Neutralizing antibodies against botulinum toxin l-hn antigen and related biological materials and applications

Abstract

The application discloses neutralizing antibodies against botulinum toxin L-HN antigen and related biological materials 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:2, the amino acid sequence of the CDR2 is SEQ ID NO:3, the amino acid sequence of the CDR3 is SEQ ID NO:4, and the sequence of the complementarity determining region is defined according to the IMGT numbering system. The antibody can effectively neutralize endotoxin, has the advantages of rapid preparation, simple structure and convenience in modification into a multispecific antibody, and is expected to be developed into a high-efficiency and safe anti-E-type botulinum toxin neutralizing nanobody, and is used for replacing serum products supplied in the market to meet the demand of human beings.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunotherapy biotechnology pharmaceuticals, and particularly relates to neutralizing antibodies against botulinum neurotoxin L-HN antigen and related biological materials and applications thereof. BACKGROUND

[0002] Botulinum neurotoxin (BoNT) is a kind of neurotoxin produced by Clostridium botulinum under anaerobic conditions, and is the most toxic biological toxin known. The illegal use of botulinum neurotoxin is also gradually increasing. According to the neutralization of specific antisera, botulinum neurotoxin is mainly divided into the traditional A-G seven serotypes and new botulinum neurotoxin. Among them, types A, B, E and F mainly cause human botulinum toxin poisoning, and types C and D can cause animal and poultry botulinum poisoning, and are also one of the causes of wild bird and fish botulinum poisoning.

[0003] The symptoms of botulism are mainly muscle paralysis and difficulty breathing, and the reaction after poisoning is fast and lethal. Botulinum toxin poisoning is generally treated with antitoxin, but the antitoxin can only neutralize free toxins in the blood or body fluid and is ineffective against toxins that have entered the cells. In addition, the production period is long, the production batch is unstable, and there are hidden dangers of allergic reactions and viral contamination. The vaccines commonly used for prevention abroad are PBT pentavalent vaccine and heptavalent vaccine, and there is no vaccine on the market in China.

[0004] Antibodies can be used to prevent and treat botulism in the short term, and have broad application prospects. Nanobody (Nb) is a novel antigen recognition tool that does not contain light chains and has a long CDR3 region, can exist independently and stably in vitro, and has a relative molecular mass of only 1 / 10 of that of traditional complete antibodies. It has the advantages of strong reconfiguration, low immunogenicity, small side effects, easy screening and expression, high affinity and stability, and has been proven to have great value as a therapeutic molecule and clinical diagnostic tool.

[0005] Although food poisoning type, infant poisoning type, trauma-induced type, adult intestinal colonization type and other botulism events have been rarely reported, but with the illegal use of botulinum toxin and the increasing use of botulinum toxin as a cosmetic preparation, the number of botulism events has also increased. Therefore, it is very important to apply nanobody technology to develop efficient and safe anti-botulinum toxin neutralizing nanobodies. SUMMARY

[0006] The technical problem to be solved by the present application is to develop a specific nanobody that can efficiently neutralize E-type botulinum toxin, provide candidate antibodies for botulism diagnosis and treatment, and lay a foundation for further development of multivalent or multispecific broad-spectrum neutralizing antibodies that can simultaneously neutralize multiple botulinum toxins.

[0007] To solve the above problems, the present application provides a nanobody targeting botulinum toxin or an antigen-binding fragment containing the nanobody.

[0008] The nanobody targeting botulinum toxin or the antigen-binding fragment containing the nanobody provided by the present application, wherein the nanobody has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 comprises SEQ ID NO: 2, the amino acid sequence of the CDR2 comprises SEQ ID NO: 3, and the amino acid sequence of the CDR3 comprises SEQ ID NO: 4.

[0009] The amino acid sequence of the CDR1 is SEQ ID NO: 2, the amino acid sequence of the CDR2 is SEQ ID NO: 3, and the amino acid sequence of the CDR3 is SEQ ID NO: 4.

[0010] The botulinum toxin is specifically botulinum toxin type E.

[0011] The above CDR is a sequence defined according to the analysis results of the IMGT system.

[0012] The nanobody described herein generally includes a VHH composed of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment contains at least a part of the nanobody, which is sufficient to endow the fragment with the ability to specifically bind to botulinum toxin type E.

[0013] The four framework regions can be FR1, FR2, FR3 and FR4.

[0014] The amino acid sequence of the FR1 is SEQ ID NO: 5 or has more than 90% identity with SEQ ID No. 5;

[0015] The amino acid sequence of the FR2 is SEQ ID NO: 6;

[0016] The amino acid sequence of the FR3 is SEQ ID NO: 7 or has more than 90% identity with SEQ ID No. 7;

[0017] The amino acid sequence of the FR4 is SEQ ID NO: 8 or has more than 90% identity with SEQ ID No. 8.

[0018] In the nanobody or antigen-binding fragment described above, the nanobody can be any one of the following:

[0019] A1) a Nanobody comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 12;

[0020] A2) a Nanobody obtained after attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 12.

[0021] The protein tag refers to a polypeptide or protein fused with a protein of interest for the expression, detection, tracing and / or purification of the protein of interest. The protein tag can be a His tag, a Flag tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, an Fc fragment of immunoglobulin G, etc.

[0022] The amino acid sequence of the Nanobody can be specifically SEQ ID NO: 1 or SEQ ID NO: 12.

[0023] In the present application, the protein tag is an Fc fragment of human immunoglobulin G (hFc).

[0024] In the above Nanobody, the Nanobody consists of the complementarity determining region and the framework region.

[0025] The present application also provides a heavy chain antibody containing the aforementioned Nanobody and an Fc region of an immunoglobulin.

[0026] In the above heavy chain antibody, the heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region includes SEQ ID NO: 1 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID NO: 1.

[0027] In the above heavy chain antibody, the amino acid sequence of the heavy chain antibody includes SEQ ID NO: 11 or SEQ ID NO: 15.

[0028] In the above heavy chain antibody, the amino acid sequence of the heavy chain antibody can be specifically SEQ ID NO: 11 or SEQ ID NO: 15.

[0029] The Fc region herein is the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. The Fc region includes native sequence Fc regions and variant Fc regions. A variant Fc region can include a human variant Fc region (an Fc region of a human immunoglobulin comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions). In one embodiment of the application, the Fc region specifically consists of two constant domains (CH2 and CH3). The immunoglobulin Fc region can be an Fc region of a human immunoglobulin. The human immunoglobulin can be a human IgGl, IgG2, IgG3, or IgG4.

[0030] The term "antibody" in the present application is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies with the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the light chain constant region is attached by a disulfide bond to the first constant region of the heavy chain, while the light chain variable region is attached to the variable region of the heavy chain. Particular amino acid residues at the interface between the variable regions of the light and heavy chains form an interface.

[0031] The term "single domain antibody (VHH)", "nanobody" in the present application has the same meaning, refers to the variable region of the heavy chain of a clone antibody, which is constructed to consist of only one heavy chain variable region, and has the minimum antigen binding fragment with complete function.

