Nano antibody TL-25 of anti-tetanus toxin L-HN fragment and application of nano antibody TL-25

Abstract

The invention discloses a nano antibody TL-25 of an anti-tetanus toxin L-HN fragment and application of the nano antibody TL-25, 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] The present invention belongs to the technical field of immunotherapy biomedicine, and specifically relates to a nanobody TL-25 against the L-HN fragment of tetanus toxin and its application. Background Art

[0002] Tetanus is an acute neurological disease caused by tetanus toxin (TeNT), secreted by Clostridium tetani. Initial symptoms often include difficulty opening the mouth and trismus, which progresses to classic signs such as a wry smile and opisthotonos. As the disease progresses, severe muscle spasms occur throughout the body, leading to severe complications such as difficulty breathing and asphyxiation, ultimately leading to death.

[0003] According to statistics, there were still 51,000 new tetanus infections and 21,000 deaths worldwide in 2021. The majority of these cases occurred in South Asia and sub-Saharan Africa, where problems such as a lack of medical resources, low vaccination coverage, and poor sanitation often lead to high infection and mortality rates. Despite widespread vaccination worldwide, neonatal tetanus remains a serious threat. The latest data from the World Health Organization shows that as of 2020, 12 countries have not yet successfully eliminated maternal and neonatal tetanus. Tetanus remains a serious public health challenge worldwide.

[0004] Passive immunotherapy has been the primary strategy for clearing tetanus toxin from tetanus patients. However, to date, only Tylenol Biopharm's fully human monoclonal antibody, Xintetob, was approved for marketing on February 11, 2025. Other antibodies under clinical development include Zhixiang Jintai's recombinant humanized monoclonal antibody GR2001 and Beike Biopharm's fully human monoclonal antibody combination formulation A82 / B86. Given the enormous unmet clinical needs in my country, the development of antibodies to prevent and treat tetanus toxin remains crucial.

[0005] Tetanus toxin poisoning can be urgently prevented with antibody preparations. Previous research by the research team has also demonstrated that the TL-HN functional domain can stimulate the production of neutralizing antibodies, providing excellent immune protection. Therefore, specific neutralizing antibodies targeting the TL-HN functional domain could be developed to prevent and treat tetanus toxin poisoning. Compared to traditional hybridoma technology, antibody preparation using phage display libraries allows for efficient screening and obtaining more specific, high-affinity antibodies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to develop a nanobody that is more effective in neutralizing tetanus toxin against the L-HN domain of tetanus toxin, which can be used to prepare a drug for the diagnosis or treatment of tetanus. The technical problem to be solved is not limited to the technical subject matter described above, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0007] To address the above technical issues, the inventors used recombinant TL-HN protein to immunize camels to construct an anti-tetanus nanobody phage display library. The recombinant TL-HN protein was then used to screen the library for specific anti-tetanus nanobodies. The specific technical solution is as follows: The present invention provides a nanobody targeting the TL-HN domain of tetanus toxin or an antigen-binding fragment containing the nanobody.

[0008] In a first aspect, the present invention provides an anti-tetanus toxin nanobody or an antigen-binding fragment thereof, wherein the nanobody or the antigen-binding fragment thereof comprises three complementary determining regions CDR1, CDR2 and CDR3, and the nanobody is TL-25; The amino acid sequence of CDR1 of TL-25 is SEQ ID NO: 6, the amino acid sequence of CDR2 of TL-25 is SEQ ID NO: 7, and the amino acid sequence of CDR3 of TL-25 is SEQ ID NO: 8; The above-mentioned Nanobody or antigen-binding fragment thereof contains, in addition to the complementary determining regions, four framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 of the TL-25 is SEQ ID NO: 2, the amino acid sequence of FR2 of the TL-25 is SEQ ID NO: 3, the amino acid sequence of FR3 of the TL-25 is SEQ ID NO: 4 or has 75% or greater identity with SEQ ID NO: 4, and the amino acid sequence of FR4 of the TL-25 is SEQ ID NO: 5.

[0009] The amino acid sequence of TL-25 in the above-mentioned Nanobody or antigen-binding fragment thereof includes SEQ ID NO: 1 or SEQ ID NO: 12.

[0010] The amino acid sequence of TL-25 is SEQ ID NO: 1 or SEQ ID NO: 12.

[0011] 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.

[0012] In a second aspect, the present invention provides an anti-tetanus toxin heavy chain antibody, wherein the heavy chain antibody comprises the aforementioned Nanobody.

[0013] The above-mentioned heavy chain antibody includes a heavy chain variable region, the amino acid sequence of which 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.

[0014] The heavy chain antibody is TL-25-hFc, and the amino acid sequence of the TL-25-hFc includes SEQ ID NO: 11 or SEQ ID NO: 15.

[0015] The constant region of the heavy chain antibody includes amino acids 131-359 of SEQ ID NO: 11 or amino acids 131-359 of SEQ ID NO: 15.

