Nanobody b12 and related biomaterials and applications

Abstract

The application discloses a nanobody B12 and related biomaterials and applications thereof, and belongs to the field of immunotherapy biotechnology and pharmaceuticals, and particularly relates to the nanobody B12 and related biomaterials and applications thereof. The nanobody or antigen-binding fragment containing the nanobody targeting botulinum toxin type B has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is SEQ ID No. 1, the amino acid sequence of CDR2 is SEQ ID No. 2, and the amino acid sequence of CDR3 is SEQ ID No. 3. The nanobody B12 is fused with the Fc segment (hFc) of human immunoglobulin to obtain a fusion protein, and the obtained B12-hFc can effectively block the infection of botulinum toxin type B, and can resist 20LD 50 The half lethal dose ED of botulinum toxin is 0.28 μg. 50 ​

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunotherapy biotechnology pharmaceuticals, and particularly relates to Nanobody B12 and related biological materials and applications thereof. BACKGROUND

[0002] Botulinum toxin, as one of the most lethal bacterial toxins, can cause severe flaccid paralysis and pose a serious threat to human life safety. Due to the high variability of botulinum toxin, it is mainly divided into seven serotypes A-G, and most of the types form a group with variant subtypes, and the main types causing human poisoning are A, B, E and F. The causes of botulism mainly include foodborne, wound, infant poisoning, adult intestinal poisoning and iatrogenic poisoning. With the increase of commercial and medical value of botulinum toxin, as one of the few potential biological threat agents with the highest risk of being used as biological weapons, botulism has a very fast onset, and vaccination after exposure is also useless, so neutralizing antibodies are urgently needed for prevention and treatment, and therefore the research on botulism has attracted increasing attention worldwide.

[0003] At present, there is no effective treatment for patients with botulinum toxin poisoning in the late exposure. Antitoxin is an important method for poisoning treatment, and abroad has bivalent and heptavalent antitoxin serum, and the A-F type monovalent antitoxin serum is produced by the Lanzhou Institute. However, this therapy has many defects, such as the possibility of causing serious serum sickness and allergic reactions, and the limited number of serum available limits its application. Monoclonal antibodies have good development prospects for treating botulism, and efforts are made to develop products that can replace antiserum to make up for the recovery of paralysis that has already occurred. At present, most of the antibody drugs for botulinum toxin are in the molecular discovery stage, and no approved drug is available, so it is urgent to develop antibody drugs to enrich the treatment means for botulism.

[0004] In recent years, nanobodies (Nanobody, Nb) have played an important role in disease diagnosis and treatment. This antibody is a naturally occurring antibody in camelids that does not contain light chains and only contains heavy chains, and has the advantages of small molecular weight, strong penetration, strong antigen binding affinity, high expression, and strong transformability, etc. These superior properties make it suitable for the development of the next generation of biological drugs, and it is accelerating the application in the treatment and development of various diseases from viral infection to cancer, and has good development prospects in the research and development of therapeutic drugs.

[0005] Botulinum toxin type B is as toxic as botulinum toxin type A, but the development of botulinum toxin type B neutralizing antibodies has not become the focus of attention, and therefore there is an urgent need in the art to apply nanobody technology to develop efficient and safe anti-botulinum toxin type B neutralizing nanobodies to replace the serum products supplied on the market to meet the needs of humans. SUMMARY

[0006] The technical problem to be solved by the present application is to develop a specific nanobody capable of efficiently neutralizing botulinum toxin, to provide a candidate antibody for botulism diagnosis and prevention, and to enrich botulism prevention means.

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

[0008] The nanobody targeting botulinum toxin type B 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 CDR1 is shown as SEQ ID No. 1, the amino acid sequence of CDR2 is shown as SEQ ID No. 2, and the amino acid sequence of CDR3 is shown as SEQ ID No. 3.

[0009] The above CDR is a sequence defined according to the analysis results of IMGT system.

[0010] 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 B.

[0011] The four framework regions can be FR1, FR2, FR3 and FR4.

[0012] The amino acid sequence of FR1 is SEQ ID No. 5;

[0013] The amino acid sequence of FR2 is SEQ ID No. 6;

[0014] The amino acid sequence of FR3 is SEQ ID No. 7;

[0015] The amino acid sequence of FR4 is SEQ ID No. 8.

[0016] In the nanobody or antigen-binding fragment described above, the nanobody can be any of the following:

[0017] A1) a nanobody with an amino acid sequence shown as SEQ ID No. 4;

[0018] A2) a nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown as SEQ ID No. 4.

[0019] The protein tag refers to a polypeptide or protein fused with the target protein for the expression, detection, tracking and / or purification of the target protein. The protein tag can be His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag, Fc fragment of immunoglobulin G, etc.

[0020] In the present application, the protein tag is the Fc fragment of human immunoglobulin G (hFc).

[0021] In the present application, the term "antibody" refers to a heterotetramer glycoprotein of about 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regular interval intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0022] In the present application, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to

[0023] The variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function.

[0024] The above antigen binding fragment can be a complete 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.

[0025] The term "Fab fragment" is a heterodimer formed by the combination of the heavy chain Fd and the complete light chain through a disulfide bond, containing only one antigen binding site. The above heavy chain Fd refers to about 1 / 2 of the H chain part in Fab (about 225 amino acid residues, including VH, CH1 and part of the hinge region).

[0026] The term "Fv fragment" refers to a vector containing VH and VL genes, respectively, co-transfected into cells to express separately, and then assembled into a functional Fv antibody; or a stop codon is set between VH and VL in the vector, two small molecular protein fragments are expressed separately, and then combined through non-covalent bonds to form Fv antibody (Fv fragment).

[0027] The term "Fab' fragment" comprises one light chain and a part of one heavy chain comprising the VH domain and the CHI domain and the region between CHI and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0028] The term "F(ab')2 fragment" comprises two light chains and two heavy chains comprising the constant region between CHI and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0029] The term "variable" in the present application denotes certain parts of the variable region of an antibody which differ in sequence among the various specific antibodies and which form the binding and specificity of the respective antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments of the variable region of the light chain and the variable region of the heavy chain called complementarity determining regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called framework regions (FR). The variable region of the heavy and light chains in nature comprises four FR regions, which approximately adopt a beta-sheet configuration and which are connected by three CDRs forming connecting loops, which in some cases can form part of the beta-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 the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991 )). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example, they are involved in antibody-dependent cellular cytotoxicity.

[0030] In some embodiments, the Nanobodies described in the present application can be truncated at the N- or C-terminus to contain only part of FR1 and / or FR4, or to lack one or both of those framework regions, as long as antigen binding and specificity are essentially maintained.

[0031] In the present application, the Nanobody is designated Nanobody B12.

[0032] The present application also provides biological materials related to the Nanobody described hereinbefore, which can be any one of the following:

[0033] B1 ) a nucleic acid molecule encoding the Nanobody or antigen binding fragment described hereinbefore;

[0034] B2) an expression cassette comprising the nucleic acid molecule of B1 );

[0035] B3) a recombinant vector comprising the nucleic acid molecule of B1 ), or a recombinant vector comprising the expression cassette of B2);

[0036] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3).

[0037] In the above biological material, the nucleic acid molecule of B1) 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.

[0038] 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 initiating transcription of the nanobody-encoding gene, but also a terminator terminating transcription of the nanobody-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0039] The recombinant vector containing the expression cassette can be constructed using an existing expression vector.

