Anti-tetanus toxin bispecific neutralizing antibody and related biological material and application thereof

Abstract

The invention discloses an anti-tetanus toxin bispecific neutralizing antibody as well as a related biological material and application thereof, and belongs to the field of immunotherapy biotechnology pharmacy. The bispecific antibody or the antigen binding fragment of the bispecific antibody comprises two humanized nano antibodies, namely T92-6-h2 and TL-25-h1, and amino acid sequences of three complementary determining regions CDR1, CDR2 and CDR3 of the T92-6-h2 are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 in sequence; the amino acid sequences of three complementary determining regions CDR1, CDR2 and CDR3 of the TL-25-h1 are SEQ ID NO: 14, ID NO: 15 and SEQ ID NO: 16 in sequence; the sequence of the complementarity determining region is defined according to an IMGT numbering system. The antibody can be combined with an L-HN functional fragment and an Hc structural domain at the same time, and can effectively block poisoning caused by lethal dose tetanus toxin.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy biomedicine technology, and specifically relates to anti-tetanus toxin bispecific neutralizing antibodies and related biomaterials and applications. Background Art

[0002] Tetanus is an acute infectious disease characterized by skeletal muscle stiffness and spasms. It is particularly common in newborns and pregnant women who have not received a vaccine containing tetanus toxoid. Non-neonatal tetanus can be contracted through trauma, hospital-acquired infections, or even open wounds caused by flea infestations. Injuries from natural disasters such as earthquakes and tsunamis can also lead to tetanus.

[0003] Humans have no natural immunity to tetanus and require active or passive immunization. Active immunization involves administering a vaccine containing tetanus toxoid to induce antibody production. However, the vaccine takes time to produce neutralizing antibodies, and even immediate administration after exposure may not provide protection. Passive immunization involves injecting tetanus antitoxin or immunoglobulin to induce antibody production, providing effective and immediate intervention. However, current passive immunization agents used in clinical practice have limitations such as high cost and potential for hypersensitivity reactions. Therefore, a suitable and safe alternative that can be expressed in large quantities in vitro is urgently needed.

[0004] In 1993, heavy chain antibodies that naturally lack light chains were first discovered in camels. Their antibodies are composed of only two heavy chains, and their antigen recognition function is mainly determined by the variable region (VHH) of the heavy chain antibody. VHH alone can recognize antigens. It has a molecular weight of only 13-15KDa, a diameter of approximately 2.5nm, and a length of 4nm, so it is also called a nanobody. Due to its small size, simple structure, high antigen binding affinity, and excellent stability under extreme conditions, nanobodies have the potential to overcome the limitations of traditional monoclonal antibodies. Over the years, nanobodies have attracted great interest in various research fields, especially in the diagnosis and treatment of diseases.

[0005] Building on research in nanobodies, the construction of bispecific antibodies targeting different epitopes of the same antigen can reduce off-target effects, lower drug dosages, or improve efficacy. Our bispecific antibody, which simultaneously targets both the Hc and L-HN domains of tetanus toxin, exhibits high binding affinity and neutralizing activity, providing complete protection against tetanus toxin-challenged mice. It has the potential to be developed as a preventive or therapeutic drug for tetanus toxin poisoning. Summary of the Invention

[0006] The primary problem to be solved by the present invention is to develop a bispecific antibody against tetanus toxin that can simultaneously target both the L-HN domain and the Hc domain, effectively neutralizing tetanus toxin. This antibody has the potential to be developed into a preventive or therapeutic drug for tetanus toxin poisoning. Other technical topics not described herein will be readily understood by those skilled in the art through the following description.

[0007] In order to solve the above problems, the present invention provides a bispecific antibody targeting the L-HN domain and the Hc domain of tetanus toxin or an antigen-binding fragment containing the antibody.

[0008] In the first aspect, the present invention provides an anti-tetanus toxin bispecific antibody or an antigen-binding fragment thereof T92-6-h2, wherein the bispecific antibody comprises a first nanobody variable region and a second nanobody variable region, wherein the first nanobody variable region and the second nanobody variable region are connected by a connecting peptide, and the amino acid sequences of CDR1, CDR2 and CDR3 in the first nanobody variable region are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the second nanobody variable region are SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0009] In the above-mentioned bispecific antibody or antigen-binding fragment thereof, the first nanobody variable region further comprises four framework regions FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the group consisting of: A1-1) amino acid sequence is SEQ ID NO: 2; A1-2) has a sequence identity of more than 75% with A1-1); The amino acid sequence comprises an amino acid sequence selected from the group consisting of: A2-1) the amino acid sequence of FR2 is SEQ ID NO: 3; A2-2) has a sequence identity of more than 75% with A2-1); The FR3 comprises an amino acid sequence selected from the group consisting of: A3-1) amino acid sequence is SEQ ID NO: 4; A3-2) Sequences with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the group consisting of: A4-1) amino acid sequence is SEQ ID NO: 5; A4-2) Sequences with more than 75% identity with A4-1); The second Nanobody variable region also comprises four framework regions FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the group consisting of: A5-1) amino acid sequence is SEQ ID NO: 10; A5-2) Sequences with more than 75% identity with A5-1); The FR2 comprises an amino acid sequence selected from the group consisting of: A6-1) amino acid sequence is SEQ ID NO: 11; A6-2) has a sequence identity of more than 75% with A6-1); The FR3 comprises an amino acid sequence selected from the group consisting of: A7-1) amino acid sequence is SEQ ID NO: 12 or; A7-2) has a sequence identity of more than 75% with A7-1); The FR4 comprises an amino acid sequence selected from the group consisting of: A8-1) amino acid sequence is SEQ ID NO: 13; A8-2) has a sequence identity of more than 75% with A8-1).

