Monoclonal antibody against human influenza virus neuraminidase, its preparation method and application

Abstract

The present application relates to a monoclonal antibody or antigen-binding fragment thereof specifically binding to human influenza virus neuraminidase (NA), related products thereof, and methods of making and uses thereof. The monoclonal antibody or antigen-binding fragment thereof of the present application can bind to human influenza virus NA protein with high affinity and neutralize human influenza virus strains with high neutralization activity, thereby inhibiting infection, and thus has great potential application value in clinical treatment, prevention and / or detection of human influenza virus infection.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, specifically to a monoclonal antibody against the neuraminidase (NA) protein of human influenza virus, its preparation method, and its application. Background Technology

[0002] Influenza virus belongs to the family Orthomyxoviridae and the genus Influenzavirus. It is an enveloped, pleomorphic, segmented, single-stranded negative-sense RNA virus. Influenza viruses are classified into four subtypes—A, B, C, and D—based on the antigenicity of their nucleoprotein (NP) and matrix protein (MP). Type A influenza virus, in particular, can be further subdivided into multiple subtypes based on the antigenicity of its surface proteins hemagglutinin (HA) and neuraminidase (NA). Currently, the HA protein includes 18 subtypes, and the NA protein includes 11 subtypes. Influenza viruses can cause acute respiratory infections, presenting with symptoms such as acute fever, cough, sore throat, body aches, and fatigue. Severe cases can lead to severe illness and even death. Furthermore, some highly pathogenic strains, such as H5N1 and H5N6, can also cause acute respiratory infectious diseases in humans with high mortality rates.

[0003] Hemagglutinin protein (HA) and influenza neuraminidase (NA) on the surface of the influenza virus are important antigenic proteins, and humans can produce antibodies against HA and NA after infection with the influenza virus. These antibodies play a crucial role in the prevention and treatment of viral infections. Therefore, screening for highly neutralizing antibodies against the HA or NA proteins of human influenza virus for effective prevention and / or treatment of human influenza virus is an important goal in the field of influenza virus control. Summary of the Invention

[0004] Purpose of the invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a monoclonal antibody with high affinity for human influenza virus NA protein and exhibiting high neutralizing activity against it, its related products, preparation methods and uses.

[0006] Solution

[0007] To achieve the above objectives, this invention, after extensive experimental research, has discovered a monoclonal antibody that specifically recognizes and targets the neuraminidase of human influenza virus, exhibiting high antigen-binding activity and highly efficient neutralization of the human influenza virus. Therefore, the monoclonal antibody of this invention is particularly suitable for the prevention and / or treatment of human influenza virus infection.

[0008] Specifically, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human influenza virus neuraminidase (NA), comprising a heavy chain variable region and / or a light chain variable region, wherein,

[0010] The heavy chain variable region includes:

[0011] The amino acid sequences are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively;

[0012] The light chain variable region includes:

[0013] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0014] Preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0015] The heavy chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and,

[0016] The light chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0017] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment comprises:

[0018] The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:7; and,

[0019] The light chain variable region has the amino acid sequence shown in SEQ ID NO:8.

[0020] In addition, the monoclonal antibody or its antigen-binding fragment further includes a constant region; preferably, the constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibody.

[0021] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:9; and preferably, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:10.

[0022] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment comprises:

[0023] A heavy chain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:11 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:11; and,

[0024] Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0025] More preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0026] The heavy chain, whose amino acid sequence is shown in SEQ ID NO:11; and,

[0027] The light chain has the amino acid sequence shown in SEQ ID NO:12.

[0028] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment further has a leader sequence at the N-terminus of its heavy chain variable region and / or light chain variable region; preferably, the leader sequence has an amino acid sequence as shown in SEQ ID NO:13, and more preferably, the leader sequence is encoded by a nucleotide sequence as shown in SEQ ID NO:26.

[0029] In some feasible implementations, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.

[0030] The monoclonal antibody or its antigen-binding fragment of the present invention can specifically bind to the human influenza virus NA protein and has a high neutralizing capacity against human influenza virus, thereby preventing and / or treating human influenza virus infection.

[0031] Secondly, the present invention provides a polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above. This polynucleotide is not limited to any particular method of its production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0032] In a feasible implementation, the polynucleotide is a polynucleotide group.

