Nano antibody for resisting tetanus toxin L-HN fragment and related biological material and application thereof

Abstract

The invention discloses an anti-tetanus toxin L-HN fragment nano antibody as well as a related biological material and application thereof, and belongs to the field of immunotherapy biotechnology pharmacy. The nano antibody or the antigen binding fragment thereof comprises three complementarity determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is SEQ ID NO: 6, the amino acid sequence of the CDR2 is SEQ ID NO: 7, the amino acid sequence of the CDR3 is SEQ ID NO: 8, and the sequences of the complementarity determining regions are defined according to an IMGT numbering system. The antibody is only combined with TL-HN protein, and 0.3125 [mu] g of the antibody can effectively block poisoning caused by tetanus toxin (10 * LD50) in a lethal dose; after being humanized, the antibody still has better affinity and neutralizing activity, and the antibody with the same dose can still effectively block poisoning caused by tetanus toxin (10 * LD50) with a lethal dose.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy biomedical technology, specifically involving nanobodies containing the L-HN fragment of anti-tetanus toxin and related biomaterials and applications. Background Technology

[0002] Tetanus is an acute infectious disease caused by tetanus toxin (TeNT) produced by Clostridium tetani, characterized by muscle spasms. TeNT is a potent neurotoxin that can invade the central nervous system; in severe cases, it can cause respiratory muscle rigidity, leading to respiratory arrest and even death. TeNT is a 150kD polypeptide chain composed of three 50kD domains. After translation by bacterial or host proteases, it is cleaved into an active double-chain form linked by a single disulfide bond: a light chain (fragment A) and a heavy chain (fragments B and C), with densities of 50kD and 100kD, respectively.

[0003] Tetanus has an extremely high mortality rate. Without medical intervention, the mortality rate for severely ill patients is close to 100%. Even with aggressive comprehensive treatment, the global mortality rate remains between 30% and 50%, making it an extremely serious and potentially fatal disease. Tetanus infection does not provide lifelong immunity; therefore, emergency tetanus vaccination is necessary after injury to prevent tetanus.

[0004] Passive immunotherapy has long been a primary strategy for clearing tetanus toxin from tetanus patients. Commonly used passive immunization agents in clinical practice include injectable tetanus antitoxin (TAT), equine tetanus antitoxin (TAT), and human tetanus immunoglobulin (HTIG). However, the production and use of animal-derived therapeutics face numerous obstacles, such as long immunization cycles and potential hypersensitivity reactions in receptors. HTIG, being a blood product, carries the risk of transmitting known and unknown infectious diseases and is difficult to obtain, hindering market demand. Therefore, specific TeNT antibodies that can be generated in vitro without an immunization process would be a better alternative to HTIG or antitoxin.

[0005] Nanobodies (Nb) are antibody molecules naturally occurring in camel-like animals. They possess characteristics such as small relative molecular mass, strong tissue penetration, high stability, and high specificity, overcoming many limitations of traditional monoclonal antibodies. For the diagnosis or treatment of tetanus, the development of high-affinity tetanus toxin-neutralizing nanobodies has become a hot topic of research.

[0006] Studies have shown that the TL-HN functional fragment, as a protective antigen, induced the highest levels of neutralizing antibodies, and among all TeNT functional fragments, the TL-HN fragment provided the best immunoprotection. Experiments demonstrated that the protective effect of TL-HN was superior to that of THc, TL, or THN fragments alone or in combination. Therefore, the TL-HN functional fragment plays an important role in the immunoprotection against tetanus toxin, laying a solid foundation for the development of TeNT neutralizing antibodies. Summary of the Invention

[0007] The technical problem to be solved by this invention is to develop a more effective nanobody for neutralizing tetanus toxin targeting the L-HN domain of tetanus toxin, which can be used in the preparation of drugs for the diagnosis or treatment of tetanus. 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.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0009] This invention provides a nanobody targeting the TL-HN domain of tetanus toxin or an antigen-binding fragment containing the nanobody.

[0010] In a first aspect, the present invention provides a nanobody or antigen-binding fragment of the anti-tetanus toxin, wherein the nanobody or antigen-binding fragment comprises three complementarity-determining regions CDR1, CDR2 and CDR3, and the nanobody is TL-16;

[0011] The amino acid sequence of CDR1 of TL-16 is SEQ ID NO:6, the amino acid sequence of CDR2 of TL-16 is SEQ ID NO:7, and the amino acid sequence of CDR3 of TL-16 is SEQ ID NO:8.

[0012] In addition to the complementarity-determining region, the above-mentioned nanobody or its antigen-binding fragment also contains four framework regions FR1, FR2, FR3 and FR4;

[0013] The amino acid sequence of FR1 of TL-16 is SEQ ID NO:2, the amino acid sequence of FR2 of TL-16 is SEQ ID NO:3, the amino acid sequence of FR3 of TL-16 is SEQ ID NO:4 or has more than 75% identity with SEQ ID NO:4, and the amino acid sequence of FR4 of TL-16 is SEQ ID NO:5.

[0014] The amino acid sequence of TL-16 in the above-mentioned nanobody or its antigen-binding fragment includes SEQ ID NO:1 or SEQ ID NO:12.

[0015] The amino acid sequence of TL-16 is SEQ ID NO:1 or SEQ ID NO:12.

[0016] 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.

[0017] Secondly, the present invention provides a heavy chain antibody against tetanus toxin, said heavy chain antibody containing the aforementioned nanobody.

[0018] The aforementioned heavy chain antibody includes a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region 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 aforementioned heavy chain antibody is TL-16-hFc, and the amino acid sequence of TL-16-hFc includes SEQ ID NO:11 or SEQ ID NO:15.

[0020] The amino acid sequence of the above heavy chain antibody is SEQ ID NO:11 or SEQ ID NO:15.

