Nanobodies against tetanus toxin l-hn fragment and related biomaterials and applications thereof

Abstract

The application discloses a nanobody against tetanus toxin L-HN fragment and related biomaterials and application thereof, and belongs to the field of immunotherapy biotechnology and pharmaceuticals. The aforementioned nanobody or 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, and the amino acid sequence of the CDR3 is SEQ ID NO: 8, and the sequence of the complementarity determining region is defined according to the IMGT numbering system. The antibody only binds to TL-HN protein, and 0.3125 μg of the antibody can effectively block poisoning caused by a lethal dose of tetanus toxin (10*LD 50 ); after humanization, the antibody still has good affinity and neutralization activity, and an equivalent dose of the aforementioned antibody can still effectively block poisoning caused by a lethal dose of tetanus toxin (10*LD 50 ).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunotherapy biological medicine, and particularly relates to nanobodies against L-HN fragments of tetanus toxin and related biomaterials and applications thereof. BACKGROUND

[0002] Tetanus is an acute infectious disease caused by tetanus neurotoxin (TeNT) produced by Clostridium tetani, which is characterized by muscle spasm. TeNT is a potent neurotoxin that can invade the central nervous system, and in severe cases, respiratory muscle rigidity can cause respiratory arrest and even endanger life. TeNT is a 150kD polypeptide chain composed of three 50kD domains, which is post-translationally cleaved by bacterial or host proteases into an active two-chain form connected by a single disulfide bond: a light chain (A fragment) and a heavy chain (B fragment and C fragment), which are 50kD and 100kD, respectively.

[0003] The mortality rate of tetanus after onset is extremely high, and the mortality rate of severe patients is close to 100% without medical intervention. Even after active comprehensive treatment, the mortality rate worldwide is still 30% to 50%, which is a very serious potentially fatal disease. Tetanus infection does not confer lifelong immunity, so tetanus immunization is needed after trauma to prevent tetanus.

[0004] Passive immunotherapy has always been the main strategy for eliminating tetanus toxin in tetanus patients. Commonly used passive immunization preparations in clinical practice include "tetanus antitoxin injection (TAT)", "horse tetanus" (anti-toxin TAT) and "human tetanus" (human tetanus immunoglobulin HTIG). However, there are many obstacles in the production and use of animal-derived therapeutic drugs, such as long immunization period, potential hypersensitivity of the recipient, etc. HTIG is a blood product, which carries the risk of transmitting known and unknown infectious diseases, and is relatively difficult to obtain, making it difficult to meet market demand. Therefore, specific TeNT antibodies produced in vitro without the need for an immunization process would be a better alternative to HTIG or antitoxin.

[0005] Nanobodies (Nbs) are naturally occurring antibody molecules in Camelidae, which have the characteristics of small relative molecular mass, strong tissue penetration, high stability, and strong specificity, overcoming many limitations of traditional monoclonal antibodies. For the diagnosis or treatment of tetanus, developing high-affinity tetanus toxin neutralizing nanobodies has become a hot spot of researchers.

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

[0007] 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.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

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

[0010] In a first aspect, the present invention provides 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-16;

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

[0012] 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;

[0013] The amino acid sequence of FR1 of the TL-16 is SEQ ID NO: 2, the amino acid sequence of FR2 of the TL-16 is SEQ ID NO: 3, the amino acid sequence of FR3 of the TL-16 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-16 is SEQ ID NO: 5.

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

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

[0016] 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.

[0017] In a second aspect, the present invention provides an anti-tetanus toxin heavy chain antibody, wherein the heavy chain antibody comprises the aforementioned Nanobody.

[0018] The above-mentioned 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.

[0019] The heavy chain antibody is TL-16-hFc, and the amino acid sequence of the TL-16-hFc includes SEQ ID NO:11 or SEQ ID NO:15.

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

[0021] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial is any one of the following:

[0022] B1) a nucleic acid molecule encoding the aforementioned Nanobody or antigen-binding fragment thereof, or the aforementioned heavy chain antibody;

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

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

[0025] B4) 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).

[0026] 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.

