Fully human monoclonal antibody MZ001 combined with H3N2 HA protein and application of fully human monoclonal antibody MZ001

Abstract

The invention discloses a fully human monoclonal antibody MZ001 combined with an H3N2HA protein and an application of the fully human monoclonal antibody MZ001. The antibody or the 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 the antigen binding fragment thereof can bind to influenza virus H3N2HA protein with high affinity, and has a good application prospect as an influenza virus diagnostic tool.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and in particular to a fully human monoclonal antibody MZ001 binding to H3N2 HA protein and its application. Background Art

[0002] Influenza virus is a single-stranded RNA virus, which can be divided into type A, type B, type C, type 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 the virus to the host cell surface receptors and the fusion of the virus membrane with the host cell membrane. It is a key factor in the process of virus infection of cells and an important part of influenza virus typing. One of the major methods for specific etiological detection of influenza A virus infection is antigen detection, which is fast and convenient, and has low sample requirements. However, in the current development of antigen detection of influenza A virus, the specific antibodies against H3N2 HA protein have the problem of poor affinity. 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 MZ001 that binds to H3N2 HA protein with high affinity and its application.

[0004] In a first aspect of the present application, an antibody or an antigen-binding fragment thereof that binds 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 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 comprising LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.

[0005] The antibodies or antigen-binding fragments thereof according to the embodiments of the present application have at least the following beneficial effects:

[0006] The antibodies or antigen-binding fragments thereof provided in the embodiments of the present application can bind to the influenza virus H3N2HA protein with high affinity and have good application prospects as diagnostic tools for influenza viruses.

[0007] In some embodiments of the present application, 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.

[0008] In some embodiments, it also includes a heavy chain constant region and a light chain constant region, 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.

[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, and 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 biomaterial is provided, the biomaterial 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 B1) or the expression cassette B2);

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

[0016] The third aspect of the present application provides a kit comprising the aforementioned antibody or antigen-binding fragment thereof.

[0017] The fourth aspect of the present application provides a 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.

[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 immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, and electrochemiluminescence.

[0020] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the SDS-PAGE diagram of the H3N2 HA protein in Example 1. The lanes from left to right are the results of Marker, +DTT reducing conditions, and -DTT non-reducing conditions, respectively.

[0022] Figure 2 This is the evaluation result of 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.

[0023] Figure 3 This is the activity verification result after biotinylation of the purified HA protein in Example 3. Volunteer serum (Serum) was used as the primary coating antibody, non-biotinylated HA and biotinylated HA (HA-biotin) were used as sandwich antigens, and HRP-streptavidin (HRP-streptavidin) was used as the secondary antibody.

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

[0025] Figure 5 The heavy chain variable region (VH) and light chain variable region (VL) of the antibody amplified by reverse transcription and nested PCR in Example 5, wherein lane M is DL5000 marker, lane 1 and lane 2 are MZ001 VH chain and MZ001 VL chain, respectively.

[0026] Figure 6 This is the SDS-PAGE gel image of the heavy chain and light chain variable regions of the MZ001 antibody constructed as a full antibody after being expressed in 293F cells and purified in Example 6. Lane M is a protein marker, and lanes 1 and 2 are the band results of MZ001 with and without DTT, respectively.

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

[0028] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination 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, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0029] 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.

[0030] In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "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 the indicated technical features or implicitly indicating the order of the indicated technical features.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those 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.

[0032] In the description of the present application, the description with reference to the terms "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 the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may 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. Accordingly, "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, antigen refers to H3N2 influenza virus HA protein, and an immunoglobulin that specifically binds to H3N2 influenza virus HA protein refers to an immunoglobulin with a K of less than 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 H3N2 influenza virus HA protein. In addition, for antigens other than H3N2 influenza virus HA protein, immunoglobulins as antibodies basically do not specifically bind to them, but this does not mean that antibodies cannot bind to other antigens, for example, immune cross-reactions are also known in the art.

[0034] Among them, K Dis the equilibrium dissociation constant between antibody and antigen, K off With K on The ratio is expressed in molar concentration (M). D It can be used to measure the binding affinity between an antibody and its antigen. 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) method, for example, SPR chip or related analysis system such as Biacore T200; biomembrane interferometry (BLI) method, for example, related detection system such as Octet Red96.