[0010] In the above-mentioned bispecific antibody or antigen-binding fragment thereof, the amino acid sequence of the connecting peptide includes amino acids 354 to 358 in SEQ ID NO:18.

[0011] In the above-mentioned bispecific antibody or antigen-binding fragment thereof, the amino acid sequence of the connecting peptide is amino acids 354 to 358 in SEQ ID NO: 18.

[0012] In the above-mentioned humanized Nanobody, the amino acid sequence of the variable region of the first Nanobody is SEQ ID NO: 1 or has an identity 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.

[0013] In the above-mentioned humanized Nanobody, the amino acid sequence of the variable region of the first Nanobody is SEQ ID NO: 9 or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID NO: 9.

[0014] The amino acid sequence of T92-6-h2 in the above-mentioned humanized Nanobody is SEQ ID NO: 18 or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID NO: 18.

[0015] The above term "antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target.

[0016] In a second aspect, the present invention provides a bispecific antibody (heavy chain antibody) against tetanus toxin, wherein the bispecific antibody comprises the aforementioned humanized Nanobody.

[0017] In the anti-tetanus toxin bispecific antibody (heavy chain antibody), its constant region is amino acids 125 to 353 of SEQ I DNO: 18.

[0018] In the anti-tetanus toxin bispecific antibody (heavy chain antibody), the amino acid sequence of the heavy chain antibody includes SEQ ID NO: 18 or has an identity of greater than 99%, greater than 95%, greater than 90%, greater than 85%, greater than 80% or greater than 75% with SEQ ID NO: 18.

[0019] In the anti-tetanus toxin bispecific antibody (heavy chain antibody), the amino acid sequence of the heavy chain antibody is SEQ ID NO: 18 or has an identity of greater than 99%, greater than 95%, greater than 90%, greater than 85%, greater than 80% or greater than 75% with SEQ ID NO: 18.

[0020] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial is any one of the following: B1) a nucleic acid molecule encoding the aforementioned bispecific antibody or antigen-binding fragment thereof, or the aforementioned heavy chain antibody; 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).

[0021] The small molecule antibody may be any of the following: F1, Fab antibody; F2, Fv antibody; F3, single-chain antibody; F4, Fab′ fragment.

[0022] The term "Fab' fragment" refers to a portion of an antibody heavy chain comprising an antibody light chain and the VH domain and the CH1 domain as well as the region between the CH1 and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab') 2 molecular.

[0023] The term "F(ab') 2 A "fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, F(ab') 2 The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0024] The term "nanobody (single-domain antibody)" (VHH) refers to a polypeptide consisting of the variable region of an antibody heavy chain. Single-domain antibodies can be prepared by genetically engineering the variable region of an antibody heavy chain (VH) to produce an antibody containing only the VH fragment. The antigen-binding ability and stability of single-domain antibodies are essentially the same as those of full-length antibodies.

[0025] The term "minimum recognition unit (MRU)" refers to a single CDR structure in the variable region, with a molecular mass of only about 1% of the complete antibody, which can bind to the corresponding antigen.

[0026] The term "Fab antibody" refers to a heterodimer formed by the heavy chain (Fd) and an intact light chain bound by disulfide bonds, containing only a single antigen-binding site. Fab antibodies can be prepared by ligating the genes encoding the heavy chain (Fd) and the complete light chain and fusing them with a bacterial protein signal peptide gene. This allows for secretory expression of the Fab antibody (Fab fragment) in Escherichia coli, with a complete three-dimensional fold and intra- and inter-chain disulfide bonds. The heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (comprising approximately 225 amino acid residues, including the VH, CH1, and part of the hinge region).

[0027] The term "Fv antibody" refers to a compound composed solely of the variable regions of the heavy and light chains of an antibody. The heavy and light chain variable regions are linked by non-covalent bonds. Fv antibodies can be prepared by constructing separate vectors containing the VH and VL genes, co-transfecting cells for expression, and then assembling into a functional Fv antibody. Alternatively, a stop codon can be inserted between the VH and VL genes in the vector to express the two small protein fragments separately, which are then non-covalently bound to form an Fv antibody (Fv fragment).

[0028] The term "single-chain antibody" (ScFv) refers to a polypeptide formed by linking the heavy and light chain variable regions of an antibody using a short peptide. ScFv can be prepared by connecting the light and heavy chain variable region genes with an oligonucleotide linker to express 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.

[0029] In a fourth aspect, the present invention provides genetic material, wherein the genetic material is any one of the following: g1) a nucleic acid molecule encoding the aforementioned Nanobody or antigen-binding fragment thereof, or the aforementioned heavy chain antibody; g2) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in g1).

[0030] In a fifth aspect, the present invention claims protection for an anti-tetanus drug, which comprises the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody.

[0031] The medicament further includes a physiologically or pharmaceutically acceptable excipient, diluent or carrier.

[0032] Herein, the above-mentioned "physiologically or pharmaceutically acceptable carriers or diluents" refer to those carriers and diluents that have no significant irritation to organisms and will not impair the biological activity and performance of the agent in the pharmaceutical composition.

