Recombinant polypeptide, composition comprising same, uses thereof, and methods therefor

Abstract

A recombinant polypeptide of the present invention comprises at least one of a recombinant receptor-binding domain of botulinum toxin type A1, a recombinant light chain domain of botulinum toxin type A, and a recombinant translocation domain of botulinum toxin type A. Also, the present invention provides a recombinant botulinum toxin type A comprising at least one of the above-described recombinant polypeptides and having increased potency, safety, or half-life compared to wild-type botulinum toxin.

Description

Recombinant polypeptide, composition containing the same, and uses and methods thereof

[0001] The present invention relates to recombinant polypeptides, and more specifically, to a recombinant polypeptide comprising a receptor binding domain of botulinum toxin type A1 containing one or more amino acid variations, or a recombinant botulinum toxin type A light chain and a recombinant receptor binding domain of botulinum toxin type A1.

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

[0003] Botulinum toxin is synthesized as a 150 kDa double-chain protein consisting of a 100 kDa heavy chain and a 50 kDa light chain linked by disulfide bonds. The heavy chain is further divided into an N-terminal domain (Hn) that helps translocate the light chain into the cell's cytosol, and a C-terminal domain (Hc) that recognizes and binds to cell surface receptors on neurons. Upon entering the cell, the light chain specifically proteolytically cleavages a portion of the soluble NSF-attachment protein receptor (SNARE), thereby inactivating neurotransmitter release. 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. Among these, botulinum toxin type A1 has undergone extensive molecular-level, preclinical, and clinical studies and is currently widely used in the pharmaceutical industry. In contrast, due to difficulties in isolating purified toxins from other botulinum toxin type A subtypes, there have been few studies or reports regarding their pharmacological characteristics. Currently, the biochemical, cellular, and in vivo properties of only some botulinum toxin type A subtypes have been elucidated. According to reported studies, various in vitro and in vivo research on botulinum toxin type A subtypes has revealed characteristics distinct from botulinum toxin type A1 in terms of mechanisms, such as efficacy, intracellular migration, and persistence. Recently, in vitro characteristics of botulinum toxin types A7 and A8 have been reported.

[0004] One objective of the present invention is to provide a recombinant polypeptide comprising one or more of a recombinant receptor binding domain and a recombinant translocation domain of botulinum toxin type A1, and a recombinant botulinum toxin comprising the same.

[0005] One object of the present invention is to provide a recombinant polypeptide comprising a recombinant receptor binding domain and a recombinant translocation domain of botulinum toxin type A1, and a composition related to a recombinant botulinum toxin comprising the same.

[0006] One objective of the present invention is to provide a pharmaceutical use of the above composition.

[0007] One object of the present invention is to provide a method for improving, preventing, or treating a disease or condition, comprising the step of administering the composition to an individual.

[0008] A recombinant polypeptide according to one embodiment of the present invention may include a receptor binding domain of botulinum toxin type A1. The receptor binding domain may include amino acid variations at one or more positions that are conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but have different amino acids at corresponding positions of botulinum toxin type A1.

[0009] The position of the above amino acid variation may include at least one selected from the group consisting of N880, W1068, and N1090.

[0010] The receptor binding domain may further include an amino acid variation at at least one position selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, Y1117, T1232, and L1278. In this specification, for example, the statement that it may include an amino acid variation at the position of N954 means that asparagine, the 954th amino acid, is substituted with, deleted from, or modified by another amino acid.

[0011] The receptor binding domain may further include at least one amino acid variant selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, Y1117F, T1232R, and L1278F.

[0012] The receptor binding domain may further include an amino acid variation at at least one position selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1232, and L1278.

[0013] The receptor binding domain may comprise at least one amino acid variant selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, and L1278F.

[0014] A recombinant polypeptide of one embodiment further comprises a translocation domain, said translocation domain may comprise a polypeptide selected from the group consisting of a translocation domain of botulinum toxin type A1, a translocation domain of botulinum toxin type A2, a translocation domain of botulinum toxin type A6, and variants thereof. For example, the translocation domain may comprise or be composed of a translocation domain of botulinum toxin type A1 or a variant thereof.

