Anti-dengue virus neutralizing antibody E412C, humanized antibody thereof, multivalent antibody thereof and application of humanized antibody and multivalent antibody

Abstract

The invention discloses an anti-dengue virus neutralizing antibody E412C, a humanized antibody thereof, a multivalent antibody of the humanized antibody and application of the multivalent antibody. The antibody provided by the invention comprises three complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is the 26th to 33rd sites of SEQ ID NO: 1, the amino acid sequence of the CDR2 is the 51st to 58th sites of SEQ ID NO: 1, and the amino acid sequence of the CDR3 is the 97th to 114 sites of SEQ ID NO: 1. The antibody provided by the invention can be used for neutralizing four serotype dengue viruses (DENV I-IV). The antibody provided by the invention can be used for preventing and treating dengue virus infection, and has important biological and medical significance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the anti-dengue virus neutralizing antibody E4_12C, its humanized antibody, its multivalent antibody, and their applications. Background Technology

[0002] Dengue virus (DENV) is a single-stranded, positive-sense RNA virus with a genome of approximately 11 kb. Mature dengue virus particles are spherical and composed of three structural proteins (C, prM, and E) and seven non-structural proteins involved in viral RNA replication (NS1, NS2A / B, NS3, NS4A / B, and NS5). The envelope protein (E protein) is responsible for viral entry into host cells and is an important target for vaccine and antibody development; the membrane protein (M protein) maintains the stability of the viral structure and ensures viral maturation; the capsid protein (C protein) encapsulates the viral RNA and participates in viral particle assembly. In addition, non-structural proteins (NS1, NS3, NS5, etc.) play crucial roles in viral replication and host immune regulation. Among them, the NS1 protein is closely related to vascular leakage syndrome and severe disease progression, and is abundant in serum during the acute phase, serving as an early diagnostic indicator; the NS3 protein possesses protease and helicase activities; and the NS5 protein is responsible for RNA polymerization and methylation. These structural features enhance the virus's transmission and immune evasion capabilities.

[0003] Dengue fever (DF) is an acute viral infectious disease caused by the dengue virus and transmitted by vector insects such as Aedes aegypti and Aedes albopictus. DF is widespread in tropical and subtropical regions worldwide where Aedes mosquitoes are present, affecting more than 100 countries and regions globally. In Latin America, the Western Pacific, Southeast Asia, and the Eastern Mediterranean, DF transmission can occur year-round. In 2025, the number of suspected and confirmed DF cases worldwide rose to over 2.53 million, with a total death toll exceeding 1,000. Brazil alone had over 1.05 million suspected and confirmed cases. It is estimated that at least 500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur globally each year (with a mortality rate of approximately 2.5%). The pathogenesis of severe dengue fever is not fully understood at present, but antibody-dependent enhancement (ADE) is generally considered to be a significant factor. ADE (Alternating Dependent Effect) refers to the phenomenon where, during a person's first viral infection, the immune system produces a large number of neutralizing antibodies against that serotype of virus, providing long-lasting protection. However, when the person is reinfected with a different serotype of virus, the antibodies produced during the initial infection can bind to other serotypes of virus and increase uptake by monocytes through their Fcγ receptors. These infected cells can then carry the virus and spread, causing systemic infection. This significantly increases the complexity of developing anti-dengue virus drugs.

[0004] In response to the increasingly severe dengue fever outbreak, Sanofi Pasteur's quadrivalent live attenuated vaccine, Dengvaxia, was approved for use in Mexico, the Philippines, and Brazil starting in late 2015. While the vaccine showed some efficacy against four dengue virus serotypes, the poor stability of the attenuated virus strain could lead to symptoms and the risk of antibody-dependent enhancement (ADE), potentially causing dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). In late 2017, Philippine health authorities ordered a halt to the use of this vaccine. Furthermore, Takeda Pharmaceutical Company of Japan's Qdenga (TAK-003), a quadrivalent live attenuated vaccine based on DENV-II, was the second dengue vaccine to be approved for marketing. It has been authorized for use in some countries, but its efficacy and safety are still being evaluated in many others, and it has not yet received authorization for use in my country. Currently, there is no widely available dengue vaccine on the market. In addition, there are currently no specific treatments for dengue virus infection; clinical treatment mainly focuses on symptomatic relief.

[0005] Neutralizing antibodies play an indispensable role in the human body's fight against viral infections. Studies have confirmed that passive immunotherapy, especially neutralizing antibodies, is effective against dengue virus infection. The E protein of dengue virus (DENV) is the most fully exposed protein on the surface of infectious viral particles and is also a major target of neutralizing antibodies. Several research teams have screened several neutralizing antibodies targeting the dengue virus E protein, but these antibodies are still in the laboratory research stage and are all holoantibodies, exhibiting varying degrees of adverse drug reaction (ADE), which may limit their further development and application. Compared to traditional antibodies, nanobodies have high affinity and are easy to genetically engineer. Furthermore, nanobodies do not contain an Fc domain, avoiding the ADE effect caused by the Fc domain. Therefore, developing multi-specific anti-dengue virus nanobodies with broad-spectrum neutralizing activity and no ADE effect is of great significance for the treatment and prevention of dengue virus infection. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide nanobodies against dengue virus. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention claims protection for a dengue virus-resistant nanobody or its antigen-binding fragment thereof, said nanobody or its antigen-binding fragment comprising three complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:1, the amino acid sequence of CDR2 is positions 51-58 of SEQ ID NO:1, and the amino acid sequence of CDR3 is positions 97-114 of SEQ ID NO:1.