[0033] As used herein, a "physiologically or pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the agent. Carrier materials herein include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, and organic acids), poorly soluble carrier materials (such as ethyl cellulose and cholesterol stearate), and enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose). Water-soluble carrier materials are preferred.

[0034] In a sixth aspect, the present invention claims protection for any of the following applications: M1) Use of the aforementioned genetic material in the preparation of the aforementioned bispecific antibody or antigen-binding fragment thereof or the aforementioned Nanobody; M2) Use of the aforementioned bispecific antibody or antigen-binding fragment thereof or the aforementioned Nanobody in the preparation of the aforementioned medicament; M3) Use of the aforementioned bispecific antibody or antigen-binding fragment thereof, or the aforementioned Nanobody, or the aforementioned genetic material, or the aforementioned drug in the preparation of a product for preventing and / or treating diseases caused by Clostridium tetani; M4) Use of the aforementioned bispecific antibody or antigen-binding fragment thereof, or the aforementioned Nanobody, or the aforementioned genetic material in the preparation of a product for detecting Clostridium tetani; M5) Use of the aforementioned bispecific antibody or antigen-binding fragment thereof, or the aforementioned nanobody, or the aforementioned genetic material, or the aforementioned drug in the preparation of a product for neutralizing neurotoxins secreted by Clostridium tetani; M6) Use of the aforementioned bispecific antibody or antigen-binding fragment thereof, or the aforementioned nanobody, or the aforementioned genetic material or drug in the preparation of a product for detecting neurotoxins secreted by Clostridium tetani.

[0035] In a seventh aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is the antibody or the antigen-binding fragment thereof, and the method comprises the step of expressing the encoding gene of the antibody or the antigen-binding fragment thereof in a mammalian cell to obtain the antibody or the antigen-binding fragment thereof.

[0036] In the above method, the mammalian cells may be isolated animal cells, such as cells in culture medium. The mammalian cells may be selected from ExpiCHO-S™ cells, Chinese hamster ovary cells (CHO), 293F cells, 293E cells, 293-6E cells, and the like.

[0037] In the above method, the mammalian cell can be FreeStyle TM HEK293-F.

[0038] In the above method, expressing the gene encoding the antibody or antigen-binding fragment thereof in mammalian cells comprises infecting, transfecting or transforming mammalian cells with the gene encoding the gene to obtain recombinant mammalian cells containing the gene encoding the gene, culturing the recombinant mammalian cells to obtain a cell culture, and isolating and purifying the antibody or antigen-binding fragment thereof from the cell culture. The antibody or antigen-binding fragment thereof can be produced from large-scale cell culture using methods known in the art. For example, Li et al., Cell culture processes for monoclonal antibody production. Mabs. 2010 September-October; 2(5): 466-477. In certain embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, NSO mouse myeloma cells, HEK293 (human embryonic kidney 293) cells, or PER.C6® cells.

[0039] The bispecific neutralizing antibody against tetanus toxin provided by the present invention can effectively neutralize tetanus toxin. Experiments have shown that the bispecific antibody T6-hFc-25 has good specificity and only binds to TL-HN recombinant protein and THc recombinant protein. 0.0488 μg of T6-hFc-25 can effectively block the lethal dose of tetanus toxin (10×LD 50 ) caused poisoning; low doses of T6-hFc-25 (25 μg / kg) had no effect on 10 × LD within 14 days. 50 TeNT challenge provided complete protection, and treatment with 0.5 μg / mouse T6-hFc-25 fusion protein remained effective 12 hours after exposure to 5 × LD50 TeNT intoxication. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of anti-tetanus toxin bispecific antibody.

[0041] Figure 2 This is the SDS-PAGE electrophoresis detection of the purified anti-tetanus bispecific antibody.

[0042] Figure 3 To detect the binding activity of anti-tetanus toxin bispecific antibodies.

[0043] Figure 4 Specificity test for anti-tetanus toxin bispecific antibodies.

[0044] Figure 5 Affinity testing of anti-tetanus toxin bispecific antibodies.

[0045] Figure 6To evaluate the neutralizing activity of anti-tetanus toxin bispecific antibodies. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0047] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0048] Recombinant TL-HN and THc protein antigens were prepared in the inventors' laboratory. The preparation methods are described in the non-patent 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 tetanus toxin. VACCINE (2023). doi:10.1016 / j.vaccine.2023.09.032," section 2.2 of Materials and Methods.

[0049] pTSE-hFc vector: recorded in the non-patent literature "Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against the protective antigen V of plague bacteria [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271", the public can obtain it from the Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0050] Construction of an anti-tetanus nanobody library and screening of a tetanus-specific phage nanobody library: Prepared in the inventors' laboratory. The preparation method is described in the Materials and Methods section of the non-patent literature, "Cheng KX, Lu JS, Guo JZ, et al. Characterization of neutralizing chimeric heavy-chain antibodies against tetanus toxin. Human Vaccines & Immunotherapeutics (2024). , doi: 10.1080 / 21645515.2024.2366641."

