Neutralizing antibody for resisting botulinum toxin L-HN antigen as well as related biological material and application thereof

Abstract

The invention discloses a neutralizing antibody for resisting a botulinum toxin L-HN antigen as well as a related biological material and application thereof, and belongs to the field of immunotherapy biotechnology pharmacy. The nano antibody or the antigen binding fragment thereof comprises three complementary determining regions CDR1, CDR2 and CDR 3, 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 sequences of the complementary determining regions are defined according to an IMGT numbering system. The antibody can effectively neutralize endotoxin, has the advantages of being rapid in preparation, simple in structure and convenient to transform into a multispecific antibody, is expected to be developed into an efficient and safe botulinum toxin type E neutralizing nano antibody, and is used for replacing serum products supplied in the market so as to meet the requirements of human beings.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy biotechnology and pharmaceuticals, and specifically relates to neutralizing antibodies against botulinum toxin L-HN antigen and related biomaterials and applications thereof. Background Art

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

[0003] The main symptoms of botulism poisoning include muscle paralysis and breathing difficulties. The reaction speed after poisoning is fast and the lethality is strong. Botulinum toxin poisoning is generally treated with antitoxins, but antitoxins can only neutralize free toxins in the blood or body fluids, and are ineffective against toxins that have entered the cells. In addition, the production period is long, the production batches are unstable, and there are hidden dangers of allergic reactions and viral contamination. The vaccines commonly used for prevention abroad are PBT pentavalent vaccines and heptavalent vaccines. There are no vaccines available on the market in China.

[0004] Antibodies can prevent and treat botulism in the short term and have broad application prospects. Nanobodies (Nb) are a novel antigen recognition tool. Compared with other types of antibodies, they do not contain light chains and have a longer CDR3 region. They can exist independently and stably in vitro. Their relative molecular mass is only 1 / 10 of that of traditional complete antibodies. They have the advantages of strong modifiability, low immunogenicity, few side effects, easy screening and expression, high affinity and stability, etc. They have been proven to have great value as therapeutic molecules and clinical diagnostic tools.

[0005] Although food poisoning, infant poisoning, trauma-induced, and adult intestinal colonization botulism poisoning incidents are rarely reported, the number of botulism poisoning incidents is increasing with the illegal use of botulinum toxin and the increasing use of botulinum toxin as a cosmetic preparation. Therefore, it is very important to apply nanoantibody technology to the development of efficient and safe anti-botulinum toxin neutralizing nanoantibodies. Summary of the invention

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

[0007] In order to solve the above problems, the present invention provides a nanobody targeting botulinum toxin or an antigen-binding fragment containing the nanobody.

[0008] The present invention provides a nanobody targeting botulinum toxin or an antigen-binding fragment containing the nanobody, wherein the nanobody has three complementary determining clusters CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 includes that shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 includes that shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 includes that shown in 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 CDRs are sequences defined based on the analysis results of the IMGT system.

[0012] The nanobodies described herein generally include a VHH consisting 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 comprises at least a portion of the nanobody that is sufficient to confer the fragment the ability to specifically bind to botulinum toxin type E.

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

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

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

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

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

[0018] In the Nanobody or antigen-binding fragment described above, the Nanobody may be any of the following:

[0019] A1) the amino acid sequence includes the Nanobody shown in SEQ ID NO: 1 or SEQ ID NO: 12;

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

[0021] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein to facilitate the expression, detection, tracing and / or purification of the target protein. 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, the Fc segment of immunoglobulin G, etc.

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

[0023] In the present invention, the protein tag is the Fc segment of human immunoglobulin G (hFc).

[0024] In the above-mentioned nanobody, the nanobody consists of the complementary determining cluster region and the framework region.

[0025] The present invention also provides a heavy chain antibody, which comprises the aforementioned nanobody and the Fc region of an immunoglobulin.

[0026] In the above-mentioned 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 a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID NO:1.

[0027] In the above-mentioned 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-mentioned heavy chain antibody, the amino acid sequence of the heavy chain antibody may specifically be SEQ ID NO:11 or SEQ ID NO:15.

[0029] The Fc region herein is a C-terminal region comprising at least a portion of the constant region of an immunoglobulin heavy chain. The Fc region includes a native sequence Fc region and a variant Fc region. The variant Fc region may include a human variant Fc region (the Fc region of a human immunoglobulin comprises an amino acid modification (e.g., substitution) at one or more amino acid positions). In a specific embodiment of the present invention, the Fc region is specifically composed of two constant domains (CH2 and CH3). The immunoglobulin Fc region may be the Fc region of a human immunoglobulin. The human immunoglobulin may be human IgG1, IgG2, IgG3, or IgG4.

[0030] The term "antibody" in the present invention is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, which consists 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, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced 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. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0031] In the present invention, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the heavy chain of a cloned antibody to construct a minimal antigen-binding fragment consisting of only one heavy chain variable region with complete functions.

[0032] The above-mentioned term "nanoantibody (single domain antibody)" (VHH)) is a polypeptide composed of the variable region of the heavy chain of an antibody. Single-domain antibodies can be prepared as follows: the variable region of the heavy chain of an antibody (VH) is expressed by a genetic engineering method to obtain an antibody containing only a VH fragment. The ability of a single-domain antibody to bind to an antigen and its stability are basically the same as those of a complete antibody. The above-mentioned term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody (parentalan tibody). The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when the activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.