[0027] 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 above-mentioned proteins in a host cell. The expression cassette may also include all regulatory sequences necessary for expressing the nucleic acid molecule for any of the above-mentioned proteins or the DNA for the above-mentioned RNA molecule. The regulatory sequences are capable of directing the coding sequence to express any of the above-mentioned proteins or the DNA for the above-mentioned RNA molecule in a suitable host cell under compatible conditions. The 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 above-mentioned 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. The promoter sequence contains transcriptional regulatory sequences that mediate expression of the protein or the DNA for the above-mentioned 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.

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

[0029] F1, Fab antibody;

[0030] F2, Fv antibody;

[0031] F3, single-chain antibody;

[0032] F4, Fab′ fragment.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The term "minimum recognition unit (MRU)" refers to a structure containing only a single CDR in the variable region, with a molecular mass of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0037] The term "Fab antibody" refers to a heterodimer formed by the heavy chain (Fd) and 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 Fab antibodies (Fab fragments) 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 a Fab (comprising approximately 225 amino acid residues, including the VH, CH1, and part of the hinge region).

[0038] The term "Fv antibody" refers to a compound composed solely of the heavy and light chain variable regions 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 to express them, and then assembling them 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).

[0039] The term "single-chain antibody" (ScFv) refers to a polypeptide composed of the heavy and light chain variable regions of an antibody linked by a short peptide. ScFv can be prepared by linking 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.

[0040] In a fourth aspect, the present invention provides genetic material, wherein the genetic material is any one of the following:

[0041] g1) a nucleic acid molecule encoding the aforementioned Nanobody or antigen-binding fragment thereof, or the aforementioned heavy chain antibody;

[0042] g2) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in g1).

[0043] 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.

[0044] The medicament further includes a physiologically or pharmaceutically acceptable excipient, diluent or carrier.

[0045] 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.

[0046] 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 (e.g., polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), and enteric-soluble carrier materials (e.g., cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred.

[0047] In a sixth aspect, the present invention claims protection for any of the following applications:

[0048] 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;

[0049] M2) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody in the preparation of the aforementioned medicament;

[0050] 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;

[0051] 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;

[0052] 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;

[0053] 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.

[0054] The anti-tetanus toxin nanobody provided by the present invention can effectively neutralize tetanus toxin. Experiments have shown that the fusion protein TL-16-hFc obtained by fusing the nanobody TL-16 with the Fc segment (hFc) of human immunoglobulin has good specificity and only binds to the TL-HN protein. 0.3125μg of TL-16-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-16-h1-hFc can still effectively block the lethal dose of tetanus toxin (10×LD 50 ) poisoning caused by. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] 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.

[0056] Figure 2 This is the SDS-PAGE electrophoresis detection of the purified anti-tetanus nanobody fusion protein.

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

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

[0059] Figure 5 Affinity detection of anti-tetanus nanobody fusion protein.

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

[0061] Figure 7 Binding activity assay for humanized anti-tetanus nanobody fusion protein.

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

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

[0064] 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.

[0065] 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.

[0066] The following examples were processed using GraphPad Prism 8 statistical software. The experimental results are expressed as mean ± standard deviation. The significance of the difference in protection between the two groups was compared using the Log-rank test. The difference was considered statistically significant when p < 0.05.

[0067] Recombinant TL-HN antigen (or recombinant TL-HN protein): Prepared in 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 tetanustoxin. VACCINE (2023). doi:10.1016 / j.vaccine.2023.09.032," in Materials and Methods, section 2.2.

[0068] pTSE-hFc vector: described in the non-patent literature "Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against protective antigens V of Yersinia pestis [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271", which is available to the public from the Academy of Military Medical Sciences of the People's Liberation Army. This biological material is only used for the relevant experiments of the invention and cannot be used for other purposes.

[0069] Example 1, construction of anti-tetanus nanobody library

[0070] 1. Camel immunization

[0071] The recombinant TL-HN antigen of tetanus was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881) and shaken to emulsify it. The emulsion was injected subcutaneously into healthy adult bactrian camels using a multi-point injection method. Booster immunization was performed every two weeks. Freund's incomplete adjuvant (Sigma, F5506) was used for immunization except for the first time when Freund's complete adjuvant was used.

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

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

[0074] 3. Nested PCR amplification of VHH gene fragments

[0075] The isolated peripheral blood mononuclear cells PBMC were subjected to total RNA extraction using the OMEGA E.Z.N.A Total RNA kit I kit (OMEGA, R6834), and then reverse transcribed to obtain cDNA using the Invitrogen Superscript III First-strandsynthesis system for RT-PCR kit (Invitrogen, 18080-051).