[0051] Example 1. Preparation of anti-tetanus toxin bispecific antibodies 1. Identify specific nanobody positive clones using phage enzyme-linked immunosorbent assay (Phage-ELISA) ELISA plates were coated with recombinant proteins TL-HN and THc, respectively, and an irrelevant antigen was coated in adjacent columns as a negative control, and the plates were coated overnight at 4°C. The plates were removed from the overnight coating, the coating solution was discarded, and the plates were washed six times with PBST. Blocking solution (3 g of skim milk powder in 100 mL of PBS) was added at 200 μL / well and incubated at 37°C for 2 h. The blocking solution was discarded, and the supernatant of the induced expressed phage was added to the corresponding ELISA wells at 100 μL / well and incubated at 37°C for 1.5 h. The plates were washed six times with PBST and freshly prepared 0.2 μg / ml HRP-labeled anti-M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) was added to the plates at 100 μL / well and incubated at 37°C for 45 min. The plates were washed six times with PBST and the color development solution (9 mL of color development solution, 1 mL of 10× OPD, 10 10 µL / well of 30% H2O2 was added to the ELISA plate and color was developed for 15-20 min in the dark. 2 M sulfuric acid stop solution (50 µL / well) was added and the plate was read at dual wavelengths of 492 / 630 nm. A clone well with an absorbance >3 times that of the antigen group / negative control group was identified as a positive clone.

[0052] The positive clones were sent to a biotechnology service company for sequence determination to obtain the DNA sequence of the inserted fragment, and finally the nanobody T92-6 that can specifically bind to THc and the nanobody TL-25 that specifically binds to TL-HN were obtained.

[0053] 2. Humanized transformation of anti-tetanus toxin nanoantibodies The antibody amino acid sequences were analyzed on the website http: / / www.abysis.org / abysis / . The spatial structure of the humanized IgG was constructed using the Swiss-model. The accessible surface area of the amino acid residues in solution was analyzed to identify amino acid residues suitable for humanization. Based on the Z-score, amino acid residues with a frequency less than 0.1 in the framework were replaced with amino acid residues from the human antibody to improve the degree of humanization. Humanized amino acid mutations were performed on the framework regions of the antibodies to generate the humanized Nanobody T92-6-h2, which specifically binds to THc, and the humanized Nanobody TL-25-h1, which specifically binds to TL-HN.

[0054] The amino acid sequence of T92-6-h2 is shown in SEQ ID NO: 1, including the framework regions (FRs: FR1, FR2, FR3, FR4) and the complementarity determining regions (CDRs: CDR1, CDR2, CDR3). The four portions of the framework regions are designated, respectively, as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; the three portions of the complementarity determining regions are designated, respectively, as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0055] The amino acid sequence of TL-25-h1 is shown in SEQ ID NO:9, including framework regions (FR: FR1, FR2, FR3, FR4) and complementarity determining regions (CDR: CDR1, CDR2, CDR3). The four portions of the framework regions are designated as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; the three portions of the complementarity determining regions are designated as SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0056] 3. Construction of eukaryotic expression plasmid for anti-tetanus toxin bispecific antibody The carboxyl terminus of the humanized nanobody T92-6-h2 (amino acid sequence of SEQ ID NO. 1) that specifically binds to the THc domain of tetanus toxin was connected to the amino terminus of the Fc segment (hFc) of human immunoglobulin. At the same time, the amino terminus of the humanized nanobody TL-25-h1 (amino acid sequence of SEQ ID NO: 9) that specifically binds to the TL-HN domain of tetanus toxin was connected to the carboxyl terminus of hFc through a linker sequence (GGGGS) to obtain the coding gene of the anti-tetanus toxin bispecific antibody T6-hFc-25. The nucleotide sequence of this bispecific antibody is SEQ ID NO: 17 and the amino acid sequence is SEQ ID NO: 18.

[0057] Conventional molecular biology techniques were used to replace 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 connecting the gene of the Fc domain of human immunoglobulin to the pCMV vector) with a nucleotide sequence having an amino acid sequence of SEQ ID NO: 1 (encoding gene of humanized Nanobody T92-6-h2); the coding sequence of the linker sequence (GGGGS), the nucleotide sequence of the amino acid sequence of SEQ ID NO: 9 (encoding gene of humanized Nanobody TL-25-h1) and a stop codon were added after the gene of the hFc domain; the other nucleotides of the pTSE-hFc vector were kept unchanged to obtain the bispecific antibody recombinant expression plasmid pTSE-T6-hFc-25 containing the coding genes of humanized Nanobody T92-6-h2 and TL-25-h1.

[0058] The recombinant expression plasmid pTSE-T6-hFc-25 is obtained by replacing the small fragment between the Sal I and Nhe I restriction sites of the pTSE-hFc expression vector with the DNA fragment represented by nucleotides 1-372 of SEQ ID NO:17 (encoding SEQ ID NO:1). Furthermore, the DNA fragment represented by nucleotides 1060-1467 of SEQ ID NO:17 (nucleotides 1060-1074 encode the linker peptide GGGGS, nucleotides 1075-1464 encode SEQ ID NO:9, and nucleotides 1465-1467 serve as the stop codon) is inserted at the terminus of the hFc fragment of the pTSE-hFc expression vector, while leaving all other nucleotides unchanged. The recombinant expression plasmid pTSE-T6-hFc-25 expresses the protein with the amino acid sequence of SEQ ID NO:18.

[0059] 4. Expression and purification of anti-tetanus toxin bispecific antibodies The bispecific antibody recombinant expression plasmid pTSE-T6-hFc-25 constructed in step 3 was transfected into the FreeStyle TMHEK293-F cells (Invitrogen, R79007) were cultured and cell viability was monitored after 48 hours. When cell viability dropped to 80-85%, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes. The supernatant was purified using the AKTA purification system and the prepacked HiTrap MabSelectSuRe antibody purification column (Situofan, 11-0034-93) to obtain the bispecific antibody T6-hFc-25. The purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 2 Shown: The molecular weight of the bispecific antibody is consistent with the expected size, with a band size of approximately 55 kDa under reducing conditions (left) and a band size of approximately 110 kDa under non-reducing conditions (right).

