titer of the volunteer serum in Example 2. HA was used as the coating antigen and the serum was diluted in a gradient of 1:200 to 409600.

[0053] Figure 3 This is the activity verification of the biotinylated purified HA protein as in Example 3. Volunteer serum was used as the primary coating antibody, unbiotinylated and biotinylated HA were used as sandwich antigens, and HRP-streptavidin was used as the secondary antibody.

[0054] Figure 4 This is the sorting result of HA-specific single memory B cells in Example 4.

[0055] Figure 5 The heavy chain variable region (VH) and light chain variable region (VL) of the antibody amplified by reverse transcription and nested PCR in Example 5, where M is the DL5000 marker, and lanes 1 to 4 represent the SEY001 VH chain, SEY001 VL chain, SEY002 VH chain, and SEY002 VL chain, respectively.

[0056] Figure 6This is an SDS-PAGE gel image of the heavy and light chain variable regions of the SEY001 and SEY002 antibodies constructed into full antibodies in Example 6, expressed in 293F cells, and then purified. M is a protein marker; lanes 1 and 2 represent the bands of SEY001 and SEY002 with and without DTT, respectively; lanes 3 and 4 represent the bands of SEY002 with and without DTT, respectively.

[0057] Figure 7 In Example 7, Octet was used to treat SEY001 ( Figure 7 A in), SEY002( Figure 7 B) The results of the affinity test of the monoclonal antibody to HA. DETAILED DESCRIPTION

[0058] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0059] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0060] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0061] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0062] Throughout the description of this application, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0063] The term "antibody" refers to an immunoglobulin that specifically binds to an antigen. Correspondingly, "antigen" refers to a target antigen to which an antibody can selectively bind, and specific types of target antigens include polypeptides, proteins, nucleic acids, lipids, carbohydrates, haptens, or other natural or synthetic compounds. Specifically, in the present application, the antigen refers to the H3N2 influenza virus HA protein, and the immunoglobulin that specifically binds to the H3N2 influenza virus HA protein refers to an immunoglobulin that specifically binds to the H3N2 influenza virus HA protein with a K of 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM or less. D Immunoglobulins that bind to the H3N2 influenza virus HA protein. Furthermore, while immunoglobulins as antibodies generally do not specifically bind to antigens other than the H3N2 influenza virus HA protein, this does not mean that the antibodies cannot bind to other antigens. For example, immune cross-reactions are also known in the art.

[0064] Among them, K D is the equilibrium dissociation constant between antibody and antigen, K off With K on The ratio of K is expressed in molar concentration (M). D It can be used to measure the binding affinity between an antibody and its antigen, K D The smaller the value, the stronger the affinity. D The value can be determined by any method known in the art, including but not limited to surface plasmon resonance (SPR) methods, for example, using an SPR chip or related analysis systems such as Biacore T200; biomembrane interferometry (BLI) methods, for example, related detection systems such as Octet Red96.

[0065] Antibody types specifically include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multivalent antibodies, etc. In addition, antibodies can be modified in various ways, such as PEGylation, glycosylation, formation of antibody molecule conjugates (such as ADCs), addition of purification tags, or fusion with other proteins.

[0066] For example, a full-length antibody comprises a multimer of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. According to the conservation of amino acid sequences, the heavy and light chains can be divided into variable regions (V) and constant regions (C). Among them, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2 and CH3), and each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable region includes three complementarity determining regions (CDRs) and is separated by framework regions (FRs), arranged in FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the amino terminal to the carboxyl terminal, so that VH contains three CDRs: HCDR1, HCDR2, HCDR3, and VL contains three CDRs: LCDR1, LCDR2 and LCDR3.

[0067] The term "complementarity determining region" is a high-frequency mutation region within the variable region. The CDRs of a heavy chain and a light chain interact together to form the antigen binding site of a full-length antibody, which is complementary to the three-dimensional structure of an antigen epitope. The high heterogeneity of CDRs determines the diversity of antibodies, thereby recognizing different antigen epitopes.

[0068] Among them, the light chain of the antibody can be divided into kappa chain and lambda chain. The heavy chain of the antibody can be generally divided into mu chain, delta chain, gamma chain, alpha chain and epsilon chain, and the antibody is divided into five types of IgM, IgD, IgG, IgA and IgE according to the heavy chain. And IgG and IgA can be further divided into different subtypes, such as IgG1, lgG2, IgG3, lgG4, IgA1 or lgA2, etc.

[0069] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, so that each antibody of the antibody population is essentially the same and binds to the same antigen epitope, except for a very small amount of antibody variants produced by mutations. It can be understood that the monoclonal antibody is monospecific or polyspecific. Specifically, the monoclonal antibody usually binds to one antigen epitope, but there is also the possibility of having polyspecificity. Correspondingly, "polyclonal antibody" refers to a collection of different antibodies against the same antigen, for example, a collection of different antibodies produced by B lymphocytes, including mixed antibodies against different antigen epitopes.