[0021] Thirdly, the present invention provides a biomaterial, said biomaterial being any of the following:

[0022] B1) Nucleic acid molecules, wherein the nucleic acid molecules encode the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody;

[0023] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0025] B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0026] 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.

[0027] 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 further comprise 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.

[0028] The small molecule antibody may be any of the following:

[0029] F1 and Fab antibodies;

[0030] F2 and Fv antibodies;

[0031] F3, single-chain antibody;

[0032] F4, Fab′ fragment.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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).

[0038] 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).

[0039] 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.

[0040] Fourthly, the present invention provides genetic material, said genetic material being any of the following:

[0041] g1) Nucleic acid molecules, wherein the nucleic acid molecules encode the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody;

[0042] g2) Expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria containing the nucleic acid molecules described in g1).

[0043] Fifthly, the present invention claims protection for a tetanus antidote comprising the aforementioned nanobody or its antigen-binding fragment or the aforementioned heavy chain antibody.

[0044] The drug also includes physiologically or pharmaceutically acceptable excipients, diluents, or carriers.

[0045] 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.

[0046] 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.

[0047] Sixthly, the present invention claims protection for any of the following applications:

[0048] M1) The application of the aforementioned genetic materials in the preparation of the aforementioned nanobodies or their antigen-binding fragments or the aforementioned heavy chain antibodies;

[0049] M2) The application of the aforementioned nanobodies or their antigen-binding fragments or the aforementioned heavy chain antibodies in the preparation of the aforementioned drugs;

[0050] M3) The use of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned genetic materials or the aforementioned drugs in the preparation of products for the prevention and / or treatment of diseases caused by Clostridium tetani;

[0051] M4) The application of the aforementioned nanobodies or their antigen-binding fragments or the aforementioned heavy chain antibodies or the aforementioned genetic materials in the preparation of products for detecting Clostridium tetani;

[0052] M5) The use of the aforementioned nanobody or its antigen-binding fragment, or the aforementioned heavy chain antibody, or the aforementioned genetic material or the aforementioned drug in the preparation of a product for neutralizing the neurotoxin secreted by Clostridium tetani;

[0053] M6) The use of the aforementioned nanobodies or their antigen-binding fragments, or the aforementioned heavy chain antibodies, or the aforementioned genetic materials or drugs in the preparation of products for detecting neurotoxins secreted by Clostridium tetani.

[0054] The tetanus toxin nanobody provided by this invention can effectively neutralize tetanus toxin. Experiments have shown that the fusion protein TL-16-hFc, obtained by fusing the nanobody TL-16 with the Fc fragment (hFc) of human immunoglobulin, exhibits good specificity, binding only to TL-HN protein. 0.3125 μg of TL-16-hFc can effectively block a lethal dose of tetanus toxin (10×LD50). 50 TL-16-h1-hFc can effectively block lethal doses of tetanus toxin (10×LD50) and poisoning caused by tetanus toxin. Even after humanization, it retains good affinity and neutralizing activity. An equivalent dose of TL-16-h1-hFc can still effectively block lethal doses of tetanus toxin (10×LD50). 50 Poisoning caused by ) Attached Figure Description

[0055] Figure 1 This image shows a partial illustration of the binding of phage clones to the target antigen after the second round of screening using Phage-ELISA. The left image shows the target antigen, and the right image shows the control antigen.

[0056] Figure 2 The purified anti-tetanus nanobody fusion protein was detected by SDS-PAGE electrophoresis.

[0057] Figure 3 To detect the binding activity of anti-tetanus nanobody fusion protein.

[0058] Figure 4For the specific detection of anti-tetanus nanobody fusion protein.

[0059] Figure 5 To detect the affinity of anti-tetanus nanobody fusion protein.

[0060] Figure 6 The purified humanized anti-tetanus nanobody fusion protein was detected by SDS-PAGE electrophoresis.

[0061] Figure 7 Detection of binding activity of humanized anti-tetanus nanobody fusion protein.

[0062] Figure 8 Specific detection of humanized anti-tetanus nanobody fusion protein.

[0063] Figure 9 Affinity detection of humanized anti-tetanus nanobody fusion protein. Detailed Implementation

[0064] 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.

[0065] 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.

[0066] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The Log-rank test was used to compare the significance of the differences in protection between the two groups. A p < 0.05 was considered statistically significant.

[0067] Recombinant TL-HN antigen (or recombinant TL-HN protein): Prepared by the inventor's laboratory. The preparation method is described in section 2.2 of the following literature: "Liu XY, Wei DK, Li ZY, Lu JS, Xie XM, Yu YZ, et al. Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanustoxin. VACCINE (2023). doi:10.1016 / j.vaccine.2023.09.032".

[0068] pTSE-hFc vector: described in the non-patent literature “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”, which can be obtained by the public from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biomaterial is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0069] Example 1: Construction of an anti-tetanus nanobody library

[0070] 1. Camel Immunity

[0071] The recombinant TL-HN antigen for tetanus was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881) and shaken to emulsify it thoroughly. The mixture was then injected subcutaneously into healthy adult Bactrian camels at multiple sites. Booster immunizations were given every two weeks. Except for the first immunization which used Freund's complete adjuvant, all subsequent immunizations used Freund's incomplete adjuvant (Sigma, F5506).

[0072] 2. Isolation of peripheral blood lymphocytes from camel blood

[0073] Approximately 150 mL of camel peripheral blood was collected after five immunizations and placed into an anticoagulant tube. Peripheral blood lymphocytes were separated from the camel blood using lymphocyte separation medium (STEMCELL, 07851). After centrifuging the mixture of whole blood and lymphocyte separation medium, the liquid in the centrifuge tube separated into four layers from top to bottom: plasma, peripheral blood mononuclear cells, glucose from the lymphocyte separation medium, and red blood cells.