[0033] The above-mentioned antigen-binding fragment can be a complete antibody, a fusion antibody, an antibody-drug conjugate, a Fab fragment, a 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 formed by the variable region VH of the heavy chain and the first constant region CH1 (Fd) and the complete light chain bound by disulfide bonds, and contains only one antigen binding site. The heavy chain Fd mentioned above refers to about 1 / 2 of the H chain portion in Fab (containing about 225 amino acid residues, including VH, CH1 and part of the hinge region).

[0035] The term "Fv fragment" refers to the construction of vectors containing VH and VL genes respectively, co-transfection of cells, expression of them separately, and then assembly into functional Fv antibodies; or a stop codon can be set between VH and VL in the vector to express two small molecule protein fragments separately, and then form Fv antibodies (Fv fragments) through non-covalent bonds.

[0036] The term "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the 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 comprising a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists 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 in the variable region, with a molecular mass of only about 1% of the complete antibody, which can bind to the corresponding antigen.

[0039] The term "variable" in the present invention means that some parts of the variable region in the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0040] In some embodiments, the Nanobodies described herein may be truncated at the N- or C-terminus so that they comprise only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as they substantially retain antigen binding and specificity.

[0041] In the present invention, the nanobody is named nanobody E22.

[0042] The present invention also provides a biomaterial related to the nanobody described above, and the biomaterial may be any of the following:

[0043] B1) a nucleic acid molecule encoding a Nanobody or antigen-binding fragment as described above;

[0044] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0045] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0046] B4) a recombinant vector containing the expression cassette described in B2);

[0047] B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0048] B6) a recombinant microorganism containing the expression cassette described in B2);

[0049] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0050] B8) A recombinant microorganism containing the recombinant vector described in B4).

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

[0052] In the above-mentioned biological material, the expression cassette described in B2) refers to a DNA capable of expressing the nanobody in a host cell, which may 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 may also include an enhancer sequence.

[0053] Existing expression vectors can be used to construct a recombinant vector containing the expression cassette.

[0054] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

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

[0056] In the above biological materials, the microorganisms may be bacteria (such as Escherichia coli), yeast, algae or fungi.

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

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

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

[0060] C2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes said Nanobody;

[0061] C3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more homology with any of the DNA sequences defined in C1) to C2) and encodes the Nanobody.

[0062] Wherein, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 2×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 1×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.5×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.1×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.1×SSC, 0.1% SDS; Hybridize in a mixed solution of SDS, 0.5M Na3PO4 and 1mM EDTA, and rinse in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0063] A person skilled in the art can easily mutate the nucleotide sequence of the gene encoding the Nanobody E22 described in B1) of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of E22 described in B1) of the present invention, as long as they encode the Nanobody and have the activity of Nanobody E22, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0064] The present invention also provides a method for preparing the above-mentioned nanoantibody, which may include the following steps: introducing a nucleic acid molecule encoding the nanoantibody described above into a recipient cell to obtain a transgenic cell expressing the nanoantibody, and culturing the transgenic cell to obtain the nanoantibody.

[0065] Furthermore, 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 any of the following:

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

[0068] C2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes said Nanobody;

[0069] C3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more homology with any of the DNA sequences defined in C1) to C2) and encodes the Nanobody.

[0070] Furthermore, the recipient cell may be a microbial cell, such as bacteria (eg, Escherichia coli), yeast, algae or fungi.

[0071] In a specific embodiment of the present invention, the receptor cell can be FreeStyle TM HEK293-F cells.

[0072] The present invention also provides a nanobody fusion protein, which is a fusion of the nanobody or antigen-binding fragment described above with another molecule, wherein the other molecule may include an immunoglobulin Fc domain, a fluorescent protein, or a VHH with different specificities.

[0073] In a specific embodiment of the present invention, the other molecule is the Fc domain of a human immunoglobulin.

[0074] In a specific embodiment of the present invention, the amino acid sequence of the Fc domain of the human immunoglobulin described above is positions 124-350 of SEQ ID NO:11.

[0075] In a specific embodiment of the present invention, the Nanobody fusion protein described above may be any of the following:

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

[0077] M2) 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 or SEQ ID NO: 15.

[0078] In the above-mentioned nanobody fusion protein, the nucleic acid molecule encoding the fusion protein may be any of the following:

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

[0080] D2) a DNA molecule that hybridizes with the DNA molecule defined in D1) under stringent conditions and encodes the fusion protein;

[0081] D3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more homology with any of the DNA sequences defined in D1) to D2) and encodes the fusion protein.

[0082] The mutation included in the 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology mentioned in D3) above refers to a mutation occurring in the framework region of the aforementioned antibody.

[0083] The present invention also provides an ELISA detection kit targeting botulinum toxin, wherein the kit comprises the nanoantibody, the biomaterial or the fusion protein described above.

