Chimeric botulinum toxin

Abstract

The present invention is a chimeric botulinum toxin in which an active substance is linked to the N-terminus of a light chain, and mutations are introduced into 1 to 10 amino acids among the amino acids making up the light chain.

Description

Chimeric botulinum toxin

[0001] The present invention relates to chimeric botulinum toxins.

[0002] Botulinum toxin, produced by Clostridium botulinum, an anaerobic gram-positive bacterium, is the most lethal neurotoxin on earth. Neurotoxins (NTXs) produced by Clostridium botulinum are classified into types A, B, C, D, E, F, and G based on differences in antigenicity, and their characteristics have been clarified.

[0003] The susceptibility of animals to NTX varies depending on the serotype. The molecular weight of NTX of all seven serotypes is approximately 150 kDa.

[0004] Botulinum toxin acts on the neuromuscular junction and autonomic ganglia and their terminals in botulism poisoning, blocking the release of acetylcholine and causing death in humans, but this activity has also been utilized positively as a useful inhibitor of neuromuscular transmission, particularly as a therapeutic agent for alleviating localized muscle tone by direct intramuscular administration to patients with diseases that cause abnormal hypertonia, such as dystonia (Non-Patent Document 1).

[0005] Although attempts have already been made to utilize botulinum neurotoxin in an effective manner, there is a need for a means to utilize the excellent effects of botulinum toxin to more efficiently deliver drugs and other substances to target sites.

[0006] Ryuji Kaji et al., "Dystonia and Botulinum Treatment," Shindan to Chiryousha, 2005

[0007] In view of the above circumstances, an object of the present invention is to provide a chimeric botulinum toxin capable of efficiently delivering an active substance to target cells.

[0008] (1. Chimeric Botulinum Toxin) As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that by linking an active substance to a botulinum toxin having a mutation introduced into a portion of its light chain, the active substance can be efficiently delivered into target cells. Based on this finding, the present inventors have conducted further research and have completed the present invention.

[0009] That is, the present invention provides the following chimeric botulinum toxins. Item 1. A chimeric botulinum toxin having an active substance linked to the N-terminus of a light chain, and having mutations introduced into 1 to 10 amino acids among the amino acids constituting the light chain. Item 2. The chimeric botulinum toxin according to Item 1, which is type A. Item 3. The chimeric botulinum toxin according to Item 1, which is type A2. Item 4. A pharmaceutical composition comprising the chimeric botulinum toxin according to any one of Items 1 to 3. Item 5. The pharmaceutical composition according to Item 4, which is for treating a disease caused by hypertonia. Item 6. Item 6. The pharmaceutical composition according to Item 5, wherein the muscle hypertonia disease is a disease caused by localized muscle hypertonia, and the disease is strabismus, blepharospasm, hemifacial spasm, spasmodic torticollis, post-stroke paralysis, infantile cerebral palsy, spasmodic dysphonia, headaches such as migraine, chronic pain such as lower back pain, stiff shoulders, muscle relaxation failure occurring at the onset of Parkinson's disease or multiple sclerosis, myofascial pain syndrome, masticatory muscle spasm, chronic anal fissure, urinary incontinence, teeth grinding, facial myokymia, tics, local dystonia, or wrinkles.

[0010] The chimeric botulinum toxin of the present invention thus constructed can efficiently deliver an active substance to target cells.

[0011] Schematic diagram of the domain structure of a botulinum toxin. Schematic diagram of the domain structure of a chimeric botulinum toxin of the present invention. SDS-PAGE analysis results of Production Examples 1 and 2. Evaluation results of 3B12scFv using ALS model cells.

[0012] In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of." Furthermore, in this specification, when a numerical range is expressed as "A to B," it means A or more and B or less.

[0013] The chimeric botulinum toxin of the present invention is a chimeric botulinum toxin in which a cargo protein or cargo peptide is linked to the N-terminus of the light chain constituting the botulinum toxin and in which mutations have been introduced into 1 to 10 amino acids among the amino acids constituting the light chain.