[0033] In some preferred embodiments, the polynucleotide group comprises:

[0034] (I) A first polynucleotide encoding the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequences shown in SEQ ID NO:14, 15, and 16 (which respectively encode the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 as HCDR1, HCDR2, and HCDR3); and,

[0035] (II) A second polynucleotide encoding the light chain variable regions LCDR1, LCDR2 and LCDR3 of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule containing nucleotide sequences as shown in SEQ ID NO:17, 18 and 19 (which may encode amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, LCDR1, LCDR2 and LCDR3).

[0036] More preferably, the polynucleotide sequence comprises:

[0037] (I) A first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:20 (which encodes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:7); and,

[0038] (II) A second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:21 (which may encode the light chain variable region of the amino acid sequence shown in SEQ ID NO:8).

[0039] Preferably, the first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention and / or the second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention further includes a nucleotide sequence located at the 5' end encoding a leader sequence. Preferably, the nucleotide sequence encoding the leader sequence is as shown in SEQ ID NO:26.

[0040] Preferably, the above-mentioned polynucleotide group further includes:

[0041] (III) A third polynucleotide encoding the heavy chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, said third polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:22 (which encodes the heavy chain constant region of the amino acid sequence shown in SEQ ID NO:9); and,

[0042] (IV) A fourth polynucleotide encoding the light chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the fourth polynucleotide is a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:23 (which may encode the light chain constant region of the amino acid sequence shown in SEQ ID NO:10).

[0043] In the most preferred embodiment, the polynucleotide group comprises:

[0044] (I) A first polynucleotide encoding a heavy chain of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide is a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:24, which encodes a heavy chain with an amino acid sequence as shown in SEQ ID NO:11; and,

[0045] (II) A second polynucleotide encoding a light chain of the monoclonal antibody of the present invention or an antigen-binding fragment thereof, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:25, which may encode a light chain with an amino acid sequence as shown in SEQ ID NO:12.

[0046] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0047] Fourthly, the present invention provides a carrier comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.

[0048] The vector of the present invention can be a cloning vector or an expression vector, such as a plasmid, a granule, a bacteriophage, etc.

[0049] In some preferred embodiments, the vector is an expression vector, preferably a eukaryotic expression vector.

[0050] Fifthly, the present invention provides a host cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above;

[0051] The host cells include, but are not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells can also be cell lines, such as the 293T cell line.

[0052] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0053] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or a host cell as described in the fifth aspect above, and a pharmaceutically acceptable carrier.

[0054] In some feasible embodiments, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation;

[0055] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0056] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0057] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

[0058] In a seventh aspect, the present invention provides a kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a host cell as described in the fifth aspect above, and / or a pharmaceutical composition as described in the sixth aspect above.

[0059] In some preferred embodiments, the kit is a detection or diagnostic kit, wherein the monoclonal antibody or its antigen-binding fragment of the present invention contained herein further includes a detectable label; in some preferred embodiments, the kit further includes a second antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment of the present invention or an anti-idiotype antibody; preferably, the second antibody further includes a detectable label; such detectable labels are well known to those skilled in the art, including but not limited to radioactive isotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (e.g., horseradish peroxidase).

[0060] Eighthly, the present invention provides a method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, the method comprising: expressing the monoclonal antibody or the antigen-binding fragment thereof in host cells as described in the fifth aspect above under conditions suitable for expression of the monoclonal antibody or the antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or the antigen-binding fragment thereof from a culture of the host cells.

[0061] In a ninth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, the pharmaceutical composition as described in the sixth aspect above, and / or the kit as described in the seventh aspect above, in any of the following aspects:

[0062] (1) Application in the preparation of products for detecting the presence or level of human influenza virus neuraminidase in a sample;

[0063] (2) Application in the preparation of products for neutralizing the virulence of human influenza virus in samples;

[0064] (3) Use in the preparation of a medicament for the prevention and / or treatment of a subject with human influenza virus infection or disease associated with said virus infection.

[0065] In a tenth aspect, the present invention provides a method for detecting the presence or level of human influenza virus neuraminidase in a sample, the method comprising using a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a host cell as described in the fifth aspect above, a pharmaceutical composition as described in the sixth aspect above, and / or a kit as described in the seventh aspect above.