[0060] Example 2. Evaluation of properties of anti-tetanus toxin bispecific antibodies 1. ELISA detection of the binding activity of bispecific antibodies with recombinant TL-HN protein or recombinant THc protein Recombinant TL-HN protein or recombinant THc protein was diluted to 2 μg / mL in carbonate coating buffer (pH 9.6) and added to the ELISA plate at 100 μL / well for overnight coating at 4°C. The next day, the plate was washed six times with PBST (0.1% Tween-20) and blocked at 37°C for 2 h at 200 μL / well of blocking buffer (3% skim milk powder). The blocking buffer was discarded and the plate was washed six times with PBST. Twenty-three gradients of bispecific antibody T6-hFc-25 (starting concentration 100 μg / mL, diluted in blocking buffer) were added at 100 μL / well. Each gradient was replicated with two wells and incubated at 37°C for 1.5 h. The plate was then washed six times with PBST and goat anti-human IgG (HRP, 1:4000) diluted in blocking buffer was added at 100 μL / well. The plate was incubated at 37°C for 45 min. The plate was washed six times with PBST and 100 μL / well of OPD substrate colorimetric solution was added and color was developed in the dark for 5-15 minutes. The color development effect was observed. After color development was complete, 100 μL of 2M sulfuric acid was added to each well to terminate the enzyme-linked reaction. The optical density was measured using a microplate reader at 492 nm / 630 nm. The concentration required for the antibody to bind to 50% of the antigen protein (EC 50 ), and evaluate the binding ability between them.

[0061] The results are as follows Figure 3 (Each point in the figure is plotted based on a specific absorbance value, and the horizontal axis is Log10.) In the figure, the horizontal axis is the logarithm of the protein molar concentration, and the vertical axis is the optical density value. The analysis shows that the half-effective concentration (EC50) of the binding of the bispecific antibody T6-hFc-25 to the recombinant TL-HN protein 50) was 1.424 nM, and the half effective concentration (EC 50 ) is 0.04592 nM.

[0062] 2. ELISA detection of the specificity of the bispecific antibody T6-hFc-25 The TL-HN, THC, TL, and THN proteins of tetanus are derived from (the TL-HN, THC, TL, and THN antigens of tetanus were prepared in the inventor's laboratory. The preparation method has been described in the non-patent literature "Liu XY, Wei DK, Li ZY, Lu JS, XieXM, 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"). The L-HN antigens of the four botulinum toxin serotypes A, B, E, and F are derived from (the L-HN antigens of the four botulinum toxin serotypes AL-HN, BL-HN, EL-HN, and FL-HN were prepared in this laboratory).Liu FJ, Shi DY, Mao YY,Xiong XH, Lu JS, Pang XB, et al. Immunological characterization and immunoprotective efficacy of functional domain antigens of botulinumneurotoxin serotype A. VACCINE (2020) 38(14):2978-2983. doi:10.1016 / j.vaccine.2020.02.060; 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 ofBotulinum Neurotoxin B Stimulates a Protective Antibody Response AgainstActive Neurotoxin. NEUROTOX RES (2021). doi:10.1007 / s12640-021-00337-x; Li Z, Lu J, Tan X, Wang R, Xu Q, Yu Y, et al. Functional EL-HN Fragment as a PotentCandidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E.Toxins (Basel) (2022) 14(2). doi:10.3390 / toxins14020135;Li ZY, Li B, Lu JS,Liu X, Tan X, Wang R, et al. Biological and Immunological Characterization ofa Functional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins(Basel) (2023) 15(3). doi:10.3390 / toxins15030200). Recombinant human Siglec-15 protein was purchased from Beijing Sino Biological Technology Co., Ltd. The experimental methods are as follows: TL-HN, TL, THC, THN, AL-HN, BL-HN, EL-HN, FL-HN, and recombinant human Siglec-15 proteins were diluted to 2 μg / mL in carbonate coating buffer and added to a 96-well plate at 100 μL / well for overnight at 4°C. The plate was washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of ELISA blocking buffer was added for blocking at 37°C for 2 h. The plate was washed 6 times with PBST, and anti-tetanus toxin bispecific antibody (T6-hFc-25) was added at 100 μL / well for incubation at 37°C for 1.5 h. The plate was then washed 6 times with PBST, and goat anti-human IgG (HRP, 1:4000) diluted in blocking buffer was added at 100 μL / well for incubation at 37°C for 45 min. The plate was washed 6 times with PBST, and 100 μL / well of peroxidase substrate was added for color development, and the plate was protected from light for 15 min. The color development was observed for 10 min. After complete color development, 100 μL of 2 M sulfuric acid was added to each well to terminate the reaction. The plate was read using a microplate reader and analyzed using GraphPad Prism 8 software.

[0063] The results are as follows Figure 4 As shown in the figure (each point is plotted based on a specific absorbance value, and the horizontal axis is Log10), T6-hFc-25 has good specificity and only binds to recombinant TL-HN protein and recombinant THc protein.