[0014] As shown in Figure 1, NTX has a structure in which a light chain (LC) with a molecular weight of about 50,000 and a heavy chain (HC) with a molecular weight of about 100,000 are linked by a disulfide bond. The heavy chain further comprises an N-terminal region (HC) with a molecular weight of approximately the same. Ｎ ) and the C-terminal region (H Ｃ ) can be divided into two domains with different functions.

[0015] As recent studies have revealed, the C-terminal region of the heavy chain of NTX binds to a toxin type-specific receptor on the surface of nerve cells, and the botulinum toxin enters nerve cells by endocytosis.

[0016] Next, the N-terminal region of the heavy chain forms a pore under the acidic conditions in the endosome, allowing the light chain to be transported to the cytoplasm and cleave the SNARE protein. As a result, the secretion of acetylcholine is inhibited due to the cleavage of the SNARE protein, which is essential for the secretion of acetylcholine, and muscle contraction is prevented, resulting in serious adverse effects on the body.

[0017] Based on differences in antigenicity, botulinum toxins are classified into seven toxin types, A, B, C, D, E, F, and G. Of these, types A, B, E, F, and G are known to cause human botulism, while types C and D cause avian or livestock botulism.

[0018] As the botulinum toxin used in preparing the chimeric botulinum toxin of the present invention, it is preferable to use type A botulinum toxin because it can deliver active substances to target cells more efficiently. Type A botulinum toxin includes subtypes A1 and A2, both of which can be suitably used. However, type A2 botulinum toxin is particularly preferable because it can deliver active substances into target cells more efficiently.

[0019] The amino acid sequences of botulinum toxin type A1 and botulinum toxin type A2 are set forth in SEQ ID NOs: 1 and 2, respectively.

[0020] Next, the structure of the chimeric botulinum toxin of the present invention will be explained with reference to FIG.

[0021] In order to inactivate the toxin activity, one or more amino acid mutations are introduced into the light chain (LC) domain of the chimeric botulinum toxin of the present invention. The number of amino acid mutations to be introduced is 1 to 10, preferably 1 to 5, more preferably 2 to 4, and particularly preferably 3.

[0022] In particular, the amino acid sequence of type A2 botulinum toxin is as shown in SEQ ID NO: 2, as described above, and it is preferable to introduce mutations into one or more, preferably three or more, amino acids located at positions 223 to 227, 242 to 257, and 359 to 370 of this sequence. For example, it is also preferable to introduce mutations into one amino acid each in the three regions of positions 223 to 227, 242 to 257, and 359 to 370 of SEQ ID NO: 2.

[0023] For example, in the case of botulinum toxin type A2, it is preferable to introduce the mutations shown in SEQ ID NO: 3. In SEQ ID NO: 3, E at position 224 is mutated to Q, R at position 363 is mutated to A, and Y at position 366 is mutated to F in the wild-type botulinum toxin type A2 shown in SEQ ID NO: 2.

[0024] The method for introducing a mutation into the light chain domain can be a wide variety of known methods, and is not particularly limited, including, for example, PCR-based gene recombination experiments using primers containing site-specific mutations.

[0025] As shown in Figure 2, an active substance is located at the N-terminus of the light chain domain. This structure can be obtained, for example, by inserting the active substance gene into the 5' end of a vector incorporating a neurotoxin gene, integrating the gene, and expressing and purifying it. The active substance is a substance that acts on target cells to exert its effect. Examples of such active substances include compounds (including low-molecular-weight compounds and high-molecular-weight compounds), peptides, or proteins that have the activity of binding to the abnormal structure of a specific peptide or protein that causes a neurodegenerative disease, or to the peptide or protein itself, and inhibiting or removing the aggregation of this abnormal structure, peptide, or protein.

[0026] More specifically, such an active substance may include, but is not limited to, αTDP-43scFv.

[0027] The method for linking an active substance to the N-terminus of the botulinum toxin light chain domain can be widely adopted and is not particularly limited. Specifically, a method can be used in which the active substance gene is inserted into a vector using a multicloning site on the 5'-end of the botulinum toxin gene.

