Human monoclonal antibody MZ001 binding to H3N2 HA protein and application thereof

Abstract

The application discloses a full human monoclonal antibody MZ001 combined with 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, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The antibody or antigen binding fragment thereof has a high affinity to the influenza virus H3N2 HA protein, and has a good application prospect as a diagnostic tool for the influenza virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to a full human monoclonal antibody MZ001 binding to H3N2 HA protein and application. BACKGROUND

[0002] Influenza virus is a single-stranded RNA virus, which can be divided into types A, B, C, D, etc. Among them, type A is the most common, including different subtypes such as H1N1, H2N2, H3N2, H5N1, H7N9, and H1N1 and H3N2 subtypes account for the vast majority of patients. Hemagglutinin HA is one of the most important membrane proteins in influenza virus, responsible for the binding of virus to host cell surface receptors and the fusion of virus membrane with host cell membrane, is a key factor in the process of virus infection of cells, and is also an important part of influenza virus typing. One of the specific pathogen detection methods for influenza A virus infection is antigen detection, which is fast and convenient, and has lower requirements for samples. However, in the development process of the antigen detection of influenza A virus, the specific antibody for H3N2 HA protein has the problem of poor affinity. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a full human monoclonal antibody MZ001 binding to H3N2 HA protein with high affinity and application.

[0004] In a first aspect of the present application, an antibody or antigen-binding fragment thereof binding to H3N2 HA protein is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 having an amino acid sequence as shown in SEQ ID NO. 1, HCDR2 having an amino acid sequence as shown in SEQ ID NO. 2, and HCDR3 having an amino acid sequence as shown in SEQ ID NO. 3, and the light chain variable region comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO. 4, LCDR2 having an amino acid sequence as shown in SEQ ID NO. 5, and LCDR3 having an amino acid sequence as shown in SEQ ID NO. 6.

[0005] According to the antibody or antigen-binding fragment thereof provided in the embodiments of the present application, at least the following beneficial effects are achieved:

[0006] The antibody or antigen-binding fragment thereof provided in the embodiments of the present application can bind to influenza virus H3N2 HA protein with high affinity, and has good application prospect as a diagnostic tool for influenza virus.

[0007] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 8.

[0008] In some embodiments, further comprising a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO. 9; and / or, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO. 10.

[0009] 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, scFv.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

[0011] In a second aspect of the present application, a biological material is provided, the biological material comprising any one of B1) to B4);

[0012] B1) a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof;

[0013] B2) an expression cassette containing the nucleic acid molecule of B1);

[0014] B3) a recombinant vector containing the nucleic acid molecule of B1) or the expression cassette of B2);

[0015] B4) a recombinant cell containing the nucleic acid molecule of B1) or the expression cassette of B2) or the recombinant vector of B3).

[0016] In a third aspect of the present application, a kit is provided, comprising the aforementioned antibody or antigen-binding fragment thereof.

[0017] In a fourth aspect of the present application, use of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned kit, in the preparation of a diagnostic product for H3N2 influenza virus is provided.

[0018] In a fifth 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.

[0019] In some embodiments, the method is any one of an immunochromatography method, an enzyme-linked immunosorbent assay, a chemiluminescence method, and an electrochemiluminescence method.

[0020] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 3 is a SDS-PAGE gel of H3N2 HA protein in Example 1. Wherein, from left to right, the lanes are Marker, +DTT reducing condition, -DTT non-reducing condition, respectively.

[0022] Figure 2 Figure 4 is the result of serum antibody titer evaluation of volunteers in Example 2. Wherein, HA is used as coating antigen, and the serum is diluted in equal proportion gradient of 1:200-409600.

[0023] Figure 3 Figure 5 is the result of activity verification of biotinylated HA protein after purification in Example 3. Wherein, the serum of volunteers (Serum) is used as coating primary antibody, and the HA without biotin and the biotinylated HA (HA-biotin) are used as sandwich antigen, respectively, and the secondary antibody is HRP-streptavidin (HRP-streptavidin).