[0070] The term "polyspecificity" refers to an antibody that can bind to two or more different antigen epitopes of the same antigen, or can bind to two or more different antigens or combinations thereof. Polyspecific antibodies can have cross-reactivity to other related antigens, for example, cross-reactivity to the same antigen from other species (homologous), or can bind to an epitope shared between two or more different antigens.

[0071] The term "epitope" refers to the portion of an antigen that is capable of specific binding to an antibody, usually consisting of amino acids or side chains with chemical activity, such as polar or nonpolar or hydrophobic groups, and optionally at least one of specific three-dimensional structure characteristics and specific charge characteristics.

[0072] Depending on the origin of the antibody, the antibody can be a human antibody, a humanized antibody or a chimeric antibody.

[0073] The term "human antibody" refers to an antibody whose variable region is derived from a human immunoglobulin sequence, and if the antibody has a constant region, the constant region is also derived from a human immunoglobulin sequence. Among them, the human immunoglobulin sequence can be a human germline sequence or a mutant form thereof, such as a mutant form generated by random mutation or site-directed mutation.

[0074] The term "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species antibody (donor antibody) and at least one FR is derived from a human antibody (recipient antibody). Among them, the non-human species includes other mammals, such as animals of Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, etc., and other animals of Primates, specifically, non-human species such as dogs, cats, pigs, sheep, cows, horses, rabbits, mice, monkeys, orangutans, chimpanzees, etc.

[0075] The term "chimeric antibody" refers to an antibody in which different parts of the antibody are derived from different animal species, for example, the variable region is derived from one species and the constant region is derived from another species, specifically, the variable region is derived from a mouse antibody and the constant region is derived from a human antibody or vice versa. Chimeric antibodies are mainly used to reduce immunogenicity in application and to improve yield in preparation, for example, where a mouse-derived antibody has a higher hybridoma yield but has a higher human immunogenicity, so that a human / mouse chimeric antibody is used. It can be understood that chimeric antibodies also include the case where different parts of the antibody are derived from different animal individuals.

[0076] In the case of known antibody complementarity determining region sequences, those skilled in the art can easily replace other part sequences (such as constant region or framework region) in the antibody with sequences from other species by techniques such as DNA recombination, to form a chimeric antibody or a humanized antibody, and substantially retain the binding specificity of the source antibody. For example, when the source antibody is a mouse antibody, its constant region or framework region can be replaced with a human antibody constant region, or vice versa, when the source antibody is a human antibody, its constant region or framework region can be replaced with a mouse antibody constant region or framework region, to reduce the immunogenicity of the antibody when used in different species or to take advantage of its specific functions, such as ADCC-related activity.

[0077] For further reducing the immunogenicity of the antibody or other purposes, other sequences in the antibody molecule except the CDR sequences can be replaced with the corresponding sequences (which can include several amino acid mutations as appropriate) of another antibody molecule (from the same or different species) while substantially retaining the binding specificity of the antibody of origin.

[0078] The term "antigen-binding fragment" refers to an antibody fragment of an immunoglobulin that retains the ability to specifically bind to the antigen bound by the full-length antibody, which can be a synthetic, enzymatically obtained or genetically engineered polypeptide. The antibody binding fragment includes at least one or more CDR fragments, and specific fragment types include Fab, Fab', F(ab')2, Fv, scFv, etc. Among them, the Fab fragment contains a variable region of one heavy chain and CH1 and one light chain, which can be, for example, the papain cleavage product of an antibody. The Fab' fragment contains a variable region of one heavy chain and CH1 and a fragment of the region between CH1 and CH2 and one light chain. The F(ab')2 fragment is composed of two Fab' fragments connected by a disulfide bond between the heavy chains, which can be, for example, the pepsin cleavage product of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The Fv fragment is composed of a heavy chain variable region and a light chain variable region. The scFv is composed of a heavy chain variable region and a light chain variable region and a linker connecting the heavy chain variable region and the light chain variable region. Through correct folding, the heavy chain variable region and the light chain variable region interact with each other through non-covalent bonds to form an Fv fragment, so that the scFv can better retain its affinity activity to the antigen.

[0079] Methods for preparing antibodies are known in the art, including but not limited to isolating cells expressing specific antibodies after immunizing animals with antigens (such as preparing antibodies by hybridoma technology), screening antibody technology using phage antibody library, transforming cells to express antibody molecules by genetic engineering technology, etc. In some specific embodiments, the present application provides a method for expressing antibodies or antigen-binding fragments thereof in host cells, which can be mammalian cells, such as mouse, rat, pig, sheep, monkey, chimpanzee or human cells. In some embodiments, the mammalian cells are selected from at least one of 293F cells, MCR-5 cells, thus providing a method for preparing antibodies or antigen-binding fragments thereof by introducing a recombinant expression vector containing the coding sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof into host cells such as 293F cells, MCR-5 cells, etc.