[0016] The constant region of the heavy chain antibody is amino acids 131-359 of SEQ ID NO: 11 or amino acids 131-359 of SEQ ID NO: 15.

[0017] The amino acid sequence of the heavy chain antibody includes SEQ ID NO:11 or SEQ ID NO:15.

[0018] The amino acid sequence of the heavy chain antibody is SEQ ID NO: 11 or SEQ ID NO: 15.

[0019] 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 Nanobody 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).

[0020] In the above-mentioned biological materials, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0021] In the above-mentioned biological materials, the expression cassette described in B2) refers to a single-stranded or double-stranded nucleic acid molecule capable of expressing the aforementioned proteins in host cells. The expression cassette may also include all regulatory sequences necessary for expressing the nucleic acid molecule for any of the aforementioned proteins or the DNA for the aforementioned RNA molecule. These regulatory sequences are capable of directing the expression of the coding sequence for any of the aforementioned proteins or the DNA for the aforementioned RNA molecule in a suitable host cell under compatible conditions. These regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligation of the regulatory sequence to the coding region of the protein-encoding nucleic acid sequence or the DNA for the aforementioned RNA molecule, a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. A promoter sequence contains transcriptional regulatory sequences that mediate expression of the protein or the DNA for the aforementioned RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence can be operably linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence can be operably linked to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the DNA encoding the protein or the RNA molecule into the cellular secretory pathway. Any signal peptide coding region that directs the expressed protein or the DNA of the RNA molecule into the secretory pathway of the selected host cell can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or RNA molecule according to the growth conditions of the host cell. Examples of regulatory sequences are systems that can turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification.

[0022] The small molecule antibody may be any of the following: F1, Fab antibody; F2, Fv antibody; F3, single-chain antibody; F4, Fab′ fragment.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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).

[0028] 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).

[0029] 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.

[0030] 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).

[0031] 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.

[0032] The medicament further includes a physiologically or pharmaceutically acceptable excipient, diluent or carrier.

[0033] 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.

[0034] 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.

[0035] 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 Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody; M2) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody in the preparation of the aforementioned medicament; M3) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned genetic material or the aforementioned drug in the preparation of a product for preventing and / or treating a disease caused by Clostridium tetani; M4) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned genetic material in the preparation of a product for detecting Clostridium tetani; M5) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody 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 Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned genetic material or drug in the preparation of a product for detecting neurotoxins secreted by Clostridium tetani.

[0036] 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.

[0037] 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.

[0038] In the above method, the mammalian cell can be FreeStyle TM HEK293-F.

[0039] 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.

[0040] The anti-tetanus toxin nanobody provided by the present invention can effectively neutralize tetanus toxin. Experiments have shown that the fusion protein TL-25-hFc, obtained by fusing the nanobody TL-25 with the Fc segment (hFc) of human immunoglobulin, has good specificity and only binds to the TL-HN protein. 0.3125μg of TL-25-hFc can effectively block the lethal dose of tetanus toxin (10×LD 50 ) poisoning; after humanization, it still has good affinity and neutralization activity, and the same dose of TL-25-h1-hFc can still effectively block the lethal dose of tetanus toxin (10 × LD 50 ) caused by poisoning. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a partial image of the binding of phage clones to the target antigen after the second round of screening using Phage-ELISA. The left image is the target antigen, and the right image is the control antigen.

[0042] Figure 2 This is the SDS-PAGE electrophoresis detection of the purified anti-tetanus nanobody fusion protein.

[0043] Figure 3 To detect the binding activity of anti-tetanus nanobody fusion protein.

[0044] Figure 4 For the specific detection of anti-tetanus nanobody fusion protein.

[0045] Figure 5 Affinity detection of anti-tetanus nanobody fusion protein.

[0046] Figure 6 The purified humanized anti-tetanus nanobody fusion protein was detected by SDS-PAGE electrophoresis.

[0047] Figure 7 Binding activity assay for humanized anti-tetanus nanobody fusion protein.

[0048] Figure 8 Specific detection of humanized anti-tetanus nanobody fusion protein.

[0049] Figure 9 Affinity detection of humanized anti-tetanus nanobody fusion protein. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] The following examples were processed using GraphPad Prism 9 statistical software. The experimental results are expressed as mean ± standard deviation. The Log-rank test was used to compare the significance of the difference in protection between the two groups. The difference was considered statistically significant when p < 0.05.

[0053] Recombinant TL-HN antigen (or recombinant TL-HN protein): Prepared by the inventors' laboratory. The preparation method is described in "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.

[0054] 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.

[0055] Example 1. Construction of anti-tetanus nanobody library 1. Camel Immunity Recombinant tetanus TL-HN antigen and Freund's complete adjuvant (Sigma, F5881) were mixed and shaken in equal volumes to fully emulsify the mixture. The mixture was then injected subcutaneously into healthy adult Bactrian camels at multiple points. Booster immunizations were performed every two weeks. Except for the first immunization with Freund's complete adjuvant, incomplete Freund's adjuvant (Sigma, F5506) was used for all other immunizations.