[0024] Figure 4 Figure 6 is the sorting result of HA-specific single memory B cells in Example 4.

[0025] Figure 5 Figure 7 is 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. Wherein, lane M is DL5000 marker, lane 1 and lane 2 are MZ001 VH chain and MZ001 VL chain, respectively.

[0026] Figure 6 Figure 8 is the SDS-PAGE gel of MZ001 antibody after the heavy chain and light chain variable region are constructed into whole antibody and expressed by 293F cells and purified in Example 6. Wherein, lane M is protein marker, lane 1 and lane 2 are MZ001 with DTT and without DTT, respectively.

[0027] Figure 7 Figure 9 is the result of detecting the affinity of MZ001 monoclonal antibody to HA using Octet in Example 7. DETAILED DESCRIPTION

[0028] The concept and the technical effects of the present application will be described below in conjunction with the embodiments so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0029] The embodiments of the present application are described below in detail, the described embodiments are exemplary and are only used to explain the present application, and cannot be understood as a limitation to the present application.

[0030] In the description of the present application, the meaning of several is more than one, the meaning of multiple is more than two, greater than, less than, more than, etc. are understood as not including the number, above, below, etc. are understood as including the number. If it is described as first, second, it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0031] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one skilled in the art to which the present application belongs. The terms used herein are only for the purpose of describing the embodiments of the present application and are not intended to limit the present application.

[0032] In the description of the present application, the description of the reference terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example" 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 the present application. In the specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0033] 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 the specific type of target antigen includes polypeptides, proteins, nucleic acids, lipids, carbohydrates, haptens or other natural or artificially synthesized compounds. Specifically, in the present application, the antigen refers to H3N2 influenza virus HA protein, and the immunoglobulin that specifically binds to H3N2 influenza virus HA protein refers to an immunoglobulin with a K D The immunoglobulin binds to H3N2 influenza virus HA protein. In addition, for other antigens other than H3N2 influenza virus HA protein, the immunoglobulin as an antibody basically does not specifically bind to them, but this does not mean that the antibody cannot bind other antigens, for example, immunological cross-reaction is also known in the art.

[0034] Wherein, K DKa is the equilibrium dissociation constant of an antibody to an antigen, and is denoted as K off The ratio of K on is denoted as molarity (M). K D can be used to measure the strength of the binding affinity between an antibody and its antigen, and the smaller the value of K D indicates the stronger the affinity. The value of K D of an antibody can be determined by any method known in the art, including but not limited to methods by surface plasmon resonance (SPR), such as using a SPR chip or related analysis system such as Biacore T200; methods by bio-layer interferometry (BLI), such as related detection system such as Octet Red96.

[0035] The type of antibody specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multivalent antibodies, etc. In addition, the antibody can also be modified in various ways, such as at least one of PEGylation, glycosylation, formation of antibody molecule conjugates (such as ADC), addition of purification tags, or fusion with other proteins, etc.

[0036] Taking a full-length antibody as an example, it comprises a multimer formed by two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. According to the conservation difference of amino acid sequences, the heavy chain and the light chain can be divided into variable region (V) and constant region (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 3 complementarity determining regions (CDRs) and is separated by framework regions (FRs), arranged as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminal to carboxyl terminal, so that VH contains three CDRs: HCDR1, HCDR2, HCDR3, and VL contains three CDRs: LCDR1, LCDR2 and LCDR3.

[0037] The term "complementarity determining region" is a high-frequency mutation region within the variable region, and the CDRs of a heavy chain and a light chain interact together to form an 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.

[0038] Among them, the light chain of the antibody is 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.

[0039] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. It is understood that a monoclonal antibody can be monospecific or multispecific. Specifically, a monoclonal antibody typically binds to one epitope, but the possibility of multispecificity exists. Correspondingly, "polyclonal antibody" refers to a collection of different antibodies against the same antigen, e.g., a collection of different antibodies produced by B lymphocytes, including a mixture of antibodies against different epitopes.