[0008] The term "antigen-binding fragment" as used above 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 (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0009] In some specific embodiments, the anti-dengue virus nanobody or its antigen-binding fragment is a dengue virus E protein nanobody or its antigen-binding fragment.

[0010] In some specific embodiments, the anti-dengue virus nanobody or its antigen-binding fragment is a nanobody or its antigen-binding fragment that binds to the dengue virus E protein.

[0011] In some specific embodiments, the anti-dengue virus nanobody or its antigen-binding fragment is a nanobody or its antigen-binding fragment that specifically binds to the dengue virus E protein.

[0012] In some specific embodiments, the dengue virus E protein is at least one of the four serotypes of dengue virus recombinant E proteins (Yiqiao Shenzhou, catalog numbers 40533-V08B2, 40532-V08H1, 40471-V08B, and 40531-V08H respectively).

[0013] In addition to the complementarity-determining region, the above-mentioned nanobody also includes four framework regions: FR1, FR2, FR3, and FR4. The FR1 comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The amino acid sequence includes the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 59-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 115-125 of SEQ ID NO:1; A4-2) is a sequence that has more than 75% similarity to A4-1).

[0014] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0015] In this document, the 80% or more of identity may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0016] In some specific embodiments, the amino acid sequence of the nanobody or its antigen-binding fragment includes SEQ ID NO:1 or SEQ ID NO:5.

[0017] Secondly, the present invention claims protection for heavy chain antibodies or multivalent nanobodies against dengue virus, said heavy chain antibodies or multivalent nanobodies including the aforementioned nanobodies.

[0018] The heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which is SEQ ID NO:1 or has a similarity of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more to SEQ ID NO:1.

[0019] The amino acid sequence of the above heavy chain antibody includes SEQ ID NO:3 or SEQ ID NO:7.

[0020] The structure of the multivalent nanobody is a first nanobody-connector-second nanobody-connector-third nanobody, wherein the third nanobody is the aforementioned nanobody. The first nanobody contains three complementarity-determining regions (CDR1, CDR2, and CDR3). The amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:11, the amino acid sequence of CDR2 is positions 51-57 of SEQ ID NO:11, and the amino acid sequence of CDR3 is positions 96-116 of SEQ ID NO:11. The second nanobody contains three complementarity-determining regions (CDR1, CDR2, and CDR3). The amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:12, the amino acid sequence of CDR2 is positions 51-58 of SEQ ID NO:12, and the amino acid sequence of CDR3 is positions 97-117 of SEQ ID NO:12.

[0021] The connector is a linker peptide.

[0022] The amino acid sequence of the linker peptide is GGGGS.

[0023] The multivalent nanobodies also include any type of purification tag.

[0024] The purification tags include His tag, GST tag, MBP tag, NusA tag, Strep tag, SUMO tag, and Flag tag, and there is no limit to the number of tags.

[0025] In certain specific embodiments of the present invention, the purification tag used is a His tag.

[0026] The aforementioned multivalent nanobody includes any one of the following: M1, the first nanobody includes, in addition to the complementarity-determining region, four framework regions FR1, FR2, FR3 and FR4; The FR1 of the first nanobody comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The FR2 of the first nanobody comprises an amino acid sequence selected from the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 of the first nanobody comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 58-95 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 of the first nanobody comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 117-127 of SEQ ID NO:1; A4-2) is a sequence with more than 75% identity with A4-1); In addition to the complementarity-determining region, the second antibody also includes four framework regions: FR1, FR2, FR3, and FR4. The FR1 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The FR2 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 59-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 118-128 of SEQ ID NO:1; A4-2) is a sequence with more than 75% identity with A4-1); M2, the amino acid sequence of the first nanobody includes SEQ ID NO:11, and the amino acid sequence of the second nanobody includes SEQ ID NO:12; M3, The amino acid sequence of the multivalent nanobody is SEQ ID NO:9.

[0027] In some specific embodiments, the anti-dengue virus heavy chain antibody or multivalent nanobody is a heavy chain antibody or multivalent nanobody of the dengue virus E protein.

[0028] In some specific embodiments, the anti-dengue virus heavy chain antibody or multivalent nanobody is a heavy chain antibody or multivalent nanobody that binds to the dengue virus E protein.

[0029] In some specific embodiments, the anti-dengue virus heavy chain antibody or multivalent nanobody is a heavy chain antibody or multivalent nanobody that specifically binds to the dengue virus E protein.

[0030] In some specific embodiments, the dengue virus E protein is at least one of the four serotypes of dengue virus recombinant E proteins (Yiqiao Shenzhou, catalog numbers 40533-V08B2, 40532-V08H1, 40471-V08B, and 40531-V08H respectively).

[0031] Thirdly, the present invention claims protection for biological materials, said biological materials comprising any of the following: B1) A nucleic acid molecule, wherein the nucleic acid molecule encodes the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody or multivalent nanobody; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0032] In the above-mentioned biological materials, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0033] In the above-mentioned biological materials, B1) the nucleic acid molecules include nucleic acid molecules SEQ ID NO:2 or SEQ ID NO:6 encoding the aforementioned nanobody or its antigen-binding fragment, nucleic acid molecules SEQ ID NO:4 or SEQ ID NO:8 encoding the aforementioned heavy chain antibody, and nucleic acid molecules SEQ ID NO:10 encoding the aforementioned multivalent nanobody.

