A fully human monoclonal antibody SEY003 binding to H3N2 HA protein and its application

Abstract

This application discloses a fully human monoclonal antibody, SEY003, that binds to the H3N2 HA protein and its applications. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The antibody or its antigen-binding fragment can bind to the influenza virus H3N2 HA protein with high affinity and can therefore be effectively used in influenza virus diagnosis.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and in particular to a fully human monoclonal antibody SEY003 that binds to H3N2 HA protein and its application. Background Art

[0002] Influenza viruses belong to the Orthomyxoviridae family and are single-stranded, negative-sense, segmented RNA viruses. Based on differences in their nucleoprotein and matrix proteins, they are classified into four types: A, B, C, and D. Currently, the main influenza A subtypes that infect humans are the H1N1 and H3N2 subtypes, and the Victoria and Yamagata lineages of influenza B. In epidemiological studies, virus typing aids in the prediction, prevention, and response to seasonal influenza. Current methods for specific pathogen detection of influenza A virus infection include antigen detection, nucleic acid detection, and pathogen isolation and culture. Antigen detection is currently a primary detection method, as it requires fewer samples, is rapid and convenient, and can cover the entire infectious period. In the development of antigen detection assays for influenza A virus, a variety of antibodies specific for different subtypes have been developed, but these antibodies have been ineffective in clinical testing, mostly due to their poor affinity. Therefore, there is a need for high-affinity antibodies to provide a potential new tool for the diagnosis of H3N2 influenza virus. Summary of the Invention

[0003] 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 SEY003 that binds to the H3N2 HA protein and its application, which has a high affinity for the H3N2 influenza virus.

[0004] 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;

[0005] The heavy chain variable region includes:

[0006] The amino acid sequence of HCDR1 is shown in SEQ ID NO.1: GFKFGDYV,

[0007] HCDR2 with the amino acid sequence shown in SEQ ID NO. 2: IRSKAYGETI, and

[0008] HCDR3 with the amino acid sequence shown in SEQ ID NO. 3: TSFLGHLEADY;

[0009] The light chain variable region includes:

[0010] The amino acid sequence is shown in SEQ ID NO.4 LCDR1: QSISTY,

[0011] LCDR2: AAS having an amino acid sequence as shown in SEQ ID NO. 5, and

[0012] The amino acid sequence of LCDR3 is shown in SEQ ID NO.6: QHTYGAPRT.

[0013] The antibodies or antigen-binding fragments thereof according to the embodiments of the present application have at least the following beneficial effects:

[0014] 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 viruses.

[0015] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7:

[0016] EVQLVESGGGLVQPGRSLRLSCTGSGFKFGDYVMSWFRQAPGKGLEWVVMSWFRQA PGKGLEWVGFIRSKAYGETIDYAASVDGRFTISRDDSKSIAYLQMISLKIEDTAVYYCTSFLG HLEADYWGQGTLVTVSS.

[0017] In some embodiments of the present application, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8:

[0018] DIQMTQSPSSSLSASVGDRVTIACRASQSISTYLNWYQQKPGKAPKPLIYAASTLQSGVP SRFSGSGSGTDFTLTISNLQPEDFATYYCQHTYGAPRTFGQGTKVESK.

[0019] In some embodiments of the present application, at least one of a heavy chain constant region and a light chain constant region is further included. In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment further includes a light chain constant region. In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region.

[0020] In some embodiments of the present application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.9:

[0021] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0022] In some embodiments of the present application, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.10:

[0023] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS.

[0024] In some embodiments of the present application, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.9, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO.10.

[0025] In some embodiments of the present application, 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.

[0026] In some embodiments of the present application, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody and a chimeric antibody.

[0027] In a second aspect of the present application, a biomaterial is provided, the biomaterial comprising any one of B1) to B4);

[0028] B1) a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof;

[0029] B2) an expression cassette containing the nucleic acid molecule of B1);

[0030] B3) a recombinant vector containing the nucleic acid molecule B1) or the expression cassette B2);

[0031] B4) A recombinant cell containing B1) a nucleic acid molecule or B2) an expression cassette or B3) a recombinant vector.