[0040] The term "multispecific" refers to an antibody that can bind to two or more different epitopes of the same antigen, or can bind to two or more different antigens or combinations thereof. A multispecific antibody can have cross-reactivity to other related antigens, e.g., cross-reactivity to the same antigen from other species (homologs), or can bind to an epitope shared between two or more different antigens.

[0041] The term "epitope" refers to a portion of an antigen that is capable of specific binding to an antibody, typically consisting of amino acids or side chains with chemically active moieties such as polar or charged moieties, and optionally at least one of specific three-dimensional structural characteristics and specific charge characteristics.

[0042] Depending on the origin of the antibody, the antibody can be a human antibody, a humanized antibody, or a chimeric antibody.

[0043] 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 produced by random mutation or site-directed mutation.

[0044] 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 the order Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, and other animals of the order Primates, such as dogs, cats, pigs, sheep, cows, horses, rabbits, mice, monkeys, orangutans, chimpanzees, and other non-human species.

[0045] The term "chimeric antibody" refers to an antibody in which different portions of the antibody are derived from different animal species, such as where the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody, or vice versa. Chimeric antibodies are used primarily to reduce immunogenicity in applications and to improve yields in production, such as where a murine antibody has a higher hybridoma yield but higher human immunogenicity, such that a human / mouse chimeric antibody is used. It is understood that chimeric antibodies also include where different portions of the antibody are derived from different animal individuals.

[0046] Where the sequences of the antibody complementarity determining regions are known, one skilled in the art can readily replace other portions of the sequence of the antibody (such as the constant region or the framework region) with sequences from other species by techniques such as DNA recombination to form a chimeric antibody or a humanized antibody, while substantially retaining the binding specificity of the source antibody. For example, where the source antibody is a murine antibody, its constant region or framework region can be replaced with a human antibody constant region, or vice versa, where the source antibody is a human antibody, its constant region or framework region can be replaced with a murine antibody constant region or framework region, to reduce the immunogenicity of the antibody when used in different species or to exploit a particular function, such as ADCC-related activity.

[0047] For further reduction of antibody immunogenicity or other purposes, other sequences in the antibody molecule, other than the CDR sequences, can be replaced with 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 source antibody.

[0048] The term "antigen-binding fragment" refers to a fragment of an immunoglobulin that retains the ability to specifically bind to an 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 a heavy chain and CH1 and a light chain, which can be, for example, a papain cleavage product of an antibody. The Fab' fragment contains a variable region of a heavy chain and CH1 and a fragment of a region between CH1 and CH2 and a 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, a pepsin cleavage product of an antibody. Two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of CH3 domains. 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. By correct folding, the heavy chain variable region and the light chain variable region form an Fv fragment by non-covalent bond interaction, so that the scFv can well retain its affinity activity to the antigen.

[0049] 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 embodiments, a method for expressing antibodies or antigen-binding fragments thereof in host cells (also known as recombinant cells) is provided, which can be mammalian cells, such as mouse, rat, pig, sheep, monkey, chimpanzee or human cells. In some of these 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 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, MCR-5 cells, etc.

[0050] The term "H3N2 influenza virus" refers to a subtype of influenza A virus, which is one of the major 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 of 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, A / Victoria / 361 / 2011, etc. Accordingly, the "H3N2 influenza virus HA protein" refers to the hemagglutinin (HA) protein of the H3N2 influenza virus, which is a type I transmembrane glycoprotein that forms a trimeric structure on the surface of the virus. The HA protein plays a key role in the binding and membrane fusion process of the virus with host cells, and is essential for the infection of host cells and intercellular transmission of influenza virus. The HA protein is composed of two subunits, HA1 and HA2, wherein HA1 is mainly responsible for binding to the sialic acid receptor on the surface of host cells, and HA2 is mainly involved in the fusion process of the virus with the cell membrane.