[0032] The term "nanobody" (single domain antibody) (VHH) as described above is a polypeptide consisting of the variable region of the heavy chain of an antibody. The single domain antibody can be prepared by expressing the variable region of the heavy chain of an antibody (VH) by genetic engineering to obtain an antibody containing only the VH fragment. The ability of the single domain antibody to bind to an antigen and its stability are substantially the same as those of a complete antibody. The term "antigen binding fragment" as described above refers to an antigen binding fragment of an antibody and an antibody analog, which generally includes at least part of the antigen binding region or variable region (e.g., one or more CDRs) of a parent antibody. The antigen binding fragment retains at least some of the binding specificity of the parent antibody. Generally, the antigen binding fragment retains at least 10% of the parent binding activity when activity is expressed on a molar basis. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target.

[0033] The antigen-binding fragment can be a whole antibody, a fusion antibody, an antibody drug conjugate, a Fab fragment, an Fv fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody (ScFv), or a minimum recognition unit (MRU) containing the nanobody.

[0034] The term "Fab fragment" is a heterodimer composed of the variable region of the heavy chain VH and the first constant region CH1 (Fd) and the entire light chain, bound by disulfide bonds, containing only one antigen-binding site. The heavy chain Fd refers to about 1 / 2 of the H chain portion in Fab (about 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0035] The term "Fv fragment" refers to a vector containing VH and VL genes, respectively, co-transfected into cells to express, respectively, and then assembled into a functional Fv antibody; or a stop codon is set between VH and VL in the vector, two small protein fragments are expressed, respectively, and then combined by non-covalent bonds to form an Fv antibody (Fv fragment).

[0036] The term "Fab' fragment" contains one light chain and a portion of one heavy chain containing a VH domain and a CH1 domain and a region between CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0037] The term "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0038] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of the whole antibody, which can bind to the corresponding antigen.

[0039] The term "variable" in the present invention refers to the fact that certain portions of the variable domains differ in sequence among antibodies, which accounts for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs that form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as participation in complement-dependent cytotoxicity.

[0040] In some embodiments, the Nanobodies described in the present invention can be truncated at the N- or C-terminus to comprise only part of FR 1 and / or FR 4, or lack one or both of those framework regions, as long as antigen binding and specificity are essentially maintained.

[0041] In the present invention, the Nanobody is designated Nanobody E22.

[0042] The present invention also provides biological materials related to the Nanobody described hereinbefore, which can be any one of the following:

[0043] B1) a nucleic acid molecule encoding the Nanobody or antigen binding fragment described hereinbefore;

[0044] B2) an expression cassette comprising the nucleic acid molecule of B1);

[0045] B3) a recombinant vector comprising the nucleic acid molecule of B1);

[0046] B4) a recombinant vector comprising the expression cassette of B2);

[0047] B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

[0048] B6) a recombinant microorganism comprising the expression cassette of B2);

[0049] B7) a recombinant microorganism comprising the recombinant vector of B3);

[0050] B8) a recombinant microorganism comprising the recombinant vector of B4).

[0051] In the above biological material, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0052] In the above biological material, the expression cassette of B2) refers to DNA capable of expressing the nanobody in a host cell, which can include not only a promoter for initiating transcription of the nanobody-encoding gene, but also a terminator for terminating transcription of the nanobody-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0053] The recombinant vector containing the expression cassette can be constructed using an existing expression vector.

[0054] In the above biological material, the vector can be a plasmid, cosmid, bacteriophage or viral vector.

[0055] In the above biological material, the recombinant vector can be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into the nanobody-hFc fusion protein expression vector pTSE-hFc.

[0056] In the above biological material, the microorganism can be bacteria (such as E. coli), yeast, algae or fungi.

[0057] In the above biological material, the nucleic acid molecule of B1) can be a nucleic acid molecule encoding the nanobody described above, wherein the CDR1-encoding gene is nucleotides 100-150 of SEQ ID NO: 9, the CDR2-encoding gene is nucleotides 175-288 of SEQ ID NO: 9, and the CDR3-encoding gene is nucleotides 286-333 of SEQ ID NO: 9.

[0058] In the above biological material, the nucleic acid molecule of B1) can be any one of the following:

[0059] C1) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 9;

[0060] C2) a DNA molecule hybridizing to the DNA molecule defined in C1) under stringent conditions and encoding the nanobody;

[0061] C3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to any one of the DNA sequences defined in C1) to C2) and encoding the nanobody.

[0062] The stringent conditions can be as follows: hybridization at 50°C in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 2xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in lxSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 0.5xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 50°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing at 65°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 65°C in a solution of 6xSSC, 0.5% SDS, followed by rinsing the membrane once each with 2xSSC, 0.1% SDS and lxSSC, 0.1% SDS.

[0063] The nucleotide sequence of the E22-encoding gene of the nanobody of B1) of the present application can be easily mutated by a person of ordinary skill in the art using known methods, such as methods of directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of E22 of B1) of the present application, as long as they encode the nanobody and have the activity of nanobody E22, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0064] The present application also provides a method for preparing the nanobody described above, which can comprise the following steps: introducing a nucleic acid molecule encoding the nanobody described above into a recipient cell to obtain a transgenic cell expressing the nanobody, and culturing the transgenic cell to obtain the nanobody.

[0065] Further, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0066] In the above method, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above can be specifically any one of the following:

[0067] C1) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 13;

[0068] C2) a DNA molecule hybridizing to the DNA molecule defined in C1) under stringent conditions and encoding the nanobody;

[0069] C3) DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with any of the DNA sequences defined in C1)-C2) and encoding said Nanobody.

[0070] Further, the recipient cell can be a microbial cell, such as a bacterium (e.g. E. coli), a yeast, an algae or a fungus.

[0071] In particular embodiments of the application, the recipient cell can be a FreeStyle TM HEK293-F cell.

[0072] The present application also provides a Nanobody fusion protein, which is a fusion of the Nanobody or antigen binding fragment as described hereinbefore with another molecule, which can include the Fc domain of an immunoglobulin, a fluorescent protein or a VHH with a different specificity.

[0073] In particular embodiments of the application, the other molecule is the Fc domain of a human immunoglobulin.

[0074] In particular embodiments of the application, the amino acid sequence of the Fc domain of a human immunoglobulin as described hereinbefore is positions 124-350 of SEQ ID NO: 11.

[0075] In particular embodiments of the application, the Nanobody fusion protein as described hereinbefore can be any one of the following:

[0076] M1) a fusion protein having an amino acid sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 15;

[0077] M2) a protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 15.

[0078] In the Nanobody fusion protein as described hereinbefore, the nucleic acid molecule encoding the fusion protein can be any one of the following:

[0079] D1) a DNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 14;

[0080] D2) a DNA molecule hybridizing to the DNA molecule defined in D1) under stringent conditions and encoding the fusion protein;

[0081] D3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with any of the DNA sequences defined in D1)-D2) and encoding the fusion protein.

[0082] The mutation contained in the above-mentioned 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology means a mutation in the framework region of the aforementioned antibody.

[0083] The present application also provides an ELISA detection kit for botulinum toxin, which comprises the nanobody, the biomaterial, or the fusion protein described above.