[0040] In the above biological material, the vector of B3) can be a plasmid, cosmid, bacteriophage or viral vector.

[0041] 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.

[0042] In the above biological material, the microorganism of B4) can be bacteria (such as Escherichia coli), yeast, algae or fungi.

[0043] 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 76-99 of SEQ ID No. 9, the CDR2-encoding gene is nucleotides 151-171 of SEQ ID No. 9, and the CDR3-encoding gene is nucleotides 286-348 of SEQ ID No. 9.

[0044] In the above biological material, the nucleic acid molecule of B1) can be any one of the following:

[0045] C1) a DNA molecule having the nucleotide sequence of SEQ ID No. 9;

[0046] C2) a DNA molecule hybridizing to the DNA molecule defined in C1) under stringent conditions and encoding the nanobody;

[0047] C3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in C1) or C2) and encoding the nanobody.

[0048] wherein the stringent conditions can be as follows: hybridization in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4 and 1 mM EDTA at 50°C, and washing in 2xSSC, 0.1% SDS at 50°C; or as follows: hybridization in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA at 50°C, and washing in lxSSC, 0.1% SDS at 50°C; or as follows: hybridization in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA at 50°C, and washing in 0.5xSSC, 0.1% SDS at 50°C; or as follows: hybridization in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA at 50°C, and washing in 0.1xSSC, 0.1% SDS at 50°C; or as follows: hybridization in a mixture of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA at 50°C, and washing in 0.1xSSC, 0.1% SDS at 65°C; or as follows: hybridization in a solution of 6xSSC, 0.5% SDS at 65°C, and washing the membrane once with 2xSSC, 0.1% SDS and lxSSC, 0.1% SDS.

[0049] The nucleotide sequence of the B12-encoding gene of the present application can be easily mutated by those of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of B12 of the present application, as long as they encode the nanobody and have the activity of nanobody B12, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0050] The present application also provides a method for preparing the above-mentioned nanobody, which can comprise the following steps: introducing a nucleic acid molecule encoding the above-mentioned nanobody into a recipient cell to obtain a transgenic cell expressing the nanobody, and culturing the transgenic cell to obtain the nanobody.

[0051] Further, the nucleic acid molecule encoding the nanobody is the above-mentioned nucleic acid molecule.

[0052] In the above-mentioned method, the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned nanobody can be specifically any one of the following:

[0053] C1) a DNA molecule having the nucleotide sequence shown in SEQ ID No. 9;

[0054] C2) a DNA molecule hybridizing to the DNA molecule defined in C1) under stringent conditions and encoding the nanobody.

[0055] C3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in C1) or C2) and encoding the Nanobody.

[0056] Further, the recipient cell can be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Specifically, it can be a bacterial cell of E. coli, Streptomyces, Salmonella typhimurium, a fungal cell such as yeast, an insect cell of Drosophila S2 or Sf9, an animal cell of CHO, COS7, 293, etc.

[0057] In a specific embodiment of the present application, the recipient cell can be a FreeStyle TM HEK293-F cell.

[0058] The present application also provides a Nanobody fusion protein, which is a fusion of the Nanobody or antigen-binding fragment as described above with another molecule, which can include the Fc domain of an immunoglobulin, a fluorescent protein, or a VHH with different specificity.

[0059] In a specific embodiment of the present application, the other molecule can be the Fc domain of a human immunoglobulin.

[0060] In a specific embodiment of the present application, the amino acid sequence of the Fc domain of a human immunoglobulin as described above is 130-356 of SEQ ID No. 11.

[0061] In a specific embodiment, the Nanobody fusion protein as described above can be any one of the following:

[0062] M1) a fusion protein with an amino acid sequence as shown in SEQ ID No. 11;

[0063] M3) a protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 11.

[0064] Herein, the fusion protein with an amino acid sequence as shown in SEQ ID No. 11 can be an anti-Botulinum toxin type B Nanobody hFc fusion protein B12-hFc.

[0065] In the Nanobody fusion protein as described above, the nucleic acid molecule encoding the fusion protein can be any one of the following:

[0066] D1) a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 10;

[0067] D2) a DNA molecule hybridizing to the DNA molecule defined in D1) under stringent conditions and encoding said fusion protein;

[0068] D3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology to any of the DNA sequences defined in D1) - D2) and encoding said fusion protein.

[0069] The present application also provides an ELISA detection kit for botulinum type B toxin, said kit comprising the nanobody, the biomaterial or the fusion protein as described above.

[0070] The present application also provides any of the following uses:

[0071] E1) use of the nanobody or antigen binding fragment as described above for the manufacture of a product for detecting botulinum type B toxin;

[0072] E2) use of the nanobody or antigen binding fragment as described above for the manufacture of a product binding to botulinum type B toxin;

[0073] E3) use of the nanobody or antigen binding fragment as described above for the manufacture of a detection reagent for botulinum type B toxin;

[0074] E4) use of the nanobody or antigen binding fragment as described above for the manufacture of a diagnostic reagent for botulinum type B toxin;

[0075] E5) use of the nanobody or antigen binding fragment as described above for the manufacture of a medicament for preventing and / or treating botulinum type B toxin;

[0076] E6) use of the biomaterial as described above for the manufacture of a product for detecting botulinum type B toxin;

[0077] E7) use of the biomaterial as described above for the manufacture of a product binding to botulinum type B toxin;

[0078] E8) use of the preparation method as described above for the manufacture of a product for detecting botulinum type B toxin;

[0079] E9) use of the preparation method as described above for the manufacture of a product binding to botulinum type B toxin;

[0080] E10) use of the kit as described above for the manufacture of a product for detecting botulinum type B toxin;

[0081] E11) use of the kit as described above for the manufacture of a product binding to botulinum type B toxin.

[0082] The product can be a medicament.

[0083] The antigen according to the present application is the Hc antigen of the botulinum toxin of serotype B, BoNT / B-Hc.

[0084] The present application not only includes the complete antibody, but also includes fragments of the nanobody having immunological activity or fusion proteins of the antibody and other sequences. Therefore, the present application also includes polypeptides such as 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 antibodies containing the nanobody described above; D2) Fab containing the nanobody described above; D3) complete antibodies containing the nanobody described above; D4) fusion antibodies containing the nanobody described above; and D5) antibody drug conjugates containing the nanobody described above.

[0085] In a specific embodiment, the fusion antibody of the nanobody of D4) can be M1) a nanobody fusion protein having an amino acid sequence as shown in SEQ ID No. 11.

[0086] As known by those skilled in the art, the conjugate and fusion antibody expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibody or fragments thereof of the present application. The present application also includes cell surface markers or antigens combined with the nanobody or fragments thereof.

[0087] The present application includes any protein or protein conjugate and fusion expression product (i.e. immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical or at least 90% homologous, preferably at least 95% homologous to the heavy chain variable region of the antibody of the present application.

[0088] The nanobody has the advantages of rapid preparation, simple structure and easy modification into a multispecific antibody. The present application immunizes a camel with an antigen, prepares a phage nanobody display library and screens to obtain a nanobody B12 capable of neutralizing botulinum toxin of serotype B, which is expected to become a highly efficient and safe nanobody for neutralizing botulinum toxin of serotype B, replacing the serum products supplied in the market and having certain application value. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 Phage-ELISA was used to identify the binding of some phage clones to the target antigen or control antigen after the third round of screening. The odd columns are the target antigen; the even columns are the control antigen.