[0064] 3. Biological layer interferometry (BLI) was used to detect the affinity between the bispecific antibody T6-hFc-25 and recombinant TL-HN protein or recombinant THc protein Add 200 μL of HBS-EP+ buffer to each well of the probe holder. Place an appropriate amount of AHC (Anti-human IgG Fc) probe into the holder and soak for 1 hour. Dilute the test antibody, T6-hFc-25, to 200 nM using HBS-EP+, and dilute the TL-HN protein to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.6 nM. Then, set up the reaction program and add the test sample to the assay plate at 200 μL / well. Place the probe holder and assay plate into the assay instrument and run the assay. Finally, import the experimental data into Data Analysis 7.0 software for analysis and calculation of affinity and other parameters.

[0065] The results are as follows Figure 5As shown, T6-hFc-25 exhibited typical binding kinetics with recombinant TL-HN or THc proteins. Using a 1:1 binding model calculated in Analysis Software 7.0, the KD values for T6-hFc-25 and recombinant TL-HN and THc proteins were all less than 1.0 pM, indicating good affinity between the antigen and antibody, suitable for subsequent development.

[0066] Example 3. Evaluation of the neutralizing activity of the anti-tetanus toxin bispecific antibody T6-hFc-25 Neutralizing activity of antibodies was determined by mixing antibodies with tetanus toxin in vitro and then injecting them into KM mice. The evaluation method was as follows: 1. Mice: KM, 4 mice per group, 18-20 g, purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0067] 2. Sample preparation: Diluent: 0.7 g KH2PO4, 2.4 g Na2HPO4·12H20, 6.8 g NaCl, 2 g gelatin, add water to 1 L, and sterilize by autoclaving.

[0068] Tetanus toxin solution: a solution obtained by dissolving tetanus toxin (purchased from China Food and Drug Inspection Institute) in diluent.

[0069] T6-hFc-25 solution: a solution obtained by dissolving the T6-hFc-25 antibody protein prepared in Example 1 in a diluent.

[0070] Human tetanus immunoglobulin TIG solution: a solution obtained by dissolving human tetanus immunoglobulin (purchased from China Food and Drug Inspection Institute) in diluent.

[0071] 3. The experimental groups are as follows: TeNT 10×LD 50 Group: KM mice were injected with tetanus toxin solution intraperitoneally, 500 μL per mouse, so that the dose of tetanus toxin was 10×LD 50 / mouse, observed for 15 days, a total of 4 mice.

[0072] TeNT + 0.039 μg-T6-hFc-25 group: Tetanus toxin solution was mixed with T6-hFc-25 solution, incubated at 37°C for 30 min, and then the mixed solution was intraperitoneally injected into KM mice, with each mouse receiving 500 μL of the solution, so that the dose of tetanus toxin TeNT protein was 10×LD 50 / mouse, so that the dose of T6-hFc-25 was 0.039 μg / mouse.

[0073] TeNT + 0.0195 μg-T6-hFc-25 group: The difference between this group and the TeNT + 10 μg-T6-hFc-25 group is that the dose of T6-hFc-25 is 0.0195 μg / mouse. Other operations are the same as those in the TeNT + 0.039 μg-T6-hFc-25 group.

[0074] TeNT + 0.00975 μg-T6-hFc-25 group: The difference between this group and the TeNT + 10 μg-T6-hFc-25 group is that the dose of T6-hFc-25 is 0.00975 μg / mouse. Other operations are the same as those in the TeNT + 0.039 μg-T6-hFc-25 group.

[0075] TeNT + 0.00488 μg-T6-hFc-25 group: The difference between this group and the TeNT + 10 μg-T6-hFc-25 group is that the dose of T6-hFc-25 is 0.00488 μg per mouse. Other procedures are the same as those of the TeNT + 0.039 μg-T6-hFc-25 group.

[0076] TeNT + 0.00244 μg-T6-hFc-25 group: The difference between this group and the TeNT + 10 μg-T6-hFc-25 group is that the dose of T6-hFc-25 is 0.00244 μg / mouse. Other operations are the same as those in the TeNT + 0.039 μg-T6-hFc-25 group.

[0077] TeNT + 0.00122 μg-T6-hFc-25 group: The difference between this group and the TeNT + 10 μg-T6-hFc-25 group is that the dose of T6-hFc-25 is 0.00122 μg / mouse. Other operations are the same as those in the TeNT + 0.039 μg-T6-hFc-25 group.

[0078] TeNT + 0.1 IU-TIG group: T6-hFc-25 solution was replaced by human tetanus immunoglobulin (TIG) solution at a dosage of 0.1 IU / mouse. Other procedures were the same as those in the TeNT + 0.039 μg-T6-hFc-25 group.

[0079] The results are as follows Figure 6 As shown, 0.00975 μg of T6-hFc-25 can completely neutralize 10×LD 50 Lethal dose of TeNT.

[0080] Example 4. Evaluation of the preventive and therapeutic effects of anti-tetanus toxin bispecific antibodies 1. Evaluate whether anti-tetanus toxin bispecific antibodies have a protective effect against Tetanus toxin challenge and whether this protective effect is dose-dependent in a mouse model The evaluation method is as follows: 1. Mice: KM, 4 mice per group, 18-20 g. Tetanus toxin solution, bispecific antibody T6-hFc-25 solution, and human tetanus immunoglobulin (TIG) solution were prepared as described in Example 3. Unrelated antibody (B-h3) solution was prepared by dissolving the unrelated antibody (B-h3) in diluent.