[0084] The present invention also provides any of the following applications:

[0085] M1) Use of the aforementioned biomaterial in the preparation of the aforementioned Nanobody or its antigen-binding fragment or the aforementioned heavy chain antibody;

[0086] M2) Use of the aforementioned Nanobody or its antigen-binding fragment or the aforementioned heavy chain antibody in the preparation of the aforementioned drug;

[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 drug in the preparation of a product for preventing and / or treating botulinum toxin poisoning;

[0088] M4) Use of the aforementioned nanoantibody or its antigen-binding fragment or the aforementioned heavy chain antibody or the aforementioned biological material in the preparation of a product for detecting botulinum toxin poisoning.

[0089] The above-mentioned product may be a medicine.

[0090] The medicament also includes a physiologically or pharmaceutically acceptable excipient, diluent or carrier.

[0091] Herein, the above-mentioned "physiologically or pharmaceutically acceptable carrier or diluent" refers to those carriers and diluents that have no significant irritation to organisms and will not impair the biological activity and performance of the agent in the pharmaceutical composition.

[0092] Herein, "physiologically or pharmaceutically acceptable excipients" refer to inert substances added to the pharmaceutical composition to further facilitate the administration of the agent. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred.

[0093] The present invention includes not only complete antibodies, but also fragments of the nanobodies or fusion proteins formed by antibodies and other sequences with immunological activity. Therefore, the present invention also includes polypeptides such as fragments, derivatives and analogs of the nanobodies that retain the same biological functions or activities as the antibodies of the present invention. The polypeptides may be as follows: D1) a single-chain antibody containing the nanobodies described above; D2) a Fab containing the nanobodies described above; D3) a complete antibody containing the nanobodies described above; D4) a fusion antibody containing the nanobodies described above; D5) an antibody-drug conjugate containing the nanobodies described above.

[0094] D4) The fusion antibody of the Nanobody may be: M1) a Nanobody fusion protein having an amino acid sequence as shown in SEQ ID NO:11.

[0095] As known to those skilled in the art, the conjugates and fusion antibody expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies of the present invention or their fragments to form conjugates. The present invention also includes cell surface markers or antigens combined with the nano antibodies or their fragments.

[0096] The present invention 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 to or at least 90% homologous to the heavy chain variable region of the antibody of the present invention, preferably at least 95% homologous.

[0097] The present invention prepares a phage nanoantibody display library by immunizing camels with antigens and screens to obtain nanoantibody molecules capable of neutralizing type E botulinum toxin. Nanoantibodies have the advantages of rapid preparation, simple structure and easy transformation into multi-specific antibodies. They are expected to be developed into highly efficient and safe anti-type E botulinum toxin neutralizing nanoantibodies to replace serum products supplied on the market to meet human needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 This is the binding status of some phage clones with target antigen or control antigen after the third round of screening by Phage-ELISA. The odd-numbered columns are target antigens, and the even-numbered columns are control antigens.

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

[0100] Figure 3 This is a test for the binding activity of the anti-botulinum toxin type E nanoantibody hFc fusion protein E22-hFc.

[0101] Figure 4 It is a specific detection of the anti-botulinum toxin type E nanoantibody hFc fusion protein E22-hFc.

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

[0103] Figure 6 This is a test for the binding activity of humanized nanobody fusion protein E22-h3 against botulinum toxin type E.

[0104] Figure 7 It is a specific detection of the humanized nanoantibody fusion protein E22-h3 against botulinum toxin type E.

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

[0106] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

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

[0108] The botulinum toxin type E (BoNT / E) and the equine botulinum antitoxin standard solution (BAT) in the following examples were purchased from the China Food and Drug Inspection Institutes.

[0109] The pTSE-hFc in the following examples is obtained by connecting the gene of the Fc domain of human immunoglobulin G to the pCMV vector. pTSE-hFc has been recorded in: Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against the protective antigen V of plague bacteria [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0110] The NEN-SCFV vector in the following embodiment is obtained by connecting the genes of the phage surface protein pill and the arabinose operon to the pET vector. The NEN-SCFV vector has been recorded 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 neutralizationability. Front. Immunol. 14: 1132822. doi: 10.3389 / fimmu.2023.1132822. Epub 2023Mar16. PMID: 37006289; PMCID: 10060833. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0111] The recombinant TL-HN antigen in the following embodiments: prepared by the inventor's laboratory. 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 tetanustoxin. VACCI NE (2023). doi: 10.1016 / j.vaccine.2023.09.032. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0112] The SARS antigen in the following examples was purchased from Sino Biological with the catalog number 40150-V08B2.

[0113] The antigen BoNT / A L-HN (AL-HN) in the following examples was prepared by our laboratory. The preparation method is described in 2.1 “Preparation of recombinant BoNT / A functional domain antigens” in the Materials and Methods on page 2 of the following document, which is the “BoNT / A·L-HN (AL-HN)” in the document: 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 botulinumneurotoxin 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.

