Recombinant botulinum toxin type A light chain, recombinant botulinum toxin, and its composition, its uses and methods thereof.

Abstract

This invention provides recombinant botulinum toxin type A light chain, recombinant botulinum toxin, and related compositions, uses, and methods, which offer increased efficacy and half-life, improved patient convenience, and the ability to provide treatment tailored to specific indications. [Solution] A recombinant botulinum toxin type A light chain is provided, which is not botulinum toxin type A4, wherein the sequence of the third domain among the first, second, third, and fourth domains of the botulinum toxin type A light chain is replaced with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

Description

Technical Field

[0001] The present invention relates to recombinant botulinum toxin, and more particularly, to recombinant botulinum toxin type A light chain, recombinant botulinum toxin, and related compositions, uses and methods.

Background Art

[0002] Botulinum toxin (BoNT) is a neurotoxic protein produced by Clostridium botulinum bacteria and related species. The botulinum toxin blocks the release of acetylcholine, a neurotransmitter secreted from the axonal terminals of the neuromuscular junction.

[0003] Botulinum toxin is synthesized by a 150 kDa double-stranded protein composed of a 100 kDa heavy chain and a 50 kDa light chain linked by a disulfide bond. The heavy chain is further divided into an N-terminal domain (Hn) that helps translocate the light chain into the cytosol of the cell, and a C-terminal domain (Hc) that recognizes and binds to cell surface receptors on nerve cells. Once inside the cell, the light chain specifically hydrolyzes (proteolytic cleavage) a part of the soluble NSF attachment protein receptor (SNARE), inactivating neurotransmitter release.

[0004] Botulinum toxin is classified into seven serotypes (botulinum toxin type A, botulinum toxin type B, botulinum toxin type C, botulinum toxin type D, botulinum toxin type E, botulinum toxin type F, and botulinum toxin type G), which are further subdivided into subtypes based on amino acid sequence variations. Of these, botulinum toxin type A1 has been extensively studied at the molecular level, as well as in preclinical and clinical studies, and is currently widely used in the pharmaceutical industry. In contrast, other subtypes of botulinum toxin type A are difficult to isolate after purification, and their pharmacological characteristics have not been studied or reported much. Currently, only a portion of these subtypes of botulinum toxin type A have had their biochemical, cellular, and in vivo properties elucidated. According to reported studies, various in vitro and in vivo studies involving botulinum toxin type A subtypes have revealed characteristics that distinguish them from botulinum toxin type A1 in terms of mechanistic aspects such as potency, intracellular transport, and persistence. Recently, the characteristics of botulinum toxin type A7 and botulinum toxin type A8 at the in vitro level have been reported.

[0005] International Publication WO2018 / 132423 discloses a botulinum neurotoxin mixture containing a light chain peptide of botulinum neurotoxin type A subtype 1 and a heavy chain peptide of botulinum neurotoxin type A subtype 2, and a pharmaceutical composition containing the same, for improved potency, cell entry kinetics, and duration of action. Furthermore, International Publication WO2017 / 214447 discloses botulinum toxin B1 with a rearranged heavy chain sequence to maintain the same level of toxic activity at an even lower volume.

[0006] However, to date, no attempts have been made to improve the potency of recombinant botulinum toxin type A light chains, or recombinant botulinum toxins containing them, by rearranging the lower domains of the botulinum neurotoxin type A subtype light chain. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International release WO2018 / 132423 [Patent Document 2] International release WO2017 / 214447 [Overview of the project] [Problems that the invention aims to solve]

[0008] The purpose of this disclosure is to provide recombinant botulinum toxin type A light chain and recombinant botulinum toxin containing the same.

[0009] Another object of this disclosure is to provide compositions relating to the recombinant botulinum toxin.

[0010] Another object of this disclosure is to provide uses for compositions relating to recombinant botulinum toxin.

[0011] A further object of this disclosure is to provide a method for improving or treating a disease, comprising the step of administering a composition relating to recombinant botulinum toxin to an individual.

[0012] Further objections to this disclosure are to provide wild-type botulinum toxin type A light chains, recombinant botulinum toxin type A light chains having increased potency or half-life compared to wild-type botulinum toxin containing the same, and methods for producing recombinant botulinum toxin containing the same. [Means for solving the problem]

[0013] One aspect of the present disclosure provides a recombinant botulinum toxin type A light chain, which is not botulinum toxin type A4, in which the sequence of the third domain is replaced in the first, second, third, and fourth domains of the botulinum toxin type A light chain with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

[0014] Another aspect of the present disclosure provides a recombinant botulinum toxin type A light chain in which the sequence of the fourth domain is further substituted with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof, in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain.

[0015] Further embodiments of the present disclosure provide recombinant botulinum toxins comprising the recombinant botulinum toxin type A light chain and the botulinum toxin heavy chain.

[0016] Further embodiments of the present disclosure provide compositions comprising the recombinant botulinum toxin and pharmaceutically acceptable excipients or additives.

[0017] Further embodiments of the present disclosure provide uses for administering compositions comprising the recombinant botulinum toxin and pharmaceutically acceptable excipients or additives to an individual to improve or treat a disease.

[0018] Further aspects of the present disclosure provide a method for producing recombinant botulinum toxin type A light chain having an increased effect or half-life compared to a wild-type botulinum toxin type A light chain, comprising the step of substituting the sequence of the third domain in the first, second, third, and fourth domains of a botulinum toxin type A light chain that is not botulinum toxin type A4 with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

[0019] Still other aspects of the present disclosure provide a method for producing a recombinant botulinum toxin type A light chain having an increased effect or half-life compared to a wild-type botulinum toxin type A light chain, the method including the step of further substituting, in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain, the sequence of the fourth domain with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof.

[0020] Still other aspects of the present disclosure provide a method for producing a recombinant botulinum toxin having an increased potency or half-life compared to a wild-type botulinum toxin, the recombinant botulinum toxin including the recombinant botulinum toxin type A light chain and a botulinum toxin heavy chain.

Advantages of the Invention

[0021] According to the present disclosure, there are provided a recombinant botulinum toxin type A light chain, a recombinant botulinum toxin, and related compositions, uses, and methods thereof, which have increased potency and half-life, enhanced patient convenience, and enable treatment for indications.