[0084] The present application also provides any one of the following uses:

[0085] Ml) use of the aforementioned biomaterial in the manufacture of the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody;

[0086] M2) use of the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody in the manufacture of the aforementioned medicament;

[0087] M3) use of the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned biomaterial or the aforementioned medicament in the manufacture of a product for preventing and / or treating botulinum toxin poisoning;

[0088] M4) use of the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned biomaterial in the manufacture of a product for detecting botulinum toxin poisoning.

[0089] The aforementioned product can be a medicament.

[0090] The medicament can further comprise a physiologically or pharmaceutically acceptable excipient, diluent, or carrier.

[0091] Herein, the aforementioned "physiologically or pharmaceutically acceptable carrier or diluent" means those carriers and diluents that have no apparent stimulating effect on an organism and do not impair the biological activity and performance of the agent in the pharmaceutical composition.

[0092] Herein, the "physiologically or pharmaceutically acceptable excipient" means an inert substance added to the pharmaceutical composition to further facilitate the administration of the agent. The carrier material herein includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, the water-soluble carrier material is preferred.

[0093] ​​​​​​​​​​​​​​​​​​​​​​​The present application not only includes the complete antibody, but also the fragments of the nanobody having immunological activity or the fusion proteins formed by the antibody and other sequences. Therefore, the present application also includes the polypeptides such as the fragments, derivatives and analogues of the nanobody which maintain the same biological function or activity as the antibody of the present application. The polypeptides can be as follows: D1) single-chain antibody containing the nanobody described above; D2) Fab containing the nanobody described above; D3) complete antibody containing the nanobody described above; D4) fusion antibody containing the nanobody described above; and D5) antibody drug conjugate containing the nanobody described above.

[0094] D4) The fusion antibody of the nanobody can be: M1) nanobody fusion protein having the amino acid sequence shown in SEQ ID NO: 11.

[0095] As known by those skilled in the art, the conjugate and fusion antibody expression products include the conjugates formed by the combination of the antibodies or fragments thereof of the present application and the drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules. The present application also includes the cell surface markers or antigens combined with the nanobody or fragments thereof.

[0096] The present application includes any protein or protein conjugate and fusion expression product (i.e. immunological conjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous, preferably at least 95% homologous to the heavy chain variable region of the antibody of the present application.

[0097] The present application prepares the phage nanobody display library by immunizing camels with the antigen and screens the nanobody molecules capable of neutralizing the botulinum toxin type E. The nanobody has the advantages of rapid preparation, simple structure and easy modification into a multi-specific antibody, and is expected to be developed into a high-efficiency and safe nanobody for neutralizing the botulinum toxin type E, which can be used to replace the serum products supplied in the market to meet the needs of human beings. BRIEF DESCRIPTION OF DRAWINGS

[0098] Figure 1 After the third round of screening, some phage clones were identified by Phage-ELISA for the binding with the target antigen or the control antigen. The odd columns are the target antigens, and the even columns are the control antigens.

[0099] Figure 2SDS-PAGE electrophoresis was used to detect the expression and purification of anti-E botulinum toxin nanobody hFc fusion protein E22-hFc and humanized nanobody fusion protein E22-h3. Lane 1 is the result of E22-hFc reduced SDS-PAGE detection, lane 2 is the result of E22-h3 reduced SDS-PAGE detection; lane 6 is the result of E22-hFc non-reduced SDS-PAGE detection, lane 7 is the result of E22-h3 non-reduced SDS-PAGE detection; lanes 3-5 are antibodies unrelated to the present patent, and M is Marker (molecular weight standard).

[0100] Figure 3 The anti-E botulinum toxin nanobody hFc fusion protein E22-hFc binding activity was detected.

[0101] Figure 4 The specificity of the anti-E botulinum toxin nanobody hFc fusion protein E22-hFc was detected.

[0102] Figure 5 The anti-E botulinum toxin nanobody hFc fusion protein E22-hFc neutralization activity was evaluated. BoNT / E is E botulinum toxin, and Ab is the hFc fusion protein E22-hFc of anti-E botulinum toxin nanobody E22 and human immunoglobulin.

[0103] Figure 6 The anti-E botulinum toxin humanized nanobody fusion protein E22-h3 binding activity was detected.

[0104] Figure 7 The specificity of the anti-E botulinum toxin humanized nanobody fusion protein E22-h3 was detected.

[0105] Figure 8 The anti-E botulinum toxin humanized nanobody fusion protein E22-h3 neutralization activity was evaluated. BoNT / E is E botulinum toxin, and Ab is the humanized nanobody fusion protein E22-h3. DETAILED DESCRIPTION

[0106] The present application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0107] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0108] Botulinum toxin type E (BoNT / E) and equine-derived botulinum antitoxin standard solution (BAT) used in the following examples were purchased from China Institute for Food and Drug Control.

[0109] pTSE-hFc used in the following examples was constructed by linking the gene of the Fc domain of human immunoglobulin G to a pCMV vector. pTSE-hFc has been described in Xie Q, Li ZY, Zhang W, et al. Screening and identification of antibodies against protective antigen V of Yersinia pestis [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only, and cannot be used for other purposes.

[0110] NEN-SCFV vector used in the following examples was constructed by linking the genes of phage surface protein pIII and arabinose operon to a pET vector. NEN-SCFV vector has been described in Chen L, Lu J, Yue J, Wang R, Du P, Yu Y, Guo J, Wang X, Jiang Y, Cheng K, Yang Z and Zheng T (2023) A humanized antihuman adenovirus 55 monoclonal antibody with good neutralization ability. Front. Immunol. 14: 1132822. doi: 10.3389 / fimmu.2023.1132822. Epub 2023 Mar 16. PMID: 37006289; PMCID: 10060833. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only, and cannot be used for other purposes.

[0111] Recombinant TL-HN antigen used in the following examples was prepared by the laboratory of the inventor. The preparation method has been described in non-patent literature: Liu XY, Wei DK, Li ZY, Lu JS, Xie XM, Yu YZ, et al. Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanus toxin. VACCINE (2023). doi: 10.1016 / j.vaccine.2023.09.032. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only, and cannot be used for other purposes.

[0112] The SARS antigen in the following examples was purchased from Yikai Shenzhou with the item number 40150-V08B2.

[0113] The antigen BoNT / A L-HN (AL-HN) in the following examples was prepared by the laboratory and the preparation method has been described in the literature Liu FJ, Shi DY, Mao YY, Xiong XH, Lu JS, Pang XB, Dong XJ, Yang ZX, Yu YZ. Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin sero type A. Vaccine. 2020 Mar 23;38(14):2978-2983. doi: 10.1016 / j.vaccine.2020.02.060. Epub 2020 Feb 26. PMID: 32113807. on page 2 in the material methods in the following literature under 2.1 “Preparation of recombinant BoNT / A functional domain antigens” as “BoNT / A L-HN (AL-HN)” in the literature.