[0056] 2. Isolation of peripheral blood lymphocytes from camel blood Approximately 150 mL of peripheral blood from camels after five immunizations was collected into anticoagulant tubes. Peripheral blood lymphocytes were isolated from the blood using lymphocyte separation solution (STEMCELL, 07851). After centrifugation of the mixture of whole blood and lymphocyte separation solution, the liquid in the centrifuge tube separated into four layers from top to bottom: plasma, peripheral blood mononuclear cells, glucose in the lymphocyte separation solution, and red blood cells.

[0057] 3. Nested PCR amplification of VHH gene fragments Total RNA was extracted from isolated peripheral blood mononuclear cells (PBMCs) using the OMEGA EZNA Total RNA kit I (OMEGA, R6834), and then reverse transcribed to obtain cDNA using the Invitrogen Superscript III First-strand synthesis system for RT-PCR kit (Invitrogen, 18080-051).

[0058] Nested PCR amplification of VHH gene fragments: 1) First round of PCR: Using the synthesized cDNA as a template, the designed IgG-specific upstream primer CALL001 and downstream primer CALLOO2 were used to PCR amplify the antibody leader sequence and CH2 sequence; 2) Using the first round of purified DNA product as a template, primers VHH-F and VHH-R were used for the second round of PCR amplification to obtain the VHH gene.

[0059] Table 1. Primer sequences used in two rounds of PCR

[0060] 4. Electroporation products The vector and amplified VHH gene were digested with restriction endonucleases Nco I and Not I. T4 ligase was used to construct the ligation product, which was then transformed into TG1 competent cells using electrofection. A tetanus-specific phage antibody library was constructed, and its capacity was determined by serial dilution. The library was found to have a capacity of 3.1×10 8 .

[0061] Example 2: Screening of anti-tetanus specific phage nanobody library The anti-tetanus specific phage nanobody library constructed in step 2 was taken and presented using helper phage M13K07. The phage was then precipitated with 20% PEG / NaCl solution (1 L solution contains 200 g PEG6000, 146.25 g NaCl) to obtain an anti-tetanus specific phage nanobody library.

[0062] The purified recombinant TL-HN protein was used as the antigen to screen the immune library constructed above using a solid-phase screening strategy (refer to the experimental protocol in Phage Display: A General Protocol / (US) Clarkson (T), (US) Lowman (HB); (US) Translated by Ma Lan et al. Chemical Industry Press, May 2008). The specific method is as follows: 1 mL of antigen diluted with 0.1 M NaHCO3 coating solution was added to each immune well and coated overnight at 4°C. The coating concentrations for each round of panning were 20 and 10 μg / mL, respectively. The immune tubes were washed twice with PBS the next day for 3 min each time, and then blocked with 2% blocking solution (bovine serum albumin) at 37°C for 2 h. After blocking, the tubes were washed 3 times with PBST (1×PBS+0.1% Tween-20) and then washed 3 times with PBS. The titer of the anti-tetanus specific phage nanoantibody library was adjusted to an appropriate concentration with blocking solution, added to the immune tubes, placed at room temperature for 2 h, and then shaken at 200 rpm for 20 min. The tubes were washed 10 times with PBST and then washed 5 times with PBS. After washing, 1 mL of elution solution (glycine-hydrochloric acid, pH 2.2) was added to each well and eluted at 400 rpm for 20 min. The eluate from the target antigen well was removed and neutralized by adding 50-60 μL of neutralizing solution (1 M Tris-HCl, pH 8.0). Escherichia coli TG1 cells in logarithmic phase were infected and allowed to stand at room temperature for 30 minutes. The cells were cultured at 37°C for 1 hour to produce and purify phages for the next round of screening. The same screening process was repeated for two rounds. The enrichment results are shown in Table 2: Table 2. Analysis of enrichment degree of anti-tetanus phage nanobody library screening

[0063] Well-separated single clones were selected from the plates containing phage after the two rounds of screening described above and inoculated into 96-well plates (250 μL / well) containing 2YT-GA medium (1 L 2YT medium containing 16 g Typtone, 10 g Yeast extract, 5 g NaCl, 100 μg / mL ampicillin, and 20% glucose). Two negative control wells (no clones) were left and cultured at 37°C until the logarithmic growth phase. M13KO7 helper phage was added at an MOI of ≈50 and 100 μL / well was added to the deep-well plates containing individual phage clones. The infection was allowed to stand at room temperature for 30 min, followed by incubation at a low speed of 150 rpm at 37°C for 1 h. The deep-well plates were centrifuged at room temperature (2000 rpm for 10 min), the supernatant discarded, and expression was induced with arabinose at a final concentration of 1 mM. The plates were cultured overnight to obtain phage particles displaying the antibody variable regions.