[0074] 3. Nested PCR amplification of the VHH gene fragment

[0075] Total RNA was extracted from isolated peripheral blood mononuclear cells (PBMCs) using the OMEGA EZNA Total RNA kit I (OMEGA, R6834), and then cDNA was obtained by reverse transcription using the Invitrogen Superscript III First-strand Synthesis System for RT-PCR kit (Invitrogen, 18080-051).

[0076] Nested PCR amplification of the VHH gene fragment: 1) First round of PCR: Using the synthesized cDNA as a template, the antibody leader sequence and CH2 sequence were amplified by PCR using the designed IgG-specific upstream primer CALL001 and downstream primer CALLOO2; 2) Using the purified DNA product from the first round as a template, the VHH gene was obtained by second round of PCR amplification using primers VHH-F and VHH-R.

[0077] Table 1. Primer sequences used in the two rounds of PCR

[0078] Primer name Primer sequence (5'-3') CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F cggCCATGGcGGTCCTGGCTGCTCTTCTACA VHH-R tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT

[0079] 4. Electro-connection products

[0080] The vector and amplified VHH gene were digested with restriction endonucleases NcoⅠ and NotⅠ. The ligation product was constructed using T4 ligase, and then transformed into E. coli TG1 competent cells using electroporation to construct a tetanus-specific phage antibody library. The library size was determined by serial dilution. The library size was found to be 3.1 × 10⁻⁶. 8 pfu.

[0081] Example 2: Screening of a tetanus-specific phage nanobody library

[0082] Take the bacterial culture of the anti-tetanus specific phage nanobody library constructed in step two above, present it using helper phage M13K07, and then precipitate the phage with 20% PEG / 2.5M NaCl solution (1L of solution contains 200g PEG6000 and 146.25g NaCl) to obtain the anti-tetanus specific phage nanobody library.

[0083] Using purified recombinant TL-HN protein as the antigen, a solid-phase screening strategy was employed (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, 2008.5) to screen the constructed immune library. The specific method is as follows:

[0084] Add 1 mL of antigen diluted with 0.1 M NaHCO3 coating buffer to each immunoassay well, and incubate overnight at 4°C. The coating concentrations for each round of panning are 20 μg / mL and 10 μg / mL, respectively. The next day, wash the immunoassay tubes twice with PBS for 3 min each time, followed by blocking with 2% blocking buffer (bovine serum albumin) at 37°C for 2 h. After blocking, wash three times with PBST (1×PBS + 0.1% Tween-20), and then wash three times with PBS. Adjust the titer of the anti-tetanus specific phage nanobody library to an appropriate concentration with blocking buffer, add it to the immunoassay tubes, and incubate at room temperature for 2 h. Then, shake gently at 200 rpm for 20 min. Wash 10 times with PBST, and then wash 5 times with PBS. After washing, add 1 mL of elution buffer (glycine-hydrochloric acid, pH 10) to each well. 2.2) Elute at 400 rpm for 20 min at room temperature. Remove the eluent from the target antigen wells and add 50-60 μL of neutralization buffer (1 M Tris-HCl, pH 8.0) for neutralization. Infect Escherichia coli TG1 cells in the logarithmic growth phase, incubate at room temperature for 30 min, and then incubate at 37°C for 1 h to generate and purify phages for the next round of screening. Repeat the same screening process twice. The enrichment results are shown in Table 2.

[0085] Table 2. Enrichment analysis of the anti-tetanus phage nanobody library

[0086] Number of filters Input amount (pfu) Output (pfu) Output / Input enrichment factor 1 <![CDATA[5×10 11 ]]> <![CDATA[4.2×10 7 ]]> <![CDATA[8.4×10 -5 ]]> --- 2 <![CDATA[1×10 11 ]]> <![CDATA[1.95×10 7 ]]> <![CDATA[1.95×10 -4 ]]> 2.3

[0087] After two rounds of screening, well-separated single clones were selected from the plates containing phages and inoculated into 96-well plates (250 μL / well) containing 2YT-GA medium (1L 2YT medium contains 16g Typtone, 10g Yeast extract, 5g NaCl, 100μg / mL ampicillin, and 20% glucose). Two negative control wells were reserved (without clones). The plates were incubated at 37°C until the logarithmic growth phase. M13KO7 helper phage was added at a ratio of MOI≈50, at a rate of 100 μL / well to the deep-well plates containing single phage clones. The plates were incubated at room temperature for 30 min, followed by incubation at 37°C for 1 h at 150 rpm. The deep-well plates were centrifuged at room temperature (2000 rpm, 10 min), the supernatant was discarded, and arabinose was added to a final concentration of 1 mM for induction of expression. The plates were incubated overnight to obtain phage particles displaying the antibody variable region.

[0088] Example 3: Identification of specific nanobody positive clones using phage-linked immunosorbent assay (Phage-ELISA).

[0089] The ELISA plate was coated with recombinant TL-HN protein as the antigen. Adjacent columns of each antigen group were used for parallel coating with irrelevant antigens as negative controls. Coating was performed overnight at 4°C. The coated ELISA plate was then discarded, washed 6 times with PBST, and blocked with blocking buffer (3g skim milk powder added to 100mL PBS) at 200μL / well, incubated at 37°C for 2h. The blocking buffer was discarded, and 100μL of the induced phage supernatant was added to each well of the ELISA plate. The plate was incubated at 37°C for 1.5h. The plate was then washed 6 times with PBST. Freshly prepared 0.2μg / ml HRP-labeled M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) was added to each well at 100μL / well, and incubated at 37°C for 45min. The plate was then washed 6 times with PBST. The chromogenic solution was prepared (9mL chromogenic solution, 1mL...) per ELISA plate. Add 10×OPD and 10μL of 30%H2O2 to the ELISA plate at a rate of 10μL / well and develop the color in the dark for 15-20 minutes; add 2M sulfuric acid stop solution (50μL / well) and read the values ​​at dual wavelengths of 492 / 630nm; for clone wells with an absorbance value >3 times that of the antigen group / negative control group, they are considered positive clone wells.