[0080] The term "H3N2 influenza virus" refers to a subtype of influenza A virus, which is one of the main pathogens of seasonal influenza and can cause acute respiratory infections in people of all ages. Specifically, the H3N2 influenza virus can be at least one representative strain selected from A / Darwin / 6 / 2021, A / Darwin / 9 / 2021, A / Guangdong / 03 / 2005, A / Guangdong / 55 / 2015, A / Hong Kong / 1 / 1968, A / Hong Kong / 4801 / 2014, A / Hong Kong / 26560 / 2009, A / Shangdong / 9 / 1993, A / Switzerland / 9715293 / 2013, A / Switzerland / 9715293 / 2013, A / Singapore / INFIMH-16-0019 / 2016, A / Thailand / 8 / 2022, and A / Victoria / 361 / 2011. Accordingly, "H3N2 influenza virus HA protein" refers to the hemagglutinin (HA) protein of the H3N2 influenza virus. As a type I transmembrane glycoprotein, it forms a trimer on the virus surface. The HA protein plays a key role in the binding and membrane fusion between the virus and host cells and is essential for influenza virus infection and intercellular transmission. The HA protein consists of two subunits, HA1 and HA2. HA1 is primarily responsible for binding to sialic acid receptors on the host cell surface, while HA2 is primarily involved in the fusion of the virus with the cell membrane.

[0081] The term "nucleic acid molecule" refers to at least one of genomic, mRNA, cDNA, or synthetically derived DNA or RNA. It is understood that those skilled in the art can mutate or modify the nucleotide sequence of a nucleic acid molecule using known methods, such as directed evolution and point mutagenesis. As long as the antibody or antigen-binding fragment thereof encoded by the mutated or modified nucleic acid molecule has the same function, it is derived from the nucleotide sequence of the embodiments of the present application and is considered a nucleic acid molecule of the embodiments of the present application.

[0082] The term "expression cassette" refers to a construct containing the necessary regulatory elements for the expression of the nucleic acid molecule contained therein in a recombinant cell. Specific regulatory elements include promoters, polyadenylation sites, etc.

[0083] The term "recombinant vector" refers to a molecule containing all elements for replication, integration, amplification and / or expression of the contained nucleic acid molecule in a recombinant cell. All elements include, but are not limited to, a promoter, a polyadenylation site, a terminator, and a replication initiation site, a transcription initiation sequence, an enhancer, a selection element, a reporter gene, etc. For example, an antibiotic resistance gene or a selection marker gene (such as ampicillin resistance gene AmpR, thymidine kinase gene TK, etc.) for screening and a coding sequence for a protein of interest can be inserted into a multiple cloning site (MCS). Alternative types of recombinant vectors include plasmids, bacteriophages, lentiviruses, adenoviruses, adeno-associated viruses, etc.

[0084] The term "recombinant cell" refers to a cell for replication, integration, amplification and / or expression of a nucleic acid molecule therein. The recombinant cell includes, but is not limited to, bacteria, fungi, insect cells, plant cells, animal cells. Among them, the bacteria can be, for example, Escherichia coli or Bacillus subtilis, etc., the fungi can be, for example, yeast or Aspergillus, etc., the insect cells can be, for example, S2 Drosophila cells or Sf9 cells, etc., the plant cells can be, for example, cells of Arabidopsis thaliana or tobacco, etc., and the animal cells can be, for example, mammalian cells such as mouse, rat, pig, sheep, monkey, chimpanzee, or human cells, and specifically 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc. It can be understood that these recombinant cells can all be non-propagating materials.

[0085] In a first aspect, the present application relates to an antibody or antigen-binding fragment thereof that binds to the HA protein of H3N2 influenza virus, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three CDRs, HCDR1, HCDR2 and HCDR3, respectively; the light chain variable region comprising three CDRs, LCDR1, LCDR2 and LCDR3, respectively. Wherein the heavy chain variable region and the light chain variable region are selected from the following groups, respectively:

[0086] A1) HCDR1 with an amino acid sequence as shown in SEQ ID NO. 1: GYNFNNYY;

[0087] A2) HCDR2 with an amino acid sequence as shown in SEQ ID NO. 2: INPDSGGT;

[0088] A3) HCDR3 with an amino acid sequence as shown in SEQ ID NO. 3: ARGYNWNSDLWWLDP;

[0089] B1) LCDR1 with an amino acid sequence as shown in SEQ ID NO. 4: QAIRND;

[0090] B2) LCDR2 with an amino acid sequence as shown in SEQ ID NO. 5: TAS;

[0091] LCDR3 having an amino acid sequence as shown in SEQ ID NO. 6: LQDYNFPIT;

[0092] A2) HCDR1 with the amino acid sequence shown in SEQ ID NO. 7: GFTVSSNY;

[0093] HCDR2 with the amino acid sequence shown in SEQ ID NO. 8: IYSGGRT;

[0094] HCDR3 with the amino acid sequence shown in SEQ ID NO. 9: ARMHSSGWYSPGWFDP;

[0095] LCDR1: QSLSSTY, the amino acid sequence of which is shown in SEQ ID NO. 10;

[0096] LCDR2: GAS having an amino acid sequence as shown in SEQ ID NO. 11;

[0097] The amino acid sequence of LCDR3 is shown in SEQ ID NO.12: QQYSSSPTWT.

