An antibody against h5 subtype and its preparation method and application

Abstract

The application discloses an antibody against H5 subtype and a preparation method and application thereof. The antibody comprises any one of the following sequences: (I) a heavy chain variable region amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region amino acid sequence shown in SEQ ID NO. 2; (II) an amino acid sequence with at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO. 1 and 2 and with the same function; and (III) an amino acid sequence obtained by deletion, substitution, insertion or addition of one or more amino acids from the amino acid sequences shown in SEQ ID NO. 1 and 2 and with the same function. The antibody of the application is a monoclonal antibody capable of effectively inhibiting H5Nx subtype highly pathogenic avian influenza pseudovirus, can play a key role in coping with H5 subtype cross-species transmission and can strongly support the design of a highly pathogenic avian influenza protective vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to an antibody targeting the H5 subtype, its preparation method, and its application. Background Technology

[0002] Since 2020, the H5 avian influenza virus has gradually formed H5N1, H5N6, and H5N8 subtypes through continuous recombination, spreading to various parts of the world via migratory birds, causing mass poultry deaths and severely impacting the global economy. More importantly, avian influenza viruses, including the H5N1, H5N6, and H5N8 subtypes, have also spread across species, infecting multiple mammals and even humans. These subtypes, including H5N1, H5N6, and H5N8, pose a serious threat to public health and safety.

[0003] Since 2000, the H5 subtype has undergone multiple mutations and recombinations, evolving into several branches. Currently, the main circulating branches are 2, 3, 4, and 4b, which exhibit significant antigenic drift compared to previous strains. None of the reported specific monoclonal antibodies targeting H5 influenza have shown the ability to inhibit existing influenza strains.

[0004] Therefore, there is still a need for a broad-spectrum antibody that can effectively inhibit existing H5 highly pathogenic avian influenza viruses in order to address the risk of transmission from existing circulating strains. Summary of the Invention

[0005] To address at least some of the problems in the prior art, the present invention provides an antibody targeting the H5 subtype, a method for its preparation, and its applications. Specifically, the present invention includes the following:

[0006] A first aspect of the present invention provides an antibody or an antigen-binding fragment thereof comprising any one of the following sequences:

[0007] (I) The amino acid sequences of the heavy chain variable region shown in SEQ ID NO.1 and the amino acid sequences of the light chain variable region shown in SEQ ID NO.2;

[0008] (II) Amino acid sequences that have at least 90% sequence identity and the same function as the amino acid sequences shown in SEQ ID NO. 1 and 2; and

[0009] (III) Amino acid sequences with the same function obtained by deleting, substituting, inserting or adding one or more amino acids of the amino acid sequences shown in SEQ ID NO.1 and 2.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG, IgM, IgE, IgD or IgA or a variant thereof; and the light chain constant region is selected from the light chain constant region of κ or λ or a variant thereof.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention includes a monoclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes Fab, Fab', F(ab)2, F(ab')2, scFv, scFv Fc fragment or a single-chain antibody ScAb.

[0012] A second aspect of the present invention provides a nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof as described in the present invention.

[0013] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0014] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or the vector described herein.

[0015] A fifth aspect of the present invention provides a method for producing the antibody or antigen-binding fragment thereof described herein, comprising culturing the host cell described herein under conditions suitable for antibody expression, and recovering the expressed antibody from the culture medium.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the nucleic acid molecule described herein, the vector described herein, or the host cell described herein.

[0017] A seventh aspect of the invention provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the carrier, the host cell, or the pharmaceutical composition of the invention in the preparation of a medicament for the prevention, treatment, detection, or diagnosis of avian influenza virus infection.

[0018] The antibody of this invention is a monoclonal antibody that can effectively inhibit the H5Nx subtype of highly pathogenic avian influenza pseudovirus. It can play a key role in responding to the cross-species transmission of the H5 subtype and strongly support the design of protective vaccines against highly pathogenic avian influenza. Attached Figure Description

[0019] Figure 1 This is a gel electrophoresis image of the H5-HA1 antigen of this invention.

[0020] Figure 2 This is a graph showing the serum titer on day 7 after immunization according to the present invention.

[0021] Figure 3 This is a graph showing the serum titer on day 21 after immunization according to the present invention.

[0022] Figure 4 This is a graph showing the serum titer on day 35 after immunization according to the present invention.

[0023] Figure 5 This is a graph showing the binding affinity of the L10 monoclonal antibody of this invention.