[0090] Some Phage-ELISA experimental results can be found in Figure 1 The left image shows the target antigen, and the right image shows the control antigen. Positive clones were sent to a biotechnology service company for sequencing to obtain the DNA sequence of the inserted fragment, ultimately yielding the camel-derived nanobody TL-16 that can specifically bind to TL-HN.

[0091] The amino acid sequence of TL-16 is shown in SEQ ID NO:1, comprising a frame region (named FR, including FR1, FR2, FR3, and FR4) and a complementarity-determining region (named CDR, including CDR1, CDR2, and CDR3). The four parts of the frame region are denoted as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; the three parts of the complementarity-determining region are denoted as SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. The nucleotide sequence of the gene encoding the aforementioned anti-tetanus nanobody TL-16 is shown in SEQ ID NO:9.

[0092] The sequence of the complementarity determination region (CDR) is defined according to the IMGT numbering system.

[0093] Example 4: Preparation of anti-tetanus nanobody-hFc fusion protein

[0094] 1. Construction of eukaryotic expression plasmid for anti-tetanus nanobody-hFc fusion protein

[0095] The C-terminus of the anti-tetanus nanobody TL-16 (nucleotide sequence SEQ ID NO:9, amino acid sequence SEQ ID NO:1) was linked to the Fc segment (hFc) of human immunoglobulin to obtain the encoding gene of the anti-tetanus nanobody-hFc fusion protein TL-16-hFc, the nucleotide sequence of which is SEQ ID NO:10 and the amino acid sequence is SEQ ID NO:11.

[0096] Using conventional molecular biology techniques, a small fragment between the Sal I and Nhe I restriction sites of the pTSE-hFc expression vector (pTSE-hFc was modified by the inventors by linking the gene of the Fc domain of human immunoglobulin to the pCMV vector) was replaced with a DNA molecule (encoding gene of TL-16 nanobody) with the nucleotide sequence SEQ ID NO:9. Other nucleotides in the pTSE-hFc vector remained unchanged, resulting in the recombinant expression plasmid pTSE-TL-16-hFc containing the encoding gene of the TL-16-hFc fusion protein. This vector expresses a protein with the amino acid sequence SEQ ID NO:11 (named TL-16-hFc, whose nucleotide sequence is SEQ ID NO:10).

[0097] The sequence of the complementarity-determining region of the aforementioned heavy chain antibody is defined according to the IMGT numbering system.

[0098] 2. Expression and purification of anti-tetanus nanobody-hFc fusion protein

[0099] The pTSE-TL-16-hFc fusion protein expression plasmid constructed in step 1 was transfected into FreeStyle using the FectoPRODNA Transfection Reagent (Polyplus, 116-001). TM In HEK293-F cells (Invitrogen, R79007), cell viability was monitored after 48 hours. When the cell viability dropped to 80-85%, the cell supernatant was collected by centrifugation at 8000 rpm for 10 min and purified using the AKTA purification system to obtain the TL-16-hFc fusion protein.

[0100] SDS-PAGE electrophoresis analysis of the purified antibody yielded the following results: Figure 2 As shown: The molecular weight of the antibody is as expected. The band size of the nanobody-hFc fusion protein under reduced conditions is about 40 kDa (left) and the band size under non-reduced conditions is about 80 kDa (right).

[0101] Example 5: Characterization of Nanobody Fusion Protein

[0102] 1. The binding activity of the nanobody-hFc fusion protein and the recombinant TL-HN protein was detected by ELISA.

[0103] The recombinant TL-HN protein was diluted to 2 μg / mL with carbonate coating buffer (pH 9.6), and 100 μL / well was added to each well of an ELISA plate. The plate was incubated overnight at 4°C. The next day, the plate was washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of blocking buffer (3% skim milk powder) was added. The plate was blocked at 37°C for 2 h. The blocking buffer was discarded, and the plate was washed 6 times with PBST. TL-16 nanobody fusion protein dilution buffer (starting concentration 10 μg / mL, diluted with blocking buffer) was added at 100 μL / well, creating 23 gradients with 2 replicates per gradient. The plate was incubated at 37°C for 1.5 h. The plate was then washed 6 times with PBST, and 100 μL / well of goat anti-human IgG (HRP, 1:4000) diluted with blocking buffer was added. The plate was incubated at 37°C for 45 min. The plate was washed 6 times with PBST, and 100 μL / well of blocking buffer was added to each well. Incubate the OPD substrate with the chromogenic buffer in the dark for 5-15 minutes, observing the color development. Once development is complete, add 50 μL of 2M sulfuric acid to each well to terminate the enzyme-linked reaction. Measure the optical density at 492 nm / 630 nm using a microplate reader. Calculate the concentration (EC50) required for antibody to bind 50% of the antigen protein. 50 ), to assess their binding ability.

[0104] The results are as follows Figure 3 As shown in the figure. The horizontal axis represents the logarithm of protein molar concentration, and the vertical axis represents the optical density value. Analysis shows that the half-maximal effective binding concentration (EC50) of TL-16-hFc fusion protein to recombinant TL-HN protein is... 50 The value is 0.01515nM.

[0105] 2. Identify the specificity of the nanobody-hFc fusion protein using ELISA.

[0106] The TL-HN, THC, TL, and THN proteins of tetanus were prepared by the inventor's laboratory according to the following literature: "Liu XY, WeiDK, Li ZY, Lu JS, Xie XM, Yu YZ, et al. Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanus toxin. VACCINE (2023). doi:10.1016 / j.vaccine.2023.09.032" (Materials Method 2.2).