[0114] The antigens BoNT / E L-HN domain (EL-HN) and BoNT / E L domain (EL), BoNT / E HN domain (EHN) in the following examples were prepared by the laboratory, the preparation method has been recorded in the following literature page 10 of the material method in 5.2 "Production of Recombinant Functional Fragment Antigens Derived from BoNT / E", which is "BoNT / E·L-HN (EL-HN), BoNT / E L (EL), BoNT / E HN (EHN)" in the literature: Li Z, Lu J, Tan X, Wang R, Xu Q, Yu Y, Yang Z. Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E. Toxins (Basel). 2022 Feb 11;14(2):135. doi: 10.3390 / toxins14020135. PMID: 35202162; PMCID: PMC8880310.

[0115] The antigen BoNT / B L-HN domain (BL-HN) in the following examples was prepared by the laboratory, the preparation method has been recorded in the following literature page 2 of the first paragraph of the material method section "Recombinant BoNT / B L-HN Fragment Preparation", which is "BoNT / B·L-HN (BL-HN)" in the literature: Li Z, Lu JS, Liu S, Wang R, Xu Q, Yu YZ, Yang ZX. 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 Aug;39(4):1044-1053. doi: 10.1007 / s12640-021-00337-x. Epub 2021 Feb 22. PMID: 33616873.

[0116] The antigen BoNT / F L-HN domain (FL-HN) in the following examples was prepared by the laboratory, and the preparation method has been described in the following literature: Li Z, Li B, Lu J, Liu X, Tan X, Wang R, Du P, Yu S, Xu Q, Pang X, Yu Y, Yang Z. Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F. Toxins (Basel). 2023 Mar 6; 15(3): 200. doi: 10.3390 / toxins15030200. PMID: 36977091; PMCID: PMC10056376. on page 13 of the material methods in 5.2 "Identification and Production of Recombinant Proteins" as "BoNT / F L-HN (FL-HN)" in the literature.

[0117] The following examples use GraphPad Prism 8 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, and the Log-rank test is used, p < 0.05 (*) indicates that the difference is significant, p < 0.01 (**) indicates that the difference is very significant, and p < 0.001 (***) indicates that the difference is extremely significant.

[0118] Example 1, Construction of Anti-E Botulinum Toxin Nanobody Library

[0119] 1. Camel immunization

[0120] The antigen BoNT / E L-HN (EL-HN) was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881), shaken and emulsified, and after complete emulsification, the healthy adult bactrian camel was injected subcutaneously by multiple point injection, and after that, the booster immunization was carried out every two weeks, except that the first time used Freund's complete adjuvant, and the rest of the immunization used Freund's incomplete adjuvant (Sigma, F5506).

[0121] 2. Isolation of peripheral blood lymphocytes from camels

[0122] Peripheral blood 120-150 mL of the camel immunized five times was collected into an anticoagulant tube to isolate peripheral blood lymphocytes (PBMC). After diluting the whole blood sample gently, it was mixed with lymphocyte separation medium (STEMCELL, 07851) to form a mixture with a clear interface between the two, to separate peripheral blood lymphocytes from the camel blood. 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 layer, PBMC layer, lymphocyte separation medium layer, and red blood cell layer.

[0123] 3. Nested PCR amplification of VHH gene segment

[0124] First, total RNA was extracted from PBMC using OMEGA E.Z.N.A Total RNA kit I kit (OMEGA, R6834). Next, the extracted RNA was reverse transcribed into cDNA using Invitrogen Superscript III First-strand synthesis system for RT-PCR kit (Invitrogen, 18080-051). In the PCR amplification stage, first-round PCR was performed using the synthesized cDNA as a template, and IgG-specific primers CALL001 (upstream primer) and CALL002 (downstream primer) designed to amplify the CH2 region sequence of the antibody. Then, the first-round PCR product was recovered and used as a template for second-round PCR amplification using another pair of designed primers VHH-F and VHH-R to amplify the VHH segment.

[0125] Table 1. Primer sequences used in two-round PCR

[0126] Primer name Primer sequence (5'-3') CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F cggCCATGGcGGTCCTGGCTGCTCTTCTACA VHH-R tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT

[0127] 4. Electroporation of ligation product

[0128] The NEN-SCFV vector and the amplified VHH gene were digested using two restriction enzymes, NcoI and NotI. Then, the cut vector and gene segment were ligated by T4 DNA ligase.

[0129] The ligation product was transformed into E. coli TG1 competent cells (BC120-01, Biomed) using electroporation technology to construct an anti-E botulinum toxin phage antibody library. By serially diluting the phage antibody library, its library capacity could be determined. The experimental results showed that the constructed antibody library capacity reached 2 x 10 8 To verify the accuracy of the library, 48 clones were randomly selected for colony PCR amplification and sequence alignment analysis. The results showed that the VHH segment insertion rate reached 100% and the sequences were all different.

[0130] Example 2, Screening of Anti-Botulinum Neurotoxin Type E Nanobody Phage Library

[0131] The constructed nanobody phage library was subjected to solid-phase screening using EL-HN as an antigen to obtain anti-Botulinum neurotoxin type E specific nanobodies.

[0132] The constructed anti-Botulinum neurotoxin type E nanobody phage library was transferred to 2YT-GA medium (1 L 2YT medium containing 16 g Tryptone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL ampicillin, 20% glucose) and incubated until the logarithmic growth phase. Then, M13KO7 helper phage was added at an MOI of about 10, and the mixture was incubated at room temperature for 30 min and then at 37°C with low-speed shaking at 150 rpm for 30 min. The deep-well plate was centrifuged for 15 min at room temperature at 4000 rpm, and the supernatant was discarded. The plate was subjected to overnight presentation using 2YT-KAA medium (1 L 2YT medium containing 16 g Tryptone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL kanamycin, 20% glucose, and 1 mM arabinose) at room temperature. The next day, the culture presentation supernatant was collected, and the phage was concentrated using 20% PEG / NaCl solution (1 L solution containing 200 g PEG6000 and 146.25 g NaCl) to obtain a high-titer antibody library presentation product for subsequent screening. The nanobody phage library was subjected to solid-phase screening to obtain specific nanobodies. EL-HN protein was coated in immunotubes using 0.05 M NaHC03 coating solution (pH = 9.6) and incubated at 4°C overnight. The next day, the immunotubes were washed twice with PBS for 3 min each, and then blocked with blocking solution (2% bovine serum albumin) at room temperature for 2 h. Then, the nanobody phage library solution was added and incubated at room temperature for 2 h. The mixture was shaken at 200 rpm for 20 min. The immunotubes were washed 10 times with PBST (1 L PBS solution containing 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, and 0.1% Tween-20) and 5 times with PBS. After washing, 1 mL elution buffer (0.1 M Glycine-HCl, pH 2.2) was added, and the mixture was shaken at 400 rpm for 20 min. The elution buffer in the antigen immunotube was removed, and 20-60 μL neutralization buffer (1 M Tris-HCl, pH 8.0) was added for neutralization. Escherichia coli TG1 in the logarithmic growth phase was infected with the neutralized elution buffer, and the mixture was incubated at room temperature for 30 min and then at 37°C with shaking at 150 rpm for 30 min. The phage was produced and purified for the next round of screening. The same screening process was repeated for 3 rounds, and the enrichment results are shown in Table 2.