[0032] The third aspect of the present application provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising culturing the aforementioned recombinant cells, isolating and purifying to obtain the antibody or the antigen-binding fragment thereof.

[0033] In a fourth aspect of the present application, a kit is provided, comprising the aforementioned antibody or antigen-binding fragment thereof.

[0034] The fifth aspect of the present application provides a use of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned kit in preparing a diagnostic product for H3N2 influenza virus.

[0035] 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.

[0036] 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

[0037] Figure 1 This is an SDS-PAGE image of the H3N2 HA protein in Example 1. The lanes from left to right represent the results of Marker, +DTT reducing conditions, and -DTT non-reducing conditions, respectively.

[0038] Figure 2 This is the result of evaluating the antibody titer of the volunteers' sera in Example 2. HA was used as the coating antigen, and the sera were diluted in a gradient of 1:200 to 409600.

[0039] Figure 3 The results are shown in Table 3. The results show that the purified HA protein after biotinylation was activated. Volunteer serum was used as the primary antibody, unbiotinylated HA and biotinylated HA (HA-biotin) were used as sandwich antigens, and HRP-streptavidin was used as the secondary antibody.

[0040] Figure 4 This is the sorting result of HA-specific single memory B cells in Example 4.

[0041] Figure 5These are the heavy chain variable regions (VH) and light chain variable regions (VL) of the antibody amplified by reverse transcription and nested PCR in Example 5. The lanes from left to right are the band results for the DL5000 marker, SEY003 VH chain, and SEY003 VL chain.

[0042] Figure 6 This is an SDS-PAGE gel image of the heavy and light chain variable regions of the SEY003 antibody constructed into a full antibody, expressed in 293F cells, and then purified in Example 6. The lanes from left to right show the protein marker, SEY003 with DTT, and SEY003 without DTT.

[0043] Figure 7 This is the result of using Octet to detect the affinity of SEY003 monoclonal antibody to HA in Example 7. DETAILED DESCRIPTION

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 with a K of 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM 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.

[0050] 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.

[0051] 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.

[0052] Taking a full-length antibody as an example, it contains two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or a multimer formed thereof. Based on the conservative differences in the amino acid sequences, the heavy and light chains can be divided into a variable region (V) and a constant region (C). Among them, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of the 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 complementary determining regions (CDRs) and is separated by framework regions (FRs), which are arranged from amino terminus to carboxyl terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Therefore, VH contains three CDRs: HCDR1, HCDR2, HCDR3, and VL contains three CDRs: LCDR1, LCDR2 and LCDR3.

[0053] The term "complementarity determining region" refers to the highly mutated region within the variable region. The CDRs of a heavy and light chain interact to form the antigen-binding site of a full-length antibody, complementing the three-dimensional structure of the antigen epitope. The high heterogeneity of CDRs determines the diversity of antibodies, allowing them to recognize different antigenic epitopes.

[0054] Antibody light chains are divided into kappa and lambda chains. Antibody heavy chains are generally classified into μ, delta, gamma, alpha, and epsilon chains, and based on these, antibodies are divided into five types: IgM, IgD, IgG, IgA, and IgE. IgG and IgA can be further divided into different subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0055] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, whereby the individual antibodies comprising the antibody population are substantially identical and bind to the same antigenic epitope, except for the presence of minimal amounts of antibody variants resulting from mutations. It is understood that monoclonal antibodies can be monospecific or multispecific. Specifically, monoclonal antibodies typically bind to a single antigenic epitope, but may also have multispecificity. Correspondingly, a "polyclonal antibody" refers to a collection of different antibodies directed against the same antigen, such as a collection of different antibodies produced by B lymphocytes, including mixed antibodies directed against different antigenic epitopes.

[0056] The term "multispecific" refers to an antibody that can bind to two or more different antigenic epitopes on the same antigen, or can bind to two or more different antigens or a combination thereof. Multispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), or may bind to epitopes shared between two or more different antigens.

[0057] The term "epitope" refers to the part of an antigen that can specifically bind to an antibody, usually composed of amino acids or chemically active groups on the side chains, such as polar, non-polar or hydrophobic groups, and optionally has at least one of a specific three-dimensional structural characteristic and a specific charge characteristic.