[0090] Figure 2SDS-PAGE electrophoresis was used to detect the anti-Botulinum toxin type B nanobody fusion protein B12-hFc after expression and purification. In a, lane 1 is the detection result of irrelevant antibody B5-hFc by reducing SDS-PAGE, lane 2 is the detection result of irrelevant antibody B9-hFc by reducing SDS-PAGE, and lane 3 is the detection result of B12-hFc by reducing SDS-PAGE; in b, lane 1 is the detection result of irrelevant antibody B5-hFc by non-reducing SDS-PAGE, lane 2 is the detection result of irrelevant antibody B9-hFc by non-reducing SDS-PAGE, and lane 3 is the detection result of B12-hFc by non-reducing SDS-PAGE; M is a protein molecular weight marker band.

[0091] Figure 3 The anti-Botulinum toxin type B nanobody fusion protein B12-hFc was detected for binding activity.

[0092] Figure 4 The anti-Botulinum toxin type B nanobody fusion protein B12-hFc was detected for specificity. 7F is the Fiber antigen of adenovirus, and AHc, BHc, EHc, and FHc are the Hc antigens of A, B, E, and F serotypes of botulinum toxin, respectively.

[0093] Figure 5 The anti-Botulinum toxin type B nanobody-hFc fusion protein B12-hFc was evaluated for neutralization activity. BoNT / B is Botulinum toxin type B, BAT-B is horse-derived Botulinum antitoxin solution, and Ab is the hFc fusion protein B12-hFc of anti-Botulinum toxin type B nanobody B12 and human immunoglobulin. DETAILED DESCRIPTION

[0094] The application will be further described in detail below with specific embodiments. The examples provided below are only intended to illustrate the application and not to limit the scope of the 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 application.

[0095] 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.

[0096] In the following examples, unless otherwise specified, the quantitative test was set up with three repeated experiments, and the average value was taken.

[0097] In the following examples, Botulinum toxin type B (BoNT / B) and horse-derived Botulinum antitoxin standard solution (BAT-B) were purchased from the China Institute for Drug Control.

[0098] The Hc antigens of the four serotypes of botulinum toxins, BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, and BoNT / F-Hc, in the following examples were prepared by the present laboratory, and the preparation method has been described in: Shi DY, Liu FJ, Li ZY, Mao YY, Lu JS, Wang R, Pang XB, Yu YZ, Yang ZX. Development and evaluation of a tetravalent botulinum vaccine. Hum Vaccin Immunother. 2022 Nov 30;18(5):2048621. doi: 10.1080 / 21645515.2022.2048621. Epub 2022 Apr 18. PMID: 35435814; PMCID: PMC9196761. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0099] The Fiber antigen of adenovirus in the following examples was prepared by the present laboratory, and the preparation method 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, Zheng T. A humanized anti-human adenovirus 55 monoclonal antibody with good neutralization ability. Front Immunol. 2023 Mar 16;14:1132822. doi: 10.3389 / fimmu.2023.1132822. PMID: 37006289; PMCID: PMC10060833. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0100] The pTSE-hFc in the following examples was modified by linking the gene of the Fc domain of human immunoglobulin G to the pCMV vector. The 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 is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0101] NEN-SCFV in the following examples is engineered by linking the genes of phage surface protein pIII and the arab sugar operon on pET vector. NEN-SCFV 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 anti-human 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 is available from the applicant for the sole use of repeating the experiments of the invention and cannot be used for other purposes.

[0102] The irrelevant control antibodies (B5, B9) in the following examples are prepared by the laboratory by immunizing camels with botulinum toxin Hc protein, isolating camel peripheral blood lymphocytes, amplifying VHH gene fragments by nested PCR, constructing specific nanobody phage library and screening to obtain nanobody molecules that specifically bind to Hc protein, two of which are named B5 and B9, which have been described in: Jiang Y, Wang R, Guo J, Cheng K, Chen L, Wang X, Li Y, Du P, Gao C, Lu J, Yu Y, Yang Z. Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B. Front Immunol. 2024 Apr 30; 15: 1380694. doi: 10.3389 / fimmu.2024.1380694 IF: 7.3Q1. PMID: 38779676; PMCID: PMC11109933. The biological material is available from the applicant for the sole use of repeating the experiments of the invention and cannot be used for other purposes.

[0103] The irrelevant control antibody (T23) in the following examples was prepared by the laboratory, and the preparation method was as follows: immunizing a camel with tetanus TL-HN protein, isolating peripheral blood lymphocytes of the camel, amplifying VHH gene fragments by nested PCR, constructing a specific nanobody phage library and screening to obtain a nanobody molecule specifically binding to TL-HN protein, and we named one of the antibodies as T23. The biological material can be obtained from the applicant, and the biological material is only used for repeating the experiments of the application and cannot be used for other purposes.

[0104] The following examples use GraphPad Prism 8 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, and One-way ANOVA test is used, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, and P<0.001 (***) indicates extremely significant difference.

[0105] Example 1, construction of anti-Botulinum toxin type B nanobody library

[0106] 1. Camel immunization

[0107] The BoNT / B-Hc antigen was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881), shaken and emulsified, and after sufficient emulsification, the healthy adult bactrian camel was injected subcutaneously by multiple point injection, and after that, the booster immunization was performed every two weeks, and except for the first time, Freund's incomplete adjuvant (Sigma, F5506) was used for each immunization.

[0108] 2. Isolation of peripheral blood lymphocytes of camels

[0109] The peripheral blood 120-150 mL of the camel after five immunizations was collected into an anticoagulant tube to isolate peripheral blood lymphocytes (PBMC). After diluting the whole blood sample gently, the mixture was mixed with lymphocyte separation medium (STEMCELL, 07851) to form a clear interface between the two, so as 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.

[0110] 3. Nested PCR amplification of VHH gene fragments

[0111] Total RNA in PBMC was extracted using OMEGA E.Z.N.A Total RNA kit I kit (OMEGA, R6834), and then cDNA was obtained by reverse transcription using Invitrogen Superscript III First-strand synthesis system for RT-PCR kit (Invitrogen, 18080-051). First round of PCR: IgG specific upstream primer CALL001 and downstream primer CALLOO2 were used to amplify CH2 region sequence of antibody; second round of PCR was performed using VHH-F and VHH-R primers to amplify VHH fragment.

[0112] Table 1. Primer sequences used in two rounds of PCR

[0113] Primer name Primer sequence (5'-3') CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F cggCCATGGcGGTCCTGGCTGCTCTTCTACA VHH-R tcccGCGGCCGCTGAGGAGAYGGTGACCWGGGT

[0114] 4. Electroporation ligation product

[0115] The vector NEN-SCFV and amplified VHH gene were digested by restriction endonuclease Ncol and Notl. Ligation product was constructed by T4 ligase.

[0116] The ligation product was transformed into E. coli TG1 competent cells (Beijing Huayueyang Biotechnology Co., Ltd., WG1220) by electroporation transformation technology to construct anti-Botulinum toxin type B specific phage antibody library. The capacity and transformation efficiency of phage antibody library were determined by dilution. The capacity of this antibody library was identified to be 4 x 1010pfu. To detect the accuracy of the library, 48 clones were randomly selected for colony PCR, and the sequence alignment results showed that the VHH fragment insertion rate was 100% and the sequences were different. 8

[0117] Example 2. Screening of anti-Botulinum toxin type B specific nanobody phage library

[0118] The nanobody phage library constructed in Example 1 was used for solid-phase screening using BoNT / B-Hc as antigen to obtain specific nanobodies against Botulinum toxin type B.