Brief Description of the Drawings

[0022] [Figure 1A] Shows the sequence comparison (ESPript3.0 (Robert and Gouet, 2014)) of botulinum toxin type A1 light chain (GenBank: CAL82360.1) and botulinum toxin type A4 light chain (GenBank: ACQ51417.1). The boundaries and starting points of each domain for domain substitution are indicated by arrows and boxes, and the following table shows the sequence ranges of each domain and the results of amino acid identity analysis between the two sequences. [Figure 1B] Shows structural information related to the domains of botulinum toxin type A1 light chain and botulinum toxin type A4 light chain, and shows the amino acid sequence differences and functional characteristics related to each domain. [Figure 1C]A schematic diagram showing a combination of a recombinant botulinum toxin type A light chain in which the domains of botulinum toxin type A1 light chain and botulinum toxin type A4 light chain are replaced. [Figure 2A] The SDS-PAGE analysis result of the recombinant botulinum toxin type A light chain purified by domain replacement. [Figure 2B] The result of confirming the site where Clover-SNAP25-mRuby2(137-206) was hydrolyzed by the recombinant botulinum toxin type A light chain with replaced domains. The presence or absence of hydrolysis of SNAP25(137-206) labeled with a fluorescent protein was confirmed under the condition where the recombinant botulinum toxin type A light chain with replaced domains was present and under the condition where it was not present. As a result, hydrolysis occurred under the condition where the recombinant botulinum toxin type A light chain with replaced domains was present. The samples were analyzed via SDS-PAGE and Coomassie blue staining. [Figure 3] A graph showing the thermal stability of the recombinant botulinum toxin type A light chain with replaced domains. The following table shows the melting temperature of each recombinant botulinum toxin light chain with replaced domains. [Figure 4] The result of the endopeptidase assay of the recombinant botulinum toxin type A light chain with replaced domains. When comparing the titer results of the recombinant botulinum toxin type A light chain with replaced domains and the wild-type botulinum toxin type A light chain, pBT103 showed a titer similar to the wild type, pBT112 increased by 1.73-fold, pBT113 increased by 1.49-fold, pBT114 increased by 1.3-fold, and pBT115 increased by 1.55-fold. [Figure 5A] The result of SDS-PAGE analysis of the purified product of the recombinant botulinum toxin type A light chain in which the light chain of botulinum toxin type A was replaced with the third domain of other botulinum toxin type A subtypes. [Figure 5B]This report analyzes the thermal stability of purified products of recombinant botulinum toxin type A light chains in which the light chain of botulinum toxin type A was replaced with the third domain of another botulinum toxin type A subtype. The solubility points of each recombinant botulinum toxin type A light chain are shown in the table below. The greatest improvement in thermal stability was observed when the third domain of the botulinum toxin type A4 light chain was introduced into the botulinum toxin type A light chain. [Figure 6A] This is a schematic diagram illustrating the recombinant botulinum toxin structure, including a recombinant botulinum toxin type A light chain with a substituted domain. [Figure 6B] This report describes the results of SDS-PAGE analysis of the purified product of recombinant botulinum toxin containing a recombinant botulinum toxin type A light chain with a substituted domain, under reducing conditions. The recombinant botulinum toxin was purified to a 150 kd form free of impurities and then analyzed by SDS-PAGE under reducing conditions. [Figure 7A] This graph shows the time course of DAS values ​​after administering Coretox (botulinum toxin type A product) and recombinant botulinum toxin (pBT142) to the right calf muscle in rats at doses of 1.2, 4, and 12 U / kg, respectively. The DAS values ​​for each measurement day were analyzed using the Mann-Whitney test (*p<0.05). [Figure 7B] This graph shows the trend line related to the highest DAS score and the calculated DAS ED50 for rats after administering Coretox and recombinant botulinum toxin (pBT142) to the right calf muscle at doses of 1.2, 4, and 12 U / kg, respectively. [Figure 7C]This graph shows the time course of CMAP values ​​after administering Coretox and recombinant botulinum toxin (pBT142) at a dose of 12 U / kg to the right calf muscle in rats. The results of the analysis of CMAP values ​​for Coretox and recombinant botulinum toxin (pBT142) on each measurement day are shown using a two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Figure 7D] This graph shows the time course of DAS values ​​after administering Coretox and recombinant botulinum toxin (pBT147) to the right calf muscle in rats at doses of 1.2, 4, and 12 U / kg, respectively. The DAS values ​​for each measurement day were analyzed using the Mann-Whitney test (*p<0.05, **p<0.01). [Figure 7E] This graph shows the trend line related to the highest DAS score and the calculated DAS ED50 for rats after administering Coretox and recombinant botulinum toxin (pBT147) to the right calf muscle at doses of 1.2, 4, and 12 U / kg, respectively. [Figure 7F] This graph shows the time course of CMAP values ​​after administering Coretox and recombinant botulinum toxin (pBT147) at a dose of 12 U / kg to the right calf muscle in rats. The results of the analysis of CMAP values ​​for Coretox and recombinant botulinum toxin (pBT147) on each measurement day using a two-tailed t-test are shown (*p<0.05, **p<0.01, ***p<0.001). [Figure 8A] This graph shows the time course of DAS values ​​after administering Coretox (botulinum toxin type A product) and recombinant botulinum toxins (pBT142 and pBT147) to the right calf muscle in mice at volumes of 1.2, 4, 12, and 40 U / kg, respectively. The DAS values ​​for each measurement day were analyzed using the Mann-Whitney test (*p<0.05). [Figure 8B] In mice, Coretox and recombinant botulinum toxin (pBT142 and pBT147) were administered to the right calf muscle at volumes of 1.2, 4, 12, and 40 U / kg, respectively. The graph shows the trend line related to the highest DAS score and the calculated DAS ED50 for each volume. [Figure 8C] This graph shows the time course of CMAP values ​​after administering Coretox and recombinant botulinum toxin (pBT142 and pBT147) at a volume of 12 U / kg each to the right calf muscle in mice. For each measurement day, the CMAP values ​​for Coretox and recombinant botulinum toxin (pBT142 and pBT147) were analyzed using a two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). [Modes for carrying out the invention]

[0023] The following provides further details regarding this disclosure, including specific examples, but these are provided solely to aid in understanding this disclosure and do not limit its scope in any way.

[0024] Example 1: Domain substitution of botulinum toxin type A light chain To determine which domain of botulinum toxin type A1 light chain or botulinum toxin type A4 light chain affects protein stability, four domains in the genes of botulinum toxin type A1 and botulinum toxin type A4 light chains were substituted. Using infusion cloning, a total of 16 domain-substituted light chain genes, including the substituted genes and the wild type, were cloned into the E. coli expression vector pET28a and named pBT102 to pBT108 and pBT109 to pBT115, respectively (Figure 1C). Subsequently, the recombinant botulinum toxin A light chain gene sequences were analyzed to confirm the domain substitutions.

[0025] Example 2: Expression and purification of domain-substituted botulinum toxin type A light chain Using the recombinant botulinum toxin type A light chain gene produced in Example 1, 14 recombinant botulinum toxin type A light chains were manufactured. The manufactured recombinant expression vectors were introduced into BL21(DE3) cells, a type of Escherichia coli (E. coli), to induce the expression of recombinant botulinum toxin type A light chains with substituted domains. This expression was induced with IPTG (isopropyl β-D-1-thiogalactopyranoside), and the final concentration was 0.5 mM. Specifically, 0.5 ml of 1 M IPTG was added to 1 L of liquid medium, and BL21(DE3) cells were cultured at 37°C for approximately 3 hours and at 18°C ​​for approximately 18 hours. The cells were then centrifuged at 4,000 rpm for 10 minutes to precipitate. The supernatant culture medium was removed, and the precipitated cells were resuspended in A buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 2 mM B-Me (pH 7.9)). The cells were then lysed on ice using an ultrasonic disperser at 35% power, 2 pulses / 1 pause for 20 minutes. The lysed cells were centrifuged at 14,000 rpm at 4°C for 1 hour. From the supernatant, from which cell debris had been removed, recombinant botulinum toxin type A light chains were separated from the supernatant using a Ni-NTA column via the binding of his tags to Ni-NTA resin. The amino acid sequences of the recombinant botulinum toxin type A light chains are shown as pBT102 (SEQ ID NO: 18), pBT103 (SEQ ID NO: 19), pBT104 (SEQ ID NO: 20), pBT105 (SEQ ID NO: 21), pBT106 (SEQ ID NO: 22), pBT107 (SEQ ID NO: 23), pBT108 (SEQ ID NO: 24), pBT109 (SEQ ID NO: 25), pBT110 (SEQ ID NO: 26), pBT111 (SEQ ID NO: 27), pBT112 (SEQ ID NO: 28), pBT113 (SEQ ID NO: 29), pBT114 (SEQ ID NO: 30), and pBT115 (SEQ ID NO: 31), respectively.