[0035] The types of antibodies specifically include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multivalent antibodies, etc. In addition, antibodies may be modified in a variety of ways, such as PEGylation, glycosylation, formation of antibody molecule conjugates (such as ADC), addition of purification tags, or fusion with other proteins.

[0036] 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 polymer formed thereof. According to the conservative differences in the amino acid sequences, the heavy chain and the light chain 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 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 a framework region (FR), which is arranged from the amino terminus to the carboxyl terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, so VH contains three CDRs: HCDR1, HCDR2, HCDR3, and VL contains three CDRs: LCDR1, LCDR2 and LCDR3.

[0037] The term "complementarity determining region" refers to the high-frequency mutation area in the variable region. The CDRs of a heavy chain and a light chain interact to form the antigen binding site of the full-length antibody, which is complementary to the three-dimensional structure of the antigen epitope. The high heterogeneity of CDR determines the diversity of antibodies, thereby recognizing different antigen epitopes.

[0038] Among them, the light chain of the antibody is divided into κ chain and λ chain. The heavy chain of the antibody can usually be divided into μ chain, δ chain, γ chain, α chain and ε chain, and 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 or IgA2.

[0039] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, whereby the individual antibodies constituting the antibody population are substantially identical and bind to the same antigenic epitope, except for antibody variants resulting from mutations that may be present in very small amounts. It is understood that monoclonal antibodies are monospecific or polyspecific. Specifically, monoclonal antibodies typically bind to one antigenic epitope, but may also have polyspecificity. Accordingly, 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.

[0040] The term "multispecific" refers to an antibody that can bind to two or more different antigenic epitopes of 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 other species (homologous), or may bind to an epitope shared between two or more different antigens.

[0041] 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 or non-polar or hydrophobic groups, and optionally has at least one of a specific three-dimensional structural feature and a specific charge feature.

[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, wherein the human immunoglobulin sequence may 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 "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). Among them, non-human species include other mammals, such as animals of the order Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, and other animals of the order Primates, specifically non-human species such as dogs, cats, pigs, sheep, cattle, horses, rabbits, mice, monkeys, orangutans, gorillas, chimpanzees, etc.

[0045] The term "chimeric antibody" refers to antibodies in which different parts are derived from different animal species, such as antibodies whose variable regions are derived from one species and whose constant regions are derived from another species, specifically, the variable regions are derived from mouse antibodies and the constant regions are derived from human antibodies or vice versa. Chimeric antibodies are mainly used to reduce immunogenicity in applications and to increase the yield during preparation, for example, where mouse antibodies have a higher hybridoma yield but higher human immunogenicity, so that human / mouse chimeric antibodies are used. It is understood that chimeric antibodies also include situations in which different parts of an antibody are derived from different animal individuals.

[0046] In the case of known antibody complementary determining region sequences, those skilled in the art can easily replace other partial sequences (such as constant regions or framework regions) in the antibody with sequences from other species by techniques such as DNA recombination, forming chimeric antibodies or humanized antibodies, and substantially retaining 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.

[0047] 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 source antibody.

[0048] The term "antigen-binding fragment" refers to an antibody fragment in an immunoglobulin that retains the ability to specifically bind to the antigen bound by the full-length antibody, and can be a synthetic, enzymatically obtained or genetically engineered polypeptide. Antibody binding fragments include 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 the variable region and CH1 of a heavy chain and a light chain, for example, it can be a papain cleavage product of an antibody. The Fab' fragment contains the variable region and CH1 of a heavy chain and the regional fragment 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, for example, it can be a pepsin cleavage product of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domain. The Fv fragment is composed of a heavy chain variable region and a light chain variable region. scFv is composed of a heavy chain variable region, a light chain variable region and a linker connecting the heavy chain variable region and the light chain variable region. Through correct folding, the heavy chain variable region and the light chain variable region interact through non-covalent bonds to form an Fv segment, so scFv can better retain its affinity activity for antigens.

[0049] Methods for preparing antibodies are known in the art, including but not limited to separating cells expressing specific antibodies after immunizing animals with antigens (e.g., preparing antibodies by hybridoma technology), screening antibody technology using phage antibody libraries, transforming cells by genetic engineering technology and making them express antibody molecules, etc. In some embodiments, a method for expressing antibodies or antigen-binding fragments thereof in host cells (also known as recombinant cells) is provided, and the host cells can be specifically mammalian cells, such as mice, rats, pigs, sheep, monkeys, orangutans or human cells. 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.