[0034] In the aforementioned biological materials, the expression cassette described in B2) refers to a cassette capable of expressing the proteins described above in host cells. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for expressing the DNA of the nucleic acid molecule or the RNA molecule of any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the expression of the coding sequence in a suitable host cell for the expression of the DNA of any of the aforementioned proteins or the RNA molecule. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence mediating the DNA expression of the protein or the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein, capable of guiding the DNA encoding the protein or the RNA molecule into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein or the DNA of the RNA molecule into the secretion pathway of the host cell used can be used in this invention. Adding regulatory sequences that can modulate the expression of proteins or RNA molecules according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are systems that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification.

[0035] The small molecule antibody may be any of the following: F1 and Fab antibodies; F2 and Fv antibodies; F3, single-chain antibody; F4, Fab′ fragment.

[0036] The term "Fab' fragment" contains a portion of an antibody light chain and an antibody heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of two Fab' fragments to form F(ab'). 2 molecular.

[0037] The term "F(ab′)" 2 The "fragment" contains two light chains and two heavy chains comprising a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, F(ab′) 2 The fragment consists of two Fab′ fragments held together by disulfide bonds between the two heavy chains.

[0038] The term "nanobody (single-domain antibody)" (VHH) refers to a polypeptide composed of the variable region of the antibody heavy chain. Single-domain antibodies can be prepared by expressing the variable region (VH) of the antibody heavy chain using genetic engineering methods to obtain antibodies containing only the VH fragment. The ability of single-domain antibodies to bind to antigens and their stability are essentially the same as those of complete antibodies.

[0039] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0040] The term "Fab antibody" refers to a heterodimer formed by the heavy chain (Fd) and the intact light chain of an antibody linked by disulfide bonds, containing only one antigen-binding site. Fab antibodies can be prepared as follows: the coding genes for the heavy chain (Fd) and the intact light chain are linked, and a bacterial protein signal peptide gene is fused. Fab antibodies (Fab fragments) can then be secreted and expressed in *E. coli*, exhibiting complete stereofolding and intra- and inter-chain disulfide bonds. The heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0041] The term "Fv antibody" refers to a compound consisting solely of the heavy chain variable region and the light chain variable region of an antibody. The heavy chain and light chain variable regions are linked by non-covalent bonds. Fv antibodies can be prepared by: constructing vectors containing the VH and VL genes separately, co-transfecting cells to express them separately, and then assembling them into a functional Fv antibody; or by setting a stop codon between the VH and VL genes in the vector to express two small protein fragments, which are then bound together non-covalently to form an Fv antibody (Fv fragment).

[0042] The term "single-chain antibody" (ScFv) refers to a polypeptide formed by linking the heavy chain and light chain variable regions of an antibody with a short peptide. ScFv can be prepared by connecting the light and heavy chain variable region genes with an oligonucleotide linker, enabling the expression of a single polypeptide chain, known as a single-chain antibody (ScFv). The polypeptide chain spontaneously folds into its native conformation, maintaining the specificity and affinity of the Fv.

[0043] Fourthly, the present invention claims protection for a medicament for treating dengue fever, the medicament comprising the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody or multivalent nanobody.

[0044] In this article, the term "physiologically or pharmaceutically acceptable carriers or diluents" refers to those carriers and diluents that do not cause significant irritation to the organism and do not impair the biological activity and properties of the reagents in the pharmaceutical composition.

[0045] In this document, "physiologically or pharmaceutically acceptable excipients" refers to inert substances added to a pharmaceutical composition to further facilitate the administration of the agent. Carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred.

[0046] Fifthly, the present invention claims protection for applications, said applications including any of the following: M1) The application of the aforementioned biomaterials in the preparation of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned multivalent nanobodies; M2) The application of the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody, or the aforementioned multivalent nanobody in the preparation of the aforementioned drug; M3) The use of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned multivalent nanobodies, or the aforementioned biomaterials, or the aforementioned drugs in the preparation of products for the prevention and / or treatment of diseases caused by dengue virus; M4) The application of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned multivalent nanobodies or the aforementioned biomaterials in the preparation of products for detecting dengue virus; M5) The use of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned multivalent nanobodies, or the aforementioned biomaterials, or the aforementioned drugs in the preparation of products for neutralizing dengue virus; M6) The use of the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody, or the aforementioned multivalent nanobody, or the aforementioned biomaterial or the aforementioned drug in the preparation of a product for inhibiting dengue virus infection.

[0047] The products include reagent kits, test strips, nucleic acid membrane strips, chips, systems, or devices.

[0048] The advantage of this invention lies in the fact that experiments have shown that the nanobody fused with the Fc fragment provided by this invention can neutralize four serotypes of dengue virus (DENV I-IV). Furthermore, multivalent antibodies integrated using the nanobody provided by this invention and two other nanobodies can also neutralize all four serotypes of dengue virus (DENV I-IV). Therefore, the heavy chain antibody or multivalent antibody provided by this invention can be used for the prevention and treatment of dengue virus infection, and has significant biological and medical value. Attached Figure Description

[0049] Figure 1 Expression, purification, and SDS-PAGE detection of E4_12C-Fc. A: Non-reducing SDS-PAGE detection; B: Reducing SDS-PAGE detection.

[0050] Figure 2 The neutralizing ability of antibody E4_12C-Fc was detected by fluorescence reduction neutralization assay.

[0051] Figure 3 The amino acid properties of the VHH of antibody E4_12C were analyzed using www.abysis.org.