[0064] Example 3: Identification of specific nanobody-positive clones using phage-ELISA The plates were coated with recombinant TL-HN protein as the antigen. The adjacent rows of each antigen group were coated with an irrelevant antigen in parallel as a negative control, and the plates were coated overnight at 4°C. The coating solution was discarded, the plates were washed six times with PBST, and blocking solution (3 g of skim milk powder in 100 mL of PBS) was added at 200 µL / well. The plates were blocked at 37°C for 2 h. The blocking solution was discarded, and 100 µL / well of the induced phage supernatant was added to the corresponding ELISA wells and incubated at 37°C for 1.5 h. The plates were washed six times with PBST with shaking, and freshly prepared 0.2 µg / ml HRP-labeled M13 mouse monoclonal antibody (SinoBioligical, 1973-MM05T-H) was added to the plates at 100 µL / well. The plates were incubated at 37°C for 45 min. The plates were washed six times with PBST with shaking, and the color development solution (9 mL of color development solution, 1 mL of 10×OPD, 10 μL 30% H2O2), add 10 μL / well to the ELISA plate and develop in the dark for 15-20 min; add 2 M sulfuric acid stop solution (50 μL / well) and read the values at dual wavelengths of 492 / 630 nm; clone wells with absorbance values >3 times that of the antigen group / negative control group are judged as positive clone wells.

[0065] Some Phage-ELISA test results are shown 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, which yielded the DNA sequence of the insert and ultimately yielded the camel-derived nanoantibody TL-25, which specifically binds to TL-HN.

[0066] The amino acid sequence of TL-25 is shown in SEQ ID NO: 1, which includes framework regions (designated FRs, including FR1, FR2, FR3, and FR4) and complementarity-determining regions (designated CDRs, including CDR1, CDR2, and CDR3). The four portions of the framework regions are sequentially designated 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 sequentially designated as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. The nucleotide sequence of the gene encoding the anti-tetanus nanobody TL-25 is shown in SEQ ID NO: 9.

[0067] The sequences of the complementarity determining regions (CDRs) are defined according to the IMGT numbering system.

[0068] Example 4. Preparation of anti-tetanus nanobody-hFc fusion protein 1. Construction of anti-tetanus nanobody-hFc fusion protein eukaryotic expression plasmid The C-terminus of the anti-tetanus nanobody TL-25 (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 gene encoding the anti-tetanus nanobody-hFc fusion protein TL-25-hFc. The nucleotide sequence of the fusion protein is SEQ ID NO: 10, and the amino acid sequence is SEQ ID NO: 11.

[0069] Using conventional molecular biology techniques, the 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 ligating the gene for the Fc domain of a human immunoglobulin to a pCMV vector) was replaced with a DNA molecule having the nucleotide sequence of SEQ ID NO: 9 (the gene encoding the TL-25 nanobody), while keeping the other nucleotides of the pTSE-hFc vector unchanged. This resulted in a recombinant expression plasmid pTSE-TL-25-hFc containing the gene encoding the TL-25-hFc fusion protein. This vector expresses a protein having the amino acid sequence of SEQ ID NO: 11 (designated TL-25-hFc, whose nucleotide sequence is SEQ ID NO: 10).

[0070] The sequences of the complementarity determining regions of the heavy chain antibodies are defined according to the IMGT numbering system.

[0071] 2. Expression and purification of anti-tetanus nanobody-hFc fusion protein The pTSE-TL-25-hFc fusion protein expression plasmid constructed in "1. Construction of anti-tetanus nanobody-hFc fusion protein eukaryotic expression plasmid" was transfected into FreeStyle using the transfection reagent FectoPRO DNA 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 minutes and purified using the AKTA purification system to obtain TL-25-hFc fusion protein.

[0072] The purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 2As shown: The molecular weight of the antibody is in line with expectations. The band size of the nanobody-hFc fusion protein under reducing conditions is approximately 40 kDa (left), and the band size under non-reducing conditions is approximately 80 kDa (right).

[0073] Example 5. Evaluation of the properties of nanobody fusion proteins 1. Detect the binding activity of nanobody-hFc fusion protein and recombinant TL-HN protein by ELISA experiment 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 the ELISA plate and coated at 4°C overnight. The next day, the plate was washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of blocking solution (3% skim milk powder) was added and blocked at 37°C for 2 h. The blocking solution was discarded, and the plate was washed 6 times with PBST. 100 μL / well of a 2-fold isocratic dilution of the nanoantibody fusion protein TL-25 (starting concentration 10 μg / mL, diluted with blocking solution) was added. A total of 23 gradients were set, with 2 replicates for each gradient, and the plate was incubated 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 solution was added at 100 μL / well and incubated at 37°C for 45 min. The plate was washed 6 times with PBST, and 100 μL / well of 100 μL of 10 μL was added to each well. OPD substrate colorimetric solution was used to develop color in the dark for 5-10 minutes. The color development effect was observed. After the color development was complete, 50 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.

[0074] The results are as follows Figure 3 (Each point in the figure is plotted based on a specific absorbance value). In the figure, the horizontal axis is the logarithmic value 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 TL-25-hFc fusion protein to recombinant TL-HN protein is 50 ) is 0.022 nM.