[0015] A recombinant polypeptide of one embodiment comprises a translocation domain of botulinum toxin type A1, wherein the translocation domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but may include an amino acid variation at one or more positions having different amino acids at corresponding positions of botulinum toxin type A1. Alternatively, a recombinant polypeptide of one embodiment comprises a translocation domain of botulinum toxin type A1 and may include an amino acid variation at position 589.

[0016] The recombinant polypeptide of one embodiment comprises a translocation domain of botulinum toxin type A1, and the position of the amino acid variant of the translocation domain may comprise at least one selected from the group consisting of D589, L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818.

[0017] The above translocation domain may include at least one amino acid variant selected from the group consisting of D589K, E734K, and E809K.

[0018] A recombinant polypeptide of one embodiment further comprises a light chain domain, and the light chain domain may comprise a polypeptide selected from the group consisting of botulinum toxin type A1, botulinum toxin type A4, and variants thereof.

[0019] The above light chain domain may include a first domain and a second domain of a botulinum toxin type A1 light chain; or variants thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or variants thereof.

[0020] The above variant light chain comprises a first domain and a second domain of a botulinum toxin type A1 light chain; and a third domain and a fourth domain of a botulinum toxin type A4 light chain; wherein leucine at position 260 of the light chain domain is substituted with phenylalanine and isoleucine at position 264 is substituted with arginine.

[0021] In one embodiment, the receptor binding domain may have a half-maximum inhibitory concentration value according to botulinum toxin heavy chain competition analysis that is lower than that of the wild-type receptor binding domain.

[0022] A recombinant polypeptide according to one embodiment of the present invention may further comprise a light chain domain and a translocation domain and may have increased efficacy, safety, or half-life compared to wild-type botulinum toxin type A. The recombinant polypeptide may comprise at least one selected from the recombinant botulinum toxins of SEQ ID NOs 8 to 32, SEQ ID NO 43, and SEQ ID NO 44.

[0023] According to one embodiment of the present invention, a recombinant polypeptide comprises a receptor binding domain of botulinum toxin type A1, and the receptor binding domain may comprise amino acid variations at two or more positions selected from N954, Y1117, T1232, R1273, and L1278. The receptor binding domain may comprise two or more amino acid variations selected from N954S, Y1117F, T1232R, R1273K, and L1278F. The receptor binding domain may comprise amino acid variations at two or more positions selected from N954, T1232, and L1278. The receptor binding domain may comprise amino acid variations at two or more positions selected from N954S, T1232R, and L1278F. The amino acid variations of the receptor binding domain are N954S and T1232R; N954S and L1278F; It may include T1232R and L1278F; T1232R and R1273K; R1273K and L1278F; N954S, T1232R and L1278F; Y1117F, R1273K and L1278F; or T1232R, R1273K and L1278F.

[0024] According to one embodiment of the present invention, the recombinant polypeptide comprises a receptor binding domain of botulinum toxin type A1, and the half-maximum inhibitory concentration value according to the botulinum toxin heavy chain competitive analysis of the variant of the receptor binding domain may be lower than that of the wild-type receptor binding domain. The variant of the receptor binding domain may comprise an amino acid variation at at least one position selected from the group consisting of N880, N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, W1068, N1090, Y1117, S1142, T1232, and L1278. The variant of the receptor binding domain may include at least one amino acid variant selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, Y1117F, S1142Y, S1142R, S1142I, S1142F, S1142H, T1232R, and L1278F. For example, the receptor binding domain may include an amino acid variant at the position of S1142. The amino acid variant may include S1142Y, S1142R, S1142I, S1142F, or S1142H.