[0052] Figure 4 To use www.abysis.org to score the sequences before and after humanization, the left figure shows the score of the sequence before humanization, and the right figure shows the score of the sequence after humanization. Figure 5 Expression, purification, and SDS-PAGE detection of the humanized antibody E4_12C_2-Fc. A: Non-reducing SDS-PAGE detection; B: Reducing SDS-PAGE detection.

[0053] Figure 6 The neutralizing ability of the humanized antibody E4_12C_2-Fc was detected by fluorescence reduction neutralization assay.

[0054] Figure 7 Expression, purification, and SDS-PAGE detection of the multivalent nanobody 8F-2C-12C. A: Non-reducing SDS-PAGE detection; B: Reducing SDS-PAGE detection.

[0055] Figure 8The neutralizing ability of the multivalent nanobody 8F-2C-12C was detected by fluorescence reduction neutralization assay. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] All experimental data were statistically analyzed using nonlinear regression equations.

[0059] The pTSE-hFc in the following examples was obtained by modifying the gene of the Fc domain of human immunoglobulin G by linking it to the pCMV vector. pTSE-hFc has been described in: "Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against protective antigen V of plague bacteria [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03):266-271." This biological material can be obtained from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biological material is only for repeating the experiments of this invention and should not be used for other purposes.

[0060] Example 1: Discovery of Antibodies I. Camel Immunity and Preparation of Antibody Library The immunized animals were healthy adult Bactrian camels. The antigen was recombinant E protein of four serotypes of dengue virus (Yiqiao Shenzhou, catalog numbers 40533-V08B2, 40532-V08H1, 40471-V08B, and 40531-V08H, respectively). The primary immunization was 1 mg of antigen plus complete Freund's adjuvant. Two weeks later, the booster immunization was performed with antigen plus incomplete Freund's adjuvant. A total of four immunizations were performed. Three days after the fifth immunization, 100 mL of anticoagulated blood was collected for the isolation of peripheral blood lymphocytes.

[0061] Peripheral blood was first diluted with sterile PBS, and then lymphocytes were separated using lymphocyte separation medium (STEMCELL, 07851). 15 mL of lymphocyte separation medium was added to every 25 mL of diluted peripheral blood. After centrifuging the mixture of whole blood and lymphocyte separation medium (800 g, 20 min), the liquid in the centrifuge tube separated into four layers from top to bottom: plasma, peripheral blood mononuclear cells, lymphocyte separation medium, and red blood cells. The peripheral blood mononuclear cells were collected.

[0062] Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the OMEGA EZNA Total RNA kit I (OMEGA, R6834). Using the extracted total RNA as a template, cDNA was obtained by reverse transcription using a first-strand cDNA synthesis kit (Thermo Scientific, K1621). Then, using the reverse-transcribed cDNA as a template, the antibody leader sequence and CH2 region sequence were amplified by PCR using the designed IgG-specific upstream primer CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3') and downstream primer CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3'). Using the recovered first-round PCR product as a template, a second round of PCR amplification was performed using the designed VHH-F (5'-cggCCATGGcGGTCCTGGCTGCTCTTCTACA-3') and VHH-R (5'-tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT-3') to amplify the VHH fragment. Finally, the prepared VHH antibody gene was double-digested with NotI and NcoI, and the ligation product was then electroporated to construct an anti-dengue virus nanobody library. The library had a capacity of 8.1 × 10⁻⁶. 8 It has good diversity.

[0063] II. Screening of anti-dengue virus nanobody (VHH) library Using recombinant E protein from four different dengue virus serotypes as antigens, the constructed nanobody phage library was screened twice using a solid-phase screening strategy (experimental protocol referenced from Phage Display: A Universal Laboratory Guide / edited by Clackson, T., and Lowman, HB.; translated by Ma Lan et al., Chemical Industry Press, May 2008). Ultimately, the nanobody clone E4_12C, capable of binding to the recombinant E protein of all four dengue virus serotypes, was obtained.

[0064] The DNA sequence of the inserted fragment in the nanobody clone E4_12C is the nanobody E4_12C. The amino acid sequence of nanobody E4_12C is SEQ ID NO:1 (the nucleotide sequence of the gene encoding nanobody E4_12C is SEQ ID NO:2), where amino acids 1-25 form the first frame region (FR1), amino acids 34-50 form the second frame region (FR2), amino acids 59-96 form the third frame region (FR3), amino acids 115-125 form the fourth frame region (FR4), amino acids 26-33 form the first complementarity-determining region (CDR1), amino acids 51-58 form the first complementarity-determining region (CDR2), and amino acids 97-114 form the third complementarity-determining region (CDR3).

[0065] The sequence of the complementarity determination region (CDR) is defined according to the Kabat numbering system.

[0066] Example 2: Preparation of Anti-Dengue Virus Heavy Chain Antibody A human-camel chimeric heavy chain antibody, named E4_12C-Fc (with IgG1 as the constant region), was prepared using the camel-derived nanobody E4_12C. The preparation process is as follows: I. Construction of Recombinant Plasmids The camel-derived E4_12C variable region (VHH) was cloned into the pTSE-hFc vector (containing the Fc domain of IgG1) using conventional molecular biology techniques. The insertion site was [insertion location missing]. Sal I and Nhe The recombinant plasmid obtained between the two restriction enzyme sites was named pTSE-E4_12C-Fc after being verified by sequencing. This plasmid is the expression vector for the antibody E4_12C-Fc.