[0133] Table 2, Anti-botulinum toxin nanobody phage library screening enrichment degree analysis

[0134] Number of screenings Input (pfu) Output (pfu) Output / input 1 5.0 x 10 11 ]] 1.2 x 10 8 ]]> 2.4 x 10 -4 ]]> 2 1.2 x 10 11 ]]> 8.6 x 10 8 ]]> 7.2 x 10 -3 ]] 3 1.2 x 10 11 ]]> 8.6 x 10 8 ]] 7.2 x 10 -3 ]]>

[0135] After the above-mentioned 3 rounds of screening, single colonies with obvious spacing and regular shape were selected from the petri dishes with good phage growth, inoculated into 96-well deep well plates containing 250 μL of 2YT-GA medium per well, and 2 wells were not inoculated with clones or inoculated with other antibody clones as negative controls; the bacterial solution was cultured at 37°C until the logarithmic growth phase, and then M13K07 helper phage was added at a ratio of MOI≈10, i.e. 100 μL of each well of the deep well plate for culturing single phage clones, and incubated at room temperature for 30 min after infection, and then incubated at 37°C with low speed 150 rpm for 30 min; the deep well plate was centrifuged for 10 min, and the supernatant was discarded, the centrifugation condition was room temperature, 2000 rpm, and the expression was induced using 1 mM of arabinose, and then incubated at 28°C with a speed of 220 rpm overnight to obtain phage particles displaying nanobodies.

[0136] Example 3, Identification of E-type botulinum toxin specific nanobodies by phage-enzyme linked immunosorbent assay (Phage-ELISA)

[0137] The EL-HN protein was coated on the enzyme-linked plate, and the concentration was diluted to 2 ng / μL with 0.05 M NaHCO3 coating solution, about 200 ng of antigen per well, and 2% BSA was used as a negative control for parallel coating in the adjacent column of the antigen well, and coated at 4°C overnight; the next day, the enzyme-linked plate was taken out, washed 6 times on the plate washing machine with PBST, and then blocked with blocking solution (30 g of skimmed milk powder was added to 1 L of PBS) at 200 μL / well, and blocked at 37°C for 2 h; the single colony bacterial solution induced overnight was centrifuged at 4°C, 3000 rpm for 10 min, and 125 μL of supernatant of each bacterial solution was added to a 96-well deep well plate with 125 μL of blocking solution, and pre-bound for 30 min; the blocked induced expression supernatant was added to the corresponding enzyme-linked plate coated with the target antigen and the control antigen at 100 μL / well, and incubated at 37°C for 1.5 h.

[0138] After washing 6 times on the plate washer with PBST oscillation, 100 μL / well of HRP-labeled anti-M13 mouse monoclonal antibody (Sino Biological, 1973-MM05T-H) diluted 1:4000 with blocking solution was added to the enzyme-linked plate, and incubated at 37°C for 45 min. After incubation, the plate was washed 6 times on the plate washer with PBST oscillation, and color developing solution (10 mL color developing solution containing 1 mL 10x OPD, 9 mL 0.2 M Na2HPO4 and 0.1 M citric acid mixed solution, and 10 μL 30% hydrogen peroxide) was prepared, and 100 μL / well was added to the enzyme-linked plate, and color developed for 15-20 min in the dark. 50 μL / well of 2 M H2SO4 was added to terminate the reaction. The absorbance values of the antigen group and the negative control group were compared using an enzyme-labeled instrument at a dual wavelength of 492 / 630 nm. If the ratio was greater than 5, the monoclonal was determined to be a positive clone. Some Phage-ELISA experimental results are shown in Table 1. Figure 1 Table 1. Phage-ELISA experimental results. Odd columns are target antigens, and even columns are control antigens. The positive clone corresponding to the bacterial solution was sent to a biotechnology service company for sequencing, and the DNA sequence of the inserted fragment was obtained, and finally a phage clone sequence E22 capable of specifically binding to BoNT / EL-HN was obtained.

[0139] The amino acid sequence of E22 is shown in SEQ ID NO: 1, which includes a framework region (FR: FR1, FR2, FR3, FR4) and a complementarity determining region (CDR: CDR1, CDR2, CDR3), wherein the four parts of the framework region are sequentially recorded as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; and the three parts of the complementarity determining region are sequentially recorded as SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. The gene encoding the above-mentioned anti-E botulinum toxin nanobody E22 has a nucleotide sequence shown in SEQ ID NO: 9.

[0140] The above-mentioned CDR is a sequence defined according to the IMGT system analysis result.

[0141] Example 4, Preparation of Anti-E Botulinum Toxin Nanobody

[0142] 1. Construction of anti-E botulinum toxin nanobody hFc fusion protein eukaryotic expression plasmid pTSE-E22-hFc

[0143] The nucleotide sequence SEQ ID NO: 9 of the obtained E22 protein was cloned into the pTSE-hFc expression vector by basic PCR amplification, enzyme digestion, ligation and other techniques, and a single clone was picked for sequencing verification. After successfully inserting the obtained VHH gene fragment into the corresponding vector, a eukaryotic expression plasmid was constructed, and the obtained recombinant plasmid was named pTSE-E22-hFc.

[0144] The recombinant vector pTSE-E22-hFc is a recombinant expression vector obtained by replacing the nucleotide sequence between the recognition sites of restriction enzymes Sal-I and Nhe-I of the pTSE-hFc vector with the DNA fragment with the nucleotide sequence of SEQ ID NO: 9, and keeping the other nucleotide sequences of the pTSE-hFc vector unchanged, which expresses a protein with the amino acid sequence of SEQ ID NO: 11 (the nucleotide sequence is SEQ ID NO: 10, named E22-hFc).

[0145] The E22-hFc protein is a nanobody hFc fusion protein against E botulinum toxin, which is obtained by linking the carboxyl terminal of the nanobody gene sequence against E botulinum toxin (SEQ ID NO: 9) with the hFc segment of human immunoglobulin.

[0146] 2. Expression and purification of nanobody hFc fusion protein against botulinum toxin

[0147] The constructed pTSE-E22-hFc expression plasmid was transfected into FreeStyle TM HEK293-F cells (Invitrogen, R79007) using transfection reagent FectoPRO DNA Transfection Reagent (Polyplus, 116-001), and after 72 hours, the cell activity was monitored every day. When the cell activity decreased from 95-100% to 80-85%, the cell supernatant was collected for purification to obtain the nanobody hFc fusion protein E22-hFc.

[0148] SDS-PAGE electrophoresis was used to analyze the purified antibody, and the results are shown in Figure 2 : The band position of the antibody is consistent with the theoretical molecular weight size, and the band size of the nanobody hFc fusion protein under reducing conditions is about 40 kDa (lane 1), and the band size under non-reducing conditions is about 80 kDa (lane 6). Figure 2 Figure 2

[0149] Example 5, Evaluation of the properties of the nanobody hFc fusion protein

[0150] ​​1. The binding activity between the nanobody hFc fusion protein E22-hFc and the BoNT / EL-HN protein was detected by ELISA.