[0026] We attempted to express a total of 16 proteins, including the recombinant botulinum toxin type A light chain and the wild-type botulinum toxin types A1 and A4. Five of these proteins were expressed at low levels or as inclusion bodies, and ultimately, 11 proteins were expressed solublely (Figure 2A). Subsequently, the 11 purified proteins were reacted with SNAP25(137-206) substrates labeled with fluorescent proteins at the N-terminus and C-terminus to confirm their protease activity. 3 μM of fluorescently labeled SNAP25(137-206) was mixed with 0.2 μM of recombinant botulinum toxin light chains with substituted domains, incubated at 37°C for 2 hours, and loaded onto an SDS-PAGE. We confirmed that SNAP25 (137-206) with a fluorescent protein tag attached is hydrolyzed by recombinant botulinum toxin type A light chain with a substituted domain via coumassi blue staining (Figure 2B).

[0027] Test Example 1: Evaluation of the thermal stability of domain-substituted botulinum toxin type A light chains. The thermal stability of recombinant botulinum toxin type A light chains with substituted domains was compared. Stability was evaluated by assessing the thermal denaturation intermediate temperature (Tm), and generally, a high thermal denaturation intermediate temperature is desirable. The thermal stability of each domain-substituted recombinant botulinum toxin type A light chain was determined using heat transfer analysis (PTS). A Protein Thermal Shift Dye Kit (Catalog No. 4461146 (Applied Biosystems)) was used, and the PTS assay was performed according to the manufacturer's method. Specifically, 10 μg of each recombinant botulinum toxin type A light chain was mixed with protein thermal shift dye and buffer to a total volume of 20 μl. Then, RT-PCR (C1000 thermal cycler equipped with a CFX96 optical reaction module (Bio-Rad)) was performed at 20°C, increasing the temperature by 1°C per 60 seconds up to 95°C, while observing the ROX signal. Based on the melting curves, the melting point (Tm: melting temperature) of each recombinant botulinum toxin light chain was determined. The melting points of each recombinant botulinum toxin type A light chain with the aforementioned domain substitution are shown in Figure 3.

[0028] The results confirmed that the thermal stability of pBT103 (SEQ ID NO: 19) and pBT104 (SEQ ID NO: 20) was increased. Compared to the melting point of wild-type botulinum toxin type A1 light chain, which is 43°C, the melting points of pBT103 and pBT104 increased to 52°C and 51°C, respectively, and pBT114 and pBT115 increased to 49°C. Therefore, compared to the melting point of wild-type botulinum toxin type A1 light chain used as a control group, pBT103 and pBT104 showed increased thermal stability by 8°C and 9°C, and pBT114 and pBT115 showed increased thermal stability by 6°C. Thus, in common, it was confirmed that the melting points of recombinant botulinum toxin type A light chains with domain substitution to include the third domain of the botulinum toxin type A4 light chain were increased.

[0029] Test Example 2: Evaluation of the potency of a domain-substituted botulinum toxin type A light chain. To confirm the in vitro activity of the domain-substituted recombinant botulinum toxin type A light chain, an enzyme-linked immunosorbent assay (ELISA)-based endopeptidase assay was performed. First, the GST-SNAP25 (137-206) substrate was diluted to a final concentration of 2 μg / mL, and then added to a 96-well plate at 100 μL per well, and reacted at 4°C for 16 hours. 2% skim milk was prepared by dissolving it in PBST (phosphate-buffered saline with Tween), and 200 μL was added to the substrate-coated wells, and reacted at 25°C for 1 hour. After the reaction, the wells were washed three times with 300 μl of PBST. Then, using Toxin Assay Buffer (a reaction buffer containing HEPES, DTT, zinc sulfate, and Tween-20), standard samples (STD (1,2,3,4,5,6 U / mL) samples) and recombinant botulinum toxin type A light chain samples with substituted domains (target concentration: 4 U / mL) were prepared. 100 μL of each sample was loaded into the wells, and the mixture was reacted at 25°C for 2 hours. After the reaction, the wells were washed three times with 300 μl of PBST. 100 μl of the primary antibody, rabbit anti-SNAP25 (190-197) pAb, was added to each well and reacted for 30 minutes. After washing three more times as described above, the secondary antibody, anti-rabbit IgG HRP conjugate, was added and reacted for 30 minutes. TMB (BioFx) solution was added at a rate of 100 μl / well, and the mixture was allowed to develop color for 30 minutes at 25°C. After the color development was complete, 50 μl / well of 3M sulfuric acid was added to interrupt the color development, and the absorbance was measured at 450 nm.

[0030] Using the optical density (OD) of the standard sample, a calibration curve was drawn between the concentration and optical density of the standard sample. The resulting linear equation was then used to determine the measured titer of the recombinant botulinum toxin type A light chain sample with substituted domains. Using the calculated titer and target concentration, the recovery rate of the recombinant botulinum toxin type A light chain sample with substituted domains was calculated, and the recovery rate relative to the target concentration was confirmed. The results are shown in Figure 4. As can be seen from Figure 4, pBT103 (SEQ ID NO: 19) showed a titer similar to that of wild-type botulinum toxin type A1 light chain, while pBT114 (SEQ ID NO: 30) showed a 1.3-fold increase in titer, and pBT115 (SEQ ID NO: 31) showed a 1.55-fold increase.

[0031] Comparative Example 1: Evaluation of expression, purification, and thermal stability of recombinant botulinum toxin type A light chain substituted with the third domain of the light chain of another botulinum toxin type A subtype. To determine whether the third domain of the A4 light chain is a crucial factor in increasing the thermal stability of recombinant botulinum toxin type A, the third domains of other botulinum toxin type A light chains (botulinum toxin type A2, A5, A7, and A8) were substituted with the third domain of the botulinum toxin type A1 light chain, and the thermal stability was then compared.

[0032] To obtain recombinant botulinum toxin type A light chains with a substituted third domain, four recombinant botulinum toxin type A light chains were purified using the purification method of Example 2. The recombinant botulinum toxin type A light chains in which the third domain of each botulinum toxin type A subtype light chain was substituted were then cloned into the Escherichia coli (E. coli) expression vector pET28a.

[0033] The vector substituted with the third domain of the botulinum toxin type A2 light chain was named pBT158 (SEQ ID NO: 32), the vector substituted with the third domain of the botulinum toxin type A5 light chain was named pBT160 (SEQ ID NO: 33), the vector substituted with the third domain of the botulinum toxin type A7 light chain was named pBT161 (SEQ ID NO: 34), and the vector substituted with the third domain of the botulinum toxin type A8 light chain was named pBT162 (SEQ ID NO: 35). Each recombinant botulinum toxin type A light chain was purified using the same method as in Example 2 (Figure 5A). The recombinant botulinum toxin light chain protein with the third domain of the botulinum toxin type A3 light chain substituted was not expressed, and the botulinum toxin type A6 light chain third domain was excluded from the test because its sequence is the same as that of the third domain of A1.