[0098] Those skilled in the art will also understand that, based on the specific antibody sequences provided herein, a small number of amino acids can be replaced, deleted, or added, and the resulting products can be verified or screened for their binding ability to the corresponding antigen or biological activity, thereby obtaining corresponding variants of the antibodies that bind to the H3N2 influenza virus HA protein provided in the examples of this application. These variants should also be included in the scope of the examples of this application.

[0099] Thus, in some embodiments, the amino acid sequence of the heavy chain variable region of A1) has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence of SEQ ID NO. 13, and the amino acid sequence of the light chain variable region has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence of SEQ ID NO. 14. In some embodiments, the amino acid sequence of the heavy chain variable region of A2) has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.16.

[0100] In some embodiments, A1) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14.

[0101] In some embodiments, A2) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof is selected from at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv. In some embodiments, the antibody or antigen-binding fragment thereof further comprises at least one of a heavy chain constant region and a light chain constant region. It is understood that the heavy chain constant region and the light chain constant region can be full-length or partial fragments thereof, such as CH1 and CH2. In some specific embodiments, the amino acid sequence of the heavy chain constant region has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO. 17. In some specific embodiments, the amino acid sequence of the light chain constant region has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence of SEQ ID NO. 18. In some specific embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO. 17. In some specific embodiments, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO. 18.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

[0104] In some specific embodiments, the antibody or antigen-binding fragment thereof further comprises a tag sequence at the N-terminus and / or C-terminus, including a tag sequence that facilitates solubility and / or purification. Specifically, the tag sequence comprises at least one of a His tag, a GGGS sequence, a FLAG tag, a Strep tag, a Nus tag, a maltose binding protein, and a GST tag. It is understood that one or more tag sequences may be included; the multiple tag sequences may comprise a combination of multiple identical tag sequences or a combination of multiple different tag sequences.

[0105] The second aspect of the present application relates to a biological material, which may be a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof; an expression cassette containing the nucleic acid molecule; a recombinant vector containing the nucleic acid molecule or the expression cassette; or a recombinant cell containing the nucleic acid molecule, the expression cassette, or the recombinant vector.

[0106] The third aspect of the present application relates to a pharmaceutical composition comprising an effective dose of an antibody or its antigen-binding fragment or a biological material. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Among them, pharmaceutically acceptable carriers include solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered, which are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drugs or active agents therein. In some embodiments, the pharmaceutical composition further comprises other substances that have a preventive or therapeutic effect on the H3N2 influenza virus or its related diseases or symptoms or complications, for example, antiviral drugs for the H3N2 influenza virus, such as neuraminidase inhibitors (NAI), hemagglutinin inhibitors (HA), RNA polymerase inhibitors, nucleoprotein inhibitors, etc. NAIs include, but are not limited to, oseltamivir, zanamivir, peramivir, and laninamivir octanoate; hemagglutinin inhibitors include, but are not limited to, nitazoxanide and arbidol; RNA polymerase inhibitors include, but are not limited to, baloxavir, RO-7, favipiravir, pimodyvir, and ZSP1273; and nucleoprotein inhibitors include, but are not limited to, naproxen and FA-6005. Other antibodies against H3N2 influenza virus, such as CR8020, CR6261, and FI6v3, or combinations thereof, may also be included.

[0107] In some embodiments, the pharmaceutical composition can be administered in the form of any one or more of powders, granules, powders, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and lavages, for example, by at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

[0108] It is understood that, for the ingredients in the pharmaceutical composition, it is possible to administer to the subject simultaneously or sequentially. Wherein, effective dose refers to the amount of the active compound (such as antibody) sufficient to cause the biological or medical reaction desired by the clinician in the subject, and specifically, effective dose can be determined by those skilled in the art according to factors such as route of administration, subject's weight, age, and condition. For example, a typical daily dose range can be 0.01mg to 100mg of active ingredient per kg body weight, such as 0.01mg, 0.02mg, 0.05mg, 0.1mg, 0.2mg, 0.5mg, 1mg, 2mg, 5mg, 10mg, 20mg, 50mg, 100mg. However, in the embodiment of the present application, it is also possible not to be limited thereto, with smaller or larger dosage.

[0109] The fourth aspect of the present application relates to a kit comprising the aforementioned antibody or antigen-binding fragment thereof.

[0110] In some embodiments, the test kit can be any one of a detection plate, a detection chip, a detection test strip, etc. for H3N2 influenza virus or H3N2 influenza virus HA protein.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof can be used to form a kit in the form of a solid (such as a lyophilized powder), a liquid (such as a solution), or directly coated on a substrate (such as a test paper or a chip).

[0112] In some embodiments, the kit detects H3N2 influenza virus or H3N2 influenza virus HA protein by any one of immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, electrochemiluminescence, etc. Enzyme-linked immunosorbent assay includes, but is not limited to, any one of direct, indirect, sandwich, and competitive methods. Immunochromatography includes, but is not limited to, any one of fluorescent microsphere immunochromatography, colloidal gold immunochromatography, latex microsphere immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof can serve as at least one of a coating antibody and a detection antibody in the kit.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof is bound to a solid substrate such as magnetic beads, microspheres, microplates, nitrocellulose membranes, or microfluidic chips.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated with a detectable label, or the kit further includes a separate detectable label capable of being conjugated to the antibody or antigen-binding fragment thereof. Specifically, the detectable label can be at least one of a radionuclide, a fluorescent substance, a chemiluminescent substance, a colored substance, an aggregation-induced luminescence substance, a micro-nanoparticle, an enzyme, and the like.

