Long-acting antibody for neutralizing botulinum toxin type A and its use

Abstract

The present invention discloses a long-acting antibody for neutralizing botulinum toxin type A and its use, belonging to the field of biomedicine. The present invention provides a long-acting antibody for neutralizing botulinum toxin type A, which is composed of a heavy chain variable region shown in SEQ ID NO:1 and an Fc region shown in SEQ ID NO:2 or SEQ ID NO:3. The present invention provides a nucleic acid molecule encoding the aforementioned antibody, a recombinant expression vector comprising the aforementioned nucleic acid molecule, and corresponding host cells, pharmaceutical compositions, kits, etc. The present invention also provides corresponding detection methods and uses.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to antibodies for neutralizing botulinum toxin type A in a long-acting manner and uses thereof. Background Art

[0002] Botulinum toxin is a neurotropic protein toxin that can cause poisoning in humans and animals. A concentration as low as 0.1~1.0µg can cause death. It is divided into four types based on the different ways the toxin enters the body: 1. Foodborne poisoning; 2. Infant infectious botulism; 3. Traumatic botulism; 4. Inhalation poisoning. There are seven serotypes of botulinum toxin (A, B, C, D, E, F, G,) based on their antigenicity. In recent years, an eighth serotype has been discovered. Although the components of the seven serotypes of botulinum toxin and their mechanisms of action are slightly different, they have similar molecular weights and similar structures.

[0003] With the widespread application of botulinum toxin type A (BoNT / A) in medical and cosmetic medicine, botulism poisoning incidents caused by improper use occur from time to time. At present, botulinum toxin type A is treated with horse anti-antibodies, but horse anti-antibodies have the disadvantages of allergic reactions, high production costs, and low-temperature storage, and antitoxins can only neutralize free toxins in body fluids and blood, and have no inhibitory effect on toxins in cells. Botulinum toxins can often enter neurons within a few hours of poisoning, and can exist in neurons for several days or even months, continuously cutting SNARE family proteins and blocking the release of acetylcholine transmitters. Intracellular toxins can only be cleared through the body's own degradation, which makes the patient's treatment cycle very long (Thanongsaksrikul J, et al. Toxins 2011; 3 (5): 469-88). Therefore, it is necessary to develop long-acting antibodies for the treatment of type A botulism. Summary of the invention

[0004] In order to solve the problem of short retention time of antibodies in the prior art, the present invention provides antibodies with increased half-life for long-term neutralization of botulinum toxin type A.

[0005] The present invention provides a long-acting antibody for neutralizing botulinum toxin type A, wherein the antibody consists of a heavy chain variable region and an Fc region, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 1, and the Fc region has an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0006] The amino acid sequence shown in SEQ ID NO: 1 is as follows: EVQLVESGGGLVQPGGSLRLSCEASGFGSWFRFDENTVNWYRQPPGKSREFDELVARYPKSGIVTYLDSVKGRFTISRDNAKKMAFLQMNSLKPEDTAVYYCNVGEFWGQGTQVTVSS.

[0007] The amino acid sequence shown in the SEQ ID NO: 2 is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0008] The amino acid sequence shown in the SEQ ID NO: 3 is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0009] Further, the antibody is afucosylated.

[0010] The present invention provides a nucleic acid molecule, which encodes the above-mentioned antibody.

[0011] The present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described above.

[0012] Furthermore, the recombinant expression vector also includes a promoter.

[0013] Furthermore, the recombinant expression vector includes a plasmid vector, a viral vector or a phage vector.

[0014] The present invention provides a host cell, wherein the host cell comprises the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector.

[0015] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody, the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector, or the above-mentioned host cell.

[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically compatible carrier.

[0017] Furthermore, the pharmaceutical composition also includes a buffer.

[0018] Furthermore, the pharmaceutical composition can be used in combination with one or more antiviral agents.

[0019] The present invention provides use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, or the aforementioned host cell in the preparation of a pharmaceutical composition for inhibiting botulinum toxin type A or for treating a disease caused by botulinum toxin type A.

[0020] The present invention provides a kit for detecting botulinum toxin type A, and the kit comprises the above-mentioned antibody.

[0021] Furthermore, the kit also includes a detectable label conjugated to the antibody.

[0022] Furthermore, the detectable label includes a fluorescent label, a radioactive isotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping agent.

[0023] The present invention provides use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, or the aforementioned host cell in preparing a product for detecting type A botulinum toxin or in preparing a product for diagnosing type A botulinum toxin infection.