[0066] In some preferred embodiments of the method, the monoclonal antibody or its antigen-binding fragment further includes a detectable marker.

[0067] In some other preferred embodiments of the method, the method further includes using a second antibody carrying a detectable label to detect the monoclonal antibody of the present invention or its antigen-binding fragment.

[0068] This method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0069] Therefore, in some specific embodiments, the present invention provides a method for diagnosing whether a subject is infected with human influenza virus, comprising: detecting the presence of human influenza virus neuraminidase in a sample from the subject using a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above; in some preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable marker; in other preferred embodiments, the method further comprises using a second antibody carrying a detectable marker to detect the monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody of the present invention.

[0070] General methods for detecting the presence or level of a target virus or antigen in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0071] In one aspect, the present invention provides a method for neutralizing the virulence of human influenza virus in a sample, comprising: contacting a sample containing human influenza virus with a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0072] The method can be used for therapeutic purposes or non-therapeutic purposes (e.g., the sample is a cell sample, not a patient or a sample from a patient).

[0073] In a twelfth aspect, the present invention provides a method for preventing and / or treating human influenza virus infection or disease associated with said virus infection in a subject, comprising: administering to the subject in need a preventive and / or therapeutically effective amount of a monoclonal antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention.

[0074] In a feasible implementation, the subject is a mammal, such as a human.

[0075] The monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition of the present invention, can be administered to a subject via any suitable route of administration, including but not limited to oral, oral, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0076] The drugs or drug compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents.

[0077] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0078] Beneficial effects

[0079] The monoclonal antibody of this invention can specifically bind to the human influenza virus NA protein with high affinity. Therefore, it has great potential to be developed into a detection antibody for human influenza virus antigen and may be able to provide a lower detection limit and improve detection sensitivity. Furthermore, the monoclonal antibody of this invention has strong neutralizing activity against human influenza virus and can effectively prevent and / or treat human influenza virus infection, which has great clinical application prospects and value. Attached Figure Description

[0080] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0081] Figure 1 The results are molecular sieve chromatography and SDS-PAGE detection results of the monoclonal antibody 18_14D described in Example 2 of this invention.

[0082] Figure 2 The results are molecular sieve chromatography and SDS-PAGE detection results of the monoclonal antibody 18_14D antigen-binding fragment described in Example 3 of this invention.

[0083] Figure 3 The results are molecular sieve chromatography and SDS-PAGE detection results of the N6 protein described in Example 4 of this invention.

[0084] Figure 4 The graph shows the binding kinetics of monoclonal antibody 18_14D to N6 protein; the horizontal axis represents time in seconds (s), and the vertical axis represents the response value in nM.

[0085] Figure 5 The study demonstrates the preventive effect of monoclonal antibody 18_14D against influenza virus infection in mice, as tested by a challenge protection assay. The left panel shows the weight change curve of mice after challenge, and the right panel shows the survival rate curve of mice after challenge. Detailed Implementation

[0086] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0087] Unless otherwise expressly stated, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0089] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity.

[0090] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, biantibodies, etc.

[0091] The “Fab” segment consists of a light chain, a heavy chain CH1, and a variable region.

[0092] The “Fab’” fragment contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab’ fragments to form the F(ab’)2 molecule.

[0093] The “F(ab')2” segment contains two light chains and two heavy chain segments containing the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0094] The “Fv” region contains variable regions from both the heavy and light chains, but lacks constant regions.

[0095] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0096] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker so short as to prevent pairing between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0097] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0098] The sequence information of the monoclonal antibody 18_14D described in the following examples is shown in Table 1 below.

[0099] Table 1. Sequence information of monoclonal antibody 18_14D

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Example 1: Antibody screening and sequence identification

[0106] With informed consent obtained from volunteers, the inventors screened a monoclonal antibody that could bind to the N6 protein from the plasma of volunteers infected with the H5N6 avian influenza virus.

[0107] Specifically, B cells were sorted from PBMCs of volunteers infected with H5N6 avian influenza virus, and CD19 was obtained by flow cytometry. + IgM – IgA – IgD – Single B cells were cultured at a density of 3 cells per well. Cells from positive wells were selected using ELISA to identify IgG-positive (secreting IgG antibodies) and antigen-positive (binding H5 or N6 proteins). These cells were lysed, and mRNA was extracted and reverse transcribed into cDNA. Using this cDNA as a template, PCR amplification was performed using primers specific to the heavy and light chains of human antibodies. The amplified products were then subjected to first-generation sequencing and high-throughput sequencing to obtain the encoding genes for the antibody heavy chain variable regions and light chain variable regions of the B cells in the positive wells.