[0119] ​The constructed anti-Botulinum toxin type B specific nanobody phage library was transferred to 2YT-GA medium (1 L 2YT medium containing 16 g Typtone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL ampicillin, 20% glucose) and cultured to the logarithmic growth phase. M13KO7 helper phage (NEW ENGLAND BioLabs, N0315S) was added at a ratio of MOI = 10. After 30 min of infection at room temperature, the culture was incubated at 37°C and 150 rpm for 30 min. The deep well plate was centrifuged at 4000 rpm for 15 min at room temperature, and the supernatant was discarded. 2YT-KAA medium (1 L 2YT medium containing 16 g Typtone, 10 g Yeast Extract, 5 g NaCl, 100 μg / mL kanamycin, 20% glucose, and 1 mM final concentration of arabinose) was used for overnight presentation. The next day, the culture presentation supernatant was collected, and the phage was concentrated using a 20% PEG / NaCl solution (1 L solution containing 200 g PEG6000, 146.25 g NaCl) to obtain a high-titer antibody library presentation product for subsequent screening.

[0120] The specific nanobody was screened using the nanobody phage library solid phase. The BoNT / B-Hc protein was coated with 0.05 M NaHCO3 solution (pH = 9.6) to the immunotube and incubated at 4°C overnight. The next day, the immunotube was washed with PBS twice for 3 min each time, 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. After that, it was combined at a low speed of 200 rpm for 20 min. Then, it was washed 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) for 3 times, and then with PBS for 5 times. After washing, 1 mL of elution solution (0.1 M Glycine-HCl, pH 2.2) was added, and the elution was performed at a shaking condition of 400 rpm for 20 min. The elution solution in the antigen immunotube was removed, and 20-60 μL of neutralization solution (1 M Tris-HCl, pH 8.0) was added for neutralization. E. coli TG1 in the logarithmic growth phase was infected, and after 30 min of incubation at room temperature, it was cultured at 37°C and 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.

[0121] Table 2, Anti-Botulinum toxin phage nanobody library screening enrichment degree analysis

[0122] Number of screenings Input (pfu) Output (pfu) Output / input 1 5.0 x 10 11 ]]> 8.4 x 10 5 ]] 1.7 x 10 -6 ]]> 2 1.2 x 10 11 ]]> 2.0 x 10 8 ]]> 1.7 x 10 -3 ]]> 3 1.2 x 10 11 ]]> 3.2 x 10 9 ]]> 2.7 x 10 -2 ]]>

[0123] After the above-mentioned 3 rounds of screening, single colonies with clear spacing and regular shape were selected from the petri dishes with good phage growth, inoculated into 96-well deep well plates containing 2YT-GA medium 250 μL per well, and the remaining 2 wells were not inoculated with clones or inoculated with other antibody clones as negative control wells; the bacterial solution was cultured at 37°C until the logarithmic growth phase, then M13KO7 helper phage was added at a ratio of MOI ≈ 10, i.e. 100 μL per well of the deep well plate in which a single phage clone was cultured, and after 30 min of infection at room temperature, it was cultured at 37°C with low speed 150 rpm for 30 min; the deep well plate was centrifuged at 2000 rpm at room temperature for 10 min, and the supernatant was discarded, and the expression was induced using 1 mM arabinose, and the culture was incubated at 28°C with a rotation speed of 220 rpm overnight to obtain phage particles displaying nanobodies.

[0124] Example 3, Phage-ELISA identification of botulinum type B specific nanobodies

[0125] The BoNT / B-Hc protein was used as an antigen to coat the enzyme-linked plate, the antigen concentration was diluted to 2 ng / μL with 0.05 M NaHCO3 coating solution, the amount was about 200 ng / well, the adjacent column of the antigen column was coated with 2% BSA antigen as a negative control, and the coating was performed at 4°C overnight; the next day, the enzyme-linked plate was taken out, washed 6 times on the plate washer with PBST, 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, 125 μL of supernatant of each bacterial solution was taken 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.

[0126] After washing 6 times with PBST on the plate washer, 100 μL / well of HRP-labeled anti-M13 mouse monoclonal antibody (Sino Bioligical, 1973-MM05T-H) diluted 4000 times with blocking solution was added to the enzyme-linked plate, and incubated at 37°C for 45 min; After washing 6 times with PBST on the plate washer, 100 μL / well of color developing solution (10 mL color developing solution containing 1 mL 10×OPD, 9 mL 0.2M Na2HPO4 and 0.1M citric acid mixed solution, and 10 μL 30% hydrogen peroxide) was added to the enzyme-linked plate, and color developed for 15-20 min in the dark; 50 μL / well of 2M H2SO4 was added to terminate the reaction; and the enzyme-labeled instrument was used to read at dual wavelengths of 492 / 630 nm. The monoclonal antibody with an antigen group to negative control group absorbance ratio greater than 5 was determined as a positive clone. Part of the Phage-ELISA experimental results are shown in Table 1. Figure 1 Table 1: Phage-ELISA experimental results

[0127] The amino acid sequence of B12 is shown in SEQ ID No. 4: including a framework region (FR: FR1, FR2, FR3, FR4) and a complementarity determining region (CDR: CDR1, CDR2, CDR3), 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. 1, SEQ ID No. 2, and SEQ ID No. 3.

[0128] SEQ ID No. 1: GFTDCRYD;

[0129] SEQ ID No. 2: IDSDGST;

[0130] SEQ ID No. 3: KLRLSRCLLPRPDDHYYGMDY;

[0131] SEQ ID No. 4:

[0132] QERLVESGGGSVQAGGSLRLSCAASGFTDCRYDMYWYRQAPGLEREFVSSIDSDGSTTYADSVKGRFTIS QDAAKHMLYLQMNSLRPEDTAMYYCKLRLSRCLLPRPDDHYYGMDYWGKGTQVTISS

[0133] SEQ ID No. 5: QERLVESGGGSVQAGGSLRLSCAAS

[0134] SEQ ID No. 6: MYWYRQAPGLEREFVSS

[0135] SEQ ID No. 7: TYADSVKGRFTISQDAAKHMLYLQMNSLRPEDTAMYYC

[0136] SEQ ID No. 8: WGKGTQVTISS

[0137] The gene encoding the above-mentioned anti-botulinum toxin type B nanobody B12 has a nucleotide sequence as shown in SEQ ID No. 9.

[0138] SEQ ID No. 9:

[0139] CAGGAGCGATTGGTGGAGTCTGGGGGAGGGTCGGTGCAGGCTGGAGGGTCTCTAAGACTCTCCTGTGCAGCCTCTGGATTTACCGACTGTAGGTACGACATGTACTGGTACCGCCAGGCTCCAGGGCTTGAGCGCGAGTTCGTCTCAAGTATTGATAGTGATGGTAGCACAACCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACGCCGCCAAGCACATGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCATGTATTACTGTAAATTACGTCTGTCACGGTGTCTCCTTCCACGCCCGGATGACCATTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCATCTCCTCA.