[0151] The experiment was repeated twice, with the following repetition each time:

[0152] Dilute EL-HN protein to 2 μg / mL with carbonate coating buffer (pH 9.6), add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4°C. Discard the coating solution and wash the plate with PBST (1 L PBS solution contains 8.0 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.27 g KH₂PO₄, and 0.1% KCl). Wash 6 times with Tween-20 solution, blot dry any remaining liquid in the plate, add 200 μL of blocking buffer (3% skim milk powder) to each well, and incubate at 37°C for 2 h. Serially dilute the antibody for binding activity assay 2-fold with the blocking buffer to an initial dilution of 12.5 μg / mL. Discard the blocking buffer, wash 6 times with PBST, blot dry any remaining liquid, add 100 μL of E22-hFc as the primary antibody to each well, and incubate at 37°C for 1.5 h. Discard the primary antibody, wash with PBST... Six times, HRP-labeled goat anti-human IgG was diluted 1:4000 with blocking buffer as a secondary antibody, and 100 μL / well was added to the plate and incubated at 37°C for 45 min. The secondary antibody was discarded, and the plate was washed six times with PBST. Peroxidase substrate chromogenic solution was added at 100 μL / well, and the plate was incubated in the dark for 15-20 min. The color development was observed, and 50 μL / well of 2M sulfuric acid was added to stop the reaction after complete development. The optical density was measured using a microplate reader at 492 nm / 630 nm dual wavelengths. The concentration required for 50% antibody binding of the antigen protein (EC50) was calculated. 50 ), to assess their binding ability.

[0153] The results are as follows Figure 3 As shown, the analysis revealed the half-maximal effective concentration (EC50) of E22-hFc binding to BoNT / EL-HN protein. 50 The value is 0.2620 nM.

[0154] 2. Identifying the specificity of nanobodies using ELISA experiments.

[0155] The experiment was repeated twice, with the following repetition each time:

[0156] AL-HN, BL-HN, EL-HN, FL-HN, TL-HN, EL, EHN, SARS antigens were coated with carbonate coating buffer and diluted to a concentration of 20 μg / mL, 100 μL / well added to a 96-well ELISA plate, coated overnight at 4°C; the next day, the coating solution in the plate was discarded, and then the overnight-coated ELISA plate was washed 6 times in a plate washer using a PBST solution containing 0.1% Tween-20, after which the remaining liquid in the wells was patted dry, and blocking solution (3% skim milk powder) was added at 200 μL / well, 37°C for 2 h; an antibody solution was prepared, and the antibody E22-hFc was adjusted to a concentration of 20 μg / mL; the blocking solution was discarded, and after 6 washes with PBST, the primary antibody E22-hFc was added at 100 μL / well, and incubated at 37°C for 1.5 h; the primary antibody was discarded, and after 6 washes with PBST, HRP-labeled goat anti-human IgG secondary antibody was added at 100 μL / well diluted with blocking solution at a ratio of 1:4000, and incubated at 37°C for 45 min; the secondary antibody was discarded, and after 6 washes with PBST, peroxidase substrate developing solution was added at 100 μL / well, and color development was observed after 15-20 min of color development in the dark, and 50 μL of 2M sulfuric acid was added to each well to terminate the reaction; the optical density value was measured using an enzyme label instrument at 492 nm / 630 nm dual wavelength, and the data results were analyzed by GraphPad Prism 8 software.

[0157] The results, as shown in Figure 4 Table 1, show that E22-hFc specifically binds to EL-HN antigen, and does not bind to other antigens or has weak binding activity.

[0158] Example 6, in vitro neutralization titer evaluation of anti-botulinum toxin nanobody

[0159] The in vitro neutralization titer of the antibody was determined by injecting KM (Kunming) mice after mixing the antibody and a lethal dose of type E botulinum toxin in vitro, 4 KM mice per group, weighing 18-20 g, purchased from Beijing Sbielof Biological Technology Co., Ltd. The experiment was repeated three times (different doses), and each repetition was as follows:

[0160] 1. Sample preparation:

[0161] Diluent: KH2PO40.7 g, Na2HPO4·12H2O 2.4 g, NaCl 6.8 g, gelatin 2 g, add water to 1 L, autoclave;

[0162] Botulinum toxin solution: dilute type E botulinum toxin (purchased from China Institute for Food and Drug Control) to 100 LD 50 / mL with diluent;

[0163] E22-hFc solution: a solution prepared by dissolving the fusion protein E22-hFc prepared in Example 4 with diluent.

[0164] BAT solution: a solution prepared by dissolving equine anti-botulinum toxin serum (purchased from China Institute for Food and Drug Control) with diluent.

[0165] 2. The specific experimental grouping scheme is as follows:

[0166] (1) BoNT / E solution 20xLD 50 Group: each KM mouse was injected intraperitoneally with 500 μL of botulinum toxin solution (100xLD 50 / mL), so that each mouse was injected with a toxin dose of 20xLD 50 .

[0167] (2) BoNT / E 20xLD 50 +E22-hFc 10 μg group: the botulinum toxin solution (100xLD 50 / mL) was mixed with the E22-hFc solution, the volume of each group was supplemented to 2.5 mL with diluent, and after uniform mixing, the mixture was incubated at 37°C for 30 min to obtain the BoNT / E+E22-hFc solution, which was injected intraperitoneally into KM mice, with each mouse injected with 500 μL, so that the dose of BoNT / E was 20xLD 50 / each and the dose of E22-hFc was 10 μg / each.

[0168] (3) BoNT / E 20xLD 50 +E22-hFc 2 μg group: the difference between this group and the BoNT / E+E22-hFc 10 μg group was that the dose of E22-hFc was 2 μg / each, and the rest of the operations were the same as those of the BoNT / E 20xLD 50 +E22-hFc 10 μg group.

[0169] (4) BoNT / E 20xLD 50 +E22-hFc 0.4 μg group: the difference between this group and the BoNT / E+E22-hFc 10 μg group was that the dose of E22-hFc was 0.4 μg / each, and the rest of the operations were the same as those of the BoNT / E 20xLD 50 +E22-hFc 10 μg group.

[0170] (5) BoNT / E 20xLD 50 +E22-hFc 0.2 μg group: the difference between this group and the BoNT / E+E22-hFc 10 μg group was that the dose of E22-hFc was 0.2 μg / each, and the rest of the operations were the same as those of the BoNT / E 20xLD50 +E22-hFc 10 μg group.

[0171] (6) BoNT / E 20xLD 50 +E22-hFc 0.1 μg group: the difference between this group and the BoNT / E + E22-hFc 10 μg group is that the dose of E22-hFc is 0.1 μg per mouse, and the rest of the operations are the same as BoNT / E 20xLD 50 +E22-hFc 10 μg group.

[0172] Four mice were injected in each experimental group, and the health and survival of the mice were monitored for 5 days.

[0173] The results are shown in Figure 5 Table 2, and it can be seen that E22-hFc at a dose of 0.2 μg or more can completely neutralize the lethal dose of 20xLD 50 BoNT / E.