[0034] As described above, when the third domain of the botulinum toxin type A1 light chain was substituted with the third domain of the botulinum toxin type A2 light chain, the third domain of the botulinum toxin type A5 light chain, the third domain of the botulinum toxin type A7 light chain, or the third domain of the botulinum toxin type A8 light chain, the recombinant botulinum toxin type A light chain in which the third domain of the botulinum toxin type A1 light chain was substituted with the third domain of the botulinum toxin type A4 had the highest solubility, meaning it exhibited the best thermal stability (Figure 5B).

[0035] Example 3: Preparation and purification of domain-substituted recombinant botulinum toxin To analyze the function of recombinant botulinum toxin involving a domain-substituted recombinant botulinum toxin type A light chain, the full-length recombinant botulinum toxin gene, including a recombinant botulinum toxin type A light chain with increased thermal stability and activity, was cloned using the pM TL80000 vector system via infusion (infusion-HD(Takara)). The cloned products were named pBT142, pBT143, pBT144, pBT145, pBT146, and pBT147, and the structure of each domain is schematically shown in Figure 6A. For the domain-substituted recombinant botulinum toxin, a detoxified Hall A-hyper strain was prepared by inactivating the toxin gene using the Clos Tron method (system) and used.

[0036] Detoxified Clostridium botulinum strains, including those in which the toxin gene is inactivated or knocked out, can be used in the production of purified recombinant botulinum toxin or purified recombinant botulinum toxin complexes. Methods for producing strains in which the toxin gene is inactivated or knocked out are known in the art, and the use of the Clos Tron method is described in the literature [Bradshaw et al. 2010, Pellett et al. 2016].

[0037] Recombinant botulinum toxins produced by the Clos-Tron method were purified to a 150 kDa form free of complex components (Figure 6A). The recombinant botulinum toxins were identified as pBT142 (SEQ ID NO: 36), pBT143 (SEQ ID NO: 37), pBT144 (SEQ ID NO: 38), pBT145 (SEQ ID NO: 39), pBT146 (SEQ ID NO: 40), and pBT147 (SEQ ID NO: 41), respectively. All of the recombinant botulinum toxins were produced in facilities authorized for toxin production under government regulations.

[0038] Test Example 3: Efficacy of recombinant botulinum toxin with substituted domains in rats Six-week-old female SD (Sprague-Dawley) rats were purchased from Orient Bio Co., Ltd., and after a one-week acclimatization and quarantine period, seven-week-old rats were used in the experiment. Sterilized laboratory animal solid feed (R40-10 (SAFE (France))) was provided as ad libitum feed, and tap water sterilized at high temperature and pressure was also provided as ad libitum water. During the acclimatization, quarantine, and experimental periods, the rats were housed under specific pathogen-free conditions with a temperature of 23±3℃, relative humidity of 55±15%, lighting for 12 hours (8am to 8pm), ventilation rate of 15 times / hour, and illumination intensity of 150-300 lux. This study was conducted after review and approval by the Animal Experiment Ethics Committee of Meditox Co., Ltd.

[0039] As a control group, 100 units of the botulinum toxin product Coretox (botulinum toxin type A, Lot No.: NSA19016 or NSA20011 (Meditox)) were used. Recombinant botulinum toxin pBT142 was prepared at 1,316 U / mL and pBT147 at 348 U / mL and used in the experiment. The potency of each recombinant botulinum toxin was measured using a mouse potency test method and the potency value derived before the efficacy test was performed. As a placebo (dummy drug) group, the botulinum toxin component was removed from the recombinant botulinum toxin, and the same excipient components were used to conduct the efficacy tests of recombinant botulinum toxin pBT142, recombinant botulinum toxin pBT147, and Coretox. Each test substance was administered at the same volume, based on the unit (U: a value indicating the biological activity of botulinum toxin, where 1 U is the median lethal dose to mice administered intraperitoneally).

[0040] Coretox was diluted with sterile physiological saline (35V3AF3, Daehan Pharmaceutical Co., Ltd.), and recombinant botulinum toxin pBT142 and recombinant botulinum toxin pBT147 were diluted to concentrations of 12, 40, and 120 U / mL, respectively, using a placebo. The day on which the test substance was administered was designated as day 0. After anesthetizing the rats with an injectable anesthetic (60 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), each test substance was administered to the right calf muscle of the rats at a rate of 0.1 mL / kg using a Hamilton syringe, according to the composition of each group in Table 1.

[0041] For efficacy evaluation, we used the DAS (digit abduction score) method, which visually assesses the degree of muscle paralysis in rats, and the compound muscle action potential (CMAP) test method, which measures the action potential of muscles in response to external electrical stimulation.

[0042] [Table 1] The DAS values ​​shown in Table 2 indicate the degree of muscle paralysis in rats based on the morphology of the toes on the administered side, while the CMAP values ​​indicate the degree of direct muscle contraction inhibition shown by nerve blockade.

[0043] [Table 2] CMAP was measured in the right calf muscle area of ​​each rat using Nicolet Viking Quest (Viasys Healthcare, Inc.). After anesthetizing with an injectable anesthetic, the hair at the measurement site was removed, and the rat was positioned in a prone position. The negative electrode was placed at the sciatic nerve site on the leg to be measured, and the positive electrode was placed approximately 1 cm away from that site, relative to the spine. The recording electrode and reference electrode were placed at the calf muscle site and Achilles tendon site, respectively, and the ground electrode was placed at the rectus femoris site. The stimulation level and duration were set to 25-30 mA and 0.2 ms, and the amplifier filter range was set to 2-10 K at 60 Hz. When measured under these conditions, the height from the base to the peak of the waveform was recorded as the CMAP measurement data. According to the test group composition in Table 1, in Experiment 1, DAS measurements for comparing the efficacy of pBT142 and Coretox were evaluated from day 1 to day 112 after administration at doses of 1.2, 4, and 12 U / kg, and CMAP measurements were measured from day 8 to day 127 in individuals administered at a dose of 12 U / kg. In Experiment 2, for comparing the efficacy of pBT147 and Coretox, DAS measurements were evaluated from day 1 to day 112 after administration at doses of 1.2, 4, and 12 U / kg, and CMAP measurements were measured from day 0 to day 126 in individuals administered at a dose of 12 U / kg.

[0044] Graphpad Prism 7.05 (GraphPad Software Inc., CA (USA)) was used for graph presentation, and SPSS software 25.0 (SPSS Inc., IL (USA)) and Excel (2013 (MS (USA))) were used for statistical analysis. Experimental results are presented as mean ± standard deviation. DAS ED 50The values ​​were calculated using a 3-indicator logistic model after fixing the minimum score of 0 and the maximum score of 4 using Graphpad Prism. Normality testing was performed using the Kolmogorov-Smirnov test. Non-parameter data were statistically analyzed using the Mann-Whitney test, and for parameter data, a two-tailed t-test was performed, with a p-value less than 0.05 considered statistically significant.