[0051] The term "nucleic acid molecule" refers to at least one of genomic, mRNA, cDNA or DNA, RNA of synthetic origin. It can be understood that those skilled in the art can mutate or modify the nucleotide sequence of the nucleic acid molecule using known methods, such as directed evolution and point mutation, as long as the antibody or antigen-binding fragment encoded by the mutated or modified nucleic acid molecule has the same function, and the nucleotide sequence derived from the embodiments of the present application is the nucleic acid molecule of the embodiments of the present application.

[0052] The term "expression cassette" refers to a construct containing regulatory elements necessary for the expression of the contained nucleic acid molecule in a recombinant cell. Specific regulatory elements include promoters, polyadenylation sites, etc.

[0053] 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. The 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 an ampicillin resistance gene AmpR, a 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.

[0054] 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.

[0055] In a first aspect, the present application relates to an antibody or antigen-binding fragment thereof binding to HA protein of H3N2 influenza virus, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1: GYTFTDHD, HCDR2: MSPNSGKT, HCDR3: ARAATFFY, LCDR1: QSLLHSYGYNS, LCDR2: LGS, and LCDR3: MQRQQTPIT, wherein the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

[0056] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 7:

[0057] QVRLVQSGAEVKKPGASVKVSCKASGYTFTDHDIHWVRQAAGQGLEWMGWMSPNS GKTAYAQKFQGRITMTRDTSISTVYLELSSLRFEDTAVYFCARAATFFYWGQGSLVTVSS.

[0058] In some embodiments, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 7:

[0059] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNSLDWFLQKPGQAPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRQQTPITFGQGTRLDIK.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof further comprises at least one of a heavy chain constant region, a light chain constant region. It is understood that the heavy chain constant region, the light chain constant region can be full length or a partial fragment thereof, such as CH1, CH2, etc.

[0061] In some embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO. 9:

[0062] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0063] In some embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO. 10:

[0064] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof is selected from at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, scFv.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof is any one of human antibody, humanized antibody and chimeric antibody.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a tag sequence at N-terminus and / or C-terminus, including a tag sequence facilitating solubility and / or purification. Specifically, the tag sequence includes at least one of His tag, GGGS sequence, FLAG tag, strep tag, nus tag, maltose binding protein, GST tag. It can be understood that one or more tag sequences can be included; multiple tag sequences can include a combination of multiple same tag sequences or a combination of multiple different tag sequences.

[0068] The second aspect of the present application relates to a biological material, which can 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; a recombinant cell containing the nucleic acid molecule or the expression cassette or the recombinant vector.

[0069] The third aspect of the present application relates to a kit, which comprises the aforementioned antibody or antigen-binding fragment thereof.

[0070] In some embodiments, the kit can be any one of a detection plate, a detection chip, a detection test paper and the like for H3N2 influenza virus or H3N2 influenza virus HA protein.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof can be in the form of solid state (such as lyophilized powder), liquid state (such as solution) or coated directly on a substrate (such as test paper or chip) to participate in the composition of the kit.

[0072] In some embodiments, the kit detects the H3N2 influenza virus or the H3N2 influenza virus HA protein by any one of an immunochromatography method, an enzyme-linked immunosorbent assay, a chemiluminescence method, an electrochemiluminescence method, etc. The enzyme-linked immunosorbent assay includes, but is not limited to, any one of a direct method, an indirect method, a sandwich method, and a competitive method, etc. The immunochromatography method includes, but is not limited to, any one of a fluorescent microsphere immunochromatography method, a colloidal gold immunochromatography method, a latex microsphere immunochromatography method, a time-resolved fluorescent microsphere immunochromatography method, a magnetic microsphere immunochromatography method, and a quantum dot immunochromatography method, etc.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof in the kit can be at least one of a coating antibody or a detection antibody.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof is bound to a solid substrate such as a magnetic bead, a microsphere, a microplate, a nitrocellulose membrane, a microfluidic chip, etc.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof is coupled with a detectable label, or the kit further comprises a separate detectable label capable of being coupled with 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 emission substance, a micro-nano particle, an enzyme, etc.