[0058] Depending on the origin of the antibody, the antibody can be a human antibody, a humanized antibody, or a chimeric antibody.

[0059] The term "human antibody" refers to antibodies whose variable regions are derived from human immunoglobulin sequences, and whose constant regions, if present, are also derived from human immunoglobulin sequences, wherein the human immunoglobulin sequences may be human germline sequences or mutant forms thereof, such as those produced by random or site-directed mutagenesis.

[0060] The term "human 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). Non-human species include other mammals, such as animals of the orders Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, and other primates, specifically dogs, cats, pigs, sheep, cattle, horses, rabbits, mice, monkeys, orangutans, gorillas, chimpanzees, and other non-human species.

[0061] The term "chimeric antibody" refers to an antibody in which different portions are derived from different animal species, such as an antibody whose variable region originates from one species and whose constant region originates from another. Specifically, the variable region may be derived from a mouse antibody and the constant region from a human antibody, or vice versa. Chimeric antibodies are primarily used to reduce immunogenicity in applications and improve production yields. For example, murine antibodies have higher hybridoma yields but higher human immunogenicity, leading to the use of human / mouse chimeric antibodies. It is understood that chimeric antibodies also include antibodies in which different portions are derived from different individual animals.

[0062] In the case of known antibody complementary determining region sequences, those skilled in the art can easily replace other partial sequences in the antibody (such as constant regions or framework regions) with sequences from other species through techniques such as DNA recombination to form chimeric antibodies or humanized antibodies, 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 conversely, 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 utilize its specific functions, such as ADCC-related activity.

[0063] In order to further reduce the immunogenicity of the antibody or for other purposes, all sequences in the antibody molecule except the CDR sequence can be replaced with the corresponding sequence of another antibody molecule (from the same or different species) (which may include several amino acid mutations as appropriate) while substantially retaining the binding specificity of the antibody from which it originated.

[0064] 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. It can be a synthetic, enzymatically obtained, or genetically engineered polypeptide. Antibody-binding fragments include at least one or more CDR fragments. Specific fragment types include Fab, Fab', F(ab')2, Fv, scFv, etc. A Fab fragment comprises the variable region and CH1 of a heavy chain, as well as a light chain, and can be, for example, a papain cleavage product of an antibody. A Fab' fragment comprises the variable region and CH1 of a heavy chain, as well as a fragment of the region between CH1 and CH2, and a light chain. A F(ab')2 fragment is composed of two Fab' fragments linked by disulfide bonds between the heavy chains, and can be, for example, a 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. An Fv fragment is composed of a heavy chain variable region and a light chain variable region. scFv is composed of the heavy chain variable region, the 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 through non-covalent bonds to form an Fv segment, so scFv can better retain its affinity activity for the antigen.

[0065] Methods for preparing antibodies are known in the art, including but not limited to isolating cells expressing specific antibodies after immunizing animals with antigens (e.g., preparing antibodies through hybridoma technology), screening antibody technologies using phage antibody libraries, transforming cells through genetic engineering technology and causing them to express antibody molecules, etc. In some embodiments, methods for expressing antibodies or antigen-binding fragments thereof in host cells (also known as recombinant cells) are provided. The host cells can specifically be mammalian cells, such as cells of mice, rats, pigs, sheep, monkeys, orangutans, or humans. In some embodiments, the mammalian cells are selected from at least one of 293F cells and MCR-5 cells, thus providing a method for preparing antibodies or antigen-binding fragments thereof by introducing a recombinant expression vector comprising a heavy chain variable region and a light chain variable region encoding an antibody or antigen-binding fragment thereof into host cells such as 293F cells and MCR-5 cells.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The term "recombinant vector" refers to a molecule containing all the elements for replication, integration, amplification and / or expression of the nucleic acid molecules contained therein in a recombinant cell. All elements include, but are not limited to, promoters, polyadenylation sites, terminators, replication initiation sites, transcription initiation sequences, enhancers, selection elements, reporter genes, and the like. For example, an antibiotic resistance gene or selection marker gene (such as the ampicillin resistance gene AmpR, the thymidine kinase gene TK, etc.) for screening and a target protein coding sequence can be inserted into the multiple cloning site (MCS). Optional recombinant vector types include plasmids, bacteriophages, lentiviruses, adenoviruses, adeno-associated viruses, and the like.