[0024] Figure 6 This is a typing detection diagram of the L10 monoclonal antibody of this invention.

[0025] Figure 7 This is a detection graph showing the neutralization assay of the L10 monoclonal antibody against H5N1 (Spain) pseudovirus according to the present invention.

[0026] Figure 8 This is a detection graph showing the neutralization assay of the L10 monoclonal antibody against H5N1 (Jiangsu) pseudovirus according to the present invention.

[0027] Figure 9 This is a detection graph showing the neutralization assay of the L10 monoclonal antibody against the H5N6 (Sichuan) pseudovirus according to the present invention.

[0028] Figure 10 This is a detection graph showing the neutralization assay of the L10 monoclonal antibody against the H5N8 (Astrakhan) pseudovirus according to the present invention.

[0029] Figure 11 This is an SDS-PAGE of the L10 monoclonal antibody of the present invention. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Antibody or antigen binding fragment

[0034] One aspect of the present invention provides an antibody or an antigen-binding fragment thereof comprising any one of the following sequences:

[0035] (I) The amino acid sequences of the heavy chain variable region shown in SEQ ID NO.1 and the amino acid sequences of the light chain variable region shown in SEQ ID NO.2;

[0036] (II) An amino acid sequence having at least 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO. 1 and 2, for example, an amino acid sequence having at least 90%, preferably at least 92%, even more preferably at least 94%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.9% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO. 1; and

[0037] (III) Amino acid sequences with the same function obtained by deleting, substituting, inserting or adding one or more amino acids of the amino acid sequences shown in SEQ ID NO.1 and 2.

[0038] In this article, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids; the heavy chain may also contain a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) in each heavy and light chain form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0039] In this document, the terms "identity" and "homology" related to sequence association are used interchangeably. Homologous sequences include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0040] In this document, the term "replacement" refers to the substitution of one or more amino acids by different amino acids. "Deletion" refers to the removal of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in the addition of one or more amino acids compared to a naturally occurring molecule. In the amino acid sequence-modified antibodies provided by this invention, the modification preferably occurs in regions other than variable regions, such as in the frame region or constant region of the antibody, and the modified antibody retains the desired functional properties of the antibody of this invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0041] In this invention, the antibodies include monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, murine antibodies, or antibodies of other species origin (e.g., rabbit, camel, or shark).

[0042] The term "monoclonal antibody," as used in this article, sometimes also called "mAb," refers to an immunoglobulin derived from a pure cell line, possessing the same structure and chemical properties, and specific for a single antigenic determinant. Unlike conventional polyclonal antibody formulations (which typically contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a homogeneous group of antibodies, but this should not be interpreted as requiring any special methods to produce the antibody.

[0043] In this invention, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0044] In this article, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from one animal species (e.g., mouse, rat), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, with the constant region combined with it derived from human cells. This variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region it is combined with. Furthermore, because the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human-derived constant region.

[0045] In this article, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the xenobiotic antibody when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity.

[0046] In this document, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. In this invention, mouse antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations.

[0047] In this document, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or a light chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2.

[0048] In this invention, the terms "binding," "immune binding," "specific binding," "targeting," and "specifically binding" are used interchangeably and generally refer to the binding of an antibody to an epitope via its antigen-binding domain, and this binding requires complementarity between the antigen-binding domain and the epitope. Therefore, when an antibody binds to an epitope via its antigen-binding domain, it is more likely to bind to a random, unrelated epitope; this is referred to as "specific binding" to the epitope. The term "specificity" in this invention is used to define the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered to have higher specificity for a specific epitope than antibody "B," or antibody "A" can be considered to bind to epitope "C" with higher specificity than binding to related epitope "D." This invention has determined the specificity of antibodies against live viruses, and its IC50... 50 The concentration was 0.199±0.006 μg / mL. In addition, animal experiments have verified its excellent preventive and therapeutic effects, and it can effectively improve lung lesions and viral titers in mice.

[0049] Unless otherwise stated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to nucleic acids or antibodies that have been extracted from their natural environment. "Isolated" nucleic acids, peptides, and proteins therefore include nucleic acids and proteins purified using standard purification methods. This term also includes nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids and / or polypeptides.

[0050] Nucleic acid molecule

[0051] In one aspect, the present invention also provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in the present invention.