[0045] After single-dose administration of Coretox and recombinant botulinum toxin pBT142 at doses of 1.2, 4, and 12 U / kg into the right calf muscle of rats, DAS evaluation results showed that after administration at a dose of 12 U / kg, the pBT142 group showed a significant increase in DAS scores compared to the Coretox-only group on day 14. In the Coretox 4 U / kg group, all individuals recovered by day 49, while in the pBT142 group at the same dose, all individuals recovered 7 days later, on day 56. Furthermore, in the Coretox 12 U / kg group, all individuals recovered 91 days after administration, while in the pBT142 group at the same dose, all individuals recovered 21 days later, on day 112 (Figure 7A). DAS ED 50 Through comparison, it was confirmed that pBT142 exhibits approximately 1.8 times stronger efficacy than Coretox (Figure 7B). Similar to the DAS results, in the CMAP trial, the 12U / kg pBT142 administration group showed significantly lower CMAP values ​​compared to the Coretox 12U / kg administration group, demonstrating stronger efficacy. The Coretox 12U / kg administration group recovered their CMAP values ​​on day 112 after administration, while the pBT142 12U / kg administration group recovered their CMAP values ​​15 days later, on day 127 (Figure 7C). From these results, it was confirmed that recombinant botulinum toxin pBT142 has extended efficacy and duration of action compared to Coretox.

[0046] After single-dose administration of Coretox and recombinant botulinum toxin pBT147 at doses of 1.2, 4, and 12 U / kg into the right calf muscle of rats, DAS scores were evaluated. After administration at a dose of 4 U / kg, DAS scores were significantly increased in the pBT147-administered groups compared to the Coretox-administered groups on days 5, 7, 14, 21, 28, 35, and 42. After administration at a dose of 12 U / kg, DAS scores were significantly increased in the pBT147-administered groups compared to the Coretox-administered groups on days 14, 21, and 28. In the Coretox 1.2 U / kg-administered group, all individuals recovered by day 28, while in the pBT147-administered group at the same dose, all individuals recovered 7 days later, on day 35. In the Coretox 4 U / kg-administered group, all individuals recovered by day 42, while in the pBT147-administered group at the same dose, all individuals recovered 28 days later, on day 70. In the Coretox 12U / kg dose group, all individuals recovered on day 84 after administration. However, in the pBT147 dose group, recovery was 28 days later, on day 112, with all individuals recovering (Figure 7D). DAS ED 50 Through comparison, it was confirmed that pBT147 exhibits approximately 1.5 times stronger efficacy than Coretox (Figure 7E). In the CMAP test, it was confirmed that the pBT147 12U / kg administration group showed significantly lower CMAP values ​​compared to the Coretox 12U / kg administration group on all measurement days. CMAP recovery was observed on day 112 after administration in the Coretox 12U / kg administration group, and 14 days later, on day 126, in the pBT147 12U / kg administration group (Figure 7F). From these results, it was confirmed that recombinant botulinum toxin pBT147 has an extended potency and duration of action compared to Coretox.

[0047] Test Example 4: Efficacy of recombinant botulinum toxin with substituted domains in mice. Female 5-week-old CD1(ICR) mice were purchased from Orient Bio Co., Ltd. After a one-week acclimatization and quarantine period, 6-week-old mice were used in the experiment. Sterilized laboratory animal solid feed (R40-10 (SAFE (France))) was provided as ad libitum feed, and tap water sterilized at high temperature and pressure was also provided as ad libitum water. During the acclimatization, quarantine, and experimental periods, the mice were housed under specific pathogen-free conditions with a temperature of 23±3℃, relative humidity of 55±15%, lighting for 12 hours (8am to 8pm), ventilation rate of 15 times / hour, and illumination intensity of 150-300 lux. This study was conducted after review and approval by the Animal Experiment Ethics Committee of Meditox Co., Ltd.

[0048] As a control group, 100 units of Coretox (botulinum toxin type A, Lot No.: NSA20015 (Meditox)) were used. Recombinant botulinum toxin pBT142 was prepared at 2,803 U / mL, and recombinant botulinum toxin pBT147 was prepared at 2,537 U / mL and used in the experiment. The potency of each recombinant botulinum toxin was measured using a mouse potency test method and the potency value derived before the efficacy test was used. For the placebo group, botulinum toxin was removed from the recombinant botulinum toxin, and the remaining excipient components were used equally. In the efficacy test, the dosage of each test substance was administered identically based on units (U: a value indicating the biological activity of botulinum toxin, where 1 U is the median lethal dose of mice administered intraperitoneally).

[0049] Coretox was diluted using sterile physiological saline (35V3AF3, Daehan Pharmaceutical Co., Ltd.), and recombinant botulinum toxins pBT142 and pBT147 were diluted to concentrations of 6, 20, 60, and 200 U / mL, respectively, using a placebo. Day 0 was designated as the day the test substance was administered. Mice were anesthetized using an injectable anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), and then, according to the group composition in Table 3, each test substance was administered to the right calf muscle of the mice using a Hamilton syringe at a rate of 0.2 mL / kg.

[0050] [Table 3] The evaluation used the DAS (digit abduction score) method, which visually assesses the degree of muscle paralysis in mice, and the compound muscle action potential (CMAP) test method, which measures the action potential of muscles in response to external electrical stimulation. The DAS values ​​shown in Table 4 indicate the degree of muscle paralysis based on the toe morphology of the side to which the mouse was administered the drug, while the CMAP values ​​indicate the degree of direct muscle contraction inhibition shown by nerve blockade.

[0051] [Table 4] CMAP was measured in the right calf muscle region of each mouse using the Nicolet Viking Quest (Viasys Healthcare, Inc.) equipment. The measurement method was the same as in Study Example 3, except for the stimulation level and duration of 7-8 mA (0.1 ms). According to the test group composition in Table 3, DAS measurements were evaluated from day 1 to day 84 after administration at doses of 1, 2, 4, 12, and 40 U / kg. CMAP measurements were taken from day 1 to day 98 in individuals administered at a dose of 12 U / kg, and statistical analysis was performed in the same manner as in Study Example 3.

[0052] After administering Coretox and two recombinant botulinum toxins, pBT142 and pBT147, as single doses to the right calf muscle of mice at volumes of 1.2, 4, 12, and 40 U / kg, DAS evaluation was performed. The results showed a significant increase in DAS scores compared to Coretox at 14, 21, and 28 days after administration in the pBT147 4 U / kg dose group. In the Coretox 1.2 U / kg dose group, all individuals recovered by 14 days, while in the pBT142 and pBT147 dose groups at the same volume, all individuals recovered 14 days later, at 28 days. In the Coretox 4 U / kg dose group, all individuals recovered 28 days after administration, but in the pBT142 and pBT147 dose groups at the same volume, all individuals recovered 14 days later, at 42 days. In the Coretox 12U / kg dose group, all individuals recovered on day 49 after administration. However, in the pBT142 and pBT147 dose groups, recovery was 14 days later, on day 63. Furthermore, in the Coretox 40U / kg dose group, all individuals recovered on day 70 after administration. However, in the pBT142 and pBT147 dose groups, recovery was 14 and 7 days later, respectively, on day 84 and day 77 (Figure 8A). DAS ED 50 Through comparison, pBT142 showed approximately 1.2 times stronger efficacy than Coretox, and pBT147 showed approximately 1.4 times stronger efficacy than Coretox (Figure 8B).

[0053] In the CMAP test, the pBT142 administration group and the pBT147 12U / kg administration group showed significantly lower CMAP values ​​compared to the Coretox 12U / kg administration group. While CMAP recovery was observed on day 84 in the Coretox 12U / kg administration group, it was observed 14 days later, on day 98, in the pBT147 12U / kg administration group (Figure 8C). As described above, the results obtained in mice indicate that recombinant botulinum toxin pBT142 and recombinant botulinum toxin pBT147 have extended efficacy and duration of action compared to Coretox.