[0076] Nested PCR amplification of VHH gene fragments: 1) First round of PCR: using synthesized cDNA as template, IgG specific upstream primer CALL001 and downstream primer CALLOO2 were used for PCR amplification of antibody leader sequence and CH2 sequence; 2) using the purified DNA product of the first round as template, primers VHH-F and VHH-R were used for second round of PCR amplification to obtain VHH gene.

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

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

[0079] 4. Electroporation products

[0080] 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 E. coli 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 pfu.

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

[0082] 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 / 2.5M NaCl solution (1 L solution contains 200 g PEG6000, 146.25 g NaCl) to obtain an anti-tetanus specific phage nanobody library.

[0083] The purified recombinant TL-HN protein was used as the antigen and the solid-phase screening strategy (for experimental protocols, refer to Phage Display: A General Protocol / (US) Clarkson (T) and Lowman (HB); translated by Ma Lan et al., Chemical Industry Press, May 2008) was used to screen the immune library constructed above. The specific method is as follows:

[0084] To each immune hole, 1 mL of antigen diluted with 0.1 M NaHC03coating solution was added, and coated at 4°C overnight. The coating concentration of each round of panning was 20, 10 μg / mL, respectively. The next day, the immune tube was washed with PBS for 2 times, 3 min each time, and then blocked with 2% blocking solution (bovine serum albumin) at 37°C for 2 h. After blocking, the tube was washed with PBST (1 x PBS + 0.1% Tween-20) for 3 times, and then washed with PBS for 3 times. The anti-tetanus specific phage nanobody library was adjusted to an appropriate concentration with blocking solution, added to the immune tube, and placed at room temperature for 2 h. Then, the tube was combined with 20 min at a low speed of 200 rpm. The tube was washed with PBST for 10 times, and then washed with PBS for 5 times. After washing, 1 mL of elution solution (glycine-hydrochloric acid, pH 2.2) was added to each hole, and the tube was shaken at 400 rpm at room temperature for 20 min. The elution solution in the hole of interest was taken out, and 50-60 μL of neutralization solution (1 M Tris-HCl, pH 8.0) was added for neutralization. E. coli TG1 cells in the logarithmic growth phase were infected, and the tube was placed at room temperature for 30 min and cultured at 37°C for 1 h. Phage was produced and purified for the next round of screening. The same screening process was repeated for 2 rounds, and the enrichment results are shown in Table 2.

[0085] Table 2. Analysis of the enrichment degree of the anti-tetanus phage nanobody library

[0086] Number of screenings Amount of input (pfu) Output (pfu) Output / Input Enrichment multiple 1 <![CDATA[5×10 11 ]]> <![CDATA[4.2×10 7 ]]> <![CDATA[8.4×10 -5 ]]> --- 2 1 x 10 11 ]]> <![CDATA[1.95×10 7 ]]> <![CDATA[1.95×10 -4 ]]> 2.3

[0087] After 2 rounds of screening, single clones with good separation were selected from the plate containing phage, inoculated into a 96-well plate containing 2YT-GA medium (1 L 2YT medium containing 16 g Typtone, 10 g Yeast extract, 5 g NaCl, 100 μg / mL ampicillin, 20% glucose) (250 μL / well), and 2 negative control wells (without cloning) were left. The tube was cultured at 37°C until the logarithmic growth phase. M13KO7 helper phage was added at a ratio of MOI ≈ 50, and the deep well plate containing the single phage clone was added with 100 μL / well. The tube was infected at room temperature for 30 min, and then cultured at a low speed of 150 rpm at 37°C for 1 h. The deep well plate was centrifuged at room temperature (2000 rpm, 10 min) to discard the supernatant, and then induced with 1 mM of arabinose to express the phage particles displaying the variable region of the antibody.