[0076] In a fourth aspect, the present application relates to use of the antibody or antigen-binding fragment thereof, or the kit in the preparation of a diagnostic product for the H3N2 influenza virus.

[0077] Specifically, the antibody or antigen-binding fragment thereof can be used in the preparation of a diagnostic product for the H3N2 influenza virus. The kit can be used in the preparation of a diagnostic product for the H3N2 influenza virus.

[0078] The present application also relates to use of the antibody or antigen-binding fragment thereof, or the pharmaceutical composition, or the kit in the diagnosis of H3N2 influenza virus infection.

[0079] The present application also relates to a method for diagnosing H3N2 influenza virus infection, comprising contacting the antibody or antigen-binding fragment thereof with a subject.

[0080] In a fifth aspect, the present application relates to a method for detecting the H3N2 influenza virus in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof as described above.

[0081] In some embodiments, the method comprises detecting the H3N2 influenza virus or the H3N2 influenza virus HA protein by any one of an immunochromatography method, an enzyme-linked immunosorbent assay, a chemiluminescence method, an electrochemiluminescence method, etc. The enzyme-linked immunosorbent assay comprises, but is not limited to, any one of a direct method, an indirect method, a sandwich method, a competitive method, etc. The immunochromatography method comprises, but is not limited to, any one of a fluorescent microsphere immunochromatography method, a colloidal gold immunochromatography method, a latex microsphere immunochromatography method, a time-resolved fluorescent microsphere immunochromatography method, a magnetic microsphere immunochromatography method, and a quantum dot immunochromatography method, etc.

[0082] In some embodiments of the present application, the method for detecting the H3N2 influenza virus in a sample is a method for non-diagnostic purposes, such as a virus monitoring analysis, etc. The transmission routes of the H3N2 influenza virus include air droplet transmission, and also include direct or indirect contact transmission through mucous membranes of the mouth, nose, eyes, etc., as well as contact with virus-contaminated objects. In addition, in a room with a large number of people and poor ventilation, the virus can also be transmitted in the form of aerosol. For example, the method can be used for the following non-diagnostic purposes:

[0083] (1) Environmental monitoring: detecting whether the H3N2 influenza virus exists in environmental samples (such as air, object surface swabs, etc.) in public places such as medical institutions, kindergartens, schools, etc., for evaluating environmental safety and developing corresponding preventive measures.

[0084] (2) In vitro culture sample detection: in virology research, detecting the presence and proliferation of the H3N2 influenza virus in laboratory cultured cell or tissue samples, for evaluating the effect of antiviral drugs or studying the biological characteristics of the virus.

[0085] (3) Production quality control: in the production process of biological products (such as vaccines, blood products, etc.), detecting whether the H3N2 influenza virus exists in raw materials, intermediate products, or finished products, to ensure product safety.

[0086] (4) Experimental animal quarantine: detecting whether the H3N2 influenza virus exists in experimental animals or their tissue samples, for quality control and management of experimental animals.

[0087] (5) Scientific research purposes: in basic research in the fields of molecular biology, immunology, etc., detecting the presence of the H3N2 influenza virus in experimental samples, for studying scientific issues such as virus-host interaction and virus evolution.

[0088] In some embodiments, the sample includes, but is not limited to, environmental samples (e.g., air, water, soil, surface swabs of objects, etc.), in vitro culture samples (e.g., cell culture supernatants, tissue cultures, etc.), biological product samples (e.g., vaccines, blood products, etc.), laboratory animal samples (e.g., tissues, body fluids of laboratory animals, etc.), research samples, etc. None of these samples involves direct diagnosis for human body.