[0108] Since each well contains 3 cells, each well can yield more than one heavy chain variable region gene sequence and more than one light chain variable region gene sequence. Furthermore, the combination of different heavy chain variable regions and light chain variable regions in the same well was explored. Through efficacy screening, a combination of heavy chain variable regions and light chain variable regions of a monoclonal antibody was obtained.

[0109] The selected monoclonal antibody was designated 18_14D. Its heavy and light chain variable regions have nucleic acid coding sequences as shown in SEQ ID NO:20 and 21, respectively, and the amino acid sequences of the heavy and light chain variable regions encoded by it are shown in SEQ ID NO:7 and 8, respectively. Further analysis of the heavy and light chain variable region sequences yielded the amino acid sequences and nucleic acid coding sequences of the three CDRs (HCDR1, HCDR2, HCDR3) of the heavy chain variable region and the three CDRs (LCDR1, LCDR2, LCDR3) of the light chain variable region. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively, and their nucleic acid coding sequences are shown in SEQ ID NO:14, 15, and 16, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and their nucleic acid coding sequences are shown in SEQ ID NO:17, 18, and 19, respectively.

[0110] Example 2: Preparation and purification of monoclonal antibody 18_14D

[0111] 1. Construction of antibody heavy / light chain recombinant expression vector

[0112] The nucleotide sequences encoding the variable regions of the heavy / light chains of antibody 18_14D obtained in Example 1 were ligated to the nucleotide sequences encoding the constant regions of the heavy / laminar chains via bridging PCR, and then cloned into the expression vector pCAGGS (purchased from Addgene) to obtain recombinant expression vectors encoding the antibody heavy and light chains, respectively. The compositions of the constructs expressing the heavy and light chains are as follows:

[0113] Heavy chain construct composition (5'-3'): CMV promoter - EcoR I restriction enzyme cleavage site - kozak sequence - leader sequence gene - VH gene - CH gene - Xho I restriction enzyme cleavage site;

[0114] Light chain (λ) construct composition (5'-3'): CMV promoter - EcoR I restriction enzyme cleavage site - kozak sequence - leader sequence gene - VL gene - CL(c) gene - Xho I restriction enzyme cleavage site;

[0115] The leader sequence is shown in SEQ ID NO:26, which encodes the amino acid sequence shown in SED ID NO:13; the CH gene sequence is shown in SED ID NO:22, which encodes the amino acid sequence shown in SEQ ID NO:9; and the CL gene sequence is shown in SEQ ID NO:23, which encodes the amino acid sequence shown in SED ID NO:10.

[0116] 2. Expression and purification of antibody 18_14D

[0117] The recombinant expression vectors encoding the heavy and light chains of the antibodies obtained above were co-transfected into HEK293F cells. The molar ratio of the heavy chain to the light chain expression plasmid was 1:1.5, and 1 μg of plasmid and 3 μg of PEI (1 mg / mL) were transfected per mL of HEK293F cells. -1 Cells were cultured in SMM 293-TII (Sinobiological) medium at 37°C and 5% CO2. Then, at 24 and 72 hours post-transfection, SMS M293-SUPI (Sinobiological) was supplemented at a ratio of 35 mL / L. On day 5, the cell supernatant was collected and filtered through a 0.22 μm membrane.

[0118] The filtered cell supernatant was adsorbed onto a Protein A affinity column (Cytiva) at 4°C. The column was washed with buffer A (20 mM Na3PO4, pH 7.4) to remove non-specifically bound proteins. The target protein was then eluted from the Protein A affinity column with buffer B (0.1 M glycine, pH 3.0). The eluent at the elution peak was collected for subsequent gel filtration chromatography. The collected eluent at the elution peak was concentrated using a 10 kDa concentrator and then filtered through a Superdex filter. TM Further purification was performed using a 200 Increase 10 / 300 GL column (Cytiva). During chromatography, the eluent from the elution peak was collected for SDS-PAGE identification of the target protein. After concentration, the target protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0119] The gel filtration chromatography and SDS-PAGE identification results of monoclonal antibody 18_14D are as follows: Figure 1 As shown; by Figure 1 It can be seen that the purity of the obtained antibody can reach more than 95%, and the heavy and light chains of the antibody can be seen at approximately 55kD and 25kD, and their molecular weight is consistent with the theoretical value.