[0114] The antigens BoNT / E L-HN domain (EL-HN), BoNT / EL domain (EL), and BoNT / EHN domain (EHN) in the following examples were prepared by our laboratory. The preparation method is described in 5.2 “Production of Recombinant Functional Fragment Antigens Derived from BoNT / E” in the Materials and Methods on page 10 of the following document, which are “BoNT / E·L-HN (EL-HN), BoNT / EL (EL), BoNT / E HN (EHN)” in the document: 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 our laboratory. The preparation method has been recorded in the first paragraph of the Materials and Methods section on page 2 of the following document, "Recombinant BoNT / B L-HN Fragment Preparation", which is the "BoNT / B·L-HN (BL-HN)" in the document: Li Z, Lu JS, Liu S, Wang R, Xu Q, Yu YZ, Yang ZX. Recombinant L-HN Fusion Antigen Derived from the L and HNDomains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin. Neurotox Res. 2021Aug;39(4):1044-1053.doi:10.1007 / s12640-021-00337-x.Epub 2021Feb 22.PMID:33616873.

[0116] The antigen BoNT / F L-HN domain (FL-HN) in the following examples was prepared by our laboratory. The preparation method has been described in 5.2 “Identification and Production of Recombinant Proteins” in the Materials and Methods on page 13 of the following document, which is “BoNT / F·L-HN (FL-HN)” in the document: 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.

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

[0118] Example 1. Construction of anti-botulinum toxin type E nanobody library

[0119] 1. Camel Immunity

[0120] The antigen BoNT / EL-HN (EL-HN) was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881), shaken and emulsified, and after sufficient emulsification, injected into healthy adult Bactrian camels by subcutaneous multi-point injection. Thereafter, booster immunizations were performed every two weeks. Except for the first immunization using Freund's complete adjuvant, all other immunizations used Freund's incomplete adjuvant (Sigma, F5506).

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

[0122] 120-150 mL of peripheral blood from camels that have undergone five immunizations was collected into anticoagulation tubes to separate peripheral blood lymphocytes (PBMC). The whole blood sample was gently diluted and mixed with lymphocyte separation solution (STEMCELL, 07851) to form a mixture with a clear interface between the two to separate peripheral blood lymphocytes from camel blood. After the mixture of whole blood and lymphocyte separation solution was centrifuged, the liquid in the centrifuge tube was divided into four layers from top to bottom: plasma layer, PBMC layer, lymphocyte separation solution layer and red blood cell layer.

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

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

[0125] Table 1. Primer sequences used in two rounds of PCR

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

[0127] 4. Electroporation products

[0128] The NEN-SCFV vector and the amplified VHH gene were digested with two restriction endonucleases, NcoI and NotI, and then the cut vector and gene fragment were connected by T4 DNA ligase.

[0129] The ligation products were transformed into E. coli TG1 competent cells (BC120-01, Biomed) using electrofection technology to construct an anti-botulinum toxin type E phage antibody library. The library capacity can be determined by serial dilution of the phage antibody library. The experimental results show that the constructed antibody library capacity reaches 2×10 8 In order to verify the accuracy of the library, 48 clones were randomly selected for colony PCR amplification and sequence comparison analysis. The results showed that the VHH fragment insertion rate reached 100% and the sequences were different.

[0130] Example 2: Screening of anti-botulinum toxin type E nanoantibody phage library

[0131] Using EL-HN as an antigen, the constructed nanobody phage library was subjected to solid phase screening to obtain anti-botulinum toxin type E specific nanobodies.

[0132] The constructed anti-botulinum toxin type E nanoantibody phage library was transferred to 2YT-GA medium (1L 2YT medium containing 16g Tryptone, 10g Yeast Extract, 5g NaCl, 100μg / mL ampicillin, 20% glucose), and after culturing to the logarithmic growth phase, M13KO7 helper phage was added at a ratio of MOI≈10. After standing at room temperature for infection for 30min, it was cultured at 37°C and a low speed of 150rpm for 30min; the deep-well plate was centrifuged for 15min and the supernatant was discarded. The centrifugation conditions were room temperature and 4000rpm; at room temperature, 2YT-KAA medium (1L 2YT medium containing 16g Tryptone, 10g Yeast Extract, 5g NaCl, 100μg / mL kanamycin, 20% glucose, and a final concentration of 1mM arabinose) was used for overnight presentation. The next day, the culture supernatant was collected and the phages were concentrated with a 20% PEG / NaCl solution (1 L of the solution contained 200 g PEG6000 and 146.25 g NaCl) to obtain high-titer antibody library products for subsequent screening. The specific nanoantibodies were screened using the nanoantibody phage library solid phase. The EL-HN protein was coated with 0.05M NaHCO3 coating solution (pH=9.6) in an immunotube and incubated at 4°C overnight. The next day, the immunotube was washed twice with PBS for 3 minutes each time. After blocking with blocking solution (2% bovine serum albumin) at room temperature for 2 hours, the nanoantibody phage library solution was added and incubated at room temperature for 2 hours; then shaken at a low speed of 200 rpm for 20 minutes; washed 10 times with PBST (1L PBS solution contains 8.0g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4, 0.1% Tween-20), and then washed 5 times with PBS. After washing, 1mL of elution solution (0.1M Glycine-HCl, pH 2.2), 400rpm shaking and eluting for 20min; taking out the eluate in the target antigen immune tube, adding 20-60μL neutralizing solution (1M Tris-HCl, pH 8.0) for neutralization; infecting Escherichia coli TG1 in the logarithmic growth phase, standing at room temperature for 30min, culturing at 37°C and 150rpm for 30min, producing and purifying phages for the next round of screening, and repeating the same screening process for 3 rounds. The enrichment results are shown in Table 2.