[0140] Example 4, Preparation of Anti-botulinum toxin type B nanobody-hFc fusion protein

[0141] 1. Construction of anti-botulinum toxin type B nanobody-hFc fusion protein eukaryotic expression plasmid pTSE-B12-hFc

[0142] According to the anti-Botulinum toxin type B nanobody gene sequence (SEQ ID No. 9), the carboxyl terminal thereof is connected with the hFc segment of human immunoglobulin to form an anti-Botulinum toxin type B nanobody-hFc fusion protein. The coding nucleotide sequence of the fusion protein is shown in SEQ ID No. 10, and the amino acid sequence of the fusion protein is shown in SEQ ID No. 11.

[0143] SEQ ID No. 10:

[0144] CAGGAGCGATTGGTGGAGTCTGGGGGAGGGTCGGTGCAGGCTGGAGGGTCTCTAAGACTCTCCTGTGCAGCCTCTGGATTTACCGACTGTAGGTACGACATGTACTGGTACCGCCAGGCTCCAGGGCTTGAGCGCGAGTTCGTCTCAAGTATTGATAGTGATGGTAGCACAACCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACGCCGCCAAGCACATGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCATGTATTACTGTAAATTACGTCTGTCACGGTGTCTCCTTCCACGCCCGGATGACCATTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCATCTCCTCAGCTAGCgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgCtgcatgaggctctgcacaGccactacacgcagaagagcctctccctgtccccgggtaaatga.

[0145] SEQ ID No. 11:

[0146] QERLVESGGGSVQAGGSLRLSCAASGFTDCRYDMYWYRQAPGLEREFVSSIDSDGSTTYADSVKGRFTISQDAAKHMLYLQMNSLRPEDTAMYYCKLRLSRCLLPRPDDHYYGMDYWGKGTQVTISSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0147] The obtained B12 gene sequence was cloned into the pTSE-hFc expression vector by basic PCR amplification, enzyme digestion, ligation, etc. 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-B12-hFc.

[0148] The structure of the recombinant vector pTSE-B12-hFc is described as follows: a DNA fragment with the sequence of SEQ ID No. 9 is inserted between the recognition sites of restriction endonucleases Sal I and Nhe I of the pTSE-hFc vector, and the other sequences of the pTSE-hFc vector remain unchanged to obtain the recombinant vector. The recombinant vector pTSE-B12-hFc can express the fusion protein B12-hFc with the amino acid sequence of SEQ ID No. 11.

[0149] 2. Expression and purification of anti-botulinum toxin nanobody fusion protein B12-hFc

[0150] The constructed pTSE-B12-hFc expression plasmid was transfected into FreeStyle 293-F cells (Invitrogen, R79007) with transfection reagent FectoPRO DNA Transfection Reagent (Polyplus, 116-001). After 72 hours, the cell supernatant was collected for purification when the cell viability decreased from 95-100% to 80-85% every day, and the nanobody-hFc fusion protein B12-hFc was obtained. TM HEK293-F cells (Invitrogen, R79007) with transfection reagent FectoPRO DNA Transfection Reagent (Polyplus, 116-001). After 72 hours, the cell supernatant was collected for purification when the cell viability decreased from 95-100% to 80-85% every day, and the nanobody-hFc fusion protein B12-hFc was obtained.

[0151] SDS-PAGE electrophoresis analysis of the purified antibody B12-hFc, where the irrelevant antibody electrophoresis band is the purified irrelevant antibody B5-hFc, B9-hFc (nanobody-hFc fusion protein against BoNT / B-Hc protein prepared by the laboratory). The results are shown in Figure 2 Figure 2: The molecular weight of the antibody B12-hFc is consistent with the expected value, and the band size of the nanobody fusion protein B12-hFc under reducing conditions is about 40 kDa (a), and the band size under non-reducing conditions is about 80 kDa (b). Figure 2 Figure 2

[0152] Example 5, Evaluation of the characteristics of the nanobody fusion protein B12-hFc

[0153] 1. ELISA experiment to detect the binding activity between the nanobody fusion protein B12-hFc and the BoNT / B-Hc protein. The experimental method is as follows:

[0154] ​​BoNT / B-Hc protein was diluted to 2 μg / mL with carbonate coating buffer (pH 9.6) and added to a 96-well ELISA plate at 100 μL / well, coated overnight at 4°C; the coating solution was discarded and the plate was washed 6 times with PBST (1 L PBS solution containing 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, 0.1% Tween-20) on a plate washer, and the residual liquid in the plate was patted dry, 200 μL / well of blocking solution (3% skim milk powder) was added, and the plate was blocked at 37°C for 2 h; the antibody to be tested for binding activity was diluted 2-fold by volume, with an initial dilution of 12.5 μg / mL, the blocking solution in the plate was discarded, and the plate was washed 6 times with PBST and the residual liquid was patted dry, 100 μL / well of B12-hFc to be tested was added to the plate, and the plate was incubated at 37°C for 1.5 h; HRP-labeled goat anti-human IgG was diluted 1:4000 with the blocking solution, the primary antibody was discarded, the plate was washed 6 times with PBST, 100 μL / well was added to the plate, and the plate was incubated at 37°C for 45 min; the secondary antibody was discarded, the plate was washed 6 times with PBST, 100 μL / well of peroxidase substrate developing solution was added, and the plate was developed in the dark for 15-20 min, the developing effect was observed, 50 μL / well of 2 M sulfuric acid was added to terminate the reaction after the developing was complete, and the optical density value was measured using an enzyme label instrument at 492 nm / 630 nm dual wavelength. The binding ability between the antibodies was evaluated by calculating the concentration required for 50% of the antigen protein to be bound (EC 50 ).

[0155] The results are shown in Table 1. Figure 3 The analysis showed that the half maximal effective concentration (EC 50 ) of B12-hFc binding to BoNT / B-Hc protein was 1.652 nM.

[0156] 2. Identification of the specificity of the nanobody fusion protein B12-hFc by ELISA experiment

[0157] The experimental method is as follows:

[0158] BoNT / A-Hc, BoNT / B-Hc, BoNT / E-Hc, BoNT / F-Hc, Fiber antigen of adenovirus (7F) were diluted to 1 μg / mL with carbonate coating buffer, 100 μL / well was added to 96-well ELISA plate, and coated overnight at 4°C; the next day, the coating solution was discarded, and the 96-well plate coated overnight was washed 6 times with PBST (0.1% Tween-20) on the plate washer, then the residual liquid in the wells was patted dry, 200 μL / well of blocking solution (3% skim milk powder) was added, and blocked at 37°C for 2 h; B12-hFc protein primary antibody solution was prepared, and the antibody was diluted to 1 μg / mL, the blocking solution was discarded, washed 6 times with PBST, 100 μL / well of B12-hFc was added, and incubated at 37°C for 1.5 h; HRP-labeled goat anti-human IgG was diluted with blocking solution at a ratio of 1:4000, the primary antibody was discarded, washed 6 times with PBST, 100 μL / well was added to the plate, and incubated at 37°C for 45 min; the secondary antibody was discarded, washed 6 times with PBST, and then 100 μL / well of peroxidase substrate developing solution was added, and developed for 15-20 min in the dark, then the developing effect was observed, 50 μL / well of 2M sulfuric acid was added to stop the reaction after complete development; the enzyme label instrument was used to measure the optical density value at 492 nm / 630 nm, and the data results were analyzed by GraphPad Prism 8 software.