[0028] (2. Pharmaceutical Composition) The present invention includes an invention relating to a pharmaceutical composition for treating hypertonic disorders.

[0029] While the chimeric botulinum toxins of the present invention can be administered alone, they are also preferably provided as various pharmaceutical compositions, which can be administered to animals and humans.

[0030] The pharmaceutical composition may be administered by any suitable route depending on the intended treatment. Specific examples of the route include oral administration and parenteral administration such as rectal, oral, subcutaneous, intramuscular, and intravenous administration.

[0031] Dosage forms include capsules, tablets, granules, powders, syrups, emulsions, suppositories, injections, etc. Liquid preparations suitable for oral administration, such as emulsions and syrups, can be prepared using water, sugars such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Capsules, tablets, powders, granules, etc. can be prepared using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin.

[0032] Preparations suitable for parenteral administration preferably consist of a sterile aqueous preparation containing an active compound that is isotonic with the blood of the recipient. For example, in the case of injections, solutions for injection may be prepared using a carrier such as a saline solution, a glucose solution, or a mixture of saline and a glucose solution.

[0033] Topical formulations are prepared by dissolving or suspending the active compound in one or more vehicles, such as mineral oil, petroleum, polyols, etc., or other bases used in topical pharmaceutical preparations. Formulations for enteral administration that can be used in the invention are prepared using conventional carriers, such as cocoa butter, hydrogenated fats, hydrogenated fatty carboxylic acids, etc., and presented as suppositories.

[0034] In the present invention, one or more auxiliary ingredients selected from glycols, oils, flavors, preservatives (including antioxidants), excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified for oral preparations, may also be added to parenteral preparations.

[0035] The effective dose and frequency of administration of the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof vary depending on the administration form, the age and body weight of the patient, the nature or severity of the symptoms to be treated, etc. Usually, the dose may be appropriately determined depending on the purpose, etc., and the frequency of administration is preferably once a day or in divided doses.

[0036] The pharmaceutical composition of the present invention can be suitably used for treating diseases associated with hypertonia.

[0037] The hypertonia disease to be treated with the pharmaceutical composition of the present invention is preferably a disease requiring rapid suppression of hypertonia, i.e., a disease requiring treatment with a fast-acting therapeutic agent. The target diseases for treatment to reduce hypertonia include strabismus, blepharospasm, hemifacial spasm, spasmodic torticollis, post-stroke paralysis, infantile cerebral palsy, spasmodic dysphonia, headaches such as migraine, chronic pain such as lower back pain, stiff shoulders, insufficient muscle relaxation occurring at the onset of Parkinson's disease or multiple sclerosis, myofascial pain syndrome, masticatory muscle spasm, chronic anal fissure, urinary incontinence, teeth grinding, facial myokymia, tics, focal dystonia, and wrinkles. Myofascial pain syndrome is a condition in which acute muscle disorders or repetitive muscle overload (overuse) causes the formation of hard, lumpy tension bands within the muscles, resulting in severe pain. It is known that excessive muscle tension in the hands and feet occurs after a stroke, or with the onset of childhood cerebral palsy, Parkinson's disease, or multiple sclerosis. Furthermore, abnormal muscle tension in the neck and shoulders can cause chronic headaches, such as migraines. Furthermore, abnormal muscle tension caused by muscle fatigue or persistent poor posture can result in chronic pain, such as lower back pain, neck pain, or back pain, as well as stiff shoulders.

[0038] Although the embodiments of the present invention have been described above, the present invention is not limited to these examples, and it goes without saying that the present invention can be embodied in various forms without departing from the spirit of the present invention.