[0075] 2. Identify the specificity of nanobody-hFc fusion protein by ELISA experiment Tetanus TL-HN, THC, TL, and THN proteins were prepared by the inventors' laboratory according to the Materials and Methods section 2.2 of the following document: 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.

[0076] AL-HN protein was prepared by the inventors' laboratory according to the materials and methods section 2.1 of the following document: 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.

[0077] The BL-HN protein was prepared by the inventors' laboratory according to the Materials and Methods section "Recombinant BoNT / B L-HN Fragment Preparation" of the following document: "Li Z, Lu JS, Liu S, Wang R, Xu Q, Yu YZ, et al. Recombinant L-HN Fusion Antigen Derived from the L and HNDomains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin. NEUROTOX RES (2021). doi:10.1007 / s12640-021-00337-x".

[0078] EL-HN protein was prepared by the inventors' laboratory according to the Materials and Methods section 5.2 of the following document: Li Z, Lu J, Tan X, Wang R, XuQ, 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.

[0079] The FL-HN protein was prepared by the inventors' laboratory according to the Materials and Methods section 5.2 of the following document: Li ZY, Li B, Lu JS, Liu X, Tan X, Wang R, et al. Biological and Immunological Characterization of aFunctional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins (Basel)(2023) 15(3). doi:10.3390 / toxins15030200).

[0080] Recombinant human Siglec-15 protein was purchased from Beijing Sino Biological Technology Co., Ltd.

[0081] The experimental method is as follows: AL-HN, BL-HN, EL-HN, FL-HN, TL-HN, THC, TL, THN, 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 antibody (TL-25-hFc fusion protein) 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. After the color development was complete, 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.

[0082] The results are as follows Figure 4 As shown in the figure (each point is drawn according to a specific absorbance value), TL-25-hFc has good specificity and only binds to TL-HN antigen.

[0083] 3. Detection of affinity between nanobody-hFc fusion protein and TL-HN protein using biological layer interferometry (BLI) The experimental method is as follows: 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 TL-25 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 using HBS-EP+. 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.

[0084] The results are as follows Figure 5As shown, TL-25-hFc and recombinant TL-HN protein exhibited typical binding kinetics. Using a 1:1 binding model in Analysis Software 7.0, the KD value for TL-25-hFc and recombinant TL-HN protein was calculated to be 17.1 nM, indicating good affinity between the antigen and antibody, suitable for subsequent development.

[0085] Example 6. Evaluation of Neutralizing Activity of Anti-Tetanus Toxin Nanobody-hFc Fusion Protein 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.

[0086] 2. Dilution solution composition: 0.7 g KH2PO4, 2.4 g Na2HPO4·12H20, 6.8 g NaCl, 2 g gelatin, add water to 1 L, and sterilize by autoclave.

[0087] Tetanus toxin (TeNT) solution: a solution obtained by dissolving tetanus toxin (purchased from China Food and Drug Inspection Institute) in the aforementioned diluent.

[0088] TL-25-hFc fusion protein solution: a solution obtained by dissolving the TL-25-hFc fusion protein prepared in Example 4 in the aforementioned diluent.

[0089] Human tetanus immunoglobulin TIG solution: a solution obtained by dissolving human tetanus immunoglobulin (purchased from China Food and Drug Inspection Institute) in the aforementioned diluent.

[0090] The experimental groups are as follows: TeNT 10×LD 50 Group: 4 KM mice were intraperitoneally injected with tetanus toxin solution, with each mouse injected with 500 μL, so that the dose of tetanus toxin was 10×LD 50 / , observe for 7 days.

[0091] TeNT + 5 μg-TL-25-hFc group: Tetanus toxin solution was mixed with TL-25-hFc fusion protein solution and incubated at 37°C for 30 min to obtain TeNT + TL-25-hFc solution. The TeNT + TL-25-hFc solution was intraperitoneally injected into four 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 TL-25-hFc fusion protein was 5 μg / mouse.

[0092] TeNT + 2.5 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein is 2.5 μg per mouse. Other procedures are the same as those in the TeNT + 5 μg-TL-25-hFc group.

[0093] TeNT + 1.25 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein is 1.25 μg / mouse, and the rest of the procedures are the same as those in the TeNT + 5μg-TL-2-hFc 5 group.

[0094] TeNT + 0.625 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein was 0.625 μg per mouse. Other procedures were the same as those in the TeNT + 5 μg-TL-25-hFc group.

[0095] TeNT + 0.3125 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein was 0.3125 μg per mouse. Other procedures were the same as those in the TeNT + 5 μg-TL-25 group.

[0096] TeNT + 0.156 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein was 0.156 μg per mouse. Other procedures were the same as those in the TeNT + 5 μg-TL-25-hFc group.

[0097] TeNT + 0.078 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25 group is that the dose of TL-25-hFc fusion protein was 0.078 μg per mouse. Other operations were the same as those in the TeNT + 5 μg-TL-25 group.