[0024] The present invention provides a method for detecting botulinum toxin type A in a sample, the method comprising contacting the above-mentioned antibody with the sample to be detected, and detecting the level of botulinum toxin type A in the sample to be detected.

[0025] Furthermore, the method is a method for non-diagnostic purposes.

[0026] Unless otherwise specified, the term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or to an antigen binding region thereof that competes with an intact antibody for specific binding, including human, humanized, chimeric, multispecific, monoclonal, polyclonal antibodies, and oligomers or antigen binding fragments thereof. The term "antibody" includes, but is not limited to, antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody.

[0027] In some embodiments, the antibody is a polypeptide, which may be collagen or gelatin, or a variant thereof or a hybrid thereof. The polypeptide may be any antibody or portion thereof, an antigen, a coagulation factor, an enzyme, a hormone or a hormone variant, a receptor or portion thereof, a regulatory protein, a structural protein, a reporter protein, or a transporter such as a serum albumin, such as bovine serum albumin and human serum albumin, or such as a transferrin, such as lactoferrin, a protein involved in the secretion process, a protein involved in the folding process, a chaperone, a peptide amino acid transporter, a glycosylation factor, a transcription factor, a synthetic peptide or oligopeptide, a protein that is an intracellular protein in its native form and is secreted by methods known in the art (e.g., fused to a signal peptide and fused to a polypeptide that is already secreted in its native form).

[0028] In some embodiments, the antibody also includes functional variants of the antibody, including but not limited to: derivatives that are substantially similar in primary structural sequence but contain chemical and / or biochemical modifications, such as those not present in the parent antibody of the present invention, such as in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic treatment, phosphorylation.

[0029] In the present invention, when a prokaryotic cell is used as a host, the recombinant expression vector generally comprises a strong promoter that enables transcription (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for starting translation and a transcription / translation termination sequence. When using Escherichia coli strains (E. coli) (for example, HB101, BL21, DH5α, Top10, JM109, etc.) as host cells, the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., (1984) 158:1018-1024) and the leftward promoter of bacteriophage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14:399-445) can be used as regulatory sites. When Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741) or any promoter that can be expressed in Bacillus can be used as a regulatory site.

[0030] When eukaryotic cells are used as hosts, the recombinant expression vector can utilize promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and usually has a polyadenylation sequence as a transcription termination sequence.

[0031] In addition, the recombinant expression vectors of the present invention also include plasmids (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), bacteriophages (for example, λgt4.λB, λ-Charon, λΔz1 and M13, etc.) or viruses (for example, SV40, etc.).

[0032] In this application, the term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or regulates (binds to a host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or regulate transcription, translation, and, if introns are present, sequences that affect RNA splicing of a coding region operably linked thereto.

[0033] In this application, unless otherwise expressly stated, the use of the singular includes the plural. In this application, unless otherwise expressly stated, the use of "or" means "and / or". In addition, the use of the term "includes" and other forms such as "comprises" are not limiting. In addition, unless otherwise expressly stated, terms such as "element" or "component" include both elements and components comprising one unit and elements and components comprising more than one subunit. In addition, the use of the term "portion" may include a portion of a portion or the entire portion.

[0034] In the present application, the "nucleic acid molecule" means genomic DNA or RNA, mRNA, cDNA or their synthetic origin or some combination, which is not associated with all or part of the polynucleotide in which the isolated polynucleotide naturally exists, or they are connected to a polynucleotide that is not connected to it in the natural state. It should be understood that for the purpose of the present invention, the "nucleic acid molecule" that "comprising" a specific nucleotide sequence does not include the entire chromosome. In addition to the specific sequence, the isolated nucleic acid molecule that "comprising" a specific nucleic acid sequence also contains sequences encoding up to 10 or even up to 20 other proteins or parts thereof, or may contain operably linked regulatory sequences that regulate the expression of the coding region of the nucleic acid sequence, and / or may contain vector sequences.

[0035] Unless otherwise explicitly stated, the left-hand end of any single-stranded polynucleotide sequence described herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of nascent RNA transcript addition from 5' to 3' is referred to as the transcription direction; the sequence region on the DNA chain having the same sequence as the RNA transcript and whose 5' is the 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence region on the DNA chain having the same sequence as the RNA transcript and whose 3' is the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0036] In this application, the term "host cell" means a cell that has been or can be transformed by a nucleic acid sequence and thereby expresses a target gene. Regardless of whether the offspring is identical to the original mother cell in morphology or genetic makeup, as long as the offspring has the target gene, the term includes the offspring of the mother cell.