[0052] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0053] Once the coding sequence of the antibody described in this invention is obtained, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0054] Vector

[0055] In this invention, a vector refers to an expression vector, including but not limited to plasmids, retroviruses, YAC, EBV-derived cell-free genes, etc. A suitable vector is one encoding a fully functional human CH or CL immunoglobulin sequence, possessing engineered, suitable restriction sites that allow easy insertion and expression of any VH or VL sequence. The resulting antibody can bind to any strong promoter, including retroviral LTRs, such as the SV-40 early promoter, Rouss sarcoma virus LTR, and Moloney's murine leukemia virus LTR. Similarly, natural Ig promoters can be used.

[0056] Preferred vectors include, but are not limited to, viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney's murine leukemia virus. DNA viral vectors are preferred. These vectors include vaccinia vectors such as smallpox or fowlpox vectors, and herpesvirus vectors such as herpes simplex virus I (HSV) vectors. The specific vector selection will depend on the target cells and the conditions under which they are treated. Introduction can be performed using standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modalities include, for example, naked DNA, CaPO4 precipitation, DEAE glucan, electroporation, protoplast fusion, liposome transfection, cell microinjection, and viral vectors.

[0057] Host cell

[0058] The host cells of this invention include prokaryotic and / or eukaryotic cells. Examples of prokaryotic host cells that can be used in this invention include, but are not limited to, *Escherichia coli* and *Bacillus subtilis*. Examples of eukaryotic host cells that can be used in this invention include, but are not limited to, yeast cells, insect cells, and mammalian cells, such as *Saccharomyces cerevisiae* cells, *Fall Armyworm* insect cells, fibroblasts (e.g., CHO cells, COS-7 cells, etc.), HEK293 cells, and 293T cells.

[0059] Pharmaceutical composition

[0060] In one aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the nucleic acid molecule described herein, the carrier described herein, or the host cell described herein. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0061] Pharmaceutically acceptable carriers for the pharmaceutical compositions of the present invention include, but are not limited to, carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances and diluents, emulsifiers, solvents, fillers, swelling agents, buffers, transport carriers, tensioning agents, solubilizers, wetting agents, complexing agents, buffering agents, antibacterial agents, and surfactants. Preferably, the pharmaceutically acceptable carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration.

[0062] Use

[0063] In one aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, or the pharmaceutical composition described herein in the preparation of a medicament for the prevention, treatment, detection, or diagnosis of avian influenza virus infection.

[0064] In this invention, the avian influenza virus includes H5 subtypes (H5Nx), such as, but not limited to, H5N1, H5N6, and H5N8.

[0065] The term "prevention or treatment" as used in this invention refers to improving a condition before or after the onset of a subject's disease or dysfunction. This degree of relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. In this invention, the term "treatment" refers to therapeutic treatment and preventative or preventative measures aimed at preventing or mitigating (reducing) undesirable physiological changes or disorders. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). Those requiring treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those who need to prevent the condition or disorder.

[0066] In this invention, "subject" includes vertebrates, such as mammals, for example, humans or veterinary mammals. In some embodiments, the subject is a human.

[0067] For diagnostic or detection purposes, the antibodies of this invention may be bound to a detectable marker, the term "detectable marker" as used herein to mean a compound that produces a detectable signal. Detectable markers may be MRI contrast agents, scintillation scanning contrast agents, X-ray imaging contrast agents, ultrasound contrast agents, or optical imaging contrast agents. Examples of detectable markers include radioactive elements, fluorophores such as fluorescein, Alexa, or anthocyanins; chemiluminescent compounds such as luminol; bioluminescent compounds such as luciferase or alkaline phosphatase; and contrast agents such as nanoparticles or gadolinium. The selection of a suitable detectable marker depends on the detection system used and is within the capabilities of those skilled in the art. For example, when the detection system is MRI, the detectable marker is preferably iron oxide nanoparticles or gadolinium; when the detection system is fluorescence imaging, the detectable marker is preferably fluorescein, Alexa, or anthocyanins; when the detection system is chemiluminescent imaging, the detectable marker is preferably luminol; when the detection system is bioluminescent imaging, the detectable marker is preferably luciferase or alkaline phosphatase; and when the detection system is nuclear imaging, the detectable marker is preferably a radioactive element.

[0068] Example 1

[0069] This example illustrates the preparation, expression, and purification of the H5-HA1 antigen.