[0050] 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 among 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. Correspondingly, "H3N2 influenza virus HA protein" refers to the hemagglutinin (HA) protein of the H3N2 influenza virus, which, as a type I transmembrane glycoprotein, forms a trimer structure on the surface of the virus. The HA protein plays a key role in the binding and membrane fusion process between the virus and the host cell, and is necessary for the influenza virus to infect host cells and spread between cells. The HA protein consists of two subunits, HA1 and HA2, of which HA1 is mainly responsible for binding to the sialic acid receptors on the surface of the host cell, while HA2 is mainly involved in the fusion process between the virus and the cell membrane.

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

[0052] The term "expression cassette" refers to a construct containing the necessary regulatory elements 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 nucleic acid molecules contained in the 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, etc. For example, antibiotic resistance genes or selection marker genes (such as ampicillin resistance gene AmpR, thymidine kinase gene TK, etc.) for screening and target protein coding sequences can be inserted into the multiple cloning site (MCS). Optional recombinant vector types include plasmids, bacteriophages, lentiviruses, adenoviruses, adeno-associated viruses, etc.

[0054] 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. Among them, bacteria, for example, can be Escherichia coli or Bacillus subtilis, fungi, for example, can be yeast or Aspergillus, insect cells, for example, can be S2 Drosophila cells or Sf9 cells, plant cells, for example, can be cells of Arabidopsis thaliana or tobacco, and animal cells, for example, can be mammalian cells, such as cells of mice, rats, pigs, sheep, monkeys, orangutans, or human beings, such as 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc. It is understood that these recombinant cells can all be non-reproductive materials.

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

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

[0057] QVRLVQSGAEVKKPGASVKVSCKASGYTFTDHDIHWVRQAAGQGLEWMGWMSPNS GKTAYAQKFQGRITMTRDTSISTVYLELSSLRFEDTAVYFCARAATFFYWGQGSLVTVSS.

[0058] In some embodiments, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8:

[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 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, CH2, etc.

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

[0062] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

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

[0064] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS.

[0065] 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.

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

[0067] 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 dissolution 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; multiple tag sequences may include a combination of multiple identical tag sequences, or a combination of multiple different tag sequences.

[0068] 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; 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 includes 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, etc. for H3N2 influenza virus or H3N2 influenza virus HA protein.

[0071] 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).

[0072] 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 method, indirect method, sandwich method and competitive method. 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.

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

[0074] In some embodiments, the antibody or antigen-binding fragment thereof is bound to a solid substrate such as magnetic beads, microspheres, microplates, nitrocellulose membranes, microfluidic chips, etc.

[0075] In some embodiments, a detectable marker is coupled to the antibody or its antigen-binding fragment, or a separate detectable marker that can be coupled to the antibody or its antigen-binding fragment is also included in the kit. Specifically, the detectable marker can be at least one of a radionuclide, a fluorescent substance, a chemiluminescent substance, a colored substance, an aggregation-induced luminescent substance, a micro-nanoparticle, an enzyme, and the like.

[0076] The fourth 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.

[0077] 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.

[0078] 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.

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

[0080] The fifth 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.

[0081] In some embodiments, the method includes detecting 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 method, indirect method, sandwich method and competitive method. 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.

[0082] In some embodiments of the present application, the method for detecting H3N2 influenza virus in a sample is a method for non-diagnostic purposes, such as virus monitoring and analysis. The transmission routes of H3N2 influenza virus include air droplet transmission, direct or indirect contact transmission through mucous membranes such as the mouth, nose, and eyes, and contact with objects contaminated by the virus. In addition, in crowded, closed or poorly ventilated rooms, it can also be transmitted in the form of aerosols. For example, this method can be used for the following non-diagnostic purposes:

[0083] (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, in order to assess environmental safety and develop appropriate preventive measures.

[0084] (2) In vitro culture sample testing: In virological research, the presence and proliferation of H3N2 influenza virus in laboratory cultured cells or tissue samples are detected to evaluate the effectiveness of antiviral drugs or study the biological characteristics of the virus.

[0085] (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.

[0086] (4) Laboratory animal quarantine: Detecting the presence of H3N2 influenza virus in laboratory animals or their tissue samples for quality control and management of laboratory animals.

[0087] (5) Scientific research purposes: In basic research in molecular biology, immunology and other related fields, the presence of H3N2 influenza virus in experimental samples can be detected to study scientific issues such as virus-host interaction and virus evolution.