[0039] Hereinafter, the embodiments of the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[0040] (Expression and Purification of Recombinant Detoxified Neurotoxin) The full-length neurotoxin gene obtained from the A2 subtype botulinum neurotoxin-producing Clostridium botulinum type A Chiba-H strain (SEQ ID NO: GenBank: CAA51824.1) was amplified with primers containing restriction enzyme sites (BamHI at the 5' end and XhoI at the 3' end) and cloned into Escherichia coli DH5α using the expression vector pETNH6 (w_BoNTA2 / pETNH6). Correct insertion of the neurotoxin gene into the vector was then confirmed by sequence analysis. Using w_BoNTA2 / pETNH6 as a template, the gene was amplified with primers for inserting three point mutations. Point mutations were then introduced into E. coli DH5α (3m_BoNTA2 / pETNH6), and sequence analysis confirmed the gene identity. Expression strains of Escherichia coli BL21 DE3codon(+) were transformed with the wild-type BoNTA2 (w_BoNTA2 / pETNH6) and mutant BoNTA2 (3m_BoNTA2 / pETNH6) plasmids, and the recombinant proteins were expressed. E. coli cells were harvested from the culture plate and cultured at 30°C with shaking in LB medium supplemented with 50 μg / ml ampicillin. After approximately 3 hours, a portion of the culture was taken and the turbidity (OD600) of the E. coli was measured. When the turbidity reached 0.7-0.9, the cells were rapidly chilled on ice. IPTG was added to the culture to a final concentration of 50 μM, and the cells were cultured overnight at 18°C ​​with shaking. After incubation, the cells were harvested, washed three times with distilled water, and stored at -80°C until purification.

[0041] The cells stored at -80°C were suspended in 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 20 mM imidazole. They were sonicated five times on ice (output 5, duty 50, 5 minutes), then centrifuged (27,000 x g, 25 minutes). The supernatant was then filtered. Since the target protein was a fusion protein with an N-terminal histidine tag, the filtrate was added to HisTrap (1 ml) for affinity chromatography. The HisTrap was equilibrated with 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 20 mM imidazole, and then added to the filtrate. After washing, the target protein was eluted using a 20-volume gradient of 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 200 mM imidazole. After confirming the presence of the approximately 150 kDa target protein in the elution fraction by SDS-PAGE, gel filtration was performed on a Superdex 200 pg column (2.6 cm diameter x 60 cm height) to remove proteins other than the target protein. The gel filtration column was equilibrated with 20 mM Na-phosphate buffer, pH 8.0, and elution was also performed with the same buffer. The approximately 150 kDa target protein fraction was identified by SDS-PAGE, and the fractions were pooled and measured for protein content. To activate the target protein, trypsin solution was added at 1 / 25 of the total protein mass and incubated at 37°C for 30 minutes. Trypsin inhibitor was then added at 1 / 12.5 mass and incubated at room temperature for 10 minutes. Furthermore, anion chromatography was performed using CaptoHiRes Q 5 / 50 (1 ml) to remove bands other than the target protein and excess trypsin and trypsin inhibitor.The CaptoHiRes Q column was equilibrated with 20 mM Na-phosphate buffer, pH 8.0. The trypsin-treated sample was loaded onto the column and washed. The target protein was eluted with a gradient of 20 mM Na-phosphate buffer, pH 8.0, to 1 M NaCl. SDS-PAGE was performed under reducing conditions to obtain purified w_BoNTA2 (Production Example 1) and purified 3m_BoNTA2 (Production Example 2), both of which have a diplex structure consisting of a 100 kDa heavy chain and a 50 kDa light chain (Figure 3). The amino acid sequences of w_BoNTA2 and 3m_BoNTA2 are shown in SEQ ID NOs: 2 and 3, respectively. After protein quantification, the purified samples were dispensed into small aliquots and stored at -80°C until use.

[0042] (In vivo toxicity evaluation test) Toxicity evaluation was performed by intraperitoneally administering the detoxified neurotoxin to mice. Purified w_BoNTA2 (Production Example 1) and 3m_BoNTA2 (Production Example 2) were diluted to 8 μg / ml and administered intraperitoneally in 0.5 ml to ddY mice (4-week-old, 20 g males). Survival was observed. Mice in the w_BoNTA2 (Production Example 1) group died the following day, while mice in the 3m_BoNTA2 (Production Example 2) group survived without any paralytic symptoms over the 4-day observation period. In this study, survival was observed at 4 μg / mouse (20 g body weight), and survival was confirmed at 0.2 mg / kg. Further, lethal activity was examined by increasing the dose of 3m_BoNTA2 (Production Example 2). Even at the maximum dose of 2 mg / kg [40 μg / mouse (20 g body weight)], no paralytic symptoms were observed over the observation period (1 week).