[0025] According to one embodiment of the present invention, the recombinant botulinum toxin comprises a variant in which one or more amino acids among the amino acids of the receptor binding domain sequence of botulinum toxin type A1 are mutated, and said amino acid variant comprises one or more selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, Y1117F, S1142Y, S1142R, S1142I, S1142F, S1142H, T1232R and L1278F, and when administered to a subject requiring treatment, at least one of efficacy and safety is superior compared to wild-type botulinum toxin type A1, and the remainder may be at least equivalent to the wild-type toxin. The above-mentioned recombinant botulinum toxin may include a light chain domain selected from the group consisting of the aforementioned botulinum toxin type A1 light chain, botulinum toxin type A4 light chain, and variants thereof. Additionally, the above-mentioned recombinant botulinum toxin may further include the aforementioned translocation domain. Compared to wild-type botulinum toxin type A1, the above-mentioned recombinant botulinum toxin has a safety margin when administered to an individual; and DAS AUC　 or CMAP AAC ; at least one of them shows a superior value, and the remainder may be at least equivalent to the wild-type toxin. For example, compared to wild-type botulinum toxin type A1, the recombinant botulinum toxin has a safety margin when administered to an individual; and DAS AUC　 or CMAP AAC ; One or more of them show a higher value, and the remainder may be at least equivalent to the wild-type toxin.

[0026] According to one embodiment of the present invention, the recombinant botulinum toxin comprises a variant of the botulinum toxin type A light chain and may comprise a receptor binding domain comprising one or more amino acid variants selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, S1142Y, S1142R, S1142I, S1142F, S1142H, Y1117F, T1232R, and L1278F. The receptor binding domain may comprise the recombinant polypeptides of SEQ ID NOs 1 to 7, 33 to 42, and 45 to 49.

[0027] The above-mentioned recombinant botulinum toxin may include a light chain domain selected from the group consisting of the aforementioned botulinum toxin type A1 light chain, botulinum toxin type A2 light chain, and variants thereof. When administered to a subject requiring treatment, the above-mentioned recombinant botulinum toxin may be superior to wild-type botulinum toxin type A1 in at least one of safety and duration of action, and the remainder may be at least equivalent to the wild-type toxin. Compared to wild-type botulinum toxin type A1, the above-mentioned recombinant botulinum toxin has a safety margin when administered to an individual; and DAS AUC　 or CMAP AAC ; It is possible that one or more of them show a higher value.

[0028] According to one embodiment of the present invention, the recombinant polypeptide may include a botulinum toxin type A1 translocation domain. The translocation domain may include one or more amino acid variations at a position that is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but has different amino acids at a corresponding position of botulinum toxin type A1.

[0029] The position of the amino acid variant in the above-mentioned translocation domain may include at least one selected from the group consisting of L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818. The above-mentioned translocation domain may include at least one amino acid variant selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V. A recombinant botulinum toxin containing the above-mentioned translocation domain may exhibit superior specific potency compared to wild-type botulinum toxin.

[0030] According to the present invention, a recombinant polypeptide comprising botulinum toxin type A1 or a subdomain thereof is provided.

[0031] Figure 1 is a graph of the intermediate temperature (Tm) of heat denaturation for botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 10, 11, 12, 13, 14, 16 and 20.

[0032] Figure 2 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of SEQ ID NO. 10 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test (*p<0.05, **p<0.01).

[0033] Figure 3 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of SEQ ID NO. 11 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test ( a p = 0.065, bp = 0.093).

[0034] Figure 4 shows a graph (A) representing the temporal change in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ NOs. 10 and 11 at a dose of 12 U / kg into the right gastrocnemius muscle in rats, and a graph (B) representing CMAP values ​​in the left gastrocnemius muscle on day 7 after administration. DAS values ​​on each measurement day were analyzed using the Mann-Whitney test, and CMAP values ​​were analyzed using a two-tailed t-test (*p<0.05, a p = 0.065).

[0035] Figure 5 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 22 and 23 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test (*p<0.05, a p =0.065, b p = 0.082).

[0036] Figure 6 is a graph showing ipsilateral and contralateral CMAP levels on day 7 after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 22 and 23 at a dose of 12 U / kg into the right gastrocnemius muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxins were analyzed according to the administration site using a two-tailed t-test (*p<0.05, **p<0.01).

[0037] Figure 7 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 14 and 15 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test (*p<0.05, **p<0.01, a p =0.065, b p = 0.093).