[0088] In some embodiments, samples include but are not limited to environmental samples (such as air, water, soil, surface wipes, etc.), in vitro culture samples (such as cell culture supernatants, tissue cultures, 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.

[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 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 cavity effusion, joint cavity 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).

[0090] The antibodies or antigen-binding fragments thereof provided in the present application can be used for the development of antibody conjugates targeting HA protein.

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

[0092] Specifically, in the examples of the present application, H3N2 HA fusion protein was used as bait to isolate a human monoclonal antibody from adult peripheral blood mononuclear cells, named MZ001. This antibody can bind to H3N2 HA protein with high specificity, so this monoclonal antibody can be used for H3N2 diagnosis and antigen quantification.

[0093] The present invention is further described below through specific examples.

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

[0095] The protein sequence of H3N2 HA (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 (18-520aa), and a trimer tag, thrombin tag, his tag and strep tag were introduced to the C-terminus. The coding gene corresponding to the above protein sequence was optimized for human codons and constructed into the vector pCAGGS plasmid. After 5-7 days of expression in suspended mammalian cells 293F, 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 column Superose6 10 / 30GL (GE healthcare). The protein was concentrated to 10 mg / mL, quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0096] The size and purity of the target protein were determined by SDS-PAGE results of the purified HA protein. Figure 1 As shown, the molecular weight of HA is about 60 kDa, and 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.

[0097] Example 2: Volunteer Serum Evaluation

[0098] 100 μL of H3N2 HA protein was added to each well at a concentration of 2 μg / mL. After overnight at 4°C, the coating of H3N2 HA protein on the microplate was completed. The plate was washed 3 times with PBST the next day. Then 2% BSA was added at 150 μL / well for blocking. After incubation at 37°C for 2 hours, the plate was washed 3 times with PBST. Subsequently, 100 μL / well of the serum of volunteers who had been vaccinated with the 4-valent 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 3 times with PBST. Then, add goat anti-human IgG (Fc specific) horseradish peroxidase-labeled secondary antibody at 1:5000 dilution at 100 μL / well, incubate at 37°C for 1 hour, and wash the plate 3 times with PBST. Add substrate TMB 100 μL / well for color development, react in the dark for 10 minutes, and then add 50 μL 2M H2SO4 to terminate the reaction. Measure OD450 and perform data processing.

[0099] 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 method, and 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 detect whether the HA protein was labeled with biotin and the activity of the labeled protein. 100 μL of volunteer serum diluted 1:1600 was added to each well, and the coating was completed after overnight at 4°C. The plate was washed 3 times with PBST the next day. Then 2% BSA was added at 150 μL / well for blocking, incubated at 37°C for 2h, and then washed 3 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 2h. Then the plate was washed 3 times with PBST, and 100 μL of HRP-streptavidin (STREP) storage solution diluted 1:5000 was added to each well, and incubated at 37°C for 45min. Then the plate was washed 3 times with PBST. 50 μL / well of substrate TMB was added for color development, and after 10 minutes of reaction in the dark, 50 μL of 2M HCl was added to terminate the reaction. OD450 was measured and 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. 7 / mL) was incubated on ice with 200nM HA protein prepared in Example 1 for half an hour; then washed twice with PBS, and then mixed with the following antibodies (all purchased from BD): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-humanCD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-humanCD38 / APC, anti-human IgG / FITC, and Streptavidin / PE. After incubation on ice for half an hour, it was washed twice with PBS. Subsequently, PBMCs were sorted with FACSAria III, and PE-Cy5 was 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.

[0106] Example 5: Single B cell PCR and cloning of human monoclonal antibody IgG1

[0107] The cells collected by sorting in Example 4 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen), and the reaction conditions were 55°C, 60min. The reverse transcription product was used as a template, and PCR was performed using HotStar TapPlus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa), and the reaction conditions were as follows: 95°C, 5min; 95°C, 30s, 55°C (heavy chain / κ chain) / 50°C (λ chain), 30s, 72°C, 90s, 35 cycles, 72°C, 7min. This was used as a template for another round of PCR (PCRb), and the conditions were as follows: 95°C, 5min; 95°C, 30s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain), 30s, 72°C, 90s, 35 cycles, 72°C, 7min. 1.2% agarose gel electrophoresis was used to separate the PCR products. The 400-500bp band was cut 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.

[0108] According to the sequencing results, a paired antibody MZ001 was obtained in total, 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.