[0107] The AL-HN protein was prepared by the inventor's laboratory according to the following literature: "Liu FJ, Shi DY, Mao YY, Xiong XH, Lu JS, Pang XB, et al. Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A. VACCINE (2020) 38(14): 2978-2983. doi: 10.1016 / j.vaccine.2020.02.060" in section 2.1.

[0108] The BL-HN protein was prepared by the inventor's laboratory according to the material method "Recombinant BoNT / BL-HN Fragment Preparation" in the following literature: "Li Z, Lu JS, Liu S, Wang R, Xu Q, Yu YZ, et al. Recombinant L-HN Fusion Antigen Derived from the L and HN Domains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin. NEUROTOX RES (2021). doi:10.1007 / s12640-021-00337-x".

[0109] The EL-HN protein was prepared by the inventor's laboratory according to the following literature: "Li Z, Lu J, Tan X, Wang R, Xu Q, Yu Y, et al. Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E. Toxins (Basel) (2022) 14(2). doi:10.3390 / toxins14020135" Material Method 5.2.

[0110] The FL-HN protein was prepared by the inventor's laboratory according to the following literature: "Li ZY, Li B, Lu JS, Liu X, Tan X, Wang R, et al. Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins (Basel) (2023) 15(3). doi:10.3390 / toxins15030200)" (Materials Method 5.2).

[0111] The recombinant human Siglec-15 protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0112] The experimental method is as follows:

[0113] The protein concentrations of AL-HN, BL-HN, EL-HN, FL-HN, TL-HN, THC, TL, THN, and recombinant human Siglec-15 were diluted to 2 μg / mL with carbonate coating buffer, and 100 μL / well was added to each well of a 96-well plate and incubated overnight at 4°C. The plates were washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of ELISA blocking buffer was added and incubated at 37°C for 2 h. The plates were then washed 6 times with PBST and incubated according to a 10... Add 0 μL / well of anti-tetanus antibody (TL-16-hFc fusion protein) and incubate at 37°C for 1.5 h. Wash the plate 6 times with PBST, then add 100 μL / well of goat anti-human IgG (HRP, 1:4000) diluted with blocking buffer and incubate at 37°C for 45 min. Wash the plate 6 times with PBST, then add 100 μL / well of peroxidase substrate chromogenic solution and incubate in the dark for 15 min. Observe the color development effect. After complete color development, add 100 μL of 2M sulfuric acid to each well to stop the reaction. Read the values ​​using a microplate reader. Analyze using GraphPad Prism 8 software.

[0114] The results are as follows Figure 4 As shown, TL-16-hFc exhibits good specificity, binding only to the TL-HN antigen.

[0115] 3. Detection of the affinity between the nanobody-hFc fusion protein and the TL-HN protein using biological layer interferometry (BLI).

[0116] The experimental method is as follows:

[0117] Add 200 μL of HBS-EP+ buffer to each well of the probe fixation box, and soak an appropriate amount of AHC (Anti-human IgG Fc) probe in the fixation box for 1 hour. Dilute the test antibody TL-16 to 200 nM and the TL-HN protein to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.6 nM with HBS-EP+. Then set the reaction program, add the test sample to the detection plate at 200 μL / well, place the probe fixation box and detection plate into the detection instrument, and run the detection program. Finally, import the experimental data into Data Analysis 7.0 software for processing and analysis, and calculate parameters such as affinity.

[0118] The results are as follows Figure 5 As shown, TL-16-hFc and recombinant TL-HN protein exhibit typical binding kinetics. Using Analysis Software 7.0 with a 1:1 binding model, the KD value of TL-16-hFc and recombinant TL-HN protein was calculated to be 10.1 nM, indicating good affinity between the antigen and antibody, suitable for further development.

[0119] Example 6: Evaluation of the neutralizing activity of the anti-tetanus toxin nanobody-hFc fusion protein

[0120] The neutralizing activity of the antibody was determined by injecting a mixture of antibody and tetanus toxin into KM mice in vitro. The evaluation method is as follows:

[0121] 1. Mice: KM, 4 mice per group, 18-20g, purchased from Beijing Spaford Biotechnology Co., Ltd.

[0122] 2. Composition of diluent: 0.7g KH2PO4, 2.4g Na2HPO4·12H2O, 6.8g NaCl, 2g gelatin, add water to 1L, and autoclave.

[0123] Tetanus toxin (TeNT) solution: This is a solution obtained by dissolving tetanus toxin (purchased from the China National Institutes for Food and Drug Control) in the aforementioned diluent.

[0124] TL-16-hFc fusion protein solution: This is a solution obtained by dissolving the TL-16-hFc fusion protein prepared in Example 4 in the aforementioned diluent.

[0125] Human tetanus immunoglobulin TIG solution: This is a solution obtained by dissolving human tetanus immunoglobulin (purchased from the China National Institutes for Food and Drug Control) in the aforementioned diluent.

[0126] The experimental groups are as follows:

[0127] TeNT 10×LD50 Group: Four KM mice were injected intraperitoneally with 500 μL of tetanus toxin solution, making the tetanus toxin dose 10 × LD50. 50 / only, observe for 7 days.

[0128] TeNT+5μg-TL-16-hFc group: Tetanus toxin solution was mixed with TL-16-hFc fusion protein solution and incubated at 37℃ for 30 min to obtain TeNT+TL-16-hFc solution. The TeNT+TL-16-hFc solution was injected intraperitoneally into 4 KM mice, with each mouse receiving 500μL, to achieve a tetanus toxin TeNT protein dose of 10×LD. 50 / animal, so that the dose of TL-16-hFc fusion protein is 5μg / animal.

[0129] TeNT+2.5μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 2.5μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16-hFc group.

[0130] TeNT+1.25μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 1.25μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-2-hFc group.

[0131] TeNT+0.625μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 0.625μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16-hFc group.

[0132] TeNT+0.3125μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 0.3125μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16 group.