[0133] Table 2. Analysis of the enrichment degree of anti-botulinum toxin nanoantibody phage library screening

[0134] Screening times Input amount (pfu) Output (pfu) Output / Input 1 <![CDATA[5.0×10 11 ]]> <![CDATA[1.2×10 8 ]]> <![CDATA[2.4×10 -4 ]]> 2 <![CDATA[1.2×10 11 ]]> <![CDATA[8.6×10 8 ]]> <![CDATA[7.2×10 -3 ]]> 3 <![CDATA[1.2×10 11 ]]> <![CDATA[8.6×10 8 ]]> <![CDATA[7.2×10 -3 ]]>

[0135] After the above three rounds of screening, single clones with obvious intervals and regular shapes were selected from the plates with good phage growth, and inoculated into 96-well deep-well plates containing 250 μL 2YT-GA medium in each well, leaving 2 wells without inoculation of clones or inoculation of clones with other antibodies as negative controls; after the bacterial solution was cultured at 37°C to the logarithmic growth phase, M13K07 helper phage was added at a ratio of MOI≈10, that is, 100 μL was added to each well of the deep-well plate where a single phage clone was cultured, and the infection was allowed to stand at room temperature for 30 minutes, and then cultured at 37°C and a low speed of 150 rpm for 30 minutes; the deep-well plate was centrifuged for 10 minutes and the supernatant was discarded. The centrifugation conditions were room temperature and 2000 rpm, and arabinose with a final concentration of 1 mM was used for induction expression, and cultured overnight at 28°C and a speed of 220 rpm to obtain phage particles displaying nanoantibodies.

[0136] Example 3: Identification of botulinum toxin type E specific nanobodies using phage-ELISA

[0137] EL-HN protein was coated on an ELISA plate and diluted to a concentration of 2 ng / μL with 0.05 M NaHCO3 coating solution. The antigen in each well was approximately 200 ng. In the adjacent columns of the antigen wells, 2% BSA was used as a negative control for parallel coating at 4°C overnight. The ELISA plate was taken out the next day, washed 6 times with PBST on a plate washer, and then blocked with blocking solution (30 g skim milk powder was added to 1 L PBS) at 200 μL / well for 2 h at 37°C. The monoclonal bacterial solution induced overnight was centrifuged at 4°C and 3000 rpm for 10 min, and 125 μL of the supernatant of each bacterial solution was taken into 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 ELISA 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 with PBST on a plate washer for 6 times, HRP-labeled anti-M13 mouse monoclonal antibody (Sino Biological, 1973-MM05T-H) was diluted with blocking solution at 1:4000, and 100 μL / well was added to the enzyme-linked plate and incubated at 37°C for 45 min; after the incubation, the plate was washed with PBST on a plate washer for 6 times, and a color developing solution (10 mL of the color developing solution contained 1 mL 10×OPD, 9 mL of a mixed solution of 0.2 M Na2HPO4 and 0.1 M citric acid, and 10 μL of 30% hydrogen peroxide) was prepared and added to the enzyme-linked plate at 100 μL / well, and the plate was color developed in the dark for 15-20 min; 2 M H2SO4 stop solution was added at 50 μL / well to terminate the reaction; an ELISA reader was used to read the plate at a dual wavelength of 492 / 630 nm, and the light absorption values ​​of the antigen group and the negative control group were compared. If the ratio was greater than 5, the monoclonal clone was determined to be a positive clone. Some Phage-ELISA test results are shown in Figure 1 , odd-numbered columns are target antigens, and even-numbered columns are control antigens. The bacterial solution corresponding to the positive clones was sent to a biotechnology service company for sequencing to obtain the DNA sequence of the inserted fragment, and finally a phage clone sequence E22 that can specifically bind to BoNT / EL-HN was obtained.

[0139] The amino acid sequence of E22 is shown in SEQ ID NO: 1: including framework regions (FR: FRi, FR2, FR3, FR4) and complementary determining regions (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, SEQ ID NO: 8; the three parts of the complementary determining region are sequentially recorded as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. The gene encoding the anti-type E botulinum toxin nanobody E22 has a nucleotide sequence as shown in SEQ ID NO: 9.

[0140] The above CDRs are sequences defined based on the analysis results of the IMGT system.

[0141] Example 4. Preparation of anti-botulinum toxin type E nanobody

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

[0143] The obtained nucleotide sequence of E22 protein SEQ ID NO:9 was cloned into the pTSE-hFc expression vector by basic PCR amplification, restriction digestion, ligation and other techniques, and a single clone was selected for sequencing verification. After the amplified VHH gene fragment was successfully inserted 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 endonucleases Sal-I and Nhe-I of the pTSE-hFc vector with a DNA fragment having a nucleotide sequence of SEQ ID NO: 9 while keeping other nucleotide sequences of the pTSE-hFc vector unchanged. The vector expresses a protein having an amino acid sequence of SEQ ID NO: 11 (whose nucleotide sequence is SEQ ID NO: 10, named E22-hFc).