[0038] Figure 8 is a graph showing ipsilateral and contralateral CMAP levels on day 7 after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 14 and 15 at a dose of 12 U / kg into the right gastrocnemius muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxins were analyzed according to the administration site using a two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001).

[0039] Figure 9 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 12 and 13 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test ( a p = 0.065).

[0040] Figure 10 is a graph showing ipsilateral and contralateral CMAP levels at 1 week (day 7) after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of SEQ ID NOs. 12 and 13 to the right gastrocnemius muscle in mice at a dose of 12 U / kg. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxins were analyzed according to the administration site using a two-tailed t-test (*p<0.05).

[0041] Figure 11 is a graph showing the temporal change in DAS values ​​after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of SEQ ID NO. 16 to the right gastrocnemius muscle in mice at doses of 1.2, 4, and 12 U / kg. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test (*p<0.05, a p =0.065, b p = 0.093).

[0042] Figure 12 is a graph showing the ipsilateral and contralateral CMAP levels at 1 week (day 6) after administering botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of SEQ ID NO. 16 at a dose of 12 U / kg into the right gastrocnemius muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxin were analyzed according to the administration site using a two-tailed t-test (**p<0.01).

[0043] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the field to which this technology belongs. For the understanding of the invention, the following definitions shall apply, wherein the singular expression includes the plural expression unless the context clearly indicates otherwise, and vice versa.

[0044] The term "and / or" includes all combinations that the associated components can define.

[0045] In interpreting the components, they are interpreted to include a margin of error even without separate explicit indication. In this specification, the term “about” is used to include a customary margin of error in the art. For example, the term “about” may mean that there is a margin of error of up to 5%, 10%, 15%, or 20% in the said number or numerical range.

[0046] As used herein, the terms 'protein' and 'polypeptide' are used interchangeably to refer to polymers of amino acid residues and their variants and synthetic analogs. These terms apply to polymers of naturally occurring amino acids, including, for example, chemical analogs of related naturally occurring amino acids, synthetically derived from one or more amino acid residues. These terms also include post-translational modifications of polypeptides, for example, glycosylation, phosphorylation, and acetylation.

[0047] The term 'Botulinum toxin (BoNT)' encompasses any polypeptide or fragment of botulinum toxin. In one embodiment, botulinum toxin refers to full-length botulinum toxin or botulinum toxin-derived fragments. In a specific embodiment, botulinum toxin can enter a neuron and execute an entire cellular mechanism that inhibits neurotransmitter release.

[0048] Unless specifically limited in this specification, 'botulinum toxin' includes all serotypes and variants or fusion proteins thereof.

[0049] For example, botulinum toxins may be selected from the group consisting of type A (BoNT / A), B (BoNT / B), C (BoNT / C), D (BoNT / D), E (BoNT / E), F (BoNT / F), G (BoNT / G), H (BoNT / H), X (BoNT / X), Enterococcus species botulinum toxin J (eBoNT / J), and mosaic botulinum toxins and / or variants thereof. Examples of mosaic toxins include BoNT / DC, BoNT / CD, and BoNT / FA. For example, type A may be used.

[0050] In one embodiment, the botulinum toxin is derived from various BoNT / A subtypes, e.g., A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT / B subtypes, e.g., B1, B2, B3, B4, B5, B6, B7, B8, Bnp, and Bbv; BoNT / C subtypes, e.g., C and CD; BoNT / D subtypes, e.g., D and DC; BoNT / E subtypes, e.g., E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT / F subtypes, e.g., F1, F2, F3, F4, F5, F6, F7; and BoNT / G subtypes, e.g., subtype G. The BoNT subtypes include chimeric BoNTs, e.g., BoNT / DC, BoNT / CD, BoNT / FA, etc.

[0051] In this specification, unless specifically limited, each domain constituting the recombinant botulinum toxin of one embodiment or the recombinant botulinum toxin (e.g., light chain, heavy chain, subdomain of the light chain, translocation domain of the heavy chain, receptor binding domain of the heavy chain, etc.) may be a recombinant polypeptide derived from any serotype.