[0098] TeNT + 0.039 μg-TL-25-hFc group: The difference between this group and the TeNT + 10 μg-TL-25-hFc group is that the dose of TL-25-hFc fusion protein was 0.039 μg per mouse. Other procedures were the same as those in the TeNT + 5 μg-TL-25-hFc group.

[0099] The results are shown in Table 3. 0.3125 μg of TL-25-hFc can completely neutralize 10×LD 50 Lethal dose of TeNT.

[0100] Table 3. Evaluation of the neutralizing activity of TL-25-hFc

[0101] Example 7 Humanization and activity evaluation of anti-tetanus toxin nanobody-hFc fusion protein 1. Analyze the antibody's amino acid sequence on the website http: / / www.abysis.org / abysis / . Based on the Z-score, replace amino acid residues in the framework with a frequency less than 0.1 with humanized sequences to improve the degree of humanization. Use the Swiss-model to construct the spatial structure of the humanized IgG and analyze the accessible surface area of the amino acid residue solution to determine which amino acid residues can be humanized. Mutate the antibody's framework regions with humanized amino acids to obtain the humanized nanobody TL-25-h1, whose amino acid sequence is shown in SEQ ID NO: 12 and nucleotide sequence is shown in SEQ ID NO: 13.

[0102] 2. Construction of eukaryotic expression vector for humanized anti-tetanus toxin nanobody-hFc fusion protein According to the anti-tetanus toxin humanized nanobody gene sequence (SEQ ID NO: 13), its C-terminus was connected to the Fc segment (hFc) of human immunoglobulin G to obtain the anti-tetanus toxin humanized nanobody-hFc fusion protein TL-25-h1-hFc with the encoding gene nucleotide sequence of SEQ ID NO: 14 and the expressed amino acid sequence of SEQ ID NO: 15.

[0103] 3. Preparation and Characterization of Humanized Nanobody-hFc Fusion Protein (1) Preparation of humanized nanobody-hFc fusion protein TL-25-h1-hFc The humanized nanobody-hFc fusion protein TL-25-h1-hFc was prepared by referring to the method in Example 4. Figure 6 As shown: The molecular weight of the fusion protein is consistent with the expected size, with the band size under reducing conditions being approximately 40 kDa (left) and the band size under non-reducing conditions being approximately 80 kDa (right).

[0104] (2) Detection of the binding activity and specificity of the humanized nanobody-hFc fusion protein TL-25-h1-hFc with the recombinant TL-HN protein The humanized nanobody-hFc fusion protein TL-25-h1-hFc was used to replace the TL-25-hFc fusion protein. The remaining operations were the same as those in Example 5, “1. Detection of the binding activity of the nanobody-hFc fusion protein to the recombinant TL-HN protein by ELISA experiment” and “2. Identification of the specificity of the nanobody-hFc fusion protein by ELISA experiment”.

[0105] The results are as follows Figure 7 、 Figure 8 Shown: The half effective concentration (EC) of TL-25-h1-hFc binding to recombinant TL-HN protein 50 ) was 0.01108 nM, and it only bound to TL-HN antigen, with good specificity. Figure 7 and Figure 8 In the figure, each point is drawn according to a specific absorbance value.

[0106] (3) Detection of affinity between humanized nanobody-hFc fusion protein TL-25-h1-hFc and recombinant TL-HN protein The humanized nanobody-hFc fusion protein TL-25-h1-hFc was used to replace the test antibody TL-25. The remaining procedures were the same as those in Example 5, "3. Detection of affinity between nanobody-hFc fusion protein and TL-HN protein by biological layer interferometry (BLI)".

[0107] The results are as follows Figure 9 As shown: The KD value of TL-25-h1-hFc and recombinant TL-HN protein reached 0.573 nM, which increased by about 10 times compared with before humanization, and the affinity between the antigen and antibody was good.

[0108] 4. Evaluation of the neutralizing activity of humanized nanobody fusion protein The humanized nanobody-hFc fusion protein TL-25-h1-hFc was used to replace the TL-25-hFc fusion protein, and the remaining operations were the same as in Example 6 "Evaluation of the neutralizing activity of nanobody-hFc fusion protein against tetanus toxin".

[0109] The results are shown in Table 4. 0.3125 μg of TL-25-h1-hFc can effectively block the lethal dose of tetanus toxin (10×LD 50 ) caused by poisoning.

[0110] Table 4. Evaluation of the neutralizing activity of TL-25-h1-hFc

[0111] Example 8. Evaluation of the preventive and therapeutic effects of humanized anti-tetanus nanobody-hFc fusion protein 1. Evaluate whether the humanized anti-tetanus nanobody-hFc fusion protein has a protective effect against Tetanus challenge and whether this protective effect is dose-dependent in a mouse model. The evaluation method is as follows: 1. Mice: KM mice (purchased from Beijing Sibeifu Biotechnology Co., Ltd.), each weighing 18-20 g. Tetanus toxin solution, nanobody TL-25-h1-hFc fusion protein solution, and TL-25-h1-hFc fusion protein solution were prepared as described in Example 6. The unrelated antibody (B-h3) solution was prepared by dissolving the unrelated antibody (B-h3) in the diluent.