[0159] The results are shown in Figure 4 Table 1, B12-hFc specifically binds to BoNT / B-Hc antigen, and does not bind to other antigens or has weak binding activity.

[0160] Example 6, Evaluation of neutralizing activity of anti-botulinum toxin nanobody-hFc fusion protein

[0161] The antibody neutralizing activity was determined by mixing the antibody with a lethal dose of type B botulinum toxin in vitro and then injecting KM (Kunming) mice (purchased from Beijing Sbielof Biotechnology Co., Ltd.) The KM mice were 4 in each group, and the weight was 18-20 g. The evaluation method is as follows:

[0162] 1. Sample preparation:

[0163] Diluent: KH2PO40.7 g, Na2HPO4·12H2O 2.4 g, NaCl 6.8 g, gelatin 2 g, water to 1 L, high pressure sterilization;

[0164] Botulinum toxin solution: type B botulinum toxin (purchased from China Institute for Food and Drug Control) was diluted to 100 LD 50 / mL with diluent;

[0165] B12-hFc solution: the solution obtained by dissolving the fusion protein B12-hFc prepared in Example 4 with diluent.

[0166] BAT-B: is a solution obtained by dissolving horse anti-botulinum toxin serum (purchased from China Institute for Food and Drug Control) with diluent.

[0167] 2. Experimental grouping scheme is as follows:

[0168] (1) BoNT / B solution 20xLD 50 Group: each KM mouse is injected intraperitoneally with 200 μL of 100xLD 50 / mL botulinum toxin solution, so that each injection solution contains 20xLD 50 / mouse.

[0169] (2) BoNT / B 20xLD 50 + B12-hFc-2 μg group: the above botulinum toxin solution is mixed with B12-hFc solution, the volume of each group is supplemented to 2.5 mL with diluent, and after uniform mixing, it is incubated at 37°C for 30 min to obtain BoNT / B + B12-hFc solution, which is injected into KM mice intraperitoneally, each mouse is injected with 500 μL, so that the dose of BoNT / B is 20xLD 50 / mouse, and the dose of B12-hFc in each injection solution is 2 μg / mouse.

[0170] (3) BoNT / B 20xLD 50 + B12-hFc-1 μg group: the difference between this group and the BoNT / B + B12-hFc-2 μg group is that the dose of B12-hFc is 1 μg / mouse, and the rest of the operations are the same as those of the BoNT / B 20xLD 50 + B12-hFc-2 μg group.

[0171] (4) BoNT / B 20xLD 50 + B12-hFc-0.5 μg group: the difference between this group and the BoNT / B + B12-hFc-2 μg group is that the dose of B12-hFc is 0.5 μg / mouse, and the rest of the operations are the same as those of the BoNT / B 20xLD 50 + B12-hFc-2 μg group.

[0172] (5) BoNT / B 20xLD 50 + B12-hFc-0.25 μg group: the difference between this group and the BoNT / B + B12-hFc-2 μg group is that the dose of B12-hFc is 0.25 μg / mouse, and the rest of the operations are the same as those of the BoNT / B 20xLD 50 + B12-hFc-2 μg group.

[0173] (6) BoNT / B 20xLD50 +B12-hFc-0.125μg group: the difference between this group and BoNT / B+B12-hFc-2μg group is that the dose of B12-hFc is 0.125 μg per mouse, and the rest of the operations are the same as BoNT / B 20×LD 50 +B12-hFc-2μg group.

[0174] (7) BoNT / B 20×LD 50 +B12-hFc-0.0625μg group: the difference between this group and BoNT / B+B12-hFc-2μg group is that the dose of B12-hFc is 0.0625 μg per mouse, and the rest of the operations are the same as BoNT / B 20×LD 50 +B12-hFc-2μg group.

[0175] (8) BoNT / B 20×LD 50 +BAT-B-0.2IU group: the botulinum toxin solution is mixed with the horse-derived botulinum antitoxin standard solution, and the volume of each group is supplemented to 2.5 mL with the diluent, and after uniform mixing, it is incubated at 37°C for 30 min to obtain a BoNT / B+BAT-B solution, which is injected into the KM mice intraperitoneally, with each mouse injected with 500 μL, so that the dose of botulinum toxin is 20×LD 50 / mouse, and the dose of horse-derived botulinum antitoxin BAT-B is 0.2 IU per mouse.

[0176] Four mice were injected in each experimental group, and the health status and survival of the mice were monitored for 7 days.

[0177] The results are shown in Table 1. Figure 5 0.25 μg of B12-hFc can completely neutralize the lethal dose of 20×LD 50 BoNT / B.

[0178] Example 7, Evaluation of the Preventive and Therapeutic Effects of Anti-botulinum Toxin Nanobody Fusion Protein B12-hFc

[0179] 1. To evaluate whether anti-botulinum toxin nanobody fusion protein B12-hFc has a protective effect on BoNT attack, and whether this protective effect shows a dose-dependent effect in a mouse model, KM mice (purchased from Beijing Sibeifeng Biotechnology Co., Ltd.) were selected, with 4 mice per group and a weight of 18-20 g. The evaluation method is as follows:

[0180] Diluent: KH2PO40.7 g, Na2HPO4·12H2O 2.4 g, NaCl 6.8 g, gelatin 2 g, add water to 1 L, autoclave;

[0181] Botulinum toxin solution: B-type botulinum toxin (purchased from China Institute for Food and Drug Control) was diluted to 40 x LD 50 / mL or 200 x LD 50 / mL with diluent;

[0182] B12-hFc solution: a solution prepared by dissolving B12-hFc prepared in Example 4 with diluent;

[0183] Horse-derived botulinum antitoxin standard (BAT-B) solution: a solution prepared by dissolving horse-derived antitoxin serum (purchased from China Institute for Food and Drug Control) with diluent;

[0184] Irrelevant antibody solution: a solution prepared by dissolving irrelevant antibody T23-hFc (a nanobody-hFc fusion protein against tetanus TL-HN protein prepared by the laboratory) with diluent.

[0185] The prevention evaluation experiment was repeated three times, and each experiment was divided into the following groups:

[0186] A, BoNT / B 20 x LD 50 / each + antibody - 0.025 mg / kg

[0187] 1) BoNT / B 20 x LD 50 / each + B12-hFc - 0.025 mg / kg group: 100 μL of B12-hFc solution (injection dose of 0.025 mg / kg) was used to treat mice via tail vein, and 500 μL of 40 x LD 50 / mL botulinum toxin solution was injected intraperitoneally into KM mice at 24, 48 and 72 hours, respectively, with an injection amount of 20 x LD 50 / each. Four mice were injected in each experimental group, and the health and survival of the mice were monitored for 7 days.

[0188] 2) BoNT / B 20 x LD 50 / each + BAT-B - 0.025 mg / kg group: 100 μL of BAT-B solution (i.e. injection dose of 0.025 mg / kg / each) was used to replace B12-hFc solution, and the rest of the operations were the same as those of BoNT / B 20 x LD 50 / each + B12-hFc - 0.025 mg / kg group.

[0189] 3) BoNT / B 20 x LD 50 / each + T23-hFc - 0.025 mg / kg group: 100 μL of T23-hFc solution (i.e. injection dose of 0.025 mg / kg) was used to replace B12-hFc solution, and the rest of the operations were the same as those of BoNT / B 20 x LD 50 / animal + B12-hFc - 0.025 mg / kg group.