[0052] In one embodiment, the term “variant” of botulinum toxin (including both wild-type toxin and recombinant toxin) may mean a botulinum toxin having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more homology with the sequence of a reference botulinum toxin. For example, the term “variant” of botulinum toxin (including both wild-type toxin and recombinant toxin) may refer to a botulinum toxin having 1 to 10, 1 to 7, 1 to 5, or 1 to 3 or 1 amino acid variation in the sequence of a reference botulinum toxin, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid variations. In one embodiment, the amino acid variation may include both conservative and non-conservative variations, and may include a conservative variation in which the amino acid is modified but the properties of the modified amino acid (hydrophilicity, hydrophobicity, etc.) are maintained.

[0053] For example, the recombinant polypeptide of the present invention may include a variant of botulinum toxin type A1 as a receptor binding domain, but if the recombinant polypeptide of one embodiment includes a light chain (and each domain thereof) and / or a translocation domain, these may include botulinum toxin type A1, serotypes other than type A1, and variants thereof.

[0054] The recombinant botulinum toxin of one embodiment may be a non-complexed botulinum toxin that does not contain complex proteins. For example, the non-complexed botulinum toxin may not contain NTNH or hemagglutinin, in which case the molecular weight of the non-complexed botulinum toxin may be about 150 kDa. In this specification, the non-complexed botulinum toxin may be referred to as 7S botulinum toxin or botulinum toxin having a molecular weight of 150 kDa.

[0055] The recombinant botulinum toxin of one embodiment may be a complex botulinum toxin composition containing complex proteins. For example, the complex botulinum toxin may contain NTNH or hemagglutinin, in which case the molecular weight of the complex botulinum toxin may be about 900 kDa or 500 kDa.

[0056] The recombinant botulinum toxin composition of one embodiment may be a composition that does not contain animal proteins. For example, the recombinant botulinum toxin composition of one embodiment may not contain a protein stabilizer derived from animals. In one embodiment, the recombinant botulinum toxin composition may not contain albumin, for example, human serum albumin or recombinant human albumin.

[0057] As used herein, the terms 'unit', 'unit(s)', or 'U' refer to an LD that is defined as the amount of botulinum toxin that kills 50% of mice injected with botulinum toxin. 50 It refers to capacity and is used interchangeably within a single product.

[0058] "Pharmaceutical composition" means a preparation containing an active ingredient. The term "preparation" means that, in addition to the active ingredient (e.g., botulinum toxin), at least one additional ingredient, e.g., albumin (e.g., human serum albumin or recombinant human albumin) and / or sodium chloride, is present in the pharmaceutical composition. The pharmaceutical composition is a preparation suitable for administration to subjects such as human patients. The pharmaceutical composition may be in a lyophilized form, e.g., a solution formed after reconstitution of a lyophilized pharmaceutical composition using saline or water, or a solution form that does not require reconstitution. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may not contain animal-derived proteins and / or albumin.

[0059] The term "active ingredient" refers to a substance of a formulation or composition that is biologically or physiologically effective. In particular, in the context of pharmaceutical formulations or compositions, the term "active ingredient" refers to a component substance that exhibits a targeted pharmaceutical effect. For example, the active ingredient of the present invention may be a non-complexed botulinum toxin without complex proteins.

[0060] "Administration" or "the act of administering" refers to the step of providing a pharmaceutical composition or an active ingredient to a subject. The pharmaceutical composition may be administered through various appropriate routes.

[0061] In one embodiment, administration of the pharmaceutical composition may be achieved by transdermal, subcutaneous, or intramuscular administration. In a specific embodiment, the composition may be achieved by administering it topically to a muscle or group of muscles. For example, the reduction of forehead wrinkles or skin wrinkles may be achieved by administering the composition transdermally or subcutaneously to the corresponding wrinkles. For example, the composition of one embodiment may be administered by subcutaneous or intramuscular injection. The composition according to one embodiment may be administered by subcutaneous or intramuscular injection via a pre-filled syringe. The composition of one embodiment may be administered transdermally using a transdermal patch or micro-needles.