[0088] Example 3. Identification of specific nanobody positive clones by phage-ELISA

[0089] The recombinant TL-HN protein was used as the antigen for coating. At the same time, the adjacent column of each antigen group was used for parallel coating with an irrelevant antigen as a negative control, and the coating was carried out at 4°C overnight. The overnight coated ELISA plate was taken, the coating solution was discarded, and the plate was washed 6 times with PBST. The blocking solution (3g skim milk powder in 100mL PBS) was added for blocking at 200μL / well and incubated at 37°C for 2h. The blocking solution in the ELISA plate was discarded, and the induced expression phage supernatant was added to the corresponding ELISA wells at 100μL / well and incubated at 37°C for 1.5h. The plate was washed 6 times with PBST and freshly prepared with 0.2μg / ml HRP-labeled M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) and added to the plate at 100μL / well and incubated at 37°C for 45min. The plate was washed 6 times with PBST and the color development solution (9mL color development solution, 1mL 10× OPD, 10 μL 30% H2O2) was added to the ELISA plate at a rate of 10 μL / well and the plate was developed in the dark for 15-20 min. 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 well.

[0090] 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, the camel-derived nanoantibody TL-16, which specifically binds to TL-HN.

[0091] The amino acid sequence of TL-16 is shown in SEQ ID NO: 1, including 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-16 is shown in SEQ ID NO. 9.

[0092] The sequences of the complementarity determining regions (CDRs) are defined according to the IMGT numbering system.

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

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

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

[0096] Using conventional molecular biology techniques, 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 connecting the gene of the Fc domain of human immunoglobulin to the pCMV vector) was replaced with a DNA molecule with a nucleotide sequence of SEQ ID NO: 9 (the gene encoding the TL-16 nanobody), while keeping the other nucleotides of the pTSE-hFc vector unchanged. The recombinant expression plasmid pTSE-TL-16-hFc containing the gene encoding the TL-16-hFc fusion protein was obtained. This vector expresses a protein with an amino acid sequence of SEQ ID NO: 11 (named TL-16-hFc, whose nucleotide sequence is SEQ ID NO: 10).

[0097] The sequences of the complementarity determining regions of the heavy chain antibodies are defined according to the IMGT numbering system.

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

[0099] The pTSE-TL-16-hFc fusion protein expression plasmid constructed in step 1 was transfected into FreeStyle TM In HEK293-F cells (Invitrogen, R79007), cell activity was monitored after 48 hours. When the cell activity 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-16-hFc fusion protein.

[0100] The purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 2 As shown: The molecular weight of the antibody is consistent 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).

[0101] Example 5. Evaluation of the properties of nanobody fusion proteins

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

[0103] The recombinant TL-HN protein was diluted to 2 μg / mL with carbonate coating buffer (pH 9.6), and 100 μL / well was added to the ELISA plate and coated overnight at 4°C. The next day, the plate was washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of blocking solution (3% skim milk powder) was added and blocked at 37°C for 2 h. The blocking solution was discarded, the plate was washed 6 times with PBST, and 100 μL / well of a 2-fold equal dilution of the nanoantibody fusion protein TL-16 (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 the plate was incubated at 37°C for 45 min. The plate was washed 6 times with PBST, and 100 μL was added to each well. OPD substrate colorimetric solution was used to develop color in the dark for 5-15 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 value was measured by 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.

[0104] The results are as follows Figure 3 As shown 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 showed that the binding half effective concentration (EC 50 ) is 0.01515nM.

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

[0106] Tetanus TL-HN, THC, TL, and THN proteins were prepared by the inventor's 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".

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

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

[0109] The EL-HN protein was prepared by the inventors’ laboratory according to the following article “Li Z, Lu J, Tan X, Wang R, Xu Q, Yu Y, et al. Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E. Toxins (Basel) (2022) 14(2). doi:10.3390 / toxins14020135” in section 5.2 of the Materials and Methods.

[0110] The FL-HN protein was prepared by the inventor's 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 a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins (Basel) (2023) 15(3). doi: 10.3390 / toxins15030200).

[0111] Recombinant human Siglec-15 protein was purchased from Beijing Sino Biological Technology Co., Ltd.

[0112] The experimental method is as follows:

[0113] 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 with carbonate coating buffer, and 100 μL / well was added to a 96-well plate and incubated at 4°C overnight. The plate was washed 6 times with PBST (0.1% Tween-20), and 200 μL / well of ELISA blocking solution was added and blocked at 37°C for 2 h. The plate was washed 6 times with PBST, and the plate was incubated at 10°C for 10 min. Anti-tetanus antibody (TL-16-hFc fusion protein) was added at 0 μL / well 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 and incubated at 37°C for 45 min. The plate was washed six times with PBST and developed with peroxidase substrate solution at 100 μL / well. The color was developed in the dark for 15 min and the color was observed. After complete color development, 100 μL of 2 M sulfuric acid was added to each well to terminate the reaction. The plate was read on a microplate reader and analyzed using GraphPad Prism 8 software.