[0089] In some embodiments, the sample includes a biological sample. In some embodiments, the biological sample includes, but is not limited to, at least one of a body fluid (e.g., blood, interstitial fluid, lymphatic fluid, cerebrospinal fluid, urine, sweat, sputum, saliva, gastric juice, intestinal juice, pancreatic juice, bile, prostatic fluid, vaginal secretion, semen, serosal cavity effusion, joint cavity effusion, bronchoalveolar lavage fluid, amniotic fluid, etc.), skin, feces, intestinal contents, a histological sample (e.g., a sample obtained by surgical, endoscopic, or percutaneous needle biopsy).

[0090] The antibody or antigen-binding fragment thereof provided in the present application can be used as a research and development of an antibody conjugate targeting the HA protein.

[0091] The antibody or antigen-binding fragment thereof provided in the present application can specifically bind to the H3N2 influenza virus HA protein, i.e., the H3N2 influenza virus HA protein as the target antigen of the antibody. Due to the antigen-binding specificity of the antibody provided in the embodiments of the present application, the antibody can be used to detect whether the H3N2 influenza virus exists in a sample (e.g., blood) from a subject. Accordingly, the embodiments of the present application provide an H3N2 detection kit which can include the antibody or antigen-binding fragment thereof of the embodiments.

[0092] Specifically, in the embodiments of the present application, a human monoclonal antibody was isolated from adult peripheral blood mononuclear cells using the H3N2 HA fusion protein as a decoy, which was named MZ001. This antibody can specifically bind to the H3N2 HA protein, and thus the monoclonal antibody can be applied to the diagnosis and antigen quantification of H3N2.

[0093] The present application is further illustrated by the following specific examples.

[0094] Example 1: Expression and purification of H3N2 HA protein

[0095] The H3N2 HA (GenBank: ARV89911.1) protein sequence was retrieved from the NCBI database. A signal peptide sequence (MNPLLILTFVAAALAAPFDDDDK, SEQ ID NO. 12) was added at the N-terminus of the HA protein (18-520 aa), and a trimerization tag, a thrombin tag, a his tag and a strep tag were introduced at 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 suspension mammalian cells 293F 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 6 10 / 30 GL (GE healthcare). The protein was concentrated to 10 mg / mL, quick-frozen in liquid nitrogen and stored at -80°C for standby.

[0096] The size and purity of the target protein were determined by SDS-PAGE of the purified HA 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.

[0097] Example 2: Evaluation of volunteer serum

[0098] 100 μL of H3N2 HA protein was added to each well at a concentration of 2 μg / mL, and coating of H3N2 HA protein on the microplate was completed after incubation at 4°C overnight. The next day, the plate was washed with PBST 3 times. Then 2% BSA was added at 150 μL / well, and after incubation at 37°C for 2 h, the plate was washed with PBST 3 times. Subsequently, the volunteer serum inoculated with a 4-valent influenza split vaccine for 6 months was added at 100 μL / well in a dilution of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, and after incubation at 37°C for 2 h, the plate was washed with PBST 3 times. Then goat anti-human IgG (Fc-specific) horseradish peroxidase-labeled secondary antibody was added at 100 μL / well at a dilution of 1:5000, and after incubation at 37°C for 1 h, the plate was washed with PBST 3 times. Color development was performed by adding 100 μL of substrate TMB per well, and after reaction in the dark for 10 min, the reaction was terminated by adding 50 μL of 2M H2SO4. The OD450 was measured, and the data were processed.

[0099] The results are shown in 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.

[0100] Example 3: HA protein biotinylation

[0101] Follow EZ-Link TM The HA protein was biotinylated using the Sulfo-NHS-LC-biotinylation kit (Thermo Fisher) to obtain biotin-labeled HA protein.

[0102] 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.

[0103] 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.

[0104] Example 4: Isolation of H3N2 HA protein-specific memory B cells

[0105] After informed consent from the volunteers, 10 mL of blood was collected and PBMCs were isolated. 7PBMCs were incubated with 200 nM of the HA protein prepared in Example 1 for 30 min on ice; 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-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and Streptavidin / PE. After incubation for 30 min on ice, they were washed twice with PBS. Subsequently, PBMCs were sorted by FACSAria III, and the PE-Cy5 - APC - APC-Cy7 + Pacific Blue + FITC + PE + cells (i.e., memory B cells) were directly collected into a 96-well plate, 1 cell / well, and sorted as Figure 4 indicated.