[0062] "Pharmaceutical composition" means a preparation containing an active ingredient. In addition to the active ingredient (e.g., botulinum toxin), the "pharmaceutical composition" may include at least one additional ingredient, for example, albumin (human serum albumin or recombinant human albumin) and / or sodium chloride. The pharmaceutical composition is a preparation suitable for administration to subjects such as mammals, including humans. Subjects to whom the composition of the present invention is administered may include humans or animals, for example, humans, pigs, dogs, cats, cattle, horses, rats, etc., without limitation.

[0063] The pharmaceutical composition may be in liquid or lyophilized form. If the pharmaceutical composition is a lyophilized preparation, it may be used by redissolving the lyophilized pharmaceutical composition, for example, using saline solution or water. It may be in solution form. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may not contain animal-derived proteins and / or albumin.

[0064] "To treat," "treating," or "treatment" means the alleviation or reduction (including partial reduction, substantial reduction, near-complete reduction, and complete reduction), resolution, or prevention (temporary or permanent) of a disease, disorder, or abnormality, thereby achieving a desired therapeutic outcome, for example, by healing injured or damaged tissue, or by altering, changing, reinforcing, improving, refining, and / or beautifying an existing or perceived disease, disorder, or abnormality. In this specification, "treatment" is a concept that includes prevention. "Prevention" means the delay of the onset of a disease, disorder, or condition. Prevention may be considered complete if the onset of a disease, disorder, or condition is delayed for a scheduled period.

[0065] In one embodiment, 'treatment' means the treatment of a disease, disorder, or medical condition in a patient such as a mammal (particularly, human), comprising one or more of the following:

[0066] (a) prevention of the occurrence of the above disease, disorder, or medical condition, i.e., prevention of the recurrence of the above disease or medical condition, or prophylactic treatment of a patient pre-disposed to the above disease or medical condition;

[0067] (b) improvement (ameliorating) of the said disease, disorder, or medical condition, including antagonizing the effect of other therapeutic agents, i.e., removal or regression of the said disease, disorder, or medical condition in the patient;

[0068] (c) suppressing the said disease, disorder, or medical condition, i.e., slowing or preventing the progression of the said disease, disorder, or medical condition in the patient; or

[0069] (d) Relief of symptoms of the above disease, disorder, or medical condition in the patient.

[0070] As used in this specification, the terms 'unit', 'unit(s)', or 'U' refer to an LD50 dose defined as the amount of botulinum toxin that kills 50% of mice injected with botulinum toxin, and are used interchangeably within a single product.

[0071] In one sun, the therapeutically effective dose of botulinum toxin is about 0.01 U / kg to about 100 U / kg, about 0.1 U / kg to about 100 U / kg, about 0.2 U / kg to about 100 U / kg, about 0.2 U / kg to about 50 U / kg, about 0.2 U / kg to about 30 U / kg, about 0.2 U / kg to about 10 U / kg, about 0.2 U / kg to about 1 U / kg, about 1.2 U / kg, about 4 U / kg, or about 12 U / kg. In other cases, based on an adult weighing 60 kg, it may be about 1 U to about 10,000 U, about 1 U to about 5,000 U, about 1 U to about 2,500 U, about 1 U to about 1,000 U, about 1 U to about 500 U, about 1 U to about 300 U, about 1 U to about 200 U, about 10 U to about 200 U, about 10 U to about 100 U, or about 10 U to about 50 U.

[0072] In the present invention, the meaning of “not containing animal protein” is that it substantially does not contain a protein derived from animals (e.g., human serum albumin).

[0073] The composition of one embodiment may be administered in a therapeutically effective amount, and as used herein, the terms “effective amount” or “therapeutic effective amount” refer to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to any medical treatment or prevention. The effective dosage level may be determined based on the severity of the disease, the activity of the drug, the patient’s age, weight, health and gender, sensitivity to the drug, the timing of administration, the route of administration and the elimination rate of the composition of the present disclosure, the duration of treatment, drugs used simultaneously or in combination with the composition of the present disclosure, and other factors known in the medical field.