[0067] The nanobody expression plasmid pTSE-E4_12C-Fc is a DNA fragment with the nucleotide sequence SEQ ID NO:2 replacing the pTSE-Fc vector. Sal I and Nhe I. A recombinant expression vector was obtained by extracting a small fragment between two restriction enzyme sites while maintaining the other nucleotide sequences of the pTSE-Fc vector unchanged. The vector expresses a protein with the amino acid sequence of SEQ ID NO:3 (named E4_12C-Fc, whose nucleotide sequence is SEQ ID NO:4).

[0068] II. Antibody Preparation 1. Antibody expression The constructed nanobody expression plasmid pTSE-E4_12C-Fc was transfected into FreeStyle using the FectoPRO DNA Transfection Reagent (Polyplus, 116-001). TM HEK293-F cells (Invitrogen, R79007). Details are as follows: FreeStyle cells were prepared one day before transfection. TM HEK293-F cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in FreeStyle 293 medium (Gibco, 12338-018). The cell density was adjusted to 1.0 × 10⁶ cells / min. 6 / mL, dispensed into 30mL cell suspensions per bottle, and cultured in a shaker at 125rpm, 5% CO2, and 37℃. The next day, prepare the transfection complex: take 24μg of antibody plasmid pTSE-E4_12C-Fc, dilute it in 3mL of Opti-MEM medium, gently mix, add 24μL of FectoPRO transfection reagent, mix well, and incubate at room temperature for 15min; then add the mixture to the FreeStyle medium prepared the previous day. TM In HEK293-F cells, gently mix and return to the cell shaker; monitor cell viability starting 48 hours after transfection, and centrifuge at 8,000 rpm for 10 min when cell viability drops to 80-85%, collect the supernatant for purification.

[0069] 2. Antibody purification The antibody expression supernatant was filtered through a 0.45 μm filter membrane to remove impurities, and a 1×PBS binding buffer was prepared using 10×PBS; HiTrap was then added. TM Install the MabSelect SuRe pre-packed column into the AKTA system. First, wash with 1×PBS binding buffer for 5-10 column volumes until the baseline stabilizes, then begin loading the sample. After loading, wash away any contaminating proteins with binding buffer, and continue washing with binding buffer for another 5-10 column volumes until the baseline is reached. Observe whether the UV280 is close to zero. If it is not close, manually zero it. Wash the pre-packed column with 0.1M citrate buffer (pH 3.0) to elute the antibody. Start collecting when the UV280 rises to 50 and stop collecting when the UV280 drops to 50.

[0070] Use Hitrap TM Desalting chromatography column will use HiTrap TMSamples purified by MabSelect SuRe affinity chromatography were desalted using 0.01M citrate buffer (pH 6.0) as the mobile phase. First, the entire system was washed with 0.01M citrate buffer (pH 6.0). After the baseline stabilized, 3-5 column volumes were washed. Then, the sample was loaded (the loading volume should not exceed one-fifth of the total column volume). The UV280 value was observed to ensure it was close to zero. If not, manual zeroing was required, followed by buffer replacement at a flow rate of 2.5 mL / min. Collection began when UV280 reached 50 and ended when it dropped back to 50. Replacement was stopped if the salt peak overlapped with the protein peak. The obtained elution peak was the purified E4_12C-Fc antibody. A portion of the sample was analyzed by SDS-PAGE for concentration determination, and the remainder was aliquoted and stored at -80℃.

[0071] Figure 1 The image shows the SDS-PAGE results of the purified E4_12C-Fc antibody. As can be seen from the figure, the purified E4_12C-Fc antibody sample has high purity and no impurities. The molecular weight of the non-reduced sample is approximately 80 kDa, and the molecular weight of the reduced sample is approximately 40 kDa. The band size is consistent with the expected molecular weight.

[0072] 3. Quantification of antibodies The purified antibody solution was filtered through a filter membrane for sterilization, and the protein concentration (1.1 mg / mL) was determined using a NanoDrop UV spectrophotometer (ThermoScientific).

[0073] Example 3: Detection of neutralizing activity of E4_12C-Fc antibody using fluorescence reduction neutralization assay I. Preparation of Dengue Virus BHK-21 cells, dengue virus type I (DENVI), dengue virus type II (DENVII), dengue virus type III (DENVIII), and dengue virus type IV (DENV IV): described in the non-patent literature "Guo J, Lu J, Du P, et al. Fluorescence Reduction Neutralization Test: A Novel, Rapid, and Efficient Method for Characterizing the Neutralizing Activity of Antibodies Against Dengue Virus. Curr Issues Mol Biol. 2025;47(3):140. Published 2025 Feb 21.doi:10.3390 / cimb47030140", are available to the public from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biological material is only for repeating the relevant experiments of this invention and shall not be used for other purposes.

[0074] 1. Pass BHK-21 cells into 75cm cells one day before virus inoculation. 2 In cell culture flasks, the cell density is increased to 75%-90% when inoculated with the virus the next day. 2. On the day of inoculation, slowly aspirate the cell culture medium from the culture flask and add 10 mL of DMEM culture medium (containing 2% FBS). 3. Using a micropipette, take appropriate amounts of the aforementioned four virus solutions (the four serotypes of dengue virus are preserved by our institute) into different cell culture bottles, so that the MOI is approximately 0.01. Shake the bottles evenly several times to disperse the virus evenly, and place them in an incubator at 37°C and 5% CO2 for 1-2 hours for adsorption, shaking the culture bottles once every 15 minutes or so during this period. 4. After adsorption is complete, discard the virus culture medium, add 15 mL of new DMEM (containing 2% FBS), and then place the cell flask in an incubator at 37°C with 5% CO2 to continue culturing; 5. Observe the cytopathic effect daily (cytopathic effects will occur after viral infection and proliferation, manifested as cell swelling and vacuolation, cell bulging out of the monocellular layer, cell shedding, etc.). 6. Harvest when 75%-100% of cells show cytopathic effects, freeze and thaw once at -80℃, centrifuge at 2,000 rpm for 5 minutes, collect the supernatant as the virus stock solution, aliquot and store at -80℃.