[0106] Example 5: Single B cell PCR and cloning of human monoclonal antibody IgGl

[0107] The cells collected by sorting in Example 4 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) under the following conditions: 55°C, 60 min. The reverse transcription product was used as a template for PCR with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCR a) under the following conditions: 95°C, 5 min; 95°C, 30 s, 55°C (heavy chain / κ chain) / 50°C (λ chain), 30 s, 72°C, 90 s, 35 cycles, 72°C, 7 min. This was used as a template for another round of PCR (PCR b) under the following conditions: 95°C, 5 min; 95°C, 30 s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain), 30 s, 72°C, 90 s, 35 cycles, 72°C, 7 min. The PCR product was separated by 1.2% agarose gel electrophoresis, and the 400-500 bp band was cut and recovered for sequencing. The agarose gel of the antibody variable region amplification is shown in Figure 5 The sequencing results were subjected to sequence analysis on the IGBLAST website.

[0108] According to the sequencing results, a total of one paired antibody MZ001 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, the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the heavy chain variable region were respectively shown as SEQ ID NO. 1-6.

[0109] In order to obtain human antibodies for subsequent evaluation, a whole anti-IgG1 construction was designed. The strategy is as follows:

[0110] Heavy chain: CMV promoter-SacI-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-KpnI;

[0111] Light chain kappa: CMV promoter-SacI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLK)-KpnI.

[0112] The correct variable region sequence and the corresponding heavy chain CH and light chain CLK constant region were connected by bridge PCR, and were cloned into the expression vector pCAGGS to obtain a recombinant plasmid containing the coding gene of the specific antibody light and heavy chain; wherein the restriction sites SacI and KpnI 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 as shown in SEQ ID NO. 9; the amino acid sequence of the light chain constant region CLK is as shown in SEQ ID NO. 10, and the amino acid sequence of the signal peptide (SP) is as shown in SEQ ID NO. 11: ETDTLLLWVLLLWVP.

[0113] Example 6: Expression and purification of monoclonal antibody

[0114] The pCAGGS plasmid containing the coding sequences of the heavy chain and the light chain was co-transfected into 293F cells. 220 mM VPA was added 24 h after transfection, and the culture was continued for 5 days. The cells were harvested, the supernatant was collected by centrifugation, and after filtration with a 0.22 μm filter, it was purified by HiTrap Protein A HP (5 ml, GE Healthcare) chromatography column and gel filtration chromatography column Superose6 10 / 30 GL (GE healthcare) at a speed of 2 mL / min, SDS-PAGE was shown as Figure 6 , and finally concentrated and stored at -80℃ for standby.

[0115] Example 7: Detection of the binding performance of the antibody

[0116] The interaction of purified antibody with the post-fusion HA protein was detected by Bio-Layer Interferometry (BLI) using Octet Red96 system (ForteBio). The antibody detection used protein A sensor loaded at a concentration of 10 μg / mL, then combined with HA protein in the range of 25-1600 nM for 180 s, dissociation for 600 s, and the regeneration of the sensor used 10 mM Glycine (pH 1.5). All proteins used in the experiment were exchanged into PBS buffer, and the detection buffer was determined by the binding of HA protein. The apparent equilibrium dissociation constant (KD value) of the interaction of HA protein with antibody was calculated using Octet Red96 evaluation software.

[0117] The results are shown in Figure 7 Figure 2, MZ001 can bind to H3N2 HA fusion protein with high affinity, K D = 0.66 nM, the association rate is 5.79 x 10 4 / s, and the dissociation rate is 3.82 x 10 -4 / s.

[0118] Example 8

[0119] This embodiment provides an H3N2 diagnostic kit, which includes H3N2 standard products of different concentration gradients, coated plates, H3N2 primary antibodies, enzyme-labeled H3N2 secondary antibodies, BSA blocking solution, color developing agent A solution, color developing agent B solution, and termination solution.