[0109] In order to obtain human antibodies for subsequent evaluation, a full anti-IgG1 construct 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 κ: CMV promoter-SacI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLκ)-KpnI.

[0112] The correct variable region sequence was analyzed and connected with the corresponding constant region of heavy chain CH and light chain CLκ by bridge PCR, and cloned into the expression vector pCAGGS to obtain a recombinant plasmid containing the light and heavy chain coding genes of a specific antibody; wherein, the light and heavy chain variable regions were connected to the vector containing the constant region using the restriction sites SacI and KpnI. 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 CLκ 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 antibodies

[0114] The pCAGGS plasmid containing the heavy chain and light chain coding sequences was 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 with a 0.22 μm filter membrane, and purified by HiTrap Protein A HP (5 ml, GE Healthcare) chromatography column and gel filtration column Superose6 10 / 30GL (GE healthcare) at a speed of 2 mL / min. SDS-PAGE was performed as shown in FIG. Figure 6 The resulting product was finally concentrated and stored at -80°C until use.

[0115] Example 7: Antibody Binding Performance Detection

[0116] The Octet Red96 system (ForteBio) was used to detect the interaction between the purified antibody and the fused HA protein by biomembrane interferometry (BLI). The antibody was detected using proteinA sensor at a concentration of 10 μg / mL, then bound to HA protein in the range of 25 to 1600 nM for 180 s, dissociated for 600 s, and the sensor was regenerated using 10 mM Glycine (pH 1.5). All proteins used in the experiment were exchanged into PBS buffer, and the detection buffer was used to measure the binding of HA protein. The apparent equilibrium dissociation constant (KD value) of the interaction between HA protein and antibody was calculated using Octet Red96 evaluation software.

[0117] The results are as follows Figure 7 As shown, MZ001 can bind to H3N2 HA fusion protein with high affinity, K D =0.66nM, the binding rate is 5.79×10 4 / Ms, the dissociation rate is 3.82×10 -4 / s.

[0118] Example 8

[0119] 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, a BSA blocking solution, a color developer A solution, a color developer B solution, a stop solution, and the like.

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

[0121] The primary antibody against H3N2 was mouse anti-H3N2 monoclonal antibody.

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

[0123] The stop solution was 2M HCl.

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

[0125] Add 75 μL of sample or H3N2 standard to each well, set up negative control and blank control, incubate at 37℃ for 1h, 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 1h, discard the liquid in the well, wash and pat dry. Then add 100 μL of color developer A solution and color developer B solution to each well, develop at 37℃ for 10min, and add 50 μL of stop solution to each well. At a wavelength of 450nm, calibrate the blank well to zero and read the OD value of each well.

[0126] The quantitative method is as follows: a standard curve is drawn according to the OD values ​​of H3N2 standards with 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.

[0127] The qualitative method is as follows: twice 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.

[0128] In Example 8, the detection limit LOD=3SD / b was calculated based on the standard curve, 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 had low detection limits. At the same time, clinical samples identified as negative and positive for nucleic acid were tested to determine the false positive and false negative of the kit. The results showed that the H3N2 diagnostic kits of Example 8 all had good specificity and sensitivity.

[0129] Example 9

[0130] This embodiment provides a H3N2 colloidal gold test paper strip, comprising a sample pad, a colloidal gold pad, a nitrocellulose membrane, a water-absorbing paper, and a PVC substrate, which are arranged in sequence in pairs. A spotting hole is provided on the sample pad.

[0131] 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 sheep anti-mouse IgG antibody, and the detection line is coated with MZ001 antibody.

[0132] During the test, add 200 μL of the sample to the spotting well, react for 15 minutes and observe the results; if both the C line and the T line have bands, it is positive; if only the C line has a band, it is negative; if there is no band on the C line, the result is invalid.

[0133] The H3N2 colloidal gold test strips of Example 9 were tested with H3N2 standards of different concentrations, and the lowest concentration of the standard when the T line appeared was determined as the detection sensitivity of the test strips. At the same time, clinical samples identified as negative and positive for nucleic acid were tested to determine the false positive and false negative situations of the test strips. The results showed that the test strips of Example 9 all had high sensitivity, specificity and sensitivity.

[0134] The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present 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 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2 and HCDR3 as shown in SEQ ID NO.3, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5 and LCDR3 as shown in SEQ ID NO.

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: The antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody and a chimeric antibody.

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

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.

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