[0145] The E22-hFc protein is an anti-botulinum toxin type E nanobody hFc fusion protein constructed by connecting its carboxyl terminus to the hFc segment of human immunoglobulin based on the anti-botulinum toxin type E nanobody gene sequence (SEQ ID NO: 9).

[0146] 2. Expression and purification of anti-botulinum toxin nanoantibody hFc fusion protein

[0147] The constructed pTSE-E22-hFc expression plasmid was transfected into FreeStyle TM In HEK293-F cells (Invitrogen, R79007), cell activity was monitored daily starting after 72 hours. When the cell activity dropped from 95-100% to 80-85%, the cell supernatant was collected for purification to obtain the nanobody hFc fusion protein E22-hFc.

[0148] The purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 2 As shown: The band position of the antibody is consistent with the theoretical molecular weight, and the band size of the nanobody hFc fusion protein under reducing conditions is about 40kDa ( Figure 2 Lane 1), the band size under non-reducing conditions is approximately 80 kDa ( Figure 2 Lane 6).

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

[0150] 1. Detect the binding activity between the nanobody hFc fusion protein E22-hFc and BoNT / EL-HN protein by ELISA experiment

[0151] The experiment was repeated twice, and each repetition was as follows:

[0152] EL-HN protein was diluted to 2 μg / mL with carbonate coating buffer (pH 9.6), and 100 μL / well was added to a 96-well ELISA plate for coating overnight at 4°C. The coating solution was discarded and the plate 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, 0.1% Tween-20) for 6 times, pat dry the residual liquid in the plate, add blocking solution (3% skim milk powder) at 200 μL / well, and block at 37°C for 2h; dilute the antibody for binding activity detection by 2 times with blocking solution, with an initial dilution of 12.5 μg / mL, discard the blocking solution in the plate, wash 6 times with PBST and pat dry the residual liquid, add the E22-hFc to be tested as the primary antibody to the plate at 100 μL / well, and incubate at 37°C for 1.5h; discard the primary antibody, wash with PBST 6 times, dilute HRP-labeled goat anti-human IgG as the secondary antibody with blocking solution at a ratio of 1:4000, add 100 μL / well to the plate, and incubate at 37°C for 45 minutes; discard the secondary antibody, wash 6 times with PBST, add 100 μL / well peroxidase substrate colorimetric solution, color for 15-20 minutes in the dark, observe the color development effect, and add 50 μL / well 2M sulfuric acid to terminate the reaction after color development is complete; use a microplate reader at 492nm / 630nm dual wavelength to measure the optical density value. By calculating the concentration required for the antibody to bind 50% of the antigen protein (EC 50 ), and evaluate the binding ability between them.

[0153] The results are as follows Figure 3 As shown, the analysis showed that the half effective concentration (EC 50 ) is 0.2620nM.

[0154] 2. Identify the specificity of nanoantibodies through ELISA experiments

[0155] The experiment was repeated twice, and each repetition was as follows:

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

[0157] The results are as follows Figure 4 As shown, E22-hFc specifically binds to EL-HN antigen, but does not bind to other antigens, or the binding activity is very weak.

[0158] Example 6. Evaluation of in vitro neutralization potency of anti-botulinum toxin nanobodies

[0159] The in vitro neutralization titer of the antibody was determined by mixing the antibody with a lethal dose of botulinum toxin type E in vitro and then injecting it into KM (Kunming) mice. Each group of 4 KM mice weighed 18-20 g and were purchased from Beijing Sibeifu Biotechnology Co., Ltd. The experiment was repeated three times (different doses), and each repetition was as follows:

[0160] 1. Sample preparation:

[0161] Diluent: KH2PO4 0.7g, Na2HPO4·12H2O 2.4g, NaCl 6.8g, gelatin 2g, add water to 1L, autoclave;

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

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

[0164] Horse botulinum antitoxin standard solution (BAT): a solution obtained by dissolving horse anti-botulinum toxin serum (purchased from China Food and Drug Inspection Institute) in diluent.

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

[0166] (1) BoNT / E solution 20×LD 50 Group: Each KM mouse was intraperitoneally injected with 500 μL of botulinum toxin solution (100×LD 50 / mL), so that the toxin dose contained in the injection solution for each mouse was 20×LD 50 .

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

[0168] (3)BoNT / E 20×LD 50 +E22-hFc 2μg group: The difference between this group and BoNT / E+E22-hFc 10μg group is that the dose of E22-hFc is 2μg / mouse, and the rest of the operation is the same as BoNT / E 20×LD 50 +E22-hFc 10 μg group.

[0169] (4)BoNT / E 20×LD 50 +E22-hFc 0.4μg group: The difference between this group and BoNT / E+E22-hFc 10μg group is that the dose of E22-hFc is 0.4μg / mouse. The rest of the operation is the same as BoNT / E 20×LD 50 +E22-hFc 10 μg group.

[0170] (5)BoNT / E 20×LD 50 +E22-hFc 0.2μg group: The difference between this group and BoNT / E+E22-hFc 10μg group is that the dose of E22-hFc is 0.2μg / mouse. The rest of the operation is the same as BoNT / E 20×LD50 +E22-hFc 10 μg group.