[0120] Example 3: Preparation of monoclonal antibody 18_14D Fab

[0121] In this embodiment, the Fab fragment of monoclonal antibody 18_14D is prepared through the following steps:

[0122] 1. Concentrate the purified monoclonal antibody 18_14D obtained in Example 2 to a concentration of 30 mg / mL or higher.

[0123] 2. Calculate the required amount of immobilized papain (agarose resin) (ThermoScientific) based on the antibody quality; add papain to a centrifuge column, centrifuge at 2000 rpm for 2 min, and discard the supernatant; resuspend in enzyme digestion buffer (20 mM Na3PO4, 10 mM EDTA and 20 mM cysteine, pH=7.0), centrifuge at 2000 rpm for 2 min, and discard the supernatant; repeat the washing process 3 times.

[0124] 3. Add the antibody from step 1 to the centrifuge column containing papain, and add enzyme digestion buffer to make the final concentration of antibody in the antibody-enzyme digestion system 20 mg / mL. Tighten the cap, invert and mix the enzyme digestion system, seal the gaps of the centrifuge column with sealing film, and place it on a rotary mixer for enzyme digestion at 37°C for 6 hours.

[0125] 4. After centrifuging the sample from step 3, take the supernatant, which is the antibody after enzyme digestion.

[0126] 5. Equilibrate the Protein A column with 20mM Na3PO4, load the enzyme-digested antibody, and collect the flow-through to obtain the Fab fragment of the antibody. Concentrate the collected Fab fragment and then perform gel filtration chromatography using a Superdex™ 200 Increase 10 / 300GL column to further purify the Fab protein. During chromatography, collect the eluent at the elution peak for SDS-PAGE identification of the target protein. After concentrating the target protein, determine its concentration, aliquot, flash-freeze in liquid nitrogen, and store at -80°C.

[0127] The gel filtration chromatography and SDS-PAGE identification results of the monoclonal antibody 18_14D Fab fragment are as follows: Figure 2 As shown; by Figure 2 It can be seen that the purity of 18_14D Fab can reach more than 95%, and the heavy chain and light chain bands of the antibody Fab fragment can be seen at about 25kD, and their molecular weight is consistent with the theoretical value.

[0128] Example 4: Expression and purification of N6 protein

[0129] (1) The expression construct containing the signal peptide, His tag, tetramer tag, thrombin restriction site and the nucleic acid coding sequence of amino acids 83-470 of N6 protein (as shown in SEQ ID NO:27) was cloned into the expression vector pCAGGS to construct a recombinant expression plasmid of N6 protein.

[0130] SEQ ID NO:27:

[0131]

[0132]

[0133] The bold part is the nucleic acid coding sequence of the signal peptide, the italic part is the nucleic acid coding sequence of the His tag, the bold + italic part is the nucleic acid coding sequence of the tetramer tag, the underlined part is the nucleic acid coding sequence of the thrombin restriction site, the bold + italic + underlined part is the stop codon, and the remaining part is the nucleic acid coding sequence of amino acid positions 83-470 of the N6 protein.

[0134] Its expressed amino acid sequence is shown in SEQ ID NO: 28:

[0135] The bold text represents the signal peptide, the italic text represents the His tag, the bold + italic text represents the tetramer tag, the underlined text represents the thrombin cleavage site, and the remaining text represents the amino acid sequence of the N6 protein from position 83 to 470.