[0070] The term "recombinant cell" refers to a cell in which a nucleic acid molecule is replicated, integrated, amplified, and / or expressed. Recombinant cells include, but are not limited to, bacteria, fungi, insect cells, plant cells, and animal cells. Examples of bacteria include Escherichia coli and Bacillus subtilis, examples of fungi include yeast and Aspergillus, examples of insect cells include S2 Drosophila cells and Sf9 cells, examples of plant cells include Arabidopsis thaliana and tobacco cells, and examples of animal cells include mammalian cells, such as mouse, rat, pig, sheep, monkey, orangutan, or human cells, specifically 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293 cells. It is understood that these recombinant cells may be non-reproductive materials.

[0071] The first aspect of the present application relates to an antibody or an antigen-binding fragment thereof that binds to the H3N2 influenza virus HA protein, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1: GFKFGDYV as shown in the amino acid sequence of SEQ ID NO.1; an HCDR2: IRSKAYGETI as shown in the amino acid sequence of SEQ ID NO.2; an HCDR3: TSFLGHLEADY as shown in the amino acid sequence of SEQ ID NO.3; an LCDR1: QSISTY as shown in the amino acid sequence of SEQ ID NO.4; an LCDR2: AAS as shown in the amino acid sequence of SEQ ID NO.5; and an LCDR3: QHTYGAPRT as shown in the amino acid sequence of SEQ ID NO.6.

[0072] In some embodiments, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8.

[0073] 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 may 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 is set forth in SEQ ID NO. 9. In some specific embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO. 10.

[0074] 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.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

[0076] In some 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.

[0077] 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.

[0078] The third aspect of the present application relates to a method for preparing an antibody or an antigen-binding fragment thereof, comprising culturing the aforementioned recombinant cells, isolating and purifying the cells, and obtaining the antibody or the antigen-binding fragment thereof.

[0079] In some embodiments, the recombinant cells can be cultured in a culture vessel of any size, such as any one or more of a culture dish, a culture plate, a culture flask, a culture bag, a bioreactor, a cell factory, and the like.

[0080] In some embodiments, the volume of the container for culturing recombinant cells is 1 mL to 10,000 L, for example, 1 mL, 2 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 200 mL, 500 mL, 1 L, 2 L, 5 L, 10 L, 20 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 2000 L, 5000 L, or 10,000 L.

[0081] In some embodiments, the recombinant cells are cultured in a serum-containing (eg, fetal bovine serum or calf serum) or serum-free medium.

[0082] In some embodiments, the culture medium includes nutrients, and the nutrients include one or more of glucose, glutamine, amino acids, nucleotides, vitamins, etc.

[0083] In some embodiments, after culturing, the step of separating the antibodies from other substances, such as cells and cell debris, is further included. In some embodiments, separation is performed by one or more methods, such as centrifugation and filtration. In some embodiments, filtration includes methods such as depth filtration and tangential flow filtration. In some embodiments, centrifugation includes methods such as simple centrifugation and continuous flow centrifugation.

[0084] In some embodiments, after the culture, the step of purifying the antibody or antigen-binding fragment thereof is further included based on the biological characteristics of the antibody (such as the charge, size, hydrophobicity, etc. of the antibody molecule). In some embodiments, the antibody purification method includes at least one of affinity chromatography, ion exchange chromatography, gel filtration chromatography, and hydrophobic interaction chromatography.

[0085] The fourth aspect of the present application relates to a kit comprising the aforementioned antibody or antigen-binding fragment thereof.

[0086] 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.

[0087] 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).

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] The fifth aspect of the present application relates to the use of an antibody or an antigen-binding fragment thereof, or a kit in the preparation of a diagnostic product for H3N2 influenza virus.

[0093] Specifically, the antibody or antigen-binding fragment thereof can be used to prepare a diagnostic product for H3N2 influenza virus. The kit can be used to prepare a diagnostic product for H3N2 influenza virus.