[0070] 1. Viruses and cells

[0071] The HA and NA gene sequences of human-infecting H5 subtype influenza viruses (A / Jiangsu / NJ210 / 2023(H5N1), A / mink / Spain / 22VIR12774-14_3869-3 / 2022(H5N1), A / Sichuan / 06689 / 2021(H5N6) and A / Astrakhan / 3212 / 2020(H5N8)) were obtained from the GISAID database, constructed into the pcDNA3.1(+) mammalian expression plasmid, and the codons were optimized and synthesized by Genewiz. The pseudovirus was successfully packaged using the mammalian expression system.

[0072] The cell lines used in this example were derived from ATCC (MDCK, CHO, 293T) and cultured in DMEM or 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. All cell lines used in this example underwent routine mycoplasma testing, and no mycoplasma was detected.

[0073] 2. Construction, expression, and purification of antigens

[0074] The gene sequence encoding H5-HA1 (residues 17-337aa) was cloned into the pcDNA3.1(+) mammalian expression vector, with a CD33 signal peptide added to the N-terminus and a 6x histidine tag introduced to the C-terminus for antigen expression. The mammalian vector was then transfected into Expi293T. TM Cells (preserved in our laboratory). 25 μg of the antigen expression plasmid was co-transfected into 293T cells using 60 μL of PEI (Yeasen, Cat. No. 40816ES02). After 8 h, the medium was replaced with complete medium, and after 12 h, it was replaced with serum-free medium for further culture. After 48 h, the supernatant was harvested for protein purification. For recombinant proteins with a His tag, Ni-NTA affinity resin (Genscript, Cat. No. L00250) was added to the supernatant; for recombinant proteins with an Fc fusion protein tag, purification was performed using Protein G affinity chromatography medium (Genscript, Cat. No. L00209). All purification steps were performed according to the manufacturer's instructions, and the eluted protein stock solution was replaced with phosphate-buffered saline (PBS). Protein concentration was determined using a Nanodrop 2000C spectrophotometer (Thermo Scientific), and protein expression purity was detected by reducing SDS-PAGE. Other point-mutated antigens were expressed using the same procedure. The H5-HA1 antigen sequence is shown in SEQ ID NO. 3.

[0075] Antigen purity was detected by SDS-PAGE gel electrophoresis, and the results are as follows: Figure 1 As shown, it meets the requirements for antigen purity.

[0076] Example 2

[0077] This example illustrates the preparation of the L10 monoclonal antibody.

[0078] 1. Mouse immunization

[0079] Six-week-old SPF-grade Balb / c female mice were immunized three times. Using different adjuvants, 50 μg of antigen was injected at multiple sites on the back for the first immunization. Subsequently, 25 μg of H5HA1 antigen was injected intraperitoneally on days 14 and 28, with a booster immunization on day 35. Immune serum was collected from mice before each immunization, and antibody titers were determined by ELISA. In short, the recombinant antigen protein was diluted to 1 ng / μL, coated onto a 96-well ELISA plate, and incubated overnight at 4°C. The next day, the plate was washed three times with PBST (0.05% Tween 20 in PBS), blocked with 2% skim milk at 37°C for 2 h, and then washed three times with PBST. Serum from immunized mice was serially diluted 10-fold with PBS and added to the coated plate, incubated at 37°C for 1 h, and washed three times with PBST. Antibody titers were detected using anti-mouse antibodies bound to horseradish peroxidase. Incubate at 37°C for 1 hour, then wash 5 times with PBST. Add 50 μL of TMB (Solarbio, Cat.no.PR1200) to each well and react in the dark for 3 minutes, then terminate the reaction with 0.5 M H₂SO₄. Finally, measure the absorbance at 450 nm using a ELISA reader (TECAN).

[0080] Serum titer test after three immunizations, on day 7 as follows Figure 2 As shown, on day 21... Figure 3 As shown. Day 35 Figure 4 As shown, the valence meets the requirements.