[0136] (2) Transfect the recombinant expression plasmid constructed above into Expi293F TM Cells were centrifuged after 5 days, and the cell supernatant was collected. The cell supernatant was filtered through a 0.22 μm filter membrane and then passed through a nickel affinity column (His Trap, Cytiva) at 4 °C. The supernatant was washed with buffer A (25 mM Tris, 150 mM NaCl, pH 8.0) and buffer B (25 mM Tris, 150 mM NaCl, pH 8.0, 20 mM imidazole) to remove non-specifically bound proteins. The target protein was then eluted from the His Trap with buffer C (25 mM Tris, 150 mM NaCl, pH 8.0, 300 mM imidazole). The eluent at the elution peak was collected for SDS-PAGE identification of the target protein and subsequent gel filtration chromatography. The eluent from the collected elution peaks was concentrated to a final volume of less than 1 ml using a 10 kD concentrator. The target protein was then further purified by gel filtration chromatography using a Superdex™ 200 Increase 10 / 300 GL column. During chromatography, the eluent from the elution peaks was collected for SDS-PAGE identification of the target protein. After concentration, the target protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0137] The results of gel filtration chromatography and SDS-PAGE identification of N6 protein are as follows: Figure 3 As shown; because the N6 protein expression construct contains a tetramer tag, it is present in Expi293F TM After being expressed in cells, it will spontaneously assemble into a tetramer form, therefore, Figure 3 The elution peaks in the gel filtration chromatography curve correspond to the tetramer of the N6 protein; however, after SDS-PAGE electrophoresis, the protein denatures, so the SDS-PAGE bands correspond to the N6 protein monomers.

[0138] Figure 3 The results showed that the purity of the obtained N6 protein was over 95%, and its molecular weight was consistent with the theoretical value, indicating that the above procedure successfully expressed the N6 protein.

[0139] Example 5: Affinity determination of the antigen-binding fragment (Fab) of monoclonal antibody 18_14D with N6 protein. In this example, the affinity between monoclonal antibody 18_14D and N6 protein was determined through the following steps:

[0140] 1. The N6 tetramer protein obtained in Example 4 was labeled with biotin (Shanghai Dibai Biotechnology Co., Ltd., H219001) according to the instructions.

[0141] 2. Dilute the biotin-labeled N6 tetramer protein to 20 μg / mL.

[0142] 3. Use PBS-T solution to serially dilute 18-14D Fab to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM.

[0143] 4. Using the ForteBio Octet Red instrument, biotin-labeled N6 tetramers were immobilized onto the SA sensor. The binding and dissociation of 18-14D Fab with N6 tetramer protein were measured, and the affinity was calculated.

[0144] The results are as follows Figure 4 As shown, by Figure 4 It can be seen that 18_14D Fab has a high affinity for the N6 protein, with an affinity of 26 nM.

[0145] Example 6: Preventive effect of monoclonal antibody 18_14D on mice infected with live influenza virus H5N6

[0146] To test the protective effect of the 18_14D antibody on mice, we conducted an animal challenge protection experiment.

[0147] The animals used in the challenge protection experiment were Balb / c mice, which were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All of them were female and 6-8 weeks old.

[0148] The virus used in the challenge protection experiment was influenza virus H5N6 (A / Yunan / DQ001 / 2015, GISAID ID: EPI_ISL_200837) (abbreviated as DQ001), described in the article "Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China, 2016, Cell Host & Microbe 20(6) DOI:10.1016 / j.chom.2016.10.022". It is available to the public from the applicant and may only be used to repeat the experiments of this invention; it may not be used for other purposes.

[0149] The specific experimental groups and challenge methods are shown in Table 1.

[0150] Table 1. Experimental groups and challenge methods for evaluating the preventive effect of 18_14D monoclonal antibody on mice.

[0151]

[0152] Within 14 days after the virus challenge, the weight change and survival of mice in each group were measured, and weight change curves and survival rate curves were plotted.

[0153] The curves showing the change in body weight and survival rate of mice in each group after challenge are as follows: Figure 5 As shown in the left and right images.

[0154] Figure 5 The results showed that, 3 hours before H5N6 challenge, mice were intraperitoneally injected with an antibody dose of 20 mg / kg, followed by challenge with the lethal dose (3LD). 50 The virus was tested, and all mice (6 / 6) survived, resulting in a 100% protection rate. Three hours before H5N6 challenge, mice were intraperitoneally injected with antibodies at doses of 1 mg / kg or 5 mg / kg, followed by challenge with a lethal dose (3LD). 50 Of the 6 mice infected with the virus, 5 survived.

[0155] The above data indicate that 18_14D antibodies can effectively prevent influenza virus infection and significantly reduce the rate of severe illness and mortality caused by influenza virus infection (at appropriate concentrations, 100% protective effect can be achieved).