[0075] II. Determination of dengue virus titer 1. Preparation of BHK-21 cells: Take BHK-21 cells in good growth condition, digest with trypsin, and adjust the cell density to 5×10⁶ cells / year using DMEM (containing 10% FBS). 5 Cells / mL, 2mL / well were seeded into 6-well cell culture plates and cultured at 37℃ in a 5% CO2 incubator. 2. On the day of the experiment, remove the 6-well plate, discard the culture medium, and add 800 μL / well of cell maintenance medium (purchased from Thermo Fisher Scientific, catalog number 11995065); dilute the virus solution to be tested 10-fold with cell maintenance medium. -1 ~10 -6 Then, add 100 μL / well to a 6-well plate, and incubate the cells with the added virus solution in a 37°C 5% CO2 incubator for 1 h. 3. After incubation, discard the supernatant, wash twice with PBS, add 2 mL of semi-solid virus culture medium per well (4% FBS 2×DMEM medium and room temperature low melting point agarose mixed 1:1), and continue to culture at 37℃ and 5% CO2 for 4-5 days until obvious cytopathic effects appear in the cells; 4. Add 1 mL of 4% paraformaldehyde solution to each well of the culture plate, place at 4℃ for 1 h to fix the cells, rinse with deionized water to remove the semi-solid culture medium, add 1 mL of 1% crystal violet solution to each well, stain at room temperature for 30 min, and rinse again with deionized water. 5. Plaque count, expressed in plaque form units (PFU / mL) to measure viral titer.

[0076] The final measured titer of dengue virus type I was 1.8 × 10⁻⁶. 5 / mL, the type II dengue virus titer is 2.6×10⁻⁶. 4 / mL, the type III dengue virus titer was 4.2×10⁻⁶. 4 / mL, the type IV dengue virus titer is 1.7×10⁻⁶. 4 / mL.

[0077] III. Detection of antibody neutralizing activity using dengue virus 1. Seed BHK-21 cells in 96-well cell culture plates (Thermo, 137101) with black borders and a black background, adding 2 × 10⁶ cells per well. 4 One cell was cultured at 37°C and 5% CO2 for 24 hours. 2. The E4_12C-Fc antibody was serially diluted three times to an initial concentration of 200 ng / μL. An appropriate amount of single serotype virus stock solution was added to each well (25 μL / well for type II and IV virus, and 12.5 μL / well for type I and III virus), with two replicates. The wells were incubated at 37℃ and 5% CO2 for 48 h. 3. Aspirate the culture medium and wash the cells once with PBS at room temperature, 10 min each time; 4. Remove any residual PBS from the wells, gently pat dry, add 4% paraformaldehyde (containing a final concentration of 0.2% Triton 100) and fix at 4°C for 30 min. Remove the fixative and allow to dry thoroughly at room temperature. 5. Wash cells once with room temperature PBS, 10 min each time; 6. Add 100 μL of 2% BSA to each well and seal overnight at 4°C; 7. Add primary antibody 4G2 (AB41349, abcam) and incubate at room temperature for 1 h, 100 μL / well, with a final primary antibody concentration of 1 μg / mL; 8. Draw out the primary antibody and wash the Thermo Fisher Scientific fluorescence detection plate (Thermo 137101) with an automated pipette using 200 μL of PBS solution per well, washing 6 times for 3 min each time; 9. Add FITC-labeled goat anti-mouse secondary antibody (AB150113, abcam; 1:3000 dilution), incubate at room temperature for 1 h, 100 μL / well; 10. Draw out the secondary antibody and wash the Thermo Fisher Scientific fluorescence detection plate with an automated pipette using 200 μL of PBS solution per well, washing 6 times for 3 min each time; 11. SpectraMax i3x multi-functional plate reader (Molecular Devices, USA) records readings (excitation light 485, 535nm); Record the experimental results, plot them using GraphPad Prism 8 software, and calculate FRNT. 50 The results showed that the E4_12C-Fc antibody could effectively neutralize four serotypes of dengue virus, and was effective against DENV I FRNT. 50 It is 1.63 μg / mL ( Figure 2 (A), for DENV II's FRNT 50 It is 1.89 μg / mL ( Figure 2 (B), for DENV III's FRNT 50 It was 34.79 μg / mL. Figure 2 (C), for DENV IV's FRNT 50 It is 1.67 μg / mL ( Figure 2 The results (D) indicate that antibody E4_12C-Fc has good broad-spectrum neutralizing activity against dengue virus.

[0078] Example 4: Humanization of Camel-Derived Nanobody E4_12C and Detection of Neutralizing Activity I. Amino acid sequence analysis of the E4_12C variable region of camel-derived nanobody use www.abysis.org The variable region amino acids of the camel-derived nanobody E4_12C obtained in Example 1 were analyzed, and the camel-derived amino acids were replaced with suitable human-derived amino acids. Figure 3 ), to carry out humanization modifications, and with the help of www.abysis.org The website's Z-score model scored the degree of humanization of the camel-derived nanobody E4_12C before and after modification. Figure 4 The results showed that the modified sequence had a high degree of humanization. The amino acid sequence of the humanized nanobody E4_12C is SEQ ID NO:5, and the nucleotide sequence of the gene encoding the humanized nanobody E4_12C_2 is SEQ ID NO:6.