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

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

[0116] The experimental method is as follows:

[0117] Add 200 μL of HBS-EP+ buffer to each well of the probe 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-16 to 200 nM and the TL-HN protein to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.6 nM 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 processing and analysis, calculating parameters such as affinity.

[0118] The results are as follows Figure 5 As shown, TL-16-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-16-hFc and recombinant TL-HN protein was calculated to be 10.1 nM, indicating good affinity between the antigen and antibody, suitable for subsequent development.

[0119] Example 6. Evaluation of Neutralizing Activity of Anti-Tetanus Toxin Nanobody-hFc Fusion Protein

[0120] 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:

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

[0122] 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.

[0123] Tetanus toxin (TeNT) solution: a solution obtained by dissolving tetanus toxin (purchased from China Food and Drug Inspection Institute) in the aforementioned diluent.

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

[0125] Human tetanus immunoglobulin TIG solution: a solution obtained by dissolving human tetanus immunoglobulin (purchased from China Food and Drug Inspection Institute) in the aforementioned diluent.

[0126] The experimental groups are as follows:

[0127] TeNT 10×LD50 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.

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

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

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

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

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

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

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

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

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

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

[0138]

[0139]

[0140] Example 7 Humanization and activity evaluation of anti-tetanus toxin nanobody-hFc fusion protein

[0141] 1. 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-16-h1, whose amino acid sequence is shown in SEQ ID NO: 12 and nucleotide sequence is shown in SEQ ID NO: 13.

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

[0143] 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-16-h1-hFc with the encoding gene nucleotide sequence of SEQ ID NO: 14 and the expressed amino acid sequence of SEQ ID NO: 15.

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

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

[0146] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was prepared by referring to the method in Example 4. Figure 6 As shown: The molecular weight of the fusion protein is consistent with expectations, 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).

[0147] (2) Detection of the binding activity and specificity of the humanized nanobody-hFc fusion protein TL-16-h1-hFc with the recombinant TL-HN protein

[0148] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was used to replace the TL-16-hFc fusion protein. The remaining operations were the same as 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”.

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

[0150] (3) Detection of affinity between humanized nanobody-hFc fusion protein TL-16-h1-hFc and recombinant TL-HN protein

[0151] The humanized nanobody-hFc fusion protein TL-16-h1-hFc was used to replace the test antibody TL-16. 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)".

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

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

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

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

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

[0157]

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

[0159] 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:

[0160] 1. Mice: KM mice (purchased from Beijing Sibeifu Biotechnology Co., Ltd.), each weighing 18-20 g. Tetanus toxin solution, nanobody TL-16-h1-hFc fusion protein solution, and TL-16-h1-hFc fusion protein solution were prepared as described in Example 6. Unrelated antibody (B-h3) solution was prepared by dissolving the unrelated antibody (B-h3) in a diluent.

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

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

[0163] (2) 125 μg / kg-TL-16-h1-hFc+TeNT group: 125 μL of TL-16-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-16-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 of the 25 μg / kg-TL-16-h1-hFc+TeNT group.

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

[0165] (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-16-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-16-h1-hFc+TeNT group.

[0166] (5) PBS group: The TL-16-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-16-h1-hFc+TeNT group.

[0167] The results are shown in Table 5. The high dose 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 protect mice from 10×LD 50 TeNT attacks provide half protection.

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

[0169]

[0170]

[0171] “ a ” indicates mice treated with TL-16-h1-hFc, TIG, B-h3, or PBS and exposed to TeNT for the indicated time.

[0172] “ b ” indicates mice received 125 μg / kg or 25 μg / kg of antibody, 0.1 IU TIG, 250 μg / kg B-h3, or PBS, respectively.

[0173] “ 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 treatment with the indicated doses of antibodies.

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

[0175] 2. Evaluating whether anti-tetanus nanobodies have a protective effect after exposure to TeNT

[0176] The evaluation method is as follows:

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

[0178] 2. The challenge dose is 5×LD 50 Each group of mice received a tail vein injection of 25 or 125 μg / kg TL-16-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:

[0179] (1) TeNT+25 μg / kg-TL-16-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-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e., injection dose of 25 μg / kg) was treated with mice via tail vein (4 mice were injected at each time point), and the death of mice was observed.