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment that specifically binds to the human influenza virus neuraminidase N6 protein, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The light chain variable region includes: The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; wherein the amino acid sequence of SEQ ID NO: 5 is DVS.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and, The light chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 2, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO: 7; and, The light chain variable region has the amino acid sequence shown in SEQ ID NO:

8.

4. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The antibody or its antigen-binding fragment also includes a constant region.

5. The monoclonal antibody or its antigen-binding fragment as described in claim 4, wherein, The constant region includes the heavy chain constant region as shown in SEQ ID NO:9 and the light chain constant region as shown in SEQ ID NO:

10.

6. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The antibody or its antigen-binding fragment comprises: Heavy chains comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; and, Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

12.

7. The monoclonal antibody or its antigen-binding fragment as described in claim 6, wherein, The antibody or its antigen-binding fragment comprises: Heavy chain, whose amino acid sequence is shown in SEQ ID NO: 11; and, The light chain has the amino acid sequence shown in SEQ ID NO:

12.

8. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

9. A polynucleotide encoding as claimed in claim 1 8. The monoclonal antibody or its antigen-binding fragment as described in any one of the claims.

10. The polynucleotide of claim 9, wherein, The polynucleotide is a polynucleotide group, which includes: (I) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:14, 15, 16; and, (II) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:17, 18, 19.

11. The polynucleotide of claim 10, wherein, The polynucleotide sequence includes: (I) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:20; and, (II) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:

21.

12. The polynucleotide of claim 11, wherein, The polynucleotide sequence also includes: (III) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:22; and, (IV) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:

23.

13. The polynucleotide of claim 11 or 12, wherein, The polynucleotide sequence includes: (I) A DNA molecule or its corresponding mRNA molecule with the nucleotide sequence shown in SEQ ID NO:24; and, (II) DNA molecules or their corresponding mRNA molecules with nucleotide sequences as shown in SEQ ID NO:

25.

14. A nucleic acid construct comprising a polynucleotide as described in any one of claims 9-13, and at least one expression regulatory element operatively linked to said polynucleotide.

15. A vector comprising a polynucleotide as described in any one of claims 9-13, or a nucleic acid construct as described in claim 14.

16. The carrier as claimed in claim 15, wherein, The carrier is an expression carrier.

17. The carrier as claimed in claim 16, wherein, The vector is a eukaryotic expression vector.

18. A host cell comprising a polynucleotide as described in any one of claims 9-13, a nucleic acid construct as described in claim 14, or a vector as described in any one of claims 15-17.

19. The host cell of claim 18, wherein, The host cell is a eukaryotic cell; 20. The host cell of claim 18, wherein, The host cell is a mammalian cell.

21. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, a polynucleotide as described in any one of claims 9-13, a nucleic acid construct as described in claim 14, a vector as described in any one of claims 15-17, or a host cell as described in any one of claims 18-20, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein, The pharmaceutical composition is in the form of a nasal spray, oral preparation, suppository, or parenteral preparation.

23. The pharmaceutical composition of claim 22, wherein, The nasal spray is selected from aerosols, sprays, and powders.

24. The pharmaceutical composition of claim 22, wherein, The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments.

25. The pharmaceutical composition of claim 22, wherein, The parenteral preparations are transdermal preparations, ointments, plasters, topical liquids, or injectable preparations.

26. A kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-8, a polynucleotide as described in any one of claims 9-13, a nucleic acid construct as described in claim 14, a vector as described in any one of claims 15-17, a host cell as described in any one of claims 18-20, and / or a pharmaceutical composition as described in any one of claims 21-25.

27. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, the method comprising: Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the host cells according to any one of claims 18-20 are made to express the monoclonal antibody or its antigen-binding fragment, and the expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cells.

28. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-8, the polynucleotide as described in any one of claims 9-13, the nucleic acid construct as described in claim 14, the vector as described in any one of claims 15-17, the host cell as described in any one of claims 18-20, the pharmaceutical composition as described in any one of claims 21-25, and / or the kit as described in claim 26 in any of the following aspects: (1) Use in the preparation of products for detecting the presence or level of human influenza virus neuraminidase N6 protein in a sample; (2) Application in the preparation of products for neutralizing the virulence of human H5N6 influenza virus in samples; (3) Use in the preparation of a medicament for the prevention and / or treatment of a subject with human H5N6 influenza virus infection or disease associated with said virus infection.

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