[0171] (6)BoNT / E 20×LD 50 +E22-hFc 0.1μg group: The difference between this group and BoNT / E+E22-hFc 10μg group is that the dose of E22-hFc is 0.1μg / mouse. The rest of the operation is the same as BoNT / E 20×LD 50 +E22-hFc 10 μg group.

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

[0173] The results are as follows Figure 5 As shown in the figure, E22-hFc above 0.2 μg can completely neutralize 20×LD 50 Lethal dose of BoNT / E.

[0174] Example 7 Humanization and activity evaluation of anti-type E botulinum toxin nanobody fusion protein E22-hFc

[0175] 1. Humanization of anti-botulinum toxin type E nanoantibody fusion protein E22-hFc

[0176] The nanobody E22-hFc fusion protein was humanized using the Swiss-model. First, the antibody was homologously modeled using the Swiss-model, and then the model was placed in DS for amino acid property analysis. The human antibody sequence with the smallest difference in the model RMSD value was used as a reference, and the amino acids in the antibody framework region were mutated to humanized amino acids, thereby obtaining the humanized nanobody E22-h3, whose amino acid sequence is SEQ ID NO: 12 and the nucleotide sequence is SEQ ID NO: 13. Finally, the modified sequence was scored with a Z-score, and the results showed that the Z-score values ​​all fell within the peak range of the human sequence set, and the sequence had a high degree of humanization.

[0177] 2. Construction of the eukaryotic expression vector pTSE-hFc-E22-h3 for humanized anti-botulinum toxin type E nanobody

[0178] According to the gene sequence of the humanized nanobody against type E botulinum toxin (SEQ ID NO: 13), its C-terminus was connected to the Fc segment (hFc) of human immunoglobulin G to obtain the anti-type E botulinum toxin nanobody fusion protein E22-h3 with the encoding gene nucleotide sequence of SEQ ID NO: 14 and the expressed amino acid sequence of SEQ ID NO: 15. E22-h3 is a mutant obtained by mutating the amino acid at position 4 of the amino acid sequence of E22 SEQ ID NO: 1 from V to L, the amino acid at position 14 from A to P, the amino acid at position 82 from D to S, the amino acid at position 116 from Q to L, and the other amino acid sequences remain unchanged.

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

[0180] The humanized nanobody fusion protein E22-h3 was prepared by referring to the method in Example 4, and the purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 2 As shown: The molecular weight of the antibody is in line with expectations, and the band size of the nanobody hFc fusion protein under reducing conditions is approximately 40 kDa ( Figure 2 Lane 2), the band size under non-reducing conditions is approximately 80 kDa ( Figure 2 Lane 7).

[0181] The binding activity and specificity of the humanized nanobody fusion protein E22-h3 and the recombinant EL-HN antigen were tested with reference to Example 5. Figure 6 , Figure 7 As shown, the analysis showed that the half effective concentration (EC 50 ) was 0.2344 nM; E22-h3 specifically bound to BoNT / EL-HN antigen, but did not bind to other antigens, or the binding activity was very weak.

[0182] 4. Evaluation of the neutralizing activity of humanized nanobodies against botulinum toxin type E

[0183] The neutralizing activity of humanized nanobodies against botulinum toxin type E was evaluated with reference to Example 6.

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

[0185] (1) BoNT / E solution 20×LD 50 Group: Each KM mouse was intraperitoneally injected with 500 μL of 100×LD 50 / mL of botulinum toxin solution, so that the toxin dose contained in each injection solution is 20×LD 50 / Only.

[0186] (2)BoNT / E 20×LD 50 +E22-h3 2μg group: Botulinum toxin solution (100×LD 50 / mL) was mixed with E22-h3 solution, and the volume of each group was supplemented to 2.5mL with diluent. After mixing evenly, the solution was incubated at 37°C for 30min to obtain BoNT / E+E22-h3 solution, which was intraperitoneally injected into KM mice, with 500μL injected per mouse, so that the dose of BoNT / E was 20×LD 50 / mouse, so that the dose of E22-h3 in each injection solution is 2 μg / mouse.

[0187] (3)BoNT / E 20×LD 50 +E22-h3 0.4μg group: The difference between this group and BoNT / E+E22-h3 2μg group is that the dose of E22-h3 is 0.4μg / mouse. The rest of the operation is the same as BoNT / E 20×LD 50 +E22-h3 2μg group.

[0188] (4)BoNT / E 20×LD 50 +E22-h3 0.08μg group: The difference between this group and BoNT / E+E22-h3 2μg group is that the dose of E22-h3 is 0.08μg / mouse. The rest of the operation is the same as BoNT / E 20×LD 50 +E22-h3 2μg group.

[0189] (5)BoNT / E 20×LD 50 +E22-h3 0.02μg group: The difference between this group and BoNT / E+E22-h3 2μg group is that the dose of E22-h3 is 0.02μg / mouse. The rest of the operation is the same as BoNT / E 20×LD 50 +E22-h3 2μg group.

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

[0191] The results are as follows Figure 8 As shown in Figure 2, E22-h3 at a dose of 0.08 μg or above can completely neutralize 20×LD 50 BoNT / E, all mice survived.