[0074] In one embodiment, botulinum toxin may be administered in a single or multiple treatment session. The dosage may be administered as a single or split injection at the injection site. In multiple treatment sessions, botulinum toxin may be administered at intervals of 6 months, 4 months, or 3 months or less. In multiple treatment sessions, the interval between administrations of botulinum toxin includes a first treatment and a second treatment, and the dosage for the second treatment may be less, more, or equal to the dosage for the first treatment.

[0075] 'Botulinum toxin' can be produced by bacteria or recombination.

[0076] Botulinum toxin includes botulinum toxin derivatives. Botulinum toxin derivatives may be derivatives comprising one or more chemical or functional modifications on a portion or part of a chain of natural botulinum toxin or recombinant native botulinum toxin having botulinum toxin activity. For example, botulinum toxin derivatives may be modified toxins having one or more deleted, modified, or substituted amino acids. Botulinum toxin may be a recombinant peptide, a fusion protein, or a hybrid neurotoxin prepared, for example, from a subunit or domain of a different toxin serotype. Botulinum toxin may also be a portion of a whole molecule having toxic activity, or a combination or conjugated molecule thereof, for example, as a portion of a fusion protein.

[0077] The term 'domain' is a three-dimensional structure that can evolve, function, and exist independently of the rest of the protein chain as a given protein sequence and conserved part.

[0078] Since domains are independently stable, domains can be swapped between one protein and another through genetic engineering to create chimeric proteins.

[0079] The term 'homology' 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, the first and second amino acid sequences exhibit 100% homology.

[0080] The term 'recombination' refers to the process in which the elements constituting genes, such as DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid), are altered from their original sequences during the process of disassembly and reassembly. DNA fragments can be artificially recombined through molecular biology experiments. DNA that has been artificially recombined in this way is called recombinant DNA.

[0081] A recombinant polypeptide according to one embodiment of the present invention may include a receptor binding domain (RBD) of botulinum toxin type A1. The receptor binding domain may include amino acid variations. The receptor binding domain may include amino acid variations at one or more locations that are conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but have different amino acids at corresponding locations of botulinum toxin type A1.

[0082] For example, the above amino acid variation may be one in which at least a portion of the sequence of botulinum toxin type A1 is substituted with an amino acid sequence common to botulinum toxin types A2 and A6. For example, asparagine, which is amino acid 880 of the receptor binding domain of botulinum toxin type A1, may be substituted with serine, which is amino acid 880 of botulinum toxin type A2 and botulinum toxin type A6.

[0083] For example, the above amino acid variation may be one in which at least part of the amino acid sequence of botulinum toxin type A1 is substituted with an amino acid other than that in the amino acid sequence common to botulinum toxin types A2 and A6. For example, asparagine, which is amino acid 880 of the receptor binding domain of botulinum toxin type A1, may be substituted with an amino acid other than serine, which is amino acid 880 of botulinum toxin type A2 and botulinum toxin type A6 (e.g., a natural amino acid, a stereoisomer thereof, or a variant thereof).

[0084] For example, asparagine at position 880 of the receptor binding domain of botulinum toxin type A1 can be conservatively substituted with other amino acids, for example, threonine or glutamine.

[0085] The recombinant polypeptide of one embodiment may comprise at least one, for example, one, two, or three sequence amino acid variations selected from the group consisting of N880, W1068, and N1090. For example, the receptor binding domain may comprise at least one, for example, one, two, or three sequence amino acid variations selected from the group consisting of N880S, W1068M, and N1090S. Each amino acid variation may be independently selected from deletions, modifications, and substitutions. For example, the amino acid variation may be a substitution.

[0086] The receptor binding domain may further include at least one sequence of amino acid variations selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, Y1117, T1232, and L1278, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve. For example, the receptor binding domain may further include at least one sequence of amino acid variations selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, Y1117F, T1232R, and L1278F, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve.

[0087] The receptor binding domain may further comprise at least one sequence of amino acid variations selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