[0094] The present application also relates to the use of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a kit in diagnosing H3N2 influenza virus infection.

[0095] 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.

[0096] 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:

[0097] (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.

[0098] (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.

[0099] (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.

[0100] (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.

[0101] (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.

[0102] 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.

[0103] 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).

[0104] The antibodies or antigen-binding fragments thereof provided in the present application can be used for the development of antibody conjugates targeting HA protein.

[0105] The antibodies or antigen-binding fragments thereof provided herein 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 embodiments 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 embodiments of this application provide an H3N2 detection kit that can include the antibodies or antigen-binding fragments thereof of the embodiments.

[0106] Specifically, in the examples of this application, a human monoclonal antibody, named SEY003, was isolated from adult peripheral blood mononuclear cells using an H3N2 HA fusion protein as bait. This antibody binds to the H3N2 HA protein with high specificity, and therefore can be used for H3N2 diagnosis and antigen quantification.

[0107] The present invention is further described below through specific examples.

[0108] Example 1: Expression and purification of H3N2 HA protein

[0109] The H3N2 HA protein sequence (GenBank: ARV89911.1) was retrieved from the NCBI database. A signal peptide sequence (MNPLLILTFVAAALAAPFDDDDK, SEQ ID NO. 12) was added to the N-terminus of the HA protein (18-520 aa), and a trimerization tag, thrombin tag, his tag, and strep tag were introduced at the C-terminus. The gene encoding the above protein sequence was codon-optimized and constructed into the pCAGGS plasmid. After expression in suspension mammalian 293F cells for 5-7 days, the supernatant was collected and filtered through a 0.22 μm filter. The protein was purified using an affinity column HisTrap HP (5 mL, GE Healthcare) and a gel filtration column Superose 6 10 / 30 GL (GE Healthcare). The protein was concentrated to 10 mg / mL, snap-frozen in liquid nitrogen, and stored at -80°C until further use.

[0110] The size and purity of the target protein were determined by SDS-PAGE of the purified HA protein. Figure 1 As shown in the figure, the molecular weight of HA is about 60 kDa. It shows the same size when DTT is added or not, indicating that HA is a trimer and the three subunits are not connected by disulfide bonds, but are in the form of a trimer through intermolecular interactions.

[0111] Example 2: Volunteer Serum Evaluation

[0112] 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 H3N2 HA protein was coated on the microplate. The next day, the plate was 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 6 months (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600) was added. After incubation at 37°C for 2 hours, the plate was washed three times with PBST. Then, add 100 μL / well of goat anti-human IgG (Fc-specific) horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5000. Incubate at 37°C for 1 hour, then wash the plate three times with PBST. Add 100 μL / well of TMB substrate for color development. After incubation in the dark for 10 minutes, add 50 μL of 2 M H₂SO₄ to terminate the reaction. Measure the OD₄50 value, and perform data processing.

[0113] The results are as follows Figure 2As shown, the antibody titer against H3N2 HA protein antigen in the volunteer's serum was detected by ELISA. The OD value results showed that the volunteer's serum contained antibodies against H3N2 HA protein and could be used as a sample for further sorting.

[0114] Example 3: HA protein biotinylation

[0115] Follow EZ-Link TM The HA protein was biotinylated using the Sulfo-NHS-LC-biotinylation kit (Thermo Fisher) to obtain biotin-labeled HA protein.

[0116] 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.

[0117] The results are as follows Figure 3 As 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.

[0118] Example 4: Isolation of H3N2 HA protein-specific memory B cells

[0119] 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 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 PE-Cy5 antibodies were collected. - APC - APC-Cy7 + Pacific Blue + FITC + PE + The cells (i.e., memory B cells) were directly collected into a 96-well plate, 1 cell / well, and sorted as follows Figure 4 shown.

[0120] Example 5: Single B cell PCR and cloning of human monoclonal antibody IgG1

[0121] 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.

[0122] According to the sequencing results, a paired antibody SEY003 was obtained. The amino acid sequence of the heavy chain variable region was SEQ ID NO.7, the amino acid sequence of the light chain variable region was SEQ ID NO.8, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the heavy chain variable region were shown in SEQ ID NOs. 1 to 6, respectively.