[0192] Example 8. Evaluation of the therapeutic effect of humanized anti-botulinum toxin nanobodies

[0193] To evaluate whether anti-botulinum toxin nanoantibodies have therapeutic effects after mice were exposed to BoNT / E, KM mice were selected, 6 mice per group, 18-20 g, purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0194] The evaluation method is as follows:

[0195] Diluent: KH2PO4 0.7g, Na2HPO4·12H2O 2.4g, NaCl 6.8g, gelatin 2g, add water to 1L, autoclave;

[0196] Botulinum toxin solution: Dilute type E botulinum toxin (purchased from China Food and Drug Inspection Institute) to 5×LD 50 / mL;

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

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

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

[0200] A. BoNT / E 5×LD 50 / pc+antibody 2.5mg / kg large group

[0201] 1) 1h group: intraperitoneal injection of 500 μL 5×LD 50 / mL botulinum toxin solution. One hour after the injection of the botulinum toxin solution, 100 μL of E22-h3 solution (i.e., the injection dose was 2.5 mg / kg) was injected into 6 mice in total. The health status and survival of the mice were monitored for 7 days.

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

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

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

[0205] 5) PBS group: E22-h3 solution was replaced with an equal volume of PBS, and the rest of the operation was the same as that of the 1h group in this group.

[0206] B. BoNT / E 5×LD 50 / pc+antibody 0.25mg / kg large group

[0207] 1) BoNT / E 5×LD 50 / mouse + E22-h3 0.25 mg / kg group: intraperitoneal injection of 500 μL 5×LD 50 / mL botulinum toxin solution. A total of 6 mice. One hour after the injection of botulinum toxin solution, 100 μL of E22-h3 solution was injected (i.e., the injection dose was 0.25 mg / kg). The health status and survival of the mice were monitored for 7 days.

[0208] 2) 2h group: The difference between this group and the 1h group of this group is that 100 μL of E22-h3 solution was injected 2 hours after the injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0209] 3) 3h group: The difference between this group and the 1h group of this group is that 100 μL of E22-h3 solution was injected 3 hours after the injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0210] 4) 6h group: The difference between this group and the 1h group of this group is that 100 μL of E22-h3 solution was injected 6 hours after the injection of botulinum toxin, and the rest of the operations were the same as the 1h group of this group.

[0211] 5) BoNT / E 5×LD 50 / rabbit + BAT 0.25 mg / kg group: E22-h3 solution was replaced with 100 μL of equine anti-botulinum toxin serum solution (i.e., injection dose of 0.25 mg / kg), and the rest of the operation was the same as that of the 1h group of this group.

[0212] The results are shown in Table 3. BoNT / E 5×LD 50 In terms of dose challenge, the E22-h3 treatment group was effective within 6 hours after exposure, and the protective effect was optimal within 3 hours. The high-dose antibody group (injection dose of 2.5 mg / kg) achieved 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 intraperitoneally injected with 5×LD 50 dose of BoNT / E; b KM mice in each experimental group received 2.5 or 0.25 mg / kg E22-h3, 0.25 mg / kg BAT, and PBS at different time points after challenge for treatment experiments; c Mice were injected with different doses of antibodies or PBS 1, 2, 3, and 6 h after challenge with the indicated doses of toxin;d The number of KM mice in each experimental group was six, and the final survival status of the mice after seven days.

[0216] SEQ ID NO: 1:

[0217] QVQVVESGGGLVQAGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTL YLQMNDLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTQVTVSS.

[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] QVQLVESGGGLVQPGGSLRLSCTASGFTFDDYAMGWFRQAPGKEREGVSCSSWSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAAWIASGGGCVGADYGYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0239] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An anti-botulinum toxin nanobody or an antigen-binding fragment thereof, characterized in that: The nanobody or its antigen-binding fragment comprises three complementary 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.

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

12.

5. An anti-botulinum toxin heavy chain antibody, characterized in that The heavy chain antibody contains the Nanobody described in any one of claims 1 to 4 and the Fc region of an immunoglobulin.

6. The heavy chain antibody according to claim 5, characterized in that The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region includes SEQ ID NO:1 or has a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID NO:

1.

7. The heavy chain antibody according to claim 5 or 6, characterized in that The amino acid sequence of the heavy chain antibody includes SEQ ID NO:11 or SEQ ID NO:

15.

8. Biomaterial, characterized in that The biological material is any of the following: B1) a nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or a heavy chain antibody as described in any one of claims 4 to 6; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in 1) or the expression cassette described in B2) or the recombinant vector described in B3).

9. A drug for preventing botulinum toxin poisoning, characterized in that: The drug comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7.

10. Application, characterized in that, The application is any of the following: M1) Use of the biomaterial of claim 8 in the preparation of a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or a heavy chain antibody as described in any one of claims 5 to 7; M2) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7 in the preparation of the drug according to claim 9; M3) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7 or the biomaterial according to claim 8 or the drug according to claim 9 in the preparation of a product for preventing and / or treating botulinum toxin poisoning; M4) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the heavy chain antibody according to any one of claims 5 to 7 or the biomaterial according to claim 8 in the preparation of a product for detecting botulinum toxin poisoning.

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