[0037] In the present application, the term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a natural protein, i.e., a protein produced by a naturally occurring and non-recombinant cell; or the macromolecule is produced by a genetically engineered or recombinant cell, and the term includes a molecule having the amino acid sequence of a natural protein, or a molecule having one or more amino acids of the natural sequence deleted, added and / or substituted. The term also includes amino acid polymers, wherein one or more amino acids are chemical analogs and polymers of the corresponding naturally occurring amino acids. The fragment may also contain modified amino acids compared to the natural protein. In certain embodiments, the fragment length is about 5-500 amino acids. For example, the fragment length may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains.

[0038] The 20 conventional (e.g., naturally occurring) amino acids and their abbreviations used herein follow conventional usage. See Immunology--A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the 20 conventional amino acids, non-natural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, consistent with standard usage and convention.

[0039] The term "naturally occurring" as used throughout this specification in connection with biological materials such as polypeptides, nucleic acids, host cells, etc., refers to a material that occurs in nature or a form of a material that occurs in nature.

[0040] The "Fc" region comprises two heavy chain fragments comprising the antibody CH1 and CH2 domains. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

[0041] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In certain embodiments, the clear description of the CDR and the determination of the residues comprising the antibody binding site are accomplished by solving the antibody structure and / or solving the antibody-ligand complex structure. In certain embodiments, this can be accomplished by any of a variety of techniques known to those skilled in the art (e.g., X-ray crystallography). In certain embodiments, different analyses can be used to determine or roughly estimate the CDR region. Examples of the methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0042] The Kabat definition is a standard for numbering residues in antibodies and is often used to identify CDR regions. The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group to model antibody structure. The AbM definition models the tertiary structure of an antibody from the primary sequence using known databases and ab initio methods.

[0043] In this application, the term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length heavy chain comprises a variable region domain VH and three constant region domains CH1, CH2 and CH3. The VH domain is located at the amino terminus of the polypeptide, while the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

[0044] In this application, the term "variable region" or "variable domain" refers to the portion of the light chain and / or heavy chain of an antibody, generally comprising the amino-terminal end of about 120-130 amino acids of the heavy chain and the amino-terminal end of about 100-110 amino acids of the light chain. In certain embodiments, different antibody variable regions vary widely in amino acid sequence, even between antibodies of the same species. The antibody variable region generally determines the specificity of a particular antibody for its target.

[0045] The term "neutralizing . . . antibody" refers to an antibody that binds to a ligand and prevents or reduces the biological effect of the ligand. This can be achieved, for example, by directly blocking the binding site on the ligand or by binding to the ligand and changing the binding ability of the ligand by indirect means (e.g., structural or energetic changes of the ligand). In certain embodiments, the term can also refer to an antigen binding protein that prevents the protein to which it binds from performing a biological function. In assessing the binding and / or specificity of an antigen binding protein (e.g., an antibody or an immunologically functional fragment thereof), an antibody or fragment may substantially inhibit the binding of a ligand to its binding partner when an excess of the antibody reduces the amount of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured by an in vitro competitive binding assay). In certain embodiments, the neutralizing capacity is characterized and / or described via a competitive assay. In certain embodiments, the neutralizing capacity is described by an IC50 or EC50 value.

[0046] As used herein, the term "pharmaceutical agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological material.

[0047] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid, or attachment of a polypeptide to a biotin moiety detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatically active streptavidin detectable by optical or colorimetric methods). In certain embodiments, the marker or label may also be a therapeutic agent.

[0048] As used herein, the term "sample" or "specimen" includes, but is not limited to, any amount of material from or originally from a living organism. The living organism includes, but is not limited to, humans, mice, monkeys, rats, rabbits, and other animals. The material includes, but is not limited to, blood, serum, urine, cells, organs, tissues, bones, bone marrow, lymph nodes, and skin.

[0049] The term "pharmaceutical composition" as used herein refers to a chemical compound, composition, agent or drug capable of inducing a desired therapeutic effect when it is properly administered to a patient. It does not necessarily require more than one type of ingredient.

[0050] As used herein, the term "treatment" includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or potential risk factors are mitigated.

[0051] As described herein, antigen binding proteins directed against botulinum toxin type A may comprise humanized antibodies and / or portions thereof.An important practical application of the described strategy is the "humanization" of the mouse humoral immune system.