[0112] 2. The experimental groups are as follows: (1) 25 μg / kg-TL-25-h1-hFc + TeNT group: KM mice were treated with 125 μL of TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) via the tail vein. 12 h, 24 h, 48 h, 3 d, 5 d, 7 d, 9 d, 12 d, and 14 d after injection, 500 μL of tetanus toxin solution (injection volume of 10 × LD) was injected into the KM mice intraperitoneally. 50 / mouse (4 mice were injected at each time point), and the death of mice was observed.

[0113] (2) 125 μg / kg-TL-25-h1-hFc + TeNT group: 125 μL of TL-25-h1-hFc fusion protein solution (concentration of 20 μg / mL, i.e., injection dose of 125 μg / kg) was used instead of 125 μL of TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) to treat mice via the tail vein. The rest of the procedures were the same as those in the 25 μg / kg-TL-25-h1-hFc + TeNT group.

[0114] (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 / mouse) was used to replace 125 μL of TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg). The rest of the procedures were the same as those of the 25 μg / kg-TL-25-h1-hFc + TeNT group.

[0115] (4) 250 μg / kg-B-h3 + TeNT group: 125 μL of irrelevant antibody (B-h3) solution (concentration of 40 μg / mL, i.e., injection dose of 250 μg / kg) was used to replace 125 μL of TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg). The rest of the procedures were the same as those of the 25 μg / kg-TL-25-h1-hFc + TeNT group.

[0116] (5) PBS group: The TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) was replaced with an equal volume of PBS. The rest of the procedures were the same as those in the 25 μg / kg-TL-25-h1-hFc + TeNT group.

[0117] The results are shown in Table 5. The high dose of antibody (125 μg / kg) had an effect on 10 × LD 50 TeNT challenge can provide complete protection to mice, and low-dose antibody (25 μg / kg) can completely protect mice from 10 × LD 50 TeNT attacks can provide 75% protection.

[0118] Table 5. Evaluation of the preventive effect of anti-tetanus toxin antibodies

[0119] “ a ” indicates mice treated with TL-25-h1-hFc, TIG, B-h3, or PBS and exposed to TeNT for the indicated time.

[0120] “ b ” indicates that mice received 125 μg / kg or 25 μg / kg of antibody, 0.1 IU TIG, 250 μg / kg of B-h3, or PBS, respectively.

[0121] “ c ” indicates that 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 being treated with the indicated doses of antibodies.

[0122] “ d " indicates that KM mice were intraperitoneally injected with 10 × LD 50 TeNT.

[0123] 2. Evaluating whether anti-tetanus nanobodies have a protective effect after exposure to TeNT The evaluation method is as follows: 1. Mice: KM, 4 mice per group, 18-20 g.

[0124] 2. The challenge dose is 5 × LD 50 Each group of mice received a tail vein injection of 25 or 125 μg / kg TL-25-h1-hFc fusion protein solution, 1 IU TIG, 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-TL-25-h1-hFc fusion protein group: KM mice were intraperitoneally injected with tetanus toxin solution at a challenge dose of 5 × LD 50 / mouse, 1, 3, 6, 12 or 24 hours after the injection of tetanus toxin solution, 125 μL of nanobody TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) was injected into the tail vein of mice (4 mice were injected at each time point), and the death of mice was observed.

[0125] (2) TeNT + 125 μg / kg-TL-25-h1-hFc fusion protein group: 125 μL of nanoantibody TL-25-h1-hFc fusion protein solution (concentration of 20 μg / mL, i.e., injection dose of 125 μg / kg) was used instead of 125 μL of nanoantibody TL-25-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg). The rest of the operations were the same as those in the TeNT + 25 μg / kg-TL-25-h1-hFc fusion protein group, and the death of mice was observed.

[0126] (3) TeNT + 1IU / mouse-TIG group: 125 μL of human tetanus immunoglobulin TIG solution (concentration of 8 IU / mL, i.e., injection dose of 1 IU / mouse) was used instead of 125 μL of nanoantibody TL-25-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 that of the TeNT + 25 μg / kg-TL-25-h1-hFc fusion protein group, and the death of mice was observed.

[0127] (4) TeNT + 250 μg / kg -B-h3 group: 125 μL of irrelevant antibody (B-h3) solution (concentration of 40 μg / mL, i.e., injection dose of 250 μg / kg) was used instead of 125 μL of nanoantibody TL-25-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 that of the TeNT + 25 μg / kg-TL-25-h1-hFc fusion protein group, and the death of mice was observed.

[0128] (5) TeNT + PBS group: Replace 125 μL of the nanoantibody TL-25-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 was the same as that of the TeNT + 25 μg / kg-TL-25-h1-hFc fusion protein group, and the death of mice was observed.