[0174] Example 7, Humanization of Anti-E botulinum toxin Nanobody Fusion Protein E22-hFc and Activity Evaluation

[0175] 1. Humanization of Anti-E botulinum toxin Nanobody Fusion Protein E22-hFc

[0176] The Swiss-model model was used to humanize the Nanobody E22-hFc fusion protein. First, the antibody was homology modeled by Swiss-model, and then the model was put into DS for amino acid property analysis. The amino acid sequence of the humanized Nanobody E22-h3 was obtained by mutating the amino acids in the framework region of the antibody to human amino acids, with the amino acid sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQ ID NO: 13. Finally, the sequence after the transformation was scored by Z-score, and the results showed that the Z-score values all fell within the peak range of the human sequence set, indicating that the sequence was highly humanized.

[0177] 2. Construction of Eukaryotic Expression Vector pTSE-hFc-E22-h3 of Anti-E botulinum toxin Humanized Nanobody

[0178] According to the anti-E botulinum toxin humanized nanobody gene sequence (SEQ ID NO: 13), the C-terminal thereof is connected with the Fc segment of human immunoglobulin G (hFc), to obtain an anti-E botulinum toxin nanobody fusion protein E22-h3 with the gene nucleotide sequence of SEQ ID NO: 14 and the expression amino acid sequence of SEQ ID NO: 15. E22-h3 is a mutant of the amino acid sequence SEQ ID NO: 1 of E22, in which the 4th amino acid is mutated from V to L, the 14th amino acid is mutated from A to P, the 82nd amino acid is mutated from D to S, the 116th amino acid is mutated from Q to L, and the other amino acid sequences remain unchanged.

[0179] 3. Preparation and property evaluation of the humanized nanobody fusion protein

[0180] The humanized nanobody fusion protein E22-h3 is prepared according to the method in Example 4, and the purified antibody is analyzed by SDS-PAGE electrophoresis, and the results are shown in Figure 2 . The molecular weight of the antibody is as expected, and the band size of the nanobody hFc fusion protein under reducing conditions is about 40 kDa (lane 2), and the band size under non-reducing conditions is about 80 kDa (lane 7). Figure 2 Figure 2

[0181] The binding activity and specificity of the humanized nanobody fusion protein E22-h3 to the recombinant EL-HN antigen are detected according to Example 5. The results are shown in Figure 6 , Figure 7 . The analysis shows that the half-effective concentration (EC 50 ) of E22-h3 binding to BoNT / EL-HN protein is 0.2344 nM; E22-h3 specifically binds to BoNT / EL-HN antigen, and does not bind to other antigens or has weak binding activity.

[0182] 4. Evaluation of the neutralization activity of the anti-E botulinum toxin humanized nanobody

[0183] The neutralization activity of the anti-E botulinum toxin humanized nanobody is evaluated according to Example 6.

[0184] The experiment is repeated three times (different doses), and each repetition is as follows:

[0185] (1) 20xLD 50 group: each KM mouse is injected intraperitoneally with 500 μL of a botulinum toxin solution at 100xLD 50 / mL, so that the toxin dose contained in each injection solution is 20xLD 50 / each.

[0186] ​​(2) BoNT / E 20xLD 50 +E22-h3 2 μg group: the botulinum toxin solution (100xLD 50 / mL) was mixed with the E22-h3 solution, the volume of each group was supplemented to 2.5 mL with the diluent, and after uniform mixing, the BoNT / E + E22-h3 solution was obtained by incubation at 37°C for 30 min. The solution was injected into KM mice intraperitoneally, 500 μL per mouse, so that the dose of BoNT / E was 20xLD 50 / mouse, and the dose of E22-h3 in the solution injected into each mouse was 2 μg / mouse.

[0187] (3) BoNT / E 20xLD 50 +E22-h3 0.4 μg group: the difference between this group and the BoNT / E + E22-h3 2 μg group was that the dose of E22-h3 was 0.4 μg / mouse, and the rest of the operations were the same as the BoNT / E 20xLD 50 +E22-h3 2 μg group.

[0188] (4) BoNT / E 20xLD 50 +E22-h3 0.08 μg group: the difference between this group and the BoNT / E + E22-h3 2 μg group was that the dose of E22-h3 was 0.08 μg / mouse, and the rest of the operations were the same as the BoNT / E 20xLD 50 +E22-h3 2 μg group.

[0189] (5) BoNT / E 20xLD 50 +E22-h3 0.02 μg group: the difference between this group and the BoNT / E + E22-h3 2 μg group was that the dose of E22-h3 was 0.02 μg / mouse, and the rest of the operations were the same as the BoNT / E 20xLD 50 +E22-h3 2 μg group.

[0190] Four mice were injected in each experimental group, and the health status and survival of the mice were monitored for 5 days.

[0191] The results are shown in Table 1. Figure 8 As shown in Table 1, E22-h3 at a dose of 0.08 μg or more can completely neutralize 20xLD 50 BoNT / E, and all the mice survived.

[0192] Example 8: Evaluation of the therapeutic effect of humanized anti-botulinum toxin nanobody

[0193] To evaluate whether the anti-botulinum toxin nanobody has a therapeutic effect after the mice are exposed to BoNT / E, KM mice were selected, 6 mice per group, 18-20 g, purchased from Beijing Sbielof Biotechnology Co., Ltd.

[0194] The evaluation method is as follows:

[0195] Diluent: KH2PO40.7 g, Na2HP04-12H2O 2.4 g, NaCl 6.8 g, gelatin 2 g, add water to 1 L, high pressure sterilization;

[0196] Botulinum toxin solution: E botulinum toxin (purchased from China Food and Drug Inspection Research Institute) is diluted with diluent to 5 x LD 50 / mL;

[0197] E22-h3 solution: a solution obtained by dissolving E22-h3 prepared in Example 7 with diluent;

[0198] Horse-derived botulinum antitoxin standard (BAT) solution: a solution obtained by dissolving horse-derived antitoxin serum (purchased from China Food and Drug Inspection Research Institute) with diluent;

[0199] The experiment is repeated twice, and each repetition is as follows:

[0200] A, BoNT / E 5 x LD 50 / only + antibody 2.5 mg / kg group

[0201] 1) 1 h group: 500 μL of 5 x LD 50 / mL botulinum toxin solution is injected intraperitoneally into KM mice. 100 μL of E22-h3 solution (i.e., the injection dose is 2.5 mg / kg) is injected 1 h after the injection of the botulinum toxin solution, and a total of 6 mice are monitored for 7 days for health and survival.

[0202] 2) 2 h group: the difference between this group and the 1 h group of the present group is that 100 μL of E22-h3 solution is injected 2 h after the injection of the botulinum toxin, and the rest of the operations are the same as those of the 1 h group of the present group.

[0203] 3) 3 h group: the difference between this group and the 1 h group of the present group is that 100 μL of E22-h3 solution is injected 3 h after the injection of the botulinum toxin, and the rest of the operations are the same as those of the 1 h group of the present group.

[0204] 4) 6 h group: the difference between this group and the 1 h group of the present group is that 100 μL of E22-h3 solution is injected 6 h after the injection of the botulinum toxin, and the rest of the operations are the same as those of the 1 h group of the present group.