[0190] 4) PBS group: replace the antibody solution with equal volume of PBS, and the rest of the operation is the same as BoNT / B 20xLD 50 / animal + B12-hFc - 0.025 mg / kg group.

[0191] B, BoNT / B 100xLD 50 / animal + antibody - 0.025 mg / kg

[0192] 1) BoNT / B 100xLD 50 / animal + B12-hFc - 0.025 mg / kg group: B12-hFc solution 100 μL (i.e. injection dose is 0.025 mg / kg) is used to treat mice through tail vein, and 500 μL 200xLD 50 / mL botulinum toxin solution is injected into KM mice through intraperitoneal injection at 24, 48 and 72 hours respectively, and the injection amount is 100xLD 50 / animal. Four animals are injected in each experimental group, and the health status and survival of the mice are monitored within 7 days.

[0193] 2) BoNT / B 100xLD 50 / animal + T23-hFc - 0.025 mg / kg group: T23-hFc solution 100 μL (i.e. injection dose is 0.025 mg / kg) is used to replace B12-hFc solution, and the rest of the operation is the same as BoNT / B 100xLD 50 / animal + B12-hFc - 0.025 mg / kg group.

[0194] C, BoNT / B 20xLD 50 / animal + antibody - 0.125 mg / kg

[0195] 1) BoNT / B 20xLD 50 / animal + B12-hFc - 0.125 mg / kg group: B12-hFc solution 100 μL (i.e. injection dose is 0.125 mg / kg) is used to treat mice through tail vein, and 500 μL 40xLD 50 / mL botulinum toxin solution is injected into KM mice through intraperitoneal injection at 24, 48 and 72 hours respectively, and the injection amount is 20xLD 50 / animal. Four animals are injected in each experimental group, and the health status and survival of the mice are monitored within 7 days.

[0196] 2) BoNT / B 20xLD 50 / only + T23-hFc - 0.125 mg / kg group: 100 μL of T23-hFc solution (i.e. injection dose of 0.125 mg / kg) was used to replace B12-hFc solution, and the rest of the procedures were the same as BoNT / B 20xLD 50 / only + B12-hFc - 0.125 mg / kg group.

[0197] D, BoNT / B 100xLD 50 / only + antibody - 0.125 mg / kg

[0198] 1) BoNT / B 100xLD 50 / only + B12-hFc - 0.125 mg / kg group: 100 μL of B12-hFc solution (i.e. injection dose of 0.125 mg / kg) was used to treat mice via tail vein, and 500 μL of 200xLD 50 / mL botulinum toxin solution was injected into KM mice via intraperitoneal injection at 24, 48 and 72 hours, respectively 50 / each. Four mice were injected in each experimental group, and the health and survival of the mice were monitored for 7 days.

[0199] 2) BoNT / B 100xLD 50 / only + BAT-B - 0.125 mg / kg group: 100 μL of BAT-B solution (i.e. injection dose of 0.125 mg / kg per mouse) was used to replace B12-hFc solution, and the rest of the procedures were the same as BoNT / B 100xLD 50 / only + B12-hFc - 0.125 mg / kg group.

[0200] 3) BoNT / B 100xLD 50 / only + T23-hFc - 0.125 mg / kg group: 100 μL of T23-hFc solution (i.e. injection dose of 0.125 mg / kg) was used to replace B12-hFc solution, and the rest of the procedures were the same as BoNT / B 100xLD 50 / only + B12-hFc - 0.125 mg / kg group.

[0201] The results are shown in Table 3. The low-dose (0.025 mg / kg) B12-hFc antibody group was effective in resisting 20xLD 50 BoNT / B attack within 3 days after injection of the antibody; the high-dose (0.125 mg / kg) B12-hFc antibody group was effective in resisting 100xLD 50 BoNT / B attack within 3 days after injection of the antibody; the high-dose (0.125 mg / kg) B12-hFc antibody group was effective in resisting 100xLD 50 BoNT / B attack within 2 days; and the high-dose (0.125 mg / kg) B12-hFc antibody group was completely protected from 100xLD

[0202] Table 3, Evaluation of the preventive effect of anti-botulinum toxin nanobody-hFc fusion proteins

[0203]

[0204] Note: a. KM mice in each experimental group treated with B12-hFc, BAT-B, T23-hFc or PBS were injected intraperitoneally with 20xLD 50 or 100xLD 50 of BoNT / B at different times; b KM mice in each experimental group were injected with 0.025 or 0.125 mg / kg B12-hFc, 0.125 or 0.125 mg / kg BAT-B, 0.025 or 0.125 mg / kg T23-hFc, or PBS for the prevention experiment; c KM mice were injected with different doses of BoNT / B at 24, 48 and 72 hours after injection of the specified dose of antibody; d The number of KM mice in each experimental group was four, and the final mouse survival after seven days was determined.

[0205] 2. To evaluate whether anti-botulinum toxin nanobody-hFc fusion proteins have a therapeutic effect after exposure to BoNT / B in mice, KM mice, 4 per group, 18-20 g, purchased from Beijing Sbi Bio-technology Co., Ltd. were used. The evaluation method is as follows:

[0206] The preparation method of the diluent, B12-hFc solution, horse-derived botulinum antitoxin standard (BAT-B) solution, and irrelevant antibody (T23-hFc) solution is as described in Step 1 of this example.

[0207] Botulinum toxin solution: B-type botulinum toxin (purchased from China Institute for Food and Drug Control) was diluted with diluent to 10 LD 50 / mL or 40 LD 50 / mL.

[0208] The experimental groups are as follows:

[0209] A, BoNT / B 5xLD 50 / each + antibody - 0.025 mg / kg

[0210] 1) BoNT / B 5xLD 50 / each + B12-hFc - 0.025 mg / kg group: KM mice were injected intraperitoneally with 500 μL of 10xLD 50 / mL botulinum toxin solution, with an injection volume of 5xLD 50mice were divided into 0.5 hour group, 1 hour group, 2 hour group and 3 hour group, 4 mice in each group. Each group of mice was injected with B12-hFc solution 100 μL (i.e. injection dose was 0.025 mg / kg) at 0.5, 1, 2 or 3 hours after injecting botulinum toxin solution, respectively. The health status and survival of mice were monitored within 7 days.

[0211] 2) BoNT / B 5xLD 50 mice + BAT-B - 0.025 mg / kg group: 100 μL of horse-derived anti-botulinum toxin serum solution (i.e. injection dose was 0.025 mg / kg) was used to replace B12-hFc solution, and the rest of the operation was the same as BoNT / B 5xLD 50 mice + B12-hFc - 0.025 mg / kg group.

[0212] 3) BoNT / B 5xLD 50 mice + T23-hFc - 0.025 mg / kg group: 100 μL of T23-hFc solution (i.e. injection dose was 0.025 mg / kg) was used to replace B12-hFc solution, and the rest of the operation was the same as BoNT / B 5xLD 50 mice + B12-hFc - 0.025 mg / kg group.

[0213] 4) PBS group: equal volume of PBS was used to replace B12-hFc solution, and the rest of the operation was the same as BoNT / B 5xLD 50 mice + B12-hFc - 0.025 mg / kg group.