[0129] The results are shown in Table 6. For 5 × LD 50 TeNT poisoning was still effective within 24 hours after exposure even when treated with only 0.5 μg / mouse (dose = 1 mg / kg antibody × 20 g / mouse) of TL-25-h1-hFc fusion protein.

[0130] Table 6. Evaluation of the therapeutic effect of anti-tetanus toxin antibodies

[0131]

[0132] “ a " indicates KM mice injected with 5 × LD 50 TeNT.

[0133] “ b ” indicates that mice were treated with the indicated dose of antibody 1 h, 3 h, 6 h, 12 h, or 24 h after exposure to TeNT.

[0134] “ c ” indicates that mice exposed to TeNT were treated with TL-25-h1-hFc, TIG, B-h3, and PBS, respectively, at the indicated times after exposure.

[0135] “ d ” indicates that mice exposed to TeNT received 25 μg / kg or 125 μg / kg TL-25-h1-hFc, 1 IU TIG, 250 μg / kg B-h3, or PBS at the specified time after exposure.

[0136] The above examples involve the following sequences: SEQ ID NO:1: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDAKNELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTQVTVSS。

[0137] SEQ ID NO:2:EVQLVESGGGLVQPGGSLRLSCAAS。

[0138] SEQ ID NO:3:MSWVRQAPGKGLEWVSA。

[0139] SEQ ID NO:4:YYKGTVKGRFTISRDDAKNELYLQMNSLQTEDTAVYYC。

[0140] SEQ ID NO:5:WGQGTQVTVSS。

[0141] SEQ ID NO:6:GFTFSNYD。

[0142] SEQ ID NO:7:LDAGGLKT。

[0143] SEQ ID NO:8:ATGFYPQYLLQHAPDVARTEDDA。

[0144] SEQ ID NO:9: 5'- gaggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgcgcggcctctggattcacattcagtaactacgacatgagctgggtccgccaggctccagggaaggggctcgagtgggtctcagctctagatgccggtggtcttaagacatactataaaggcaccgtgaagggccgattcaccatctccagagacgacgccaagaacgagttgtatctgcaaatgaacagcctgcaaactgaggacactgccgtgtattactgcgccacgggtttctatccacagtacttactccaacacgcaccagacgtcgcgcgcaccgaagacgacgcctggggccaggggacccaggtcaccgtctcctca-3'。

[0145] SEQ ID NO:10:

[0146] SEQ ID NO:11: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDAKNELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK。

[0147] SEQ ID NO:12: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS。

[0148] SEQ ID NO:13: 5'-gaagtgcagctggtggagagcggcggcggcctggtgcagcccggcggtagcctgcggctgtcctgcgccgccagcggcttcaccttcagcaactacgacatgagctgggtgagacaggcccccggcaagggcctggagtgggtgagcgccctggacgccggcggcctgaaaacctactacaagggcaccgtgaagggccggttcaccatctctcgtgatgatagtaagaacacactgtatctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccaccggcttttatccccagtacctgcttcagcacgccccagacgtggccagaaccgaggacgacgcctggggccagggcaccctggtgaccgtgagcagc-3'。

[0149] SEQ ID NO:14: 5'-gaagtgcagctggtggagagcggcggcggcctggtgcagcccggcggtagcctgcggctgtcctgcgccgccagcggcttcaccttcagcaactacgacatgagctgggtgagacaggcccccggcaagggcctggagtgggtgagcgccctggacgccggcggcctgaaaacctactacaagggcaccgtgaagggccggttcaccatctctcgtgatgatagtaagaacacactgtatctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccaccggcttttatccccagtacctgcttcagcacgccccagacgtggccagaaccgaggacgacgcctggggccagggcaccctggtgaccgtgagcagcgctagcgacaaaactcacacatgcccaccgtgccca gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgctgcatgaggctctgcacagccactacacgcagaagagcctctccctgtccccgggtaaatga-3'。

[0150] SEQ ID NO:15: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSALDAGGLKTYYKGTVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0151] 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 nanobody or antigen-binding fragment thereof against tetanus toxin, characterized in that: The nanobody or antigen-binding fragment thereof 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 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) has a sequence identity of more than 75% with A4-1).

3. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The amino acid sequence of the Nanobody or antigen-binding fragment thereof includes 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 antigen-binding fragment thereof includes 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 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.

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 includes any one of the following: B1) a nucleic acid molecule encoding the Nanobody 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; 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. A medicament for treating tetanus, 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 includes any of the following: M1) Use of the biomaterial of claim 8 in the preparation 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; 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 medicament according to claim 9; M3) 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 medicament according to claim 9 in the preparation of a product for preventing and / or treating a disease caused by Clostridium tetani; M4) 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 genetic material according to claim 8 in the preparation of a product for detecting Clostridium tetani; M5) 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 medicament according to claim 9 in the preparation of a product for neutralizing neurotoxins 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.

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