[0052] In certain embodiments, humanized antibodies are substantially non-immunogenic in humans. In certain embodiments, humanized antibodies have substantially the same affinity for a target as antibodies of another species from which they are derived. In certain embodiments, amino acids of the variable domains of antibodies that can be modified without reducing the natural affinity of the antigen binding domain while reducing its immunogenicity are identified.

[0053] In certain embodiments, antibody variants include glycosylation variants, wherein the number and / or type of glycosylation sites have been altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, protein variants contain more or less N-linked glycosylation sites than native proteins. N-linked glycosylation sites are characterized by Asn-X-Ser or Asn-X-Thr sequences, wherein the amino acid residue designated as X can be any amino acid residue except proline. The sequence is generated by replacing amino acid residues, providing potential new sites for N-linked sugar chains. Alternatively, the substitution that eliminates the sequence will remove existing N-linked sugar chains. Also provided is the rearrangement of N-linked sugar chains, wherein one or more N-linked glycosylation sites (usually naturally occurring glycosylation sites) are eliminated, while one or more new N-linked sites are generated. In addition, preferred antibody variants include cysteine ​​variants, wherein one or more cysteine ​​residues are missing or replaced by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody must be refolded into a biologically active conformation (e.g., after separation of insoluble inclusion bodies). Cysteine ​​variants typically have fewer cysteine ​​residues than the native protein, and the number is usually an even number to minimize interactions caused by unpaired cysteines.

[0054] As is known, antibodies can be expressed in cell lines other than hybridoma cell lines. Suitable mammalian host cells can be transformed using sequences encoding specific antibodies. Polynucleotides can be introduced into host cells by any known method, including, for example, packaging polynucleotides into viruses (or viral vectors) and transducing host cells with the viruses (or vectors) or by transfection procedures known in the art. The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including transfection mediated by dextran, calcium phosphate precipitation, 1,5-dimethyl-1,5-diazoundecamethylene polybrene (polybrene) mediated transfection, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes and directly microinjecting DNA into the nucleus. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is the SDS-PAGE electrophoresis picture of HM, HM-Fc5 and HM-Fc6.

[0056] Figure 2 The figure is the neutralization result of the antibody in the intoxicated human FcRn transgenic mouse model, where A: the survival curve of transgenic mice challenged with BoNT / A (n=5); B~D: the protective survival curves of the three antibodies in the LD100 of BoNT / A challenged mice. The antibody doses were 500μg / kg (B), 5μg / kg (C) and 0.5μg / kg (D) (n=10).

[0057] Figure 3 The pharmacokinetic (A) and pharmacodynamic (B) results of antibody HM in human FcRn transgenic mice, where A: the curve of antibody concentration change over time in transgenic mice, initially injected with 500 μg / mouse (n=5). B: On the 50th day after antibody injection, 10 times the lethal dose of toxin was injected into hFCRN mice, and the survival curves of each group of mice in the three antibody groups (N=5) were different. DETAILED DESCRIPTION

[0058] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0060] Example 1

[0061] In the early stage of the laboratory, the Fc region (SEQ ID NO:4) was modified by computer-aided design, and the gene sequences of two improved Fc region mutants Fc5 and Fc6 (SEQ ID NO:2, SEQ ID NO:3) were obtained. The design introduced two enzyme cleavage sites NotI and PmeI at both ends of the target gene. These genes were synthesized by Azenta Life Science, and then the mutant Fc regions were cloned into antibody expression vectors and named HM (SEQ ID NO:1+SEQ ID NO:4), HM-Fc5 (SEQ ID NO:1+SEQ ID NO:2) and HM-Fc6 (SEQ ID NO:1+SEQ ID NO:3).

[0062] SEQ ID The amino acid sequence of NO:4 is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0063] Thaw ExpiCHO-S™ cells and transfer them to a 125 mL shake flask in a 37°C, 8% CO 2 Pre-warm ExpiCHO™ Expression Medium in the incubator until the cell density reaches 4 × 10 6 ~6×10 6 mL -1 The cells were diluted to 0.2×10 6 ~0.3×10 6 mL -1 , continue incubation.

[0064] The day before transfection, the cell density was adjusted to 3 × 10 6 On the day of transfection, the concentration was adjusted to 6 × 10 6 mL -1 Plasmid DNA (100 μg 100 -1 mL -1 ) and ExpiFectamine™ CHO Transfection Reagent (320 μL) were diluted in cold OptiPRO™ SFM medium. After 5 minutes of mixing and incubation at room temperature, the complex was slowly added to the cell suspension and mixed gently. 18-22 hours after transfection, ExpiFectamine™ CHO Enhancer (600 μL) and ExpiCHO™ Feed (24 mL) were added.