[0116] The fifth aspect of the present application relates to the use of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a kit in the preparation of a product for diagnosis, prevention or treatment of H3N2 influenza virus.

[0117] Specifically, antibodies or antigen-binding fragments thereof can be used to prepare diagnostic products for H3N2 influenza virus. Antibodies or antigen-binding fragments thereof, when combined with other substances that have preventive or therapeutic effects against H3N2 influenza virus or its related diseases, symptoms, or complications, can be used to prepare products for the prevention or treatment of H3N2 influenza virus. The specific binding properties of antibodies or antigen-binding fragments to the H3N2 influenza virus HA protein enable targeted delivery of other substances with preventive or therapeutic effects, thereby improving efficacy, reducing dosage, and shortening the onset of action. Pharmaceutical compositions can be used to prepare products for the prevention or treatment of H3N2 influenza virus. Kits can be used to prepare diagnostic products for H3N2 influenza virus.

[0118] The present application also relates to the use of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition, or a kit for diagnosing, preventing, or treating H3N2 influenza virus infection. Specifically, the antibody or antigen-binding fragment thereof can be used to diagnose, prevent, or treat H3N2 influenza virus infection, the pharmaceutical composition can be used to prevent or treat H3N2 influenza virus infection, and the kit can be used to diagnose H3N2 influenza virus infection.

[0119] The present application also relates to a method for diagnosing, preventing, or treating H3N2 influenza virus infection, comprising contacting an antibody or antigen-binding fragment thereof, or a pharmaceutical composition, or a kit with a subject. Specifically, a method for diagnosing, preventing, or treating H3N2 influenza virus infection comprises contacting an antibody or antigen-binding fragment thereof with a subject; a method for preventing or treating H3N2 influenza virus infection comprises administering a pharmaceutical composition to a subject; and a method for diagnosing H3N2 influenza virus infection comprises administering a kit to a subject.

[0120] The sixth aspect of the present application relates to a method for detecting H3N2 influenza virus in a sample, which comprises contacting the sample with the aforementioned antibody or antigen-binding fragment thereof.

[0121] In some embodiments of the present application, the method for detecting H3N2 influenza virus in a sample is a non-diagnostic method, such as virus monitoring and analysis. The H3N2 influenza virus can be transmitted through airborne droplets, direct or indirect contact with mucous membranes such as the mouth, nose, and eyes, and contact with objects contaminated by the virus. In addition, it can also be transmitted through aerosols in crowded, enclosed, or poorly ventilated rooms. For example, this method can be used for the following non-diagnostic purposes:

[0122] (1) Environmental monitoring: Detecting the presence of H3N2 influenza virus in environmental samples (such as air, surface wipes, etc.) in public places such as medical institutions, kindergartens, and schools, to assess environmental safety and formulate corresponding preventive measures.

[0123] (2) In vitro culture sample testing: In virological research, the presence and proliferation of H3N2 influenza virus in laboratory cultured cell or tissue samples are detected to evaluate the effect of antiviral drugs or study the biological characteristics of the virus.

[0124] (3) Production quality control: During the production process of biological products (such as vaccines, blood products, etc.), detect whether there is H3N2 influenza virus contamination in raw materials, intermediates or finished products to ensure product safety.

[0125] (4) Laboratory animal quarantine: Detecting the presence of H3N2 influenza virus in laboratory animals or their tissue samples for the purpose of quality control and management of laboratory animals.

[0126] (5) Scientific research purposes: In basic research in molecular biology, immunology and other related fields, the presence of H3N2 influenza virus in experimental samples is detected, and it is used to study scientific issues such as virus-host interaction and virus evolution.

[0127] In some embodiments, samples include, but are not limited to, environmental samples (such as air, water, soil, surface swabs, etc.), in vitro culture samples (such as cell culture supernatants, tissue culture, etc.), biological product samples (such as vaccines, blood products, etc.), experimental animal samples (such as experimental animal tissues, body fluids, etc.), scientific research samples, etc. None of these samples involve direct use in human diagnosis.

[0128] In some embodiments, the sample comprises a biological sample. In some embodiments, the biological sample includes but is not limited to at least one of body fluids (e.g., blood, tissue fluid, lymph, cerebrospinal fluid, urine, sweat, sputum, saliva, gastric juice, intestinal juice, pancreatic juice, bile, prostatic fluid, vaginal secretions, semen, serous effusion, joint effusion, bronchoalveolar lavage fluid, amniotic fluid, etc.), skin, feces, intestinal contents, and histological samples (e.g., samples obtained by surgery, endoscopy, or percutaneous puncture biopsy).

[0129] The antibodies or antigen-binding fragments thereof provided in the examples of this application can be used for the development of antibody conjugates targeting HA protein.