[0133] TeNT+0.156μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 0.156μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16-hFc group.

[0134] TeNT+0.078μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16 group is that the dose of TL-16-hFc fusion protein is 0.078μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16 group.

[0135] TeNT+0.039μg-TL-16-hFc group: The difference between this group and the TeNT+10μg-TL-16-hFc group is that the dose of TL-16-hFc fusion protein is 0.039μg / animal, and the rest of the operation is the same as the TeNT+5μg-TL-16-hFc group.

[0136] The results are shown in Table 3. 0.3125 μg of TL-16-hFc can completely neutralize 10×LD. 50 Lethal dose of TeNT.

[0137] Table 3. Evaluation of neutralizing activity of TL-16-hFc

[0138]

[0139]

[0140] Example 7: Humanization and Activity Evaluation of Anti-tetanus Toxin Nanobody-hFc Fusion Protein

[0141] 1. The amino acid sequence of the antibody was analyzed on the website http: / / www.abysis.org / abysis / . Based on the Z-score, amino acid residues with a frequency less than 0.1 in the frame were replaced with human sequences to improve the degree of humanization. The spatial structure of the humanized IgG was constructed using the Swiss-model, and the accessible surface area of ​​the amino acid residue solution was analyzed to determine which amino acid residues could be humanized. Humanized amino acid mutations were performed on the amino acids in the frame region of the antibody to obtain the humanized nanobody TL-16-h1, whose amino acid sequence is SEQ ID NO:12 and nucleotide sequence is SEQ ID NO:13.

[0142] 2. Construction of a eukaryotic expression vector for humanized anti-tetanus toxin nanobody-hFc fusion protein

[0143] Based on the gene sequence of the humanized antitetracheotoxin nanobody (SEQ ID NO:13), its C-terminus was linked to the Fc segment (hFc) of human immunoglobulin G to obtain the antitetracheotoxin humanized nanobody-hFc fusion protein TL-16-h1-hFc, with the encoding gene nucleotide sequence of SEQ ID NO:14 and the expressed amino acid sequence of SEQ ID NO:15.

[0144] 3. Preparation and Characterization Evaluation of Humanized Nanobody-hFc Fusion Protein

[0145] (1) Preparation of humanized nanobody-hFc fusion protein TL-16-h1-hFc

[0146] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was prepared according to the method in Example 4, and the results are as follows. Figure 6 As shown: The molecular weight of the fusion protein is as expected, with a band size of approximately 40 kDa under reducing conditions (left) and approximately 80 kDa under non-reducing conditions (right).

[0147] (2) The binding activity and specificity of humanized nanobody-hFc fusion protein TL-16-h1-hFc to recombinant TL-HN protein were detected.

[0148] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was used to replace the TL-16-hFc fusion protein. The remaining operations were the same as in Example 5: "1. Detect the binding activity of the nanobody-hFc fusion protein to the recombinant TL-HN protein by ELISA" and "2. Identify the specificity of the nanobody-hFc fusion protein by ELISA".

[0149] The results are as follows Figure 7 , Figure 8 As shown: the half-maximal effective concentration (EC50) of TL-16-h1-hFc binding to recombinant TL-HN protein. 50 The concentration was 0.01595 nM, and it only bound to the TL-HN antigen, showing good specificity.

[0150] (3) The affinity between humanized nanobody-hFc fusion protein TL-16-h1-hFc and recombinant TL-HN protein was detected.

[0151] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was used to replace the test antibody TL-16, and the remaining operations were the same as in Example 5, "3. Detection of the affinity between the nanobody-hFc fusion protein and the TL-HN protein using biological layer interferometry (BLI)".

[0152] The results are as follows Figure 9 As shown, the KD value of TL-16-h1-hFc and recombinant TL-HN protein reached 0.09096 nM, which is about 10 times higher than before humanization, indicating good affinity between antigen and antibody.

[0153] 4. Evaluation of the neutralizing activity of humanized nanobody fusion protein

[0154] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was used instead of the TL-16-hFc fusion protein, and the remaining operations were the same as in Example 6, "Evaluation of the neutralizing activity of the anti-tetanus toxin nanobody-hFc fusion protein".

[0155] The results are shown in Table 4. 0.3125 μg of TL-16-h1-hFc can effectively block lethal doses of tetanus toxin (10×LD50). 50 Poisoning caused by )

[0156] Table 4. Evaluation of neutralizing activity of TL-16-h1-hFc

[0157]

[0158] Example 8: Evaluation of the preventive and therapeutic effects of humanized anti-tetanus nanobody-hFc fusion protein

[0159] I. To evaluate whether the humanized anti-tetanus nanobody-hFc fusion protein has a protective effect against TeNT attack, and whether this protective effect shows a dose-dependent effect in mouse models, the evaluation method is as follows:

[0160] 1. Mice: KM mice (purchased from Beijing Spaford Biotechnology Co., Ltd.), 18-20g per mouse. The preparation methods for the tetanus toxin solution, the nanobody TL-16-h1-hFc fusion protein solution, and the TL-16-h1-hFc fusion protein solution are as described in Example 6. The irrelevant antibody (B-h3) solution was prepared by dissolving the irrelevant antibody (B-h3) in a diluent.

[0161] 2. The experimental groups are as follows:

[0162] (1) 25 μg / kg-TL-16-h1-hFc+TeNT group: KM mice were treated with 125 μL of TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) via the tail vein. 500 μL of tetanus toxin solution was injected intraperitoneally into KM mice at 12 h, 24 h, 48 h, 3 d, 5 d, 7 d, 9 d, 12 d, and 14 d after injection. The injection dose was 10 × LD50. 50 / mouse (4 mice were injected at each time point), and the mortality of the mice was observed.