[0079] II. Preparation of recombinant humanized E4_12C_2-Fc heavy chain antibody using conventional molecular biology methods The expression plasmid h-pTSE-E4_12C_2-Fc for the humanized nanobody E4_12C_2-Fc is formed by replacing the pTSE-Fc vector with a DNA fragment whose nucleotide sequence is SEQ ID NO:6. Sal I and Nhe I. A recombinant expression vector was obtained by extracting a small fragment between two restriction enzyme sites while maintaining the other nucleotide sequences of the pTSE-Fc vector unchanged. The vector expresses a protein with the amino acid sequence of SEQ ID NO:7 (named humanized E4_12C_2-Fc, whose nucleotide sequence is SEQ ID NO:8).

[0080] The expression plasmid of the humanized nanobody E4_12C_2-Fc was used for expression, purification, and SDA-PAGE detection as described in Example 2. Figure 5 The size of the protein bands basically conforms to the theoretical molecular weight of the protein.

[0081] III. The method for detecting the dengue virus neutralizing activity of the E4_12C_2-Fc antibody is the same as in Example 3. The results show that the neutralizing activity remains basically unchanged after antibody humanization, and it can still effectively neutralize four serotypes of dengue virus, including DENV I FRNT. 50 It is 1.59 μg / mL ( Figure 6 (A), for DENV II's FRNT 50 0.08 μg / mL ( Figure 6 (B), for DENV III's FRNT 50 It is 0.27 μg / mL ( Figure 6 (C), for DENV IV's FRNT 50 It is 0.71 μg / mL ( Figure 6 (D).

[0082] Example 5: Design, construction, and neutralization activity detection of multivalent nanobody 8F-2C-12C I. Preparation of recombinant plasmid 8F-2C-12C multivalent nanobody using conventional molecular biology methods To avoid the ADE effect caused by the Fc domain, the multivalent nanobody 8F-2C-12C was redesigned. The structure is a tandem of three antibody VHHs: 2E1_8F_1, 2E2_2C_1, and E4_12C_2. The Fc domain was removed, and the VHHs are linked by a flexible peptide (GGGGS) to increase the antibody molecular weight and prolong the half-life. The 8F-2C-12C antibody gene (SEQ ID NO:10) was obtained through whole-gene synthesis, with nucleotides 1 to 381 being 2E1_8F_1, nucleotides 397 to 780 being 2E2_2C_1, and nucleotides 796 to 1170 being E4_12C_2. The 8F-2C-12C antibody recombinant plasmid was obtained by replacing the small fragment between the sal I and Nhe I restriction sites of the pTSE-hFc enzyme in the plasmid with the recombinant gene, while maintaining the other nucleotide sequences of pTSE-hFc unchanged. The amino acid sequence expressed by this recombinant plasmid is antibody 8F-2C-12C as described in SEQ ID NO:9. A stop codon was added to the 3' end of the recombinant gene in SEQ ID NO:10 to prevent the recombinant plasmid from expressing the Fc fragment.

[0083] In antibody 8F-2C-12C, amino acids 1-127 are nanobody 2E1_8F_1, amino acids 133-260 are nanobody 2E2_2C_1, and amino acids 266-390 are nanobody E4_12C_2. Nanobody 2E1_8F_1 and nanobody 2E2_2C_1, as well as nanobody 2E2_2C_1 and nanobody E4_12C_2, are linked by flexible peptides (GGGGS). Nanobody E4_12C_2 has a His×6 tag at its C-terminus.

[0084] II. Preparation of multivalent nanobody 8F-2C-12C antibody The recombinant plasmid of the 8F-2C-12C antibody was used for expression and purification according to Example 2, with the purification column replaced by HiTrap. TM MabSelect VH3, replace the binding buffer with a pH 7.4 phosphate buffer (20mM Na3PO4, 0.15M NaCl), and keep the other steps the same.

[0085] Figure 7In the figure, lane "1" represents the 8F-2C-12C antibody, and lane "2" represents 8F-2C-12C (mercaptoethanol). As shown in the figure, the purified 8F-2C-12C antibody sample has a single band without any impurities. The molecular weight of both the non-reducing sample (lane "1") and the reducing sample (lane "2") is approximately 50 kDa, consistent with the expected molecular weight.

[0086] III. Fluorescence Reduction Neutralization Assay for Detecting the Neutralizing Activity of 8F-2C-12C Antibody The specific method for detecting the neutralizing activity of the 8F-2C-12C antibody is described in Example 3. The results showed that even after removing the Fc domain, the 8F-2C-12C antibody could still effectively neutralize all four serotypes of dengue virus. Compared to the parent antibody, the neutralizing activity of the multivalent nanobody 8F-2C-12C was not weakened, and even improved. It also showed increased neutralization activity against DENV I FRNT. 50 0.06 μg / mL ( Figure 8 (A), for DENV II's FRNT 50 0.22 μg / mL Figure 8 (B), for DENV III's FRNT 50 It is 0.37 μg / mL ( Figure 8 (C), for DENV IV's FRNT 50 It is 0.49 μg / mL ( Figure 8 (D).