[0130] The antibodies or antigen-binding fragments thereof provided in the examples of this application can specifically bind to the H3N2 influenza virus HA protein, i.e., the H3N2 influenza virus HA protein serves as the target antigen of the antibodies. Due to the antigen-binding specificity of the antibodies provided in the examples of this application, the antibodies can be used to detect the presence of the H3N2 influenza virus in a sample (e.g., blood) from a subject. Accordingly, the examples of this application provide an H3N2 detection kit that can include the antibodies or antigen-binding fragments thereof of the examples.

[0131] Specifically, in the embodiments of the present application, two human monoclonal antibodies, named SEY001 and SEY002, were isolated from human peripheral blood mononuclear cells using H3N2 HA fusion protein as a decoy. The antibodies can specifically bind to H3N2 HA protein, and thus can be applied to the diagnosis and antigen quantification of H3N2.

[0132] The present application is further illustrated by the following specific examples.

[0133] Example 1: Expression and purification of H3N2 HA protein

[0134] The H3N2 HA (GenBank: ARV89911.1) protein sequence was retrieved from the NCBI database. A signal peptide sequence (MNPLLILTFVAAALAAPFDDDDK) was added to the N terminus of the HA protein (18-520 aa), and a trimer tag, thrombin tag, his tag and strep tag were introduced to the C terminus. The coding gene corresponding to the above protein sequence was optimized for human codons and constructed into the vector pCAGGS plasmid. After expression in 293F suspension mammalian cells for 5-7 days, the supernatant was collected and filtered with a 0.22 μm filter membrane. The protein was purified using an affinity column HisTrap HP (5 mL, GE healthcare) and a gel filtration chromatography column Superose 610 / 30 GL (GE healthcare). The protein was concentrated to 10 mg / mL, frozen in liquid nitrogen and stored at -80°C for standby.

[0135] SDS-PAGE of the purified HA protein determined the size and purity of the target protein. As shown in Figure 1 , the molecular weight of HA was about 60 kDa, and the same size was shown with or without DTT, indicating that HA was a trimer and the three subunits were not connected by disulfide bonds, but existed in the form of a trimer through intermolecular interaction.

[0136] Example 2: Evaluation of volunteer serum

[0137] 100 μL of H3N2 HA protein solution was added to each well at a concentration of 2 μg / mL. After incubation at 4°C overnight, the microplate was coated with H3N2 HA protein. The next day, the plates were washed three times with PBST. Then, 150 μL / well of 2% BSA was added for blocking, incubated at 37°C for 2 hours, and washed three times with PBST. Subsequently, 100 μL / well of serially diluted serum from volunteers who had received the quadrivalent influenza split vaccine for four months (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12400) was added. After incubation at 37°C for 2 hours, the plates were washed three times with PBST. Then, 100 μL / well of goat anti-human IgG (Fc-specific) horseradish peroxidase-conjugated secondary antibody was added at a dilution of 1:5000. After incubation at 37°C for 1 hour, the plates were washed three times with PBST. Add 100 μL / well of TMB substrate for color development. After 10 min of reaction in the dark, add 50 μL of 2M H2SO4 to terminate the reaction. Measure OD450 and perform data processing.

[0138] The results are as follows Figure 2 As shown, the antibody titer against H3N2 HA protein antigen in the volunteer's serum was detected by ELISA. Analysis of the OD value showed that the volunteer's serum contained antibodies against H3N2 HA protein and could be used as a sample for further sorting.

[0139] Example 3: HA protein biotinylation

[0140] Follow EZ-Link TM The HA protein was biotinylated using the Sulfo-NHS-LC-biotinylation kit (Thermo Fisher) to obtain biotin-labeled HA protein.

[0141] ELISA was used to determine whether the HA protein was biotinylated and the activity of the labeled protein. 100 μL of volunteer serum diluted 1:1600 was added to each well and coated overnight at 4°C. The next day, the plates were washed three times with PBST. 150 μL / well of 2% BSA was added for blocking, incubated at 37°C for 2 hours, and then washed three times with PBST. 100 μL of unlabeled or labeled HA protein at a concentration of 2 μg / mL was added to each well and incubated at 37°C for 2 hours. The plates were then washed three times with PBST. 100 μL of HRP-streptavidin (STREP) stock solution diluted 1:5000 was added to each well and incubated at 37°C for 45 minutes. The plates were then washed three times with PBST. The substrate TMB (50 μL / well) was added for color development. After reacting in the dark for 10 minutes, 50 μL of 2M HCl was added to terminate the reaction. The OD450 value was measured and the data was processed.

[0142] The results are as follows Figure 3As shown in the figure, it can be seen that the final absorbance value of the labeled HA protein is significantly higher than that of the unlabeled HA protein, indicating that the HA protein was successfully labeled according to the method provided by the kit and the activity of the labeled protein was not affected in any way.