[0163] (2) 125μg / kg-TL-16-h1-hFc+TeNT group: 125μL of TL-16-h1-hFc fusion protein solution (concentration of 20μg / mL, i.e., injection dose of 125μg / kg) was used to replace 125μL of TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e., injection dose of 25μg / kg) in mice via tail vein treatment. The remaining procedures were the same as those in the 25μg / kg-TL-16-h1-hFc+TeNT group.

[0164] (3) 0.1 IU-TIG+TeNT group: 125 μL of human tetanus immunoglobulin TIG solution (concentration of 0.8 IU / mL, i.e. injection dose of 0.1 IU / animal) was used instead of 125 μL of TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg). The rest of the operation was the same as the 25 μg / kg-TL-16-h1-hFc+TeNT group.

[0165] (4) 250μg / kg-B-h3+TeNT group: replace 125μL of TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e. injection dose of 250μg / kg) with 125μL of irrelevant antibody (B-h3) solution (concentration of 40μg / mL, i.e. injection dose of 25μg / kg), and the rest of the operation is the same as 25μg / kg-TL-16-h1-hFc+TeNT group.

[0166] (5) PBS group: Replace the TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg) with an equal volume of PBS. The rest of the operation is the same as the 25 μg / kg-TL-16-h1-hFc+TeNT group.

[0167] The results are shown in Table 5. High-dose antibody (125 μg / kg) showed efficacy against 10×LD50 within 12 days. 50 TeNT challenge provides comprehensive protection in mice; low-dose antibody (25 μg / kg) provides protection against 10×LD50 within 3 days. 50 TeNT attacks can provide half protection.

[0168] Table 5. Evaluation of the preventive effect of anti-tetanus toxin antibodies

[0169]

[0170]

[0171] “ a "This means that mice were treated with TL-16-h1-hFc, TIG, B-h3 or PBS and exposed to TeNT for a specified period of time."

[0172] “ b "This indicates that the mice were treated with 125 μg / kg or 25 μg / kg of antibody, 0.1 IU TIG, 250 μg / kg of B-h3, or PBS, respectively."

[0173] “ c "This indicates that mice were challenged with TeNT at 12h, 24h, 48h, 3d, 5d, 7d, 9d, 12d, and 14d after being treated with a specified dose of antibody.

[0174] “ d "This indicates that KM mice were injected intraperitoneally with 10×LD10 ... 50 TeNT.

[0175] II. Evaluation of whether anti-tetanus nanobodies have a protective effect after exposure to TeNT.

[0176] The evaluation method is as follows:

[0177] 1. Mice: KM, 4 mice per group, 18-20g.

[0178] 2. The challenge dose was 5 × LD50. 50 Each group of mice was injected with 25 or 125 μg / kg TL-16-h1-hFc fusion protein solution, 1 IU TIG, 250 μg / kg B-h3, or PBS via tail vein 1, 3, 6, 12, or 24 hours after injection of tetanus toxin solution. Mice mortality was observed. The experimental groups are as follows:

[0179] (1)TeNT+25μg / kg-TL-16-h1-hFc fusion protein group: KM mice were intraperitoneally injected with tetanus toxin solution, and the challenge dose was 5×LD. 50 Four mice were injected with 125 μL of TL-16-h1-hFc nanobody fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg) via tail vein at 1, 3, 6, 12 or 24 hours after injection of tetanus toxin solution, and the mortality of the mice was observed.

[0180] (2) TeNT+125μg / kg-TL-16-h1-hFc fusion protein group: 125μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 20μg / mL, i.e., injection dose of 125μg / kg) was used instead of 125μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e., injection dose of 25μg / kg). The rest of the operation was the same as that of the TeNT+25μg / kg-TL-16-h1-hFc fusion protein group. The mortality of mice was observed.

[0181] (3) TeNT+1IU / mouse-TIG group: 125μL of human tetanus immunoglobulin TIG solution (concentration of 8IU / mL, i.e., injection dose of 1IU / mouse) was used to replace 125μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e., injection dose of 25μg / kg). The rest of the operation was the same as the TeNT+25μg / kg-TL-16-h1-hFc fusion protein group. The mortality of mice was observed.

[0182] (4) TeNT+250μg / kg-B-h3 group: 125μL of unrelated antibody (B-h3) solution (concentration of 40μg / mL, i.e. injection dose of 250μg / kg) was used instead of 125μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e. injection dose of 25μg / kg). The rest of the operation was the same as that of the TeNT+25μg / kg-TL-16-h1-hFc fusion protein group. The mortality of mice was observed.

[0183] (5) TeNT+PBS group: Replace 125μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 4μg / mL, i.e. injection dose of 25μg / kg) with an equal volume of PBS. The rest of the operation is the same as the TeNT+25μg / kg-TL-16-h1-hFc fusion protein group. The mortality of mice was observed.

[0184] The results are shown in Table 6. For 5×LD 50 TeNT poisoning was effectively treated with only 0.5 μg / mouse (dose = 1 mg / kg antibody × 20 g / mouse) of TL-16-h1-hFc fusion protein within 24 hours of exposure.

[0185] Table 6. Evaluation of the therapeutic effect of anti-tetanus toxin antibodies

[0186]

[0187]

[0188] “ a "This indicates that KM mice were injected with 5×LD50TeNT."

[0189] “ b "This means that mice were treated with a specified dose of antibody at 1h, 3h, 6h, 12h or 24h after exposure to TeNT."

[0190] “ c"This indicates that mice exposed to TeNT were treated with TL-16-h1-hFc, TIG, B-h3, and PBS at specified times after exposure."

[0191] “ d "This indicates that mice exposed to TeNT were treated with 25 μg / kg or 125 μg / kg TL-16-h1-hFc, 1 IU TIG, 250 μg / kg B-h3 or PBS at specific times after exposure."

[0192] The sequences involved in the above embodiments are:

[0193] SEQ ID NO:1:

[0194] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNAK NELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS.