[0120] The preparation process of the pre-coated plate is as follows: the purified H3N2 primary antibody is diluted with a carbonate buffer solution at pH 9.6 to obtain a mixture with a concentration of 5 μg / mL, and the 96-well plate is coated with 0.1 mL per well, and then incubated at 4°C for 21 h. The liquid in the wells is discarded, and the plate is washed and dried; then 0.1 mL of BSA blocking solution is added to each well, and the plate is incubated at 37°C for 2 h. The liquid in the wells is discarded, the plate is washed and dried, and a pre-coated plate is obtained.

[0121] The H3N2 primary antibody is a mouse anti-H3N2 monoclonal antibody.

[0122] The enzyme-labeled H3N2 secondary antibody is labeled on the H3N2 secondary antibody by the sodium periodate oxidation method using HRP, and the H3N2 secondary antibody is MZ001.

[0123] The termination solution is 2M HCl.

[0124] The use method of the kit is as follows:

[0125] Add 75 μL of sample or H3N2 standard to each well, set negative control and blank control, incubate at 37℃ for 1 h, discard the liquid in the well, wash and pat dry. Then add 25 μL of enzyme-labeled H3N2 secondary antibody to each well, incubate at 37℃ for 1 h, discard the liquid in the well, wash and pat dry. Then add 100 μL of color developing agent A and color developing agent B to each well, color develop at 37℃ for 10 min, add 50 μL of stop solution to each well. Zero the blank well at 450 nm, read the OD value of each well.

[0126] The quantitative method is as follows: draw a standard curve according to the OD values of H3N2 standards of different concentrations and determine the linear range, and substitute the OD value of the sample into the standard curve to obtain the H3N2 concentration in the sample.

[0127] The qualitative method is as follows: set the 2 times of the OD value of the negative control as the critical OD value, and if the OD value of the sample is greater than the critical OD value, it is determined that the H3N2 antigen is positive, otherwise it is negative.

[0128] The detection limit LOD = 3SD / b is calculated according to the standard curve in Example 8, where SD is the standard deviation of the blank control and b is the slope of the standard curve, and the results show that all of them have a lower detection limit. At the same time, the clinical samples identified as negative and positive by nucleic acid were detected to determine the false positive and false negative of the kit. The results show that the H3N2 diagnostic kit of Example 8 has good specificity and sensitivity.

[0129] Example 9

[0130] The present embodiment provides an H3N2 colloidal gold test strip, which comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, a water-absorbing paper and a PVC substrate arranged in sequence and abutted with each other. The sample pad is provided with a sample hole.

[0131] The colloidal gold pad is coated with colloidal gold-labeled mouse anti-H3N2 monoclonal antibody, 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 MZ001 antibody.

[0132] During detection, 200 μL of sample is added to the sample hole, and the result is observed after 15 min of reaction; both the C line and the T line appear as positive; only the C line appears as negative; and no band on the C line is invalid.

[0133] The H3N2 colloidal gold test strip of Example 9 is detected by using H3N2 standards of different concentrations, respectively, to determine the lowest concentration of the standard that causes the T line to appear a band, which is the detection sensitivity of the test strip. At the same time, the clinical samples identified as negative and positive by nucleic acid are detected to determine the false positive and false negative of the test strip. The results show that the test strip of Example 9 has high sensitivity, specificity and sensitivity.

[0134] The application has been described in detail by combining with the embodiments above, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without 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 LGS, 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. 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); B4) A recombinant cell containing the nucleic acid molecule described in B1), wherein the recombinant cell is selected from bacteria, fungi, and animal cells, and the recombinant cell is a non-reproductive material.

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

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the kit according to claim 7, in the preparation of a diagnostic product for H3N2 influenza virus.

9. 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 5.

10. The method according to claim 9, characterized in that The method is any one of immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, and electrochemiluminescence.

Images