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

[0181] (3) TeNT + 1 IU / 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 nanobody TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg), and the rest of the operations were the same as in the TeNT + 25 μg / kg - TL-16-h1-hFc fusion protein group, and the mice were observed for death.

[0182] (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 nanobody TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg), and the rest of the operations were the same as in the TeNT + 25 μg / kg - TL-16-h1-hFc fusion protein group, and the mice were observed for death.

[0183] (5) TeNT + PBS group: an equal volume of PBS was used instead of 125 μL of nanobody TL-16-h1-hFc fusion protein solution (concentration of 4 μg / mL, i.e. injection dose of 25 μg / kg), and the rest of the operations were the same as in the TeNT + 25 μg / kg - TL-16-h1-hFc fusion protein group, and the mice were observed for death.

[0184] The results are shown in Table 6. For 5xLD 50 TeNT intoxication, even with only 0.5 μg / mouse (dose = 1 mg / kg antibody x 20 g / mouse) of TL-16-h1-hFc fusion protein treatment was effective within 24 hours after exposure.

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

[0186]

[0187]

[0188] “ a ” indicates that KM mice were injected with 5xLD50 TeNT.

[0189] “ b ” indicates that the mice were treated with the indicated dose of antibody 1 h, 3 h, 6 h, 12 h or 24 h after exposure to TeNT.

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

[0191] “ d ” indicates that mice exposed to TeNT received 25 μg / kg or 125 μg / kg TL-16-h1-hFc, 1 IU TIG, 250 μg / kg B-h3, or PBS at the specified time after exposure.

[0192] The above examples involve the following sequences:

[0193] SEQ ID NO: 1:

[0194] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNAK NELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS.

[0195] SEQ ID NO:2: EVQLVESGGGLVQPGGSLRLSCAAS.

[0196] SEQ ID NO:3: MSWVRQAPGKGLEWVSA.

[0197] SEQ ID NO:4:YYKGTVKGRFTISRDNAKNELYLQMNSLQTEDTAVYYC.

[0198] SEQ ID NO: 5: WGQGTLVTVSS.

[0199] SEQ ID NO:6: GFTFNNYD.

[0200] SEQ ID NO:7: LDADGLKT.

[0201] SEQ ID NO:8: ATGFYPQYLLQHAPDVARTEDDA.

[0202] SEQ ID NO:9:

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

[0204] SEQ ID NO:10:

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

[0206] SEQ ID NO:11:

[0207] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNAKNELYLQMNSLQTEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK。

[0208] SEQ ID NO:12

[0209] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSS。

[0210] SEQ ID NO:13

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

[0212] SEQ ID NO:14

[0213] 5'-GAA GTC GAC TTA GAG ATC GGT GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAActggctttacctttaataactacgacatgagctgggtgcggcaggcccccggcaagggcctggaatgggtgagcgccctggacgccgacggcctgaaaacctactacaagggcaccgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgcgccaccggcttctacccccagtacttactgcagcacgcccccgacgtggcccggaccgaggacgacgcctggggccagggcaccctggtgaccgtgagcagcgctagcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgctgcatgaggctctgcacagccactacacgcagaagagcctctccctgtccccgggtaaatga-3'.

[0214] SEQ ID NO:15

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYDMSWVRQAPGKGLEWVSALDADGLKTYYKGTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGFYPQYLLQHAPDVARTEDDAWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0216] 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 amino acid sequence of FR1 is SEQ ID NO: 2; The amino acid sequence of FR2 is SEQ ID NO: 3; The amino acid sequence of FR3 is SEQ ID NO: 4 or has greater than 75% identity with SEQ ID NO: 4; The amino acid sequence of FR4 is SEQ ID NO:

5.

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

1.

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

12.

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

6. The heavy chain antibody according to claim 5, characterized in that The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1 or has 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 is 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 4, or the heavy chain antibody according to any one of claims 5 to 7; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3).

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

10. Application, characterized in that, The application is any of the following: M1) Use of the biomaterial of claim 8 in the preparation of a Nanobody or antigen-binding fragment thereof 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 biomaterial 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 tetanus toxin 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 tetanus toxin secreted by Clostridium tetani.

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