[0065] After 8 days of culture, antibody protein was harvested from the culture supernatant, initially purified using a HiTrap MabSelet Protein A column, eluted with 150 nM acetic acid on an Akta purification system, and stored in PBS. Antibody concentration was determined by BSA concentration assay, while preliminary characterization of the antibody was accomplished by SDS electrophoresis.

[0066] Figure 1The SDS-PAGE electrophoresis diagram of HM, HM-Fc5 and HM-Fc6 shows that the antibody purity is excellent. Lane 1: Marker; Lane 2: HM reduction electrophoresis band; Lane 3: HM-Fc5 reduction electrophoresis band; Lane 4: HM-Fc6 reduction electrophoresis band; Lane 5: HM non-reduction electrophoresis band; Lane 6: HM-Fc5 non-reduction electrophoresis band; Lane 7: HM-Fc6 non-reduction electrophoresis band.

[0067] Example 2

[0068] 1. Neutralization effect of antibodies in the intoxicated human FcRn transgenic mouse model

[0069] In order to further verify the neutralizing effect of antibodies on toxins, 16-18g human FcRn transgenic mice (8 weeks old) were selected for BoNT / A attack dose test. The BoNT / A toxin stored in the laboratory was diluted to a series of concentrations, and 100μL of toxin was injected intraperitoneally into each mouse. The survival status and mortality of the mice were then observed. The survival curve of the mice is shown in Figure 2. Figure 2 As shown in A, a dose of 500 ng / kg BoNT / A can be set as the lethal dose (LD100) of BoNT / A in the human FcRn transgenic mouse model.

[0070] The neutralizing activity of antibodies (HM, HM-Fc5, and HM-Fc6) was verified in human FcRn transgenic mice by BoNT / A challenge test. Different concentrations of antibodies (500 μg / kg, 5 μg / kg, and 0.5 μg / kg) were incubated with 200 μL volume of BoNT / A LD100 at room temperature for 30 minutes and then intraperitoneally injected into mice. The PBS+BoNT / A group was used as the control group to observe the survival of the poisoned mice. Figure 2 As shown in BC, all mice in the 500μg / kg and 5μg / kg groups survived, while the low-concentration 0.5μg / kg antibody group only protected about 50% of the mice ( Figure 2 D), reflecting the dose-dependent protective effect of the antibody in vivo.

[0071] 2. Pharmacokinetic and pharmacodynamic results of antibody HM in human FcRn transgenic mice

[0072] In human FcRn transgenic mice, we determined the in vivo pharmacokinetic and pharmacodynamic characteristics of the antibody. Mice were injected with 500 μg of antibody on day 1, and the antibody concentration in the serum was measured at different time points by sandwich ELISA. Figure 3 As shown in A, the half-life of HM-Fc5 and HM-Fc6 is longer than that of the parent antibody HM. On day 50 after injection, 10 times the lethal dose of BoNT / a was intraperitoneally injected into mice. Figure 3As shown in B, both HM-Fc5 and HM-Fc6 can effectively protect mice from death, while all mice in the HM group died within 36 hours, and all mice in the control group that were not vaccinated with antibodies died within 16 hours. The results show that HM-Fc5 and HM-Fc6 can effectively prolong the in vivo residence time under the same initial conditions, thereby shortening the medication interval.

Claims

1. A long-acting antibody for neutralizing botulinum toxin type A, the antibody comprising a heavy chain variable region and an Fc region, the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, and the Fc region having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. A nucleic acid molecule encoding the antibody according to claim 1.

3. A recombinant expression vector comprising the nucleic acid molecule according to claim 2. The recombinant expression vector according to claim 3 , further comprising a promoter.

5. A host cell, comprising the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 or 4.

6. A pharmaceutical composition, comprising the antibody of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4.

7. Use of the antibody of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4 in the preparation of a pharmaceutical composition for inhibiting botulinum toxin type A or treating a disease caused by botulinum toxin type A.

8. A kit for detecting botulinum toxin type A, comprising the antibody according to claim 1.

9. Use of the antibody of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4 in the preparation of a product for detecting botulinum toxin type A or in the preparation of a product for diagnosing botulinum toxin type A infection.

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