[0054] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as they are commonly used in the art to which this disclosure pertains. For the understanding of this disclosure, the following definitions apply, where a singular term includes a plural form, and vice versa.

[0055] As used herein, the terms "protein" and "polypeptide" are interchangeable to mean polymers of amino acid residues, their variants, and synthetic analogs. These terms apply to naturally occurring amino acid polymers, including non-naturally occurring amino acids synthesized from one or more amino acid residues, such as chemical analogs of the relevant naturally occurring amino acids. These terms also include post-translational modifications of polypeptides, such as glycosylation, phosphorylation, and acetylation.

[0056] The term "botulinum toxin (BoNT)" encompasses any polypeptide or fragment of botulinum toxin. In specific examples, "botulinum toxin" refers to full-length botulinum toxin or botulinum toxin-derived fragments. In specific examples, "botulinum toxin" can enter neurons and perform an overall cellular mechanism that inhibits neurotransmitter release.

[0057] The term "domain" refers to a given protein sequence and conserved portion, a tertiary structure that can evolve and function independently of the rest of the protein chain. To achieve independent stability, a domain can be subjected to a domain swap between one protein and another through genetic engineering, thereby creating a chimeric protein.

[0058] The term "percent identity" refers to the degree of amino acid sequence identity between polypeptides. For example, if the first amino acid sequence is identical to the second amino acid sequence, then the first and second amino acid sequences exhibit 100% identity. The percentage identity of two amino acid sequences can be determined using the algorithm in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87: 2264-68, 1990, or using a modified version of the algorithm in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90: 5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) in Altschul, et al. J. Mol. Biol. 215: 403-10, 1990. BLAST protein searches are performed using the XBLAST program (score=50, wordlength=3) to obtain homologous amino acid sequences for a protein molecule of interest. If there is a gap between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402, 1997. When using the BLAST program and the Gapped BLAST program, the default variables of each program (e.g., XBLAST and NBLAST) may be used.

[0059] The term "recombination" refers to the process in which elements that make up genes, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are rearranged in a way that differs from their original sequence during the disintegration and recombination processes. Through molecular biology experiments, DNA sections can be artificially rearranged. DNA that has been artificially rearranged in this way is called recombinant DNA.

[0060] The term "variant" encompasses any modification of a nucleic acid sequence or amino acid sequence, such as cleavage, addition, deletion, substitution, and any combination thereof of nucleic acids or amino acids. That is, "variants of recombinant botulinum toxin type A light chain domain" can encompass cleavage, addition, deletion, substitution, and any combination thereof of nucleic acids or amino acids constituting the recombinant botulinum neurotoxin type A light chain domain.

[0061] The term "substitution" also includes any amino acid other than the wild-type residue normally found at any given sequence position. Such substitutions are also substitutions by nonpolar (hydrophobic) amino acids, such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Such substitutions are also substitutions by polar (hydrophilic) amino acids, such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Such substitutions are also substitutions by electrically charged amino acids, such as negatively charged amino acids, such as aspartic acid and glutamic acid, and positively charged amino acids, such as lysine, arginine, and histidine.

[0062] The term "wild type" refers to the naturally occurring, typical phenotype of a species. Generally, this wild type is also the product of the normal allele at the locus, as opposed to the one produced by the non-standard mutant allele.

[0063] The term "individual" can also be male or female. The individual can be a fully developed individual (e.g., an adult) or an individual undergoing development (e.g., a child, infant, or fetus). The individual can also be a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow.

[0064] Most botulinum toxin preparations currently on the market contain botulinum toxin type A1 as the active ingredient, except for Myobloc, which uses botulinum toxin type B1. The duration of effect of botulinum toxin type A1 preparations is approximately 3 months on average, and it is known to be even shorter for botulinum toxin type B1 preparations. Due to the characteristics of botulinum toxin preparations, which must be administered by intramuscular injection, there is a need to develop products with a longer duration of action than currently available preparations in terms of patient convenience.

[0065] The inventors divided the light chain sequences of botulinum toxin types A1 and A4 into a total of four domains, as shown in Figure 1, based on the domain characteristics shown in Figure 2, in the first, second, third, and fourth domains.

[0066] The first domain of the botulinum toxin type A1 light chain (SEQ ID NO: 1) and the first domain of the botulinum toxin type A4 light chain (SEQ ID NO: 5) are regions containing an α-exosite, which is the site that primarily interacts with the substrate SNAP25. The first domain of the light chains of botulinum toxins type A1 and A4 is also the amino acid at position 1, or the amino acids at positions 90 to 150 (for example, the amino acids at positions 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150).

[0067] The second domain of the botulinum toxin type A1 light chain (SEQ ID NO: 2) and the second domain of the botulinum toxin type A4 light chain (SEQ ID NO: 6) are the parts primarily involved in the enzymatic activity of the light chain, containing 170 loops, and are the parts that bind to the cofactor zinc. The second domain of the light chain of botulinum toxin types A1 and A4 is also composed of amino acids at positions 91-151 (e.g., amino acids at positions 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146 and 151) or amino acids at positions 210-270 (e.g., amino acids at positions 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265 and 270).

[0068] The third domain of the botulinum toxin type A1 light chain (SEQ ID NO: 3) and the third domain of the botulinum toxin type A4 light chain (SEQ ID NO: 7) contain 250 loops and 370 loops, respectively, which stabilize substrate binding, and are involved in both the α-exosite and β-exosite, which are substrate binding sites. The third domain of the light chain of botulinum toxin types A1 and A4 is also amino acid 211-271 (e.g., amino acids 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, 261, 266 and 271) or amino acid 386-446 (e.g., amino acids 386, 391, 396, 401, 406, 411, 416, 421, 426, 431, 436, 441 and 446).

[0069] The fourth domain of the botulinum toxin type A1 light chain (SEQ ID NO: 4) and the fourth domain of the botulinum toxin type A4 light chain (SEQ ID NO: 8) are the parts involved in the structural flexibility of the botulinum toxin type A light chain. The fourth domain of the botulinum toxin type A1 and A4 light chains is also amino acids 362-425 (e.g., amino acids 362, 367, 372, 377, 382, ​​387, 392, 397, 402, 407, 412, 417, 422, and 425) or amino acids 410-448 (e.g., amino acids 410, 414, 417, 420, 425, 430, 435, 440, 445, and 448).

[0070] According to one aspect of this disclosure, a recombinant botulinum toxin type A light chain is provided in which, in the first, second, third, and fourth domains of the botulinum toxin type A light chain, which is not botulinum toxin type A4, the sequence of the third domain is substituted with the sequence of the third domain of the botulinum toxin type A4 light chain or a variant thereof.

[0071] In one specific example, a botulinum toxin type A light chain that is not botulinum toxin type A4 is also a botulinum toxin type A1 light chain (SEQ ID NO: 9), a botulinum toxin type A2 light chain (SEQ ID NO: 10), a botulinum toxin type A3 light chain (SEQ ID NO: 11), a botulinum toxin type A5 light chain (SEQ ID NO: 13), a botulinum toxin type A6 light chain (SEQ ID NO: 14), a botulinum toxin type A7 light chain (SEQ ID NO: 15), or a botulinum toxin type A8 light chain (SEQ ID NO: 16). For example, a botulinum toxin type A light chain is also a botulinum toxin type A1 light chain.