[0205] 5) PBS group: an equal volume of PBS is used instead of the E22-h3 solution, and the rest of the operations are the same as those of the 1 h group of the present group.

[0206] B, BoNT / E 5 x LD 50 / only + antibody 0.25 mg / kg group

[0207] 1) BoNT / E 5xLD 50 / mouse + E22-h3 0.25 mg / kg group: 500 μL of 5xLD 50 / mL botulinum toxin solution was injected intraperitoneally into KM mice. A total of 6 mice were used. 100 μL of E22-h3 solution (i.e. injection dose of 0.25 mg / kg) was injected 1 h after injection of botulinum toxin solution. The mice were monitored for 7 days for health and survival.

[0208] 2) 2h group: This group was different from the 1h group of this group in that 100 μL of E22-h3 solution was injected 2h after injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0209] 3) 3h group: This group was different from the 1h group of this group in that 100 μL of E22-h3 solution was injected 3h after injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0210] 4) 6h group: This group was different from the 1h group of this group in that 100 μL of E22-h3 solution was injected 6h after injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0211] 5) BoNT / E 5xLD 50 / mouse + BAT 0.25 mg / kg group: 100 μL of equine anti-botulinum toxin serum solution (i.e. injection dose of 0.25 mg / kg) was used instead of E22-h3 solution, and the rest of the operations were the same as the 1h group of this group.

[0212] The results are shown in Table 3. BoNT / E 5xLD 50 dose challenge, the E22-h3 treatment group was effective within 6 hours of exposure, and the protection effect was best within 3 hours, and the high-dose group of antibody (injection dose of 2.5 mg / kg) could achieve complete protection.

[0213] Table 3, Evaluation of the therapeutic effect of E22-h3 antibody

[0214]

[0215] Note: a KM mice in each experimental group treated with E22-h3, BAT or PBS were first injected intraperitoneally with 5xLD 50 dose of BoNT / E; b KM mice in each experimental group were treated with 2.5 or 0.25 mg / kg E22-h3, 0.25 mg / kg BAT, PBS at different time points after challenge; c KM mice were injected with different doses of antibody or PBS at 1, 2, 3 and 6 hours after challenge with the specified dose of toxin;d The number of KM mice in each experimental group was six, and the final mouse survival after seven days.

[0216] SEQ ID NO: 1:

[0217] QVQVVESGGGLVQAGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTLYLQMNDLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTQVTVSS.

[0218] SEQ ID NO: 2: GFTFDDYA.

[0219] SEQ ID NO: 3: SSWSTYYA.

[0220] SEQ ID NO: 4: IASGGGCVGADYGY.

[0221] SEQ ID NO: 5: QVQVVESGGGLVQAGGSLRLSCTAS.

[0222] SEQ ID NO: 6: MGWFRQAPGKEREGVSC.

[0223] SEQ ID NO: 7: DSVKGRFTISRDNAKNTLYLQMNDLKPEDTAMYYCAAW.

[0224] SEQ ID NO: 8: WGQGTQVTVSS.

[0225] SEQ ID NO: 9:

[0226] 5'-CAGGTGCAGGTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCAGGGGGGTCTCTGAGACTCTCCTGTACAGCCT CTGGATTCACTTTTGATGATTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCTCATGTAGTAGTTGGAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACGACCTGAAACCTGAGGACACGGCCATGTATTACTGTGCGGCCTGGATCGCTAGTGGTGGTGGCTGCGTAGGAGCTGACTACGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA-3'.

[0227] SEQ ID NO: 10:

[0228] 5'-CAGGTGCAGGTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCAGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTTGATGATTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCTCATGTAGTAGTTGGAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACGACCTGAAACCTGAGGACACGGCCATGTATTACTGTGCGGCCTGGATCGCTAGTGGTGGTGGCTGCGTAGGAGCTGACTACGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCTAGCgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgctgcatgaggctctgcacaGccactacacgcagaagagcctctccctgtccccgggtaaatga-3'

[0229] SEQ ID NO: 11:

[0230] QVQVVESGGGLVQAGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTLYLQMNDLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0231] SEQ ID NO: 12

[0232] QVQLVESGGGLVQPGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTLVTVSS.

[0233] SEQ ID NO: 13

[0234] 5'-CAAGTGCAACTGGTTGAGAGCGGCGGAGGACTGGTTCAACCCGGAGGAAGCCTGAGACTGAGCTGTACAGCCAGCGGCTTCACATTTGACGACTACGCCATGGGCTGGTTTAGACAGGCTCCCGGCAAGGAAAGAGAGGGAGTGAGCTGTTCTAGCTGGAGCACCTATTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGATACCGCCATGTACTATTGCGCCGCCTGGATTGCTTCCGGAGGAGGATGTGTGGGCGCCGATTATGGATATTGGGGACAGGGCACACTTGTGACAGTGAGCTCT-3'.

[0235] SEQ ID NO: 14

[0236]

[0237] SEQ ID NO: 15

[0238] QVQLVESGGGLVQPGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0239] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the present application is intended to include any change, use or improvement of the application, including changes made by using conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. A nanobody or antigen-binding fragment thereof against botulinum toxin, characterized in that, The nanobody 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: 2, the amino acid sequence of the CDR2 is SEQ ID NO: 3, and the amino acid sequence of the CDR3 is SEQ ID NO:

4.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The nanobody comprises four framework regions FR1, FR2, FR3 and FR4 in addition to the complementarity determining regions; The amino acid sequence of the FR1 is SEQ ID NO: 5 or has more than 90% identity with SEQ ID NO: 5; The amino acid sequence of the FR2 is SEQ ID NO: 6; The amino acid sequence of the FR3 is SEQ ID NO: 7 or has more than 90% identity with SEQ ID NO: 7; The amino acid sequence of the FR4 is SEQ ID NO: 8 or has more than 90% identity with SEQ ID NO:

8.

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

12.

5. A heavy chain antibody against botulinum toxin, characterized in that, The heavy chain antibody comprises the nanobody according to any one of claims 1 to 4 and an Fc region of an immunoglobulin.

6. The heavy chain antibody according to claim 5, characterized in that, The heavy chain antibody comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is 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 comprises SEQ ID NO: 11 or SEQ ID NO:

15.

8. A biomaterial, characterized in that, The biological material is any one of the following: B1) a nucleic acid molecule encoding the nanobody or the 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 comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1) or the expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1) or the expression cassette of B2) or the recombinant vector of B3).

9. A medicament against botulinum toxin poisoning, characterized in that, The medicament comprises the nanobody or the 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. Use, characterized in that, The use is any one of the following: M1) the use of the biological material of claim 8 for the preparation of the nanobody or the 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; M2) the use of the nanobody or the 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 for the preparation of the medicament of claim 9; M3) the use of the nanobody or the 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 biological material of claim 8 or the medicament of claim 9 for the preparation of a product for the prevention and / or treatment of botulinum toxin poisoning. M4) Use of a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a heavy-chain antibody according to any one of claims 5 to 7 or a biomaterial according to claim 8 for the manufacture of a product for the detection of botulinum toxin intoxication.

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