[0081] 2. Preparation of hybridoma cells and screening and purification of monoclonal antibodies

[0082] Mice immunized with Mn adjuvant were selected for spleen cell fusion. After humane sacrifice, the spleens were harvested under sterile conditions and homogenized using a 70 μm filter (BD FALCON, Cat. no. 352350). The spleens were then fused with SP2 / 0 cells using PEG 1450 (Sigma, Cat. no. P7181). The fused hybridoma cells were cultured in 96-well plates containing 20% ​​FBS, 1×HAT, and 1% penicillin / streptomycin in 1640 medium. Two weeks later, surviving hybridoma cells were screened and labeled. The antibody binding capacity in the supernatant of surviving cells was measured using ELISA. Optimal detection results were shown below. Figure 5As shown. The monoclonal cells with the strongest binding capacity in the supernatant were subcloned using limiting dilution and sequenced to obtain SEQ ID NO. 1 and 2. Hybridoma cells secreting L10 monoclonal antibodies were amplified and cultured. The cell supernatant was collected, centrifuged at 4000 rpm for 4 min, and cell debris was removed. Antibody purification was performed using Protein G affinity chromatography, and the purified antibody storage medium was replaced with phosphate-buffered saline (PBS). The antibody concentration was determined using a Nanodrop 2000C spectrophotometer (ThermoScientific), and the antibody expression purity was detected by reducing SDS-PAGE. The results are shown below. Figure 11 As shown, L10 was genotyped using an antibody typing kit, and the results were as follows. Figure 6 As shown.

[0083] Example 3

[0084] This embodiment illustrates the preparation of pseudoviruses and the determination of their titers and neutralization.

[0085] pcDNA3.1-HA,NA, and pNL4-3.luc.RE- were co-transfected into 293T cells via PEI (Yeasen, Cat. No. 40816ES02) and cultured at 37°C for 8 h. The culture medium was then replaced with DMEM complete medium containing 10% FBS and cultured for another 48 h. The cell supernatant was collected, aliquoted, and stored at -80°C for later use. For pseudovirus titer determination, the pseudovirus was serially diluted twofold and co-incubated with target cells (MDCK) at 37°C for 12 h. The culture medium was then replaced with DMEM complete medium containing 10% FBS and cultured for another 48 h. The cell supernatant was removed, and 50 μL of cell lysis buffer was added to each well. The cells were incubated at 200 rpm for 60 min, and the fluorescence intensity (RLU) was measured using a microplate reader. The pseudoviruses selected for antibody testing (Spain, Jiangsu, Sichuan, and Astrakhan) were used for neutralization assays. For the neutralization assay of pseudovirus, pseudovirus diluted to 10,000-30,000 RLU was co-incubated with serially diluted antibody (J14 as an irrelevant control antibody) for 30 min. The mixture was then added to target cells MDCK and cultured at 37°C for 12 h. The culture medium was then replaced with DMEM complete medium containing 10% FBS and cultured for another 48 h. The fluorescence intensity (RL U) of the pseudovirus was detected, and the data were processed using GraphPad Prism 9.

[0086] The neutralization assay results of L10 against H5N1 (Spain) pseudovirus are as follows: Figure 7 As shown, IC 50 The result was 0.075 ± 0.024 μg / mL; the neutralization assay of L10 against H5N1 (Jiangsu) pseudovirus was as follows: Figure 8 As shown, IC50 The result of the neutralization assay for H5N6 (Sichuan) pseudovirus with L10 was 0.101 ± 0.031 μg / mL; Figure 9 As shown, IC 50 The result of the neutralization assay for H5N8 (Astrakhan) pseudovirus with L10 was 0.031 ± 0.010 μg / mL; Figure 10 As shown, IC 50 It was 0.022 ± 0.008 μg / mL.

[0087] 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. 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. An antibody or its antigen-binding fragment, characterized in that, It can target and bind to the hemagglutinin of H5 subtype avian influenza virus, which contains the heavy chain variable region amino acid sequence shown in SEQ ID NO.1 and the light chain variable region amino acid sequence shown in SEQ ID NO.

2.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from IgG, IgM, IgE, IgD or IgA domains or variants thereof; and the light chain constant region is selected from κ or λ light chain constant regions or variants thereof.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies, or murine antibodies.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include Fab, Fab', F(ab)2, or F(ab')2.

5. A nucleic acid molecule, characterized in that, Its encoding is the antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. A carrier, characterized in that, It contains the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that, It comprises the nucleic acid molecule according to claim 5, or the vector according to claim 6.

8. A pharmaceutical composition, characterized in that, It comprises an antibody or its antigen-binding fragment according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a vector according to claim 6, or a host cell according to claim 7.

9. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, It includes culturing the host cells according to claim 7 under conditions suitable for antibody expression, and recovering the expressed antibody from the culture medium.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the prevention, treatment, detection, or diagnosis of avian influenza virus infection, characterized in that, The avian influenza virus in question is of the H5N1, H5N6, or H5N8 subtype.

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