[0195] SEQ ID NO:2: EVQLVESGGGLVQPGGSLRLSCAAS.

[0196] SEQ ID NO: 3: MSWVRQAPGKGLEWVSA.

[0197] SEQ ID NO:4:YYKGTVKGRFTISRDNAKNELYLQMNSLQTEDTAVYYC.

[0198] SEQ ID NO:5: WGQGTLVTVSS.

[0199] SEQ ID NO:6:GFTFNNYD.

[0200] SEQ ID NO:7:LDADGLKT.

[0201] SEQ ID NO:8: ATGFYPQYLLQHAPDVARTEDDA.

[0202] SEQ ID NO:9:

[0203] 5'-gaggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgcgcggcct ctggattcacattcaacaactacgacatgagctgggtccgccaggctccagggaaggggctcgagtgggtctcagctctagacgccgatggtctgaagacatactataaaggcaccgtgaagggccgattcaccatctccagggacaacgccaagaatgagttgtatctgcaaatgaacagcctgcaaactgaggacactgccgtgtattactgcgccacgggtttctatccacagtacttactccagcacgcaccagacgtcgcgcgcaccgaagacgacgcctggggccaggggaccctggtcaccgtctcctca-3'。

[0204] SEQ ID NO:10:

[0205] 5'-gaggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgcgcggcct ctggattcacattcaacaactacgacatgagctgggtccgccaggctccagggaaggggctcgagtgggtctcagctctagacgccgatggtctgaagacatactataaaggcaccgtgaagggccgattcaccatctccagggacaacgccaagaatgagttgtatctgcaaatgaacagcctgcaaactgaggacactgccgtgtattactgcgccacgggtttctatccacagtacttactccagcacgcaccagacgtcgcgcgcaccgaagacgacgcctggggccaggggaccctggtcaccgtctcctcagctagcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgctgcatgaggctctgcacaGccactacacgcagaagagcctctccctgtccccgggtaaatga-3'。

[0206] SEQ ID NO:11:

[0207] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNAKNELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK。

[0208] SEQ ID NO:12

[0209] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS。

[0210] SEQ ID NO:13

[0211] 5'-gaagtgcagctggtggagtctggaggaggactggtgcagcctggaggctctctgagactgtcttgtgccgcct ctggctttacctttaataactacgacatgagctgggtgcggcaggcccccggcaagggcctggaatgggtgagcgccctggacgccgacggcctgaaaacctactacaagggcaccgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgccaccggcttctacccccagtacttactgcagcacgcccccgacgtggcccggaccgaggacgacgcctggggccagggcaccctggtgaccgtgagcagc-3'。

[0212] SEQ ID NO:14

[0213] 5'-Gaagtgcagctggtggagtctggaggaggactggtgcagcctggaggctctctgagactgtcttgtgccgcctctggctttacctttaataactacgacatgagctgggtgcggcaggcccccggcaagggcctggaatgggtgagcgccctggacgccgacggcctgaaaacctactacaagggcaccgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgccaccggcttctacccccagtacttactgcagcacgcccccgacgtggcccggaccgaggacgacgcctggggccagggcaccctggtgaccgtgagcagcgctagcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgctgcatgaggctctgcacagccactacacgcagaagagcctctccctgtccccgggtaaatga-3'.

[0214] SEQ ID NO:15

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0216] 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 thereof against tetanus toxin, characterized in that: The nanobody or its antigen-binding fragment comprises three complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is SEQ ID NO:6, the amino acid sequence of the CDR2 is SEQ ID NO:7, and the amino acid sequence of the CDR3 is SEQ ID NO:

8.

2. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The Nanobody comprises, in addition to the complementarity determining regions, four framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 is SEQ ID NO: 2; The amino acid sequence of FR2 is SEQ ID NO: 3; The amino acid sequence of FR3 is SEQ ID NO:4 or has more than 75% identity with SEQ ID NO:4; The amino acid sequence of FR4 is SEQ ID NO:

5.

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

1.

4. The nanobody or antigen-binding fragment thereof according to claim 2, characterized in that The amino acid sequence of the Nanobody or its antigen-binding fragment is SEQ ID NO:

12.

5. An anti-tetanus toxin heavy chain antibody, characterized in that The heavy chain antibody comprises the Nanobody described in any one of claims 1-4.

6. The heavy chain antibody according to claim 5, characterized in that The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:1 or has a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID NO:

1.

7. The heavy chain antibody according to claim 5 or 6, characterized in that The amino acid sequence of the heavy chain antibody includes SEQ ID NO:11 or SEQ ID NO:

15.

8. Biomaterial, characterized in that The biological material is any of the following: B1) a nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or a heavy chain antibody as described in any one of claims 4 to 6; 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) A recombinant microorganism containing the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3).

9. An anti-tetanus drug, characterized in that The drug comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7.

10. Application, characterized in that, The application is any of the following: M1) Use of the biomaterial of claim 8 in the preparation of a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or a heavy chain antibody as described in any one of claims 5 to 7; M2) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7 in the preparation of the drug according to claim 9; M3) Use of a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a heavy chain antibody according to any one of claims 5 to 7 or a biomaterial according to claim 8 or a drug according to claim 9 in the preparation of a product for preventing and / or treating a disease caused by Clostridium tetani; M4) Use of a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or a heavy chain antibody as described in any one of claims 5 to 7 or a genetic material as described in claim 8 in the preparation of a product for detecting Clostridium tetani; M5) Use of a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or a heavy chain antibody as described in any one of claims 5 to 7 or a biomaterial as described in claim 8 or a drug as described in claim 9 in the preparation of a product for neutralizing a neurotoxin secreted by Clostridium tetani; M6) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7 or the biomaterial according to claim 8 or the drug according to claim 9 in the preparation of a product for detecting neurotoxins secreted by Clostridium tetani.

Images