[0081] 2. KM mice were injected via the tail vein with 25 and 125 μg / kg T6-hFc-25 solution, 0.1 IU TIG, 250 μg / kg B-h3, and PBS, respectively. Tetanus toxin solution was injected intraperitoneally 12 h, 24 h, 48 h, 3 d, 5 d, 7 d, 9 d, 12 d, and 14 d after injection, respectively. The challenge dose was 10 × LD 50 / mouse, and observe the death of mice. The experimental groups are as follows: (1) 25 μg / kg-T6-hFc-25 + TeNT group: 125 μL of T6-hFc-25 solution (i.e., injection dose of 25 μg / kg) was injected into the tail vein of mice. After 12 h, 24 h, 48 h, 3 d, 5 d, 7 d, 9 d, 12 d, and 14 d, 500 μL of tetanus toxin solution (injection volume of 10 × LD) was injected into the abdominal cavity of KM mice. 50 / mouse, and observe the death of mice.

[0082] (2) 125 μg / kg-T6-hFc-25 + TeNT group: 125 μL of T6-hFc-25 solution (i.e., injection dose of 125 μg / kg) was injected into the mice via the tail vein. The rest of the procedures were the same as those in the 25 μg / kg-T6-hFc-25 + TeNT group.

[0083] (3) 0.1 IU-TIG + TeNT group: The T6-hFc-25 solution was replaced with 125 μL of human tetanus immunoglobulin TIG solution (i.e., the injection dose was 0.1 IU / mouse), and the rest of the procedures were the same as those of the 25 μg / kg-T6-hFc-25 + TeNT group.

[0084] (4) 250 μg / kg-B-h3 + TeNT group: The T6-hFc-25 solution was replaced with 125 μL of irrelevant antibody (B-h3) solution (i.e., the injection dose was 250 μg / kg), and the rest of the procedures were the same as those in the 25 μg / kg-T6-hFc-25 + TeNT group.

[0085] (5) PBS group: The T6-hFc-25 solution was replaced with an equal volume of PBS, and the rest of the procedures were the same as those in the 25 μg / kg-T6-hFc-25 + TeNT group.

[0086] The results are shown in Table 1. Low dose T6-hFc-25 (25 μg / kg) had an adverse effect on 10 × LD 50 TeNT attack can provide complete protection.

[0087] Table 1 Evaluation of the preventive effect of anti-tetanus toxin bispecific antibodies

[0088]

[0089] a Mice were treated with T6-hFc-25, TIG, B-h3, or PBS and exposed to TeNT for the indicated times.

[0090] b Mice were treated with 125 or 25 μg / kg nanobody, 0.1 IU TIG, 250 μg / kg B-h3, or PBS.

[0091] c Mice were challenged with TeNT 12 h, 24 h, 48 h, 3 d, 5 d, 7 d, 9 d, 12 d, and 14 d after treatment with the indicated doses of antibodies.

[0092] d KM mice were intraperitoneally injected with 10 × LD 50 TeNT.

[0093] 2. Evaluating whether anti-tetanus bispecific antibodies are protective after exposure to Tetanus The evaluation method is as follows: 1. Mice: KM, 4 mice per group, 18-20 g.

[0094] 2. KM mice were intraperitoneally injected with tetanus toxin solution at a dose of 5 × LD 50 Each group of mice received a tail vein injection of 25 or 125 μg / kg T6-hFc-25 solution, 1 IUTIG, 250 μg / kg B-h3, or PBS 1, 3, 6, 12, or 24 hours after the injection of tetanus toxin solution. The mice were observed for mortality. The experimental groups were as follows: (1) TeNT + 25 μg / kg-T6-hFc-25 group: 125 μL of T6-hFc-25 solution (i.e., injection dose of 50 μg / kg) was injected into the mice via the tail vein, and the death of the mice was observed.

[0095] (2) TeNT + 125 μg / kg-T6-hFc-25 group: 125 μL of T6-hFc-25 solution (i.e., injection dose of 125 μg / kg) was injected into the mice via the tail vein, and the death of the mice was observed.

[0096] (3) TeNT + 1 IU-TIG group: 125 μL of human tetanus immunoglobulin TIG solution (i.e., injection dose of 1 IU / mouse) was used instead of T6-hFc-25 solution, and mice were treated via the tail vein, and the death of mice was observed.

[0097] (4) TeNT + 250 μg / kg -B-h3 group: The T6-hFc-25 solution was replaced with 125 μL of irrelevant antibody (B-h3) solution (i.e., the injection dose was 250 μg / kg), and the mice were treated via the tail vein, and the death of the mice was observed.

[0098] (5) TeNT + PBS group: The T6-hFc-25 solution was replaced with an equal volume of PBS, and the rest of the procedures were the same as those in the TeNT + 25 μg / kg-T6-hFc-25 group.

[0099] The results are shown in Table 2. For TeNT intoxication at 5 × LD50, treatment with 0.5 μg / mouse (dose = 1 mg / kg antibody × 20 g / mouse) of T6-hFc-25 was still effective within 12 hours after exposure.

[0100] Table 2 Evaluation of the therapeutic effect of anti-tetanus toxin bispecific antibodies

[0101] a KM mice injected with 5 × LD 50 TeNT.

[0102] b Mice were treated with the indicated doses of antibodies 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h after exposure to TeNT.

[0103] c TeNT-exposed mice were treated with T6-hFc-25, TIG, B-h3, and PBS at the indicated times after exposure.