[0123] In order to obtain human antibodies for subsequent evaluation, a fully anti-IgG1 construct was designed. The strategy is as follows:

[0124] Heavy chain: CMV promoter-SacI-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-KpnI;

[0125] Light chain κ: CMV promoter-SacI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLκ)-KpnI.

[0126] The correct variable region sequence was analyzed and ligated with the corresponding constant regions of the heavy chain CH and light chain CLκ via bridge PCR, then cloned into the expression vector pCAGGS to generate a recombinant plasmid containing the genes encoding the light and heavy chains of the specific antibody. The restriction sites SacI and KpnI were used to ligate the light and heavy chain variable regions into the vector containing the constant regions. The amino acid sequence of the heavy chain constant region CH is shown in SEQ ID NO. 9; the amino acid sequence of the light chain constant region CLκ is shown in SEQ ID NO. 10; and the amino acid sequence of the signal peptide (SP) is shown in SEQ ID NO. 11: ETDTLLLWVLLLWVP.

[0127] Example 6: Expression and purification of monoclonal antibodies

[0128] The pCAGGS plasmids containing the heavy and light chain coding sequences were co-transfected into 293F cells. 220 mM MVPA was added 24 h after transfection and culture was continued for 5 days. The cells were harvested, the supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and purified at 2 mL / min through a HiTrap Protein A HP column (5 ml, GE Healthcare) and a gel filtration column Superose 6 10 / 30 GL (GE Healthcare). The results were analyzed by SDS-PAGE. Figure 6 The product was finally concentrated and stored at -80°C until use.

[0129] Example 7: Antibody Binding Performance Detection

[0130] The interaction between the purified antibodies and the fused HA protein was detected by biomembrane interferometry (BLI) using the Octet Red96 system (ForteBio). Antibodies were loaded at a concentration of 10 μg / mL using a protein A sensor. Binding to HA protein ranging from 25 to 1600 nM was performed for 180 s, followed by dissociation for 600 s. The sensor was regenerated using 10 mM glycocein (pH 1.5). All proteins used in the experiment were exchanged into PBS buffer, and the detection buffer was used to measure HA protein binding. The apparent equilibrium dissociation constant (KD value) of the HA protein-antibody interaction was calculated using Octet Red96 evaluation software.

[0131] The results are as follows Figure 7 As shown, SEY003 can bind to H3N2 HA fusion protein with high affinity. D =3.26nM, the binding rate is 4.27×10 4 / Ms, and the dissociation rate was 1.39×10 -4 / s.

[0132] Example 8

[0133] 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.

[0134] 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.

[0135] The primary antibody against H3N2 was a mouse anti-H3N2 monoclonal antibody.

[0136] The enzyme-labeled H3N2 secondary antibody was labeled with HRP by sodium periodate oxidation method and the H3N2 secondary antibody was SEY003.

[0137] The stop solution was 2M HCl.

[0138] The kit is used as follows:

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In Example 8, 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 Example 8 all had good specificity and sensitivity.

[0143] Example 9

[0144] 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.

[0145] 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 SEY003 antibody.

[0146] During the test, add 200 μL of sample to the spotting hole, 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 a band, it is negative; if there is no C line band, the result is invalid.

[0147] The H3N2 colloidal gold test strip of Example 9 was 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 negative and positive for nucleic acid were also tested to determine the number of false positives and false negatives. The results demonstrated that the test strip of Example 9 exhibited high sensitivity, specificity, and sensitivity.

[0148] 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: It includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes 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 includes LCDR1 as shown in SEQ ID NO.4, LCDR2 with an amino acid sequence of AAS, and LCDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. 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.9; and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

10.

4. 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.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a human antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a human antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a chimeric antibody.

8. 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 7; 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).

9. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The method comprises culturing the recombinant cell according to claim 8, isolating and purifying the antibody or the antigen-binding fragment thereof.

10. A kit, characterized in that The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the kit according to claim 10, in the preparation of a diagnostic product for H3N2 influenza virus.

12. A method for detecting H3N2 influenza virus in a sample for non-diagnostic purposes, characterized in that: The method comprises contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

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