[0072] In one specific example, the third domain of the botulinum toxin type A4 light chain is also the sequence of SEQ ID NO: 7. In another specific example, a variant of the third domain of the botulinum toxin type A4 light chain involves both the α-exosite and β-exosite, which are the same substrate binding sites as the third domain of the botulinum toxin type A4 light chain, and contains an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the sequence of SEQ ID NO: 3.

[0073] Another aspect of the present disclosure provides a recombinant botulinum toxin type A light chain in which the sequence of the fourth domain is further substituted with the sequence of the fourth domain of a botulinum toxin type A4 light chain or a variant thereof, in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain.

[0074] In one specific example, the botulinum toxin type A4 light chain domain 4 is also the sequence of SEQ ID NO: 8. In another specific example, a variant of the botulinum toxin type A4 light chain domain 4 is involved in the structural flexibility of the botulinum toxin type A light chain in the same way as the botulinum toxin type A4 light chain domain 4 and contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 4.

[0075] Further aspects of this disclosure provide a recombinant botulinum toxin comprising a recombinant botulinum toxin type A light chain and a botulinum toxin heavy chain.

[0076] In one specific example, the botulinum toxin heavy chain is also a botulinum toxin type A heavy chain, a botulinum toxin type B heavy chain, a botulinum toxin type C heavy chain, a botulinum toxin type D heavy chain, a botulinum toxin type E heavy chain, a botulinum toxin type F heavy chain, or a botulinum toxin type G heavy chain.

[0077] In one specific example, the botulinum toxin heavy chain is also a botulinum toxin type A heavy chain. For example, the botulinum toxin heavy chain is also a botulinum toxin type A1 heavy chain, a botulinum toxin type A2 heavy chain, a botulinum toxin type A3 heavy chain, a botulinum toxin type A4 heavy chain, a botulinum toxin type A5 heavy chain, a botulinum toxin type A6 heavy chain, a botulinum toxin type A7 heavy chain, or a botulinum toxin type A8 heavy chain. For example, the botulinum toxin heavy chain is also a botulinum toxin type A1 heavy chain (SEQ ID NO: 17).

[0078] In one specific example, the recombinant botulinum toxin is also a recombinant botulinum toxin in which a recombinant botulinum toxin type A light chain and a botulinum toxin heavy chain are linked by disulfide bonds. For example, recombinant botulinum toxin is a relatively inactive single polypeptide chain of about 150 kDa weight with a high degree of amino acid sequence identity among botulinum toxin types, and is produced by Clostridium bacteria. The single polypeptide is then broken down into a heavy chain of about 100 kDa and a light chain of 50 kDa, and the heavy and light chains can form disulfide bonds.

[0079] Further embodiments of the present disclosure provide compositions comprising recombinant botulinum toxin and pharmaceutically acceptable excipients or additives.

[0080] In one specific example, pharmaceutically acceptable excipients or additives may also be stabilizers, ionic compounds, surfactants, buffers, lyophilization protectants, or combinations thereof, such as amino acids (e.g., methionine), salts (e.g., NaCl), buffers, nonionic surfactants (e.g., polysorbates (e.g., polysorbate 20)), sugars (e.g., disaccharides such as sucrose), sugar alcohols (e.g., sorbitol), or combinations thereof.

[0081] In one specific example, the composition may also be free of albumin or animal-derived components. For example, compositions free of albumin or animal-derived components include those disclosed in WO2009-008595A1 or WO2012-134240A2, the contents of which are incorporated herein by reference as a whole.

[0082] In one specific example, the composition may be formulated into any form, such as a solid or liquid formulation, for example, a lyophilized powder, liquid, or pre-filled syringe formulation.

[0083] According to yet another aspect of this disclosure, a composition comprising the recombinant botulinum toxin and a pharmaceutically acceptable excipient or additive is provided for use in administering to an individual to improve or treat a disease.

[0084] In one specific example, a composition comprising recombinant botulinum toxin, a pharmaceutically acceptable excipient, or additive is administered to an individual for the purpose of improving wrinkles, angular jawlines, jawline prominence, wounds, skin softening, scars, acne, pores, elasticity, or keloids.

[0085] In one specific example, a composition comprising recombinant botulinum toxin, a pharmaceutically acceptable excipient, or an additive is administered to an individual for the treatment of facial spasms, blepharospasm, torticollis, blepharospasm, cervical dystonia, midpharyngeal dystonia, spasmodic dysphonia, migraines, anal pruritus, or hyperhidrosis.

[0086] In one specific example, the composition may be administered transdermally, subcutaneously, or intramuscularly. In another specific example, the composition may be administered topically to a muscle or a group of muscles. In yet another specific example, the reduction of forehead wrinkles and skin wrinkles may also be achieved by administering the composition transdermally or subcutaneously to the wrinkles.

[0087] Further embodiments of this disclosure provide a method for improving or treating a disease by administering to an individual a composition further comprising the recombinant botulinum toxin and a pharmaceutically acceptable excipient or additive. In one specific example, the method also includes the step of administering the composition to an individual for improving wrinkles, angular jaw, jawline, wounds, skin softening, scars, acne, pores, elasticity, or keloids.

[0088] In one specific example, the method also includes the step of administering the composition to an individual, and is for treating facial spasms, blepharospasm, torticollis, blepharospasm, cervical muscle tone disorders, mid-pharyngeal muscle tone disorders, spasmodic dysphonia, migraines, anal pruritus, or hyperhidrosis.

[0089] Further aspects of this disclosure provide a method for producing recombinant botulinum toxin type A light chain having increased potency or half-life compared to wild-type botulinum toxin type A1 light chain, comprising the step of substituting the sequence of the third domain in the first, second, third, and fourth domains of a botulinum toxin type A light chain that is not botulinum toxin type A4 with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

[0090] In one specific example, the method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to a wild-type botulinum toxin type A1 light chain also includes the step of substituting the sequence of the third domain of the botulinum toxin type A1 light chain with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

[0091] For example, this method also includes the steps of: substituting four domains of the botulinum toxin type A1 light chain and the botulinum toxin type A4 light chain genes with each other; cloning genes containing the wild-type botulinum toxin type A light chain and the recombinant botulinum toxin type A light chain with substituted domains, introducing them into Escherichia coli (E. coli), and inducing the expression of the recombinant botulinum toxin type A light chain with substituted domains; and producing a recombinant botulinum toxin type A light chain (pBT103 (SEQ ID NO: 19)) in which the sequence of the third domain of the botulinum toxin type A1 light chain is substituted with the sequence of the third domain of botulinum toxin type A4 or a variant thereof, thereby producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to wild-type botulinum toxin.

[0092] A further aspect of the present disclosure provides a method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to wild-type botulinum toxin type A1, comprising the step of further substituting the sequence of the fourth domain in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof.

[0093] In one specific example, the method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to wild-type botulinum toxin type A1 also includes the step of further substituting the sequence of the fourth domain of the recombinant botulinum toxin type A light chain with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof.