[0087] SEQ ID NO:11 (where amino acids 1-25 form the first frame region (FR1), amino acids 34-50 form the second frame region (FR2), amino acids 58-95 form the third frame region (FR3), amino acids 117-127 form the fourth frame region (FR4), amino acids 26-33 form the first complementarity-determining region (CDR1), amino acids 51-57 form the first complementarity-determining region (CDR2), and amino acids 96-116 form the third complementarity-determining region (CDR3)): QVQLVESGGGVVQPGGSLRLSCAASRYPNKLYRMGWFRQAPGKGLEGVAALDSRNRTHYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAMYYCAAVSGVRVGSPPELDGSQYMYWGQGTLVTVSS.

[0088] SEQ ID NO:12 (where amino acids 1-25 form the first frame region (FR1), amino acids 34-50 form the second frame region (FR2), amino acids 59-96 form the third frame region (FR3), amino acids 118-128 form the fourth frame region (FR4), amino acids 26-33 form the first complementarity-determining region (CDR1), amino acids 51-58 form the first complementarity-determining region (CDR2), and amino acids 97-117 form the third complementarity-determining region (CDR3)): QVQLVESGGGLVQPGGSLRLSCAASSYTYGTNCIGWFRQAPGKGLEGVALIYSGAGSTYYADSVKGRFTVSRDNSKNTVYLQMNSLRAEDTAMYFCAAKLRSIYGCAAPLALNSYNAWGQGTLVTVSS.

[0089] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A nanobody or antigen-binding fragment against dengue virus, characterized in that, The nanobody or its antigen-binding fragment comprises three complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:1, the amino acid sequence of CDR2 is positions 51-58 of SEQ ID NO:1 and the amino acid sequence of CDR3 is positions 97-114 of SEQ ID NO:

1.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, In addition to the complementarity-determining region, the nanobody also includes four framework regions: FR1, FR2, FR3, and FR4. The FR1 comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The amino acid sequence includes the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 59-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 115-125 of SEQ ID NO:1; A4-2) is a sequence that has more than 75% similarity to A4-1).

3. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the nanobody or its antigen-binding fragment includes SEQ ID NO:1 or SEQ ID NO:

5.

4. A heavy chain antibody or multivalent nanobody against dengue virus, characterized in that, The heavy chain antibody includes the nanobody described in any one of claims 1-3.

5. The heavy chain antibody or multivalent nanobody according to claim 4, characterized in that, The amino acid sequence of the heavy chain antibody includes SEQ ID NO:3 or SEQ ID NO:

7.

6. The heavy chain antibody or multivalent nanobody according to claim 4, characterized in that, The structure of the multivalent nanobody is a first nanobody-connector-second nanobody-connector-third nanobody, wherein the third nanobody is the nanobody according to any one of claims 1-3. The first nanobody contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:11, the amino acid sequence of CDR2 is positions 51-57 of SEQ ID NO:11, and the amino acid sequence of CDR3 is positions 96-116 of SEQ ID NO:

11. The second nanobody contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is positions 26-33 of SEQ ID NO:12, the amino acid sequence of CDR2 is positions 51-58 of SEQ ID NO:12, and the amino acid sequence of CDR3 is positions 97-117 of SEQ ID NO:

12.

7. The heavy chain antibody or multivalent nanobody according to claim 6, characterized in that, The multivalent nanobody includes any of the following: M1, the first nanobody includes, in addition to the complementarity-determining region, four framework regions FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The amino acid sequence includes the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 58-95 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 117-127 of SEQ ID NO:1; A4-2) is a sequence with more than 75% identity with A4-1); In addition to the complementarity-determining region, the second antibody also includes four framework regions: FR1, FR2, FR3, and FR4. The FR1 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-25 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The FR2 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A2-1)FR2 is positions 34-50 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 59-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 of the second antibody comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 118-128 of SEQ ID NO:1; A4-2) is a sequence with more than 75% identity with A4-1); M2, the amino acid sequence of the first nanobody includes SEQ ID NO:11, and the amino acid sequence of the second nanobody includes SEQ ID NO:12; M3, The amino acid sequence of the multivalent nanobody is SEQ ID NO:

9.

8. A biomaterial, characterized in that, The biomaterial includes any one of the following: B1) A nucleic acid molecule, wherein the nucleic acid molecule encodes a nanobody or its antigen-binding fragment as described in any one of claims 1-3, or a heavy chain antibody or multivalent nanobody as described in any one of claims 4-7; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

9. A drug for treating dengue fever, characterized in that, The pharmaceutical composition comprises any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7.

10. Application, characterized in that, The application includes any of the following: M1) The use of the biomaterial of claim 8 in the preparation of the nanobody or antigen-binding fragment of any one of claims 1-3, or the heavy chain antibody or multivalent nanobody of any one of claims 4-7; M2) The use of any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7, in the preparation of the drug of claim 9; M3) The use of any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7, or the biomaterials of claim 8, or the medicaments of claim 9 in the preparation of products for the prevention and / or treatment of diseases caused by dengue virus; M4) The use of any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7, or the biomaterials of claim 8 in the preparation of products for detecting dengue virus; M5) The use of any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7, or the biomaterials of claim 8, or the medicaments of claim 9, in the preparation of products for neutralizing dengue virus; M6) The use of any of the nanobodies or antigen-binding fragments of claims 1-3, or any of the heavy chain antibodies or multivalent nanobodies of claims 4-7, or the biomaterial of claim 8, or the medicament of claim 9, in the preparation of products for inhibiting dengue virus infection.

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