[0214] B, BoNT / B 20xLD 50 mice + antibody - 0.025 mg / kg

[0215] 1) BoNT / B 20xLD 50 mice + B12-hFc - 0.025 mg / kg group: 500 μL of 40xLD 50 mL botulinum toxin solution was injected intraperitoneally to KM mice, and the injection amount was 20xLD 50 mice were divided into 0.5 hour group, 1 hour group, 2 hour group and 3 hour group, 4 mice in each group. Each group of mice was injected with B12-hFc solution 100 μL (i.e. injection dose was 0.025 mg / kg) at 0.5, 1, 2 or 3 hours after injecting botulinum toxin solution, respectively. The health status and survival of mice were monitored within 7 days.

[0216] 2) BoNT / B 20xLD 50 / only + T23-hFc - 0.025 mg / kg group: 100 μL of T23-hFc solution (i.e., the injection dose was 0.025 mg / kg) was used to replace the B12-hFc solution, and the rest of the procedures were the same as BoNT / B 5xLD 50 / only + B12-hFc - 0.025 mg / kg group.

[0217] C, BoNT / B 5xLD 50 / only + antibody - 0.125 mg / kg

[0218] 1) BoNT / B 5xLD 50 / only + B12-hFc - 0.125 mg / kg group: 500 μL of 10xLD 50 / mL botulinum toxin solution was injected into KM mice intraperitoneally, and the injection dose was 5xLD 50 / only. The injected mice were divided into 0.5 hour group, 1 hour group, 2 hour group, and 3 hour group, 4 mice in each group. Each group of mice was injected with 100 μL of B12-hFc solution (i.e., the injection dose was 0.125 mg / kg) at 0.5, 1, 2, or 3 hours after the injection of the botulinum toxin solution, respectively. The health status and survival of the mice were monitored for 7 days.

[0219] 2) BoNT / B 5xLD 50 / only + T23-hFc - 0.125 mg / kg group: 100 μL of T23-hFc solution (i.e., the injection dose was 0.125 mg / kg) was used to replace the B12-hFc solution, and the rest of the procedures were the same as BoNT / B 5xLD 50 / only + B12-hFc - 0.125 mg / kg group.

[0220] D, BoNT / B 20xLD 50 / only + antibody - 0.125 mg / kg

[0221] 1) BoNT / B 20xLD 50 / only + B12-hFc - 0.125 mg / kg group: 500 μL of 40xLD 50 / mL botulinum toxin solution was injected into KM mice intraperitoneally, and the injection dose was 20xLD 50 / only. The injected mice were divided into 0.5 hour group, 1 hour group, 2 hour group, and 3 hour group, 4 mice in each group. Each group of mice was injected with 100 μL of B12-hFc solution (i.e., the injection dose was 0.125 mg / kg) at 0.5, 1, 2, or 3 hours after the injection of the botulinum toxin solution, respectively. The health status and survival of the mice were monitored for 7 days.

[0222] 2) BoNT / B 20xLD 50 / only+BAT-B-0.125mg / kg group: replace B12-hFc solution with 100 μL of equine anti-botulinum toxin serum solution (i.e. injection dose is 0.125 mg / kg / only), and the rest of the operation is the same as BoNT / B 20xLD 50 / only+B12-hFc-0.125mg / kg group.

[0223] 3) BoNT / B 20xLD 50 / only+T23-hFc-0.125mg / kg group: replace B12-hFc solution with 100 μL of T23-hFc solution (i.e. injection dose is 0.125 mg / kg), and the rest of the operation is the same as BoNT / B 20xLD 50 / only+B12-hFc-0.125mg / kg group.

[0224] Table 4, therapeutic effect evaluation of anti-botulinum toxin nanobody-hFc fusion protein

[0225]

[0226]

[0227] Note: a KM mice in each experimental group treated with B12-hFc, BAT-B, T23-hFc or PBS were first injected intraperitoneally with 5xLD 50 or 20xLD 50 dose of BoNT / B; b KM mice in each experimental group were treated with 0.025 or 0.125 mg / kg B12-hFc, 0.125 or 0.125 mg / kg BAT-B, 0.025 or 0.125 mg / kg T23-hFc, PBS at different time points after infection; c KM mice were injected with different doses of antibodies or PBS at 0.5, 1, 2 and 3 hours after the mice were attacked with the specified dose of toxin; d The number of KM mice in each experimental group was four, and the final mouse survival after seven days.

[0228] The results are shown in Table 4, and the high-dose BoNT / B attack (20xLD 50 ), the B12-hFc treatment group was effective within 2 hours after exposure, and the protection effect was best within 1 hour, and 2.5 μg of antibody could achieve complete protection; for low-dose BoNT / B attack (5xLD 50 ), the B12-hFc treatment group was effective within 3 hours after exposure, and the protection effect was best within 1 hour, and 0.5 μg of antibody could achieve complete protection.

[0229] 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 special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A nanobody targeting botulinum toxin type B, characterized in that, The nanobody has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown as SEQ ID No. 1, the amino acid sequence of the CDR2 is shown as SEQ ID No. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID No.

3.

2. The Nanobody according to claim 1, characterized in that, The nanobody is as follows A1) or A2): A1) a nanobody with an amino acid sequence shown as SEQ ID No. 4; A2) a nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown as SEQ ID No.

4.

3. A biological material related to the nanobody of claim 1 or 2, which is any one of the following: B1) a nucleic acid molecule encoding the nanobody of claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1); B4) a recombinant microorganism containing the nucleic acid molecule of B1).

4. The biomaterial of claim 3, wherein, The nucleic acid molecule of B1) is a nucleic acid molecule encoding the nanobody of claim 1 or 2, wherein the gene encoding the CDR1 is nucleotides 76-99 of SEQ ID No. 9, the gene encoding the CDR2 is nucleotides 151-171 of SEQ ID No. 9, and the gene encoding the CDR3 is nucleotides 286-348 of SEQ ID No.

9.

5. A method for the production of a Nanobody according to claim 1 or 2, comprising the steps of: The nucleic acid molecule encoding the nanobody of claim 1 or 2 is introduced into a recipient cell to obtain a transgenic cell expressing the nanobody, and the transgenic cell is cultured to obtain the nanobody.

6. The biomaterial according to claim 3 or 4, characterized in that, The nucleic acid molecule of B1) is a DNA molecule with a nucleotide sequence shown as SEQ ID No.

9.

7. A Nanobody-hFc fusion protein characterized in that, The nanobody-hFc fusion protein is a fusion of the nanobody of claim 1 or 2 and the Fc domain of human immunoglobulin.

8. The Nanobody-hFc fusion protein according to claim 7, characterized in that, The fusion protein is any one of the following: M1) a fusion protein with an amino acid sequence shown as SEQ ID No. 11; M2) a protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown as SEQ ID No.

11.

9. An ELISA test kit for targeting botulinum toxin type B, characterized by, The kit contains the nanobody of claim 1 or 2 or the fusion protein of claim 7 or 8.

10. Any one of the following applications: E1) use of the nanobody of claim 1 or 2 in the preparation of a product for detecting botulinum type B toxin; E2) use of the nanobody of claim 1 or 2 in the preparation of a drug for preventing and / or treating botulinum type B toxin infection; E3) use of the biological material of claim 3 or 4 in the preparation of a product for detecting botulinum type B toxin; E4) use of the preparation method of claim 5 in the preparation of a product for detecting botulinum type B toxin; E5) use of the kit of claim 9 in the preparation of a product for detecting botulinum type B toxin.

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