[0104] d TeNT-exposed mice were treated with 25 μg / kg or 125 μg / kg T6-hFc-25, 1 IU TIG, 250 μg / kg B-h3, or PBS at the specified time after exposure.

[0105] The above examples involve the following sequences: SEQ ID NO: 1: QVQLVESGGGLVQPGGSLRLSCAASGYDYIREYMGWFRQAPGKGLEEVAIIYIYGGNTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAERREMARISSGNVKYWGQGTLVTVSS.

[0106] SEQ ID NO:2: QVQLVESGGGLVQPGGSLRLSCAAS.

[0107] SEQ ID NO:3: MGWFRQAPGKGLEEVAI.

[0108] SEQ ID NO:4: DYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC.

[0109] SEQ ID NO: 5: WGQGTLVTVSS.

[0110] SEQ ID NO: 6: GYDYIREY.

[0111] SEQ ID NO:7: IYIYGGNT.

[0112] SEQ ID NO:8: AAERREMARISSGNVKY.

[0113] SEQ ID NO:9: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS.

[0114] SEQ ID NO:10: EVQLVESGGGLVQPGGSLRLSCAAS.

[0115] SEQ ID NO: 11: MSWVRQAPGKGLEWVSA. <h2 style=";text-align:left;direction:ltr">

[0116] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12:YYKGTVKGRFTISRDSKNTLYLQMNSLRAEDTAVYYC。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0117] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13:WGQGTLVTVSS。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0118] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14:GFTFSNYD。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0119] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15:LDAGGLKT。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0120] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16:ATGFYPQYLLQHAPDVARTEDDA。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0121] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17:<h2 style=";text-align:left;direction:ltr">

[0122] SEQ ID NO: 18: QVQLVESGGGLVQPGGSLRLSCAASGYDYIREYMGWFRQAPGKGLEEVAIIYIYGGNTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAERREMARISSGNVKYWGQGTLVTV SSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSEVQLVESG GGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS.

[0123] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A bispecific antibody or antigen-binding fragment thereof against tetanus toxin, characterized in that: The bispecific antibody comprises the variable region of a first nanobody and the variable region of a second nanobody, the variable region of the first nanobody and the variable region of the second nanobody are connected by a connecting peptide, the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the first nanobody are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the second nanobody are SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The first Nanobody variable region also comprises four framework regions FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the group consisting of: A1-1) amino acid sequence is SEQ ID NO: 2; A1-2) has a sequence identity of more than 75% with A1-1); The amino acid sequence comprises an amino acid sequence selected from the group consisting of: A2-1) the amino acid sequence of FR2 is SEQ ID NO: 3; A2-2) has a sequence identity of more than 75% with A2-1); The FR3 comprises an amino acid sequence selected from the group consisting of: A3-1) amino acid sequence is SEQ ID NO: 4; A3-2) Sequences with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the group consisting of: A4-1) amino acid sequence is SEQ ID NO: 5; A4-2) Sequences with more than 75% identity with A4-1); The second Nanobody variable region also comprises four framework regions FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the group consisting of: A5-1) amino acid sequence is SEQ ID NO: 10; A5-2) Sequences with more than 75% identity with A5-1); The FR2 comprises an amino acid sequence selected from the group consisting of: A6-1) amino acid sequence is SEQ ID NO: 11; A6-2) has a sequence identity of more than 75% with A6-1); The FR3 comprises an amino acid sequence selected from the group consisting of: A7-1) amino acid sequence is SEQ ID NO: 12 or; A7-2) has a sequence identity of more than 75% with A7-1); The FR4 comprises an amino acid sequence selected from the group consisting of: A8-1) amino acid sequence is SEQ ID NO: 13; A8-2) has a sequence identity of more than 75% with A8-1).

3. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein: The amino acid sequence of the connecting peptide includes amino acids 354 to 358 in SEQ ID NO:

18.

4. An anti-tetanus toxin heavy chain antibody, characterized in that The heavy chain antibody comprises the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The heavy chain antibody according to claim 4, characterized in that The constant region is amino acids 125 to 353 of SEQ ID NO:

18.

6. The heavy chain antibody according to claim 4 or 5, characterized in that The amino acid sequence of the heavy chain antibody includes SEQ ID NO: 18 or has a homology of greater than 99%, greater than 95%, greater than 90%, greater than 85%, greater than 80% or greater than 75% with SEQ ID NO:

18.

7. Biomaterial, characterized in that The biological material includes any one of the following: B1) a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to claim 4 or 5; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) 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).

8. An anti-tetanus drug, characterized in that The drug comprises the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to claim 4 or 5.

9. Application, characterized in that The application includes any of the following: M1) Use of the biomaterial according to claim 7 in the preparation of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6; M2) Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6, in the preparation of the medicament according to claim 8; M3) Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6, or the biomaterial according to claim 7, or the medicament according to claim 8 in the preparation of a product for preventing and / or treating diseases caused by Clostridium tetani; M4) Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6, or the biomaterial according to claim 7 in the preparation of a product for detecting Clostridium tetani; M5) Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6, or the biomaterial according to claim 7, or the medicament according to claim 8 in the preparation of a product for neutralizing neurotoxins secreted by Clostridium tetani; M6) Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6, or the biomaterial according to claim 7, or the drug according to claim 8 in the preparation of a product for detecting neurotoxins secreted by Clostridium tetani.

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