[0043] (Expression and Purification of Anti-TDP-43 scFv (3B12 scFv)) Based on the report by Tamaki et al. (Tamaki Y., et.al. Scientific Reports | (2018) 8:6030 DOI:10.1038 / s41598-018-24463-3), we connected the heavy and light chain sequences of the variable region of an antibody that binds to abnormally conformed TDP-43 but not to normal TDP-43, promoting its degradation. A plasmid was constructed containing a gene containing a signal sequence at the 5' end of the variable region sequence to promote protein expression into the periplasm and a histidine tag at the 3' end. The expressed protein of approximately 29 kDa was purified in the same manner as the recombinant neurotoxin described above. Because the target protein, approximately 29 kDa, has a smaller molecular weight than the neurotoxin, a Superdex 75 pg (2.6 cm diameter x 60 cm height) was used for gel filtration. Expression of the target protein was confirmed using a polyclonal antibody against a histidine tag added to the C-terminus.

[0044] (Effect of 3B12scFv using ALS model cells) Based on the report by Tamaki et al. (Tamaki Y., et.al. Scientific Reports | (2018) 8:6030 DOI:10.1038 / s41598-018-24463-3), the human TDP-43 gene was cloned and point mutations were introduced into two sites in the nuclear localization signal region of TDP-43, R83L and K84Q, and two sites in the RRM domain, C173S and C175S. A GFP sequence was added to the 3' end of the plasmid TDP-43mNLS, C173 / C175S_GFP, which was constructed in the mammalian cell expression plasmid pcDNA3.1 (TDP-43mNLS, C173 / C175S_GFP / pcDNA3.1). To transiently express TDP-43mNLS, C173 / C175S_GFP / pcDNA3.1 in human embryonic kidney HEK293 cells, we used the gene transfer reagent Lipofectamine 3000 to confirm the formation of abnormal TDP-43 (aggregates) and generate ALS model cells. To examine the effect of 3B12scFv on ALS model cells, ALS model cells were treated with 20 mM digitonin at 37°C for 5 minutes and then washed with medium (D-MEM). 3B12scFv was added at 50 μg / ml (approximately 2 μM), and the medium was replaced with maintenance medium (D-MEM-10% FCS). GFP fluorescence was observed for TDP-43 aggregates. As shown in Figure 4, TDP-43 aggregates (gray; lower left arrow) that were widely distributed in the cytoplasm of ALS model cells were localized to the nucleus (white; center arrow) after introduction of 3B12scFv into the cells, returning to normal distribution.

Claims

1. A chimeric botulinum toxin in which an active substance is linked to the N-terminus of the light chain and mutations are introduced into 1 to 10 amino acids among the amino acids constituting the light chain.

2. The chimeric botulinum toxin of claim 1, which is type A.

3. The chimeric botulinum toxin of claim 1, which is type A2.

4. A pharmaceutical composition comprising the chimeric botulinum toxin according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, which is for treating diseases of hypertonia.

6. The pharmaceutical composition according to claim 5, wherein the muscle hypertonia disease is a disease caused by localized muscle hypertonia, and the disease is strabismus, blepharospasm, hemifacial spasm, spasmodic torticollis, post-stroke paralysis, infantile cerebral palsy, spasmodic dysphonia, headaches such as migraine, chronic pain such as lower back pain, stiff shoulders, muscle relaxation failure occurring at the onset of Parkinson's disease or multiple sclerosis, myofascial pain syndrome, masticatory muscle spasm, chronic anal fissure, urinary incontinence, teeth grinding, facial myokymia, tics, local dystonia, or wrinkles.

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