[0094] For example, this method also includes the steps of: substituting four domains of the genes for botulinum toxin type A1 light chain and botulinum toxin type A4 light chain with each other; cloning genes containing the wild-type botulinum toxin type A light chain and the recombinant botulinum toxin type A light chain with substituted domains, introducing them into Escherichia coli (E. coli), and inducing the expression of the recombinant botulinum toxin type A light chain with substituted domains; and producing a recombinant botulinum toxin type A light chain (pBT104 (SEQ ID NO: 20)) in which the sequences of the third domain and the fourth domain of the botulinum toxin type A1 light chain are substituted with the sequences of the third and fourth domains of botulinum toxin type A4 or their variants, thereby producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to wild-type botulinum toxin type A1.

[0095] Further aspects of the present disclosure provide cells comprising nucleic acids or vectors comprising a polynucleoside encoding the recombinant botulinum toxin type A light chain and a polynucleoside encoding the botulinum toxin heavy chain. The cells also express recombinant botulinum toxin comprising the recombinant botulinum toxin type A light chain and the botulinum toxin heavy chain.

[0096] A further aspect of the present disclosure provides a method for producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin, comprising the step of culturing cells comprising a nucleic acid or vector comprising a polynucleoside encoding the recombinant botulinum toxin type A light chain and a polynucleoside encoding the botulinum toxin heavy chain. The method also further comprises the step of separating the recombinant botulinum toxin from the culture.

[0097] In one specific example, the botulinum toxin heavy chain is also botulinum toxin type A1 heavy chain, botulinum toxin type A2 heavy chain, botulinum toxin type A3 heavy chain, botulinum toxin type A4 heavy chain, botulinum toxin type A5 heavy chain, botulinum toxin type A6 heavy chain, botulinum toxin type A7 heavy chain, or botulinum toxin type A8 heavy chain. In one specific example, the botulinum toxin heavy chain is also botulinum toxin type A1 heavy chain (SEQ ID NO: 17).

[0098] For example, this method also includes the steps of: cloning full-length recombinant botulinum toxin genes (pBT142, pBT147) containing domain-substituted recombinant botulinum toxin type A light chains (pBT103, pBT104) and botulinum toxin type A1 heavy chains; introducing the cloned toxin genes into an inactivated detoxified bacterial strain, culturing the inactivated detoxified bacterial strain to which the toxin genes have been introduced, and producing domain-substituted botulinum toxin [Bradshaw et al. 2010, Pellett et al. 2016]; and purifying and isolating the recombinant toxin in 150 kDa form that does not contain complex components from the culture, thereby producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin.

Claims

1. A recombinant botulinum toxin type A light chain, which is not botulinum toxin type A4, in which the sequence of the third domain is replaced in the first, second, third, and fourth domains of the botulinum toxin type A light chain with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

2. The recombinant botulinum toxin type A light chain according to claim 1, wherein in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain, the sequence of the fourth domain is further substituted with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof.

3. The recombinant botulinum toxin type A light chain according to claim 1 or 2, wherein the botulinum toxin type A light chain is botulinum toxin type A1, A2, A3, A5, A6, A7, or A8.

4. The recombinant botulinum toxin type A light chain according to claim 3, wherein the botulinum toxin type A light chain is botulinum toxin type A1.

5. Recombinant botulinum toxin comprising a recombinant botulinum toxin type A light chain and a botulinum toxin heavy chain according to any one of claims 1 to 4.

6. The recombinant botulinum toxin according to claim 5, wherein the botulinum toxin type A heavy chain is botulinum toxin type A, B, C, D, E, F, or G.

7. The recombinant botulinum toxin according to claim 6, wherein the botulinum toxin type A heavy chain is botulinum toxin type A1, A2, A3, A4, A5, A6, A7, or A8.

8. The recombinant botulinum toxin according to claim 7, wherein the botulinum toxin type A heavy chain is botulinum toxin type A1.

9. A composition comprising recombinant botulinum toxin type A according to any one of claims 5 to 8, and a pharmaceutically acceptable excipient or additive.

10. The composition according to claim 9, wherein the pharmaceutically acceptable excipients or additives are stabilizers, ionic compounds, surfactants, buffers, lyophilization protectants, or a combination thereof.

11. The composition according to claim 10, wherein the pharmaceutically acceptable excipients or additives are amino acids, salts, buffers, nonionic surfactants, sugars, sugar alcohols, or combinations thereof.

12. The composition according to any one of claims 9 to 11, which does not contain albumin or animal-derived components.

13. The composition according to any one of claims 9 to 12, in the form of a freeze-dried powder, a liquid, or a pre-filled syringe formulation.

14. A composition according to any one of claims 9 to 13, for the purpose of improving wrinkles, angular jaw, prominent jawline, scars, skin softening, scars, acne, pores, elasticity, or keloid symptoms.

15. A composition according to any one of claims 9 to 13, for the treatment of facial spasms, blepharospasm, torticollis, blepharospasm, cervical muscle tone disorders, midpharyngeal muscle tone disorders, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis.

16. A composition according to any one of claims 9 to 15, for transdermal, subcutaneous, or intramuscular administration.

17. Uses of the composition according to any one of claims 9 to 13, for the purpose of improving wrinkles, angular jaw, prominent jawline, scars, skin softening, scars, acne, pores, elasticity, or keloid symptoms.

18. Uses of the composition according to any one of claims 9 to 13 for the treatment of facial spasms, blepharospasm, torticollis, blepharospasm, cervical muscle tone disorders, midpharyngeal muscle tone disorders, spasmodic dysphonia, migraines, anal pruritus, or hyperhidrosis.

19. A method for improving wrinkles, angular jaw, jawline, scars, skin softening, scars, acne, pores, elasticity, or keloids, comprising the step of administering a composition according to any one of claims 9 to 13 to an individual.

20. A method for treating facial spasms, blepharospasm, torticollis, blepharospasm, cervical muscle tone disorder, midpharyngeal muscle tone disorder, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis, comprising the step of administering a composition according to any one of claims 9 to 13 to an individual.

21. A method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to a wild-type botulinum toxin type A light chain, comprising the step of substituting the sequence of the third domain in the first, second, third, and fourth domains of a botulinum toxin type A light chain that is not botulinum toxin type A4 with the sequence of the third domain of botulinum toxin type A4 or a variant thereof.

22. A method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to a wild-type botulinum toxin type A light chain, comprising the step of further substituting the sequence of the fourth domain in the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof, according to claim 21.

23. A method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to a wild-type botulinum toxin type A light chain according to claim 21 or 22, wherein the botulinum toxin type A light chain is botulinum toxin type A1, A2, A3, A5, A6, A7, or A8.

24. A method for producing a recombinant botulinum toxin type A light chain having increased potency or half-life compared to the wild-type botulinum toxin type A light chain according to claim 23, wherein the botulinum toxin type A light chain is botulinum toxin type A1.

25. A method for producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin, comprising the step of culturing cells containing a nucleic acid molecule or vector containing a polynucleoside encoding a recombinant botulinum toxin type A light chain and a polynucleoside encoding a botulinum toxin heavy chain, as described in any one of claims 1 to 4.

26. A method for producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin according to claim 25, wherein the botulinum toxin heavy chain is botulinum toxin type A, B, C, D, E, F, or G.

27. A method for producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin according to claim 26, wherein the botulinum toxin heavy chain is botulinum toxin type A1, A2, A3, A4, A5, A6, A7, or A8.

28. A method for producing recombinant botulinum toxin having increased potency or half-life compared to wild-type botulinum toxin according to claim 27, wherein the botulinum toxin heavy chain is botulinum toxin type A1.

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