[0143] Example 4: Isolation of H3N2 HA protein-specific memory B cells

[0144] After informed consent from the volunteers, 10 mL of blood was collected and PBMCs were isolated. 7 / mL) was incubated on ice with 200nM HA protein prepared in Example 1 for half an hour; then washed twice with PBS, and then mixed with the following antibodies (all purchased from BD): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-humanCD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-humanCD38 / APC, anti-human IgG / FITC, and Streptavidin / PE. After incubation on ice for half an hour, the cells were washed twice with PBS. Subsequently, PBMCs were sorted using FACSAria III, and cells with PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ (i.e., memory B cells) were collected and directly collected into a 96-well plate, 1 cell / well, and sorted as shown in FIG. Figure 4 shown.

[0145] Example 5: Single B cell PCR and cloning of human monoclonal antibody IgG1

[0146] The cells collected from the sorting in Example 4 were reverse transcribed using Superscript III reverse transcriptase (Invitrogen) at 55°C for 60 minutes. This reverse transcription product was used as a template for PCR amplification of the antibody variable region sequence (PCRa) using HotStar TapPlus enzyme (QIAgen). The reaction conditions were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C (heavy chain / κ chain) / 50°C (λ chain), 30 seconds, 72°C for 90 seconds, 35 cycles, and 72°C for 7 minutes. This product was used as a template for another round of PCR (PCRb) using the following conditions: 95°C for 5 minutes; 95°C for 30 seconds, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain), 30 seconds, 72°C for 90 seconds, 35 cycles, and 72°C for 7 minutes. 1.2% agarose gel electrophoresis was used to separate the PCR products. The 400-500bp band was excised and recovered and sent to a sequencing company for sequencing. The antibody variable region amplification agarose gel was shown in the figure below. Figure 5 The sequencing results were analyzed on the IGBLAST website.

[0147] Based on the sequencing results, a total of two paired antibodies were obtained: SEY001 and SEY002.

[0148] Among them, the amino acid sequence of the heavy chain variable region of SEY001 is SEQ ID NO.13, the amino acid sequence of the light chain variable region is SEQ ID NO.14, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the heavy chain variable region are shown in SEQ ID NOs.1 to 6, respectively.

[0149] The amino acid sequence of the heavy chain variable region of SEY002 is SEQ ID NO.15, the amino acid sequence of the light chain variable region is SEQ ID NO.16, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the heavy chain variable region are shown in SEQ ID NOs.7 to 12, respectively.

[0150] In order to obtain human antibodies for subsequent evaluation, a fully anti-IgG1 construct was designed. The strategy is as follows:

[0151] Heavy chain: CMV promoter-SacI-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-KpnI;

[0152] Light chain κ: CMV promoter-SacI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLκ)-KpnI.

[0153] The correct variable region sequence and the corresponding constant region of heavy chain CH and light chain CLKappa were connected by bridge PCR, and were cloned into the expression vector pCAGGS to obtain a recombinant plasmid containing the coding gene of the light and heavy chains of the specific antibody; wherein the restriction sites SacI and Kpnl were used to connect the light and heavy chain variable regions into the vector containing the constant region. The amino acid sequence of the heavy chain constant region CH is shown in SEQ ID NO. 17; the amino acid sequence of the light chain constant region CLKappa is shown in SEQ ID NO. 18; the amino acid sequence of the signal peptide (SP) is shown in SEQ ID NO. 19: ETDTLLLWVLLLWVP.

[0154] Example 6: Expression and purification of monoclonal antibody

[0155] The pCAGGS plasmid containing the coding sequences of the heavy and light chains was co-transfected into 293F cells. 220 mM VPA was added 24 h after transfection, and the cells were cultured for another 5 days. The cells were harvested, the supernatant was collected by centrifugation, and then filtered through a 0.22 pm filter. The purified antibody was purified by HiTrap Protein A HP (5 ml, GE Healthcare) chromatography column and gel filtration chromatography column Superose 610 / 30 GL (GE healthcare) at a speed of 2 mL / min. SDS-PAGE is shown in Figure 6 , and finally concentrated and stored at -80°C for standby.

[0156] Example 7: Detection of the binding performance of the antibody

[0157] The interaction between the purified antibody and the HA protein after fusion was detected by the Bio-Layer Interferometry (BLI) technology using the Octet Red96 system (ForteBio). The antibody was detected using a protein A sensor loaded at a concentration of 10 pg / mL, then combined with HA protein in the range of 25-1600 nM for 180 s, and dissociated for 600 s. The regeneration of the sensor used 10 mM Glycine (pH 1.5). All the proteins used in the experiment were exchanged into PBS buffer, and the detection buffer was used to detect the binding of the HA protein. The apparent equilibrium dissociation constant (KD value) of the interaction between the HA protein and the antibody was calculated using the Octet Red96 evaluation software.

[0158] The results are shown in Figure 7 , SEY001 can bind to H3N2 HA fusion protein with high affinity, K D = 36 nM, the binding rate is 2.21 x 10 4 / Ms, and the dissociation rate is 8.05 x 10 -4 / s; SEY002 can also bind to H3N2 HA fusion protein with high affinity, K D =33nM, the binding rate is 0.51×10 4 / Ms, and the dissociation rate was 1.69×10 -4 / s.

[0159] Example 8

[0160] This embodiment provides an H3N2 diagnostic kit, including H3N2 standards of different concentration gradients, a coating plate, an H3N2 primary antibody, an enzyme-labeled H3N2 secondary antibody, BSA blocking solution, a color developer A solution, a color developer B solution, a stop solution, and the like.

[0161] The preparation process of the pre-coated plate is as follows: the purified H3N2 primary antibody is diluted with pH 9.6 carbonate buffer to obtain a mixture with a concentration of 5 μg / mL, and a 96-well plate is coated with 0.1 mL per well. After incubation at 4°C for 21 hours, the liquid in the wells is discarded, and the plates are washed and patted dry. Then, BSA blocking solution is added to each well, 0.1 mL per well, and the plates are incubated at 37°C for 2 hours. The liquid in the wells is discarded, and the plates are washed and patted dry to obtain the pre-coated plates.

[0162] The primary antibody against H3N2 was a mouse anti-H3N2 monoclonal antibody.

[0163] The enzyme-labeled H3N2 secondary antibody was labeled with HRP by sodium periodate oxidation method and the H3N2 secondary antibody was SEY001.

[0164] The stop solution was 2M HCl.

[0165] The kit is used as follows:

[0166] Add 75 μL of sample or H3N2 standard to each well, along with negative and blank controls, and incubate at 37°C for 1 hour. Discard the wells, wash, and pat dry. Then, add 25 μL of enzyme-labeled H3N2 secondary antibody to each well, incubate at 37°C for 1 hour, discard the wells, wash, and pat dry. Add 100 μL of chromogen A and B to each well, develop at 37°C for 10 minutes, and add 50 μL of stop solution to each well. Zero the blank well and read the OD value of each well at 450 nm.

[0167] The quantitative method is as follows: a standard curve is drawn based on the OD values ​​of H3N2 standards of different concentrations and the linear range is determined. The OD value of the sample is substituted into the standard curve to obtain the H3N2 concentration in the sample.

[0168] The qualitative method is as follows: 2 times the OD value of the negative control is set as the critical OD value. When the OD value of the sample is greater than the critical OD value, it is determined to be H3N2 antigen positive, otherwise it is negative.

[0169] Example 9

[0170] This embodiment provides an H3N2 diagnostic kit, which differs from Example 8 in that the H3N2 secondary antibody is SEY002.

[0171] In Examples 8 and 9, the limit of detection (LOD) was calculated based on the standard curve as 3SD / b, where SD is the standard deviation of the blank control and b is the slope of the standard curve. The results showed that all three assays had low limits of detection. Clinical samples identified as negative and positive for nucleic acid were also tested to determine the number of false positives and false negatives reported by the kits. The results demonstrated that the H3N2 diagnostic kits in Examples 8 and 9 all had good specificity and sensitivity.

[0172] Example 10

[0173] This embodiment provides an H3N2 colloidal gold test strip, comprising a sample pad, a colloidal gold pad, a nitrocellulose membrane, a blotting paper, and a PVC substrate, which are arranged in sequence.

[0174] Among them, the colloidal gold pad is coated with colloidal gold-labeled mouse anti-H3N2 monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line (C line) and a detection line (T line). The quality control line is coated with goat anti-mouse IgG antibody, and the detection line is coated with SEY001 antibody.

[0175] During the test, add 200 μL of sample to the spotting well, react for 15 minutes and observe the results; if both C line and T line have bands, it is positive; if only C line has bands, it is negative; if there is no C line band, the result is invalid.

[0176] Example 11

[0177] This embodiment provides an H3N2 colloidal gold test strip, which differs from Example 10 in that the H3N2 secondary antibody is SEY002.

[0178] The H3N2 colloidal gold test strips of Examples 10-11 were tested using H3N2 standards of varying concentrations. The lowest concentration of the standard at which a T-line band appeared was determined as the test strip's detection sensitivity. Clinical samples identified as nucleic acid negative and positive were also tested to determine the occurrence of false positives and false negatives. The results demonstrated that the test strips of Examples 10-11 all exhibited high sensitivity, specificity, and sensitivity.

[0179] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. An antibody or antigen-binding fragment thereof that binds to H3N2 HA protein, characterized in that: comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are any one of the following A1) or A2); A1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2, and HCDR3 as shown in SEQ ID NO. 3, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, and LCDR3 as shown in SEQ ID NO. 6; A2) The heavy chain variable region includes HCDR1 as shown in SEQ ID NO.7, HCDR2 as shown in SEQ ID NO.8, and HCDR3 as shown in SEQ ID NO.9, and the light chain variable region includes LCDR1 as shown in SEQ ID NO.10, LCDR2 as shown in SEQ ID NO.11, and LCDR3 as shown in SEQ ID NO.

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region and the light chain variable region are any one of the following A1) or A2); A1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; A2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is selected from at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein It also includes at least one of a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.17; and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

18.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is any one of a human antibody and a chimeric antibody.

6. Biomaterial, characterized in that The biological material includes any one of B1) to B4); B1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A recombinant cell containing the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3).

7. A pharmaceutical composition, characterized in that Comprising an effective dose of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the biomaterial according to claim 6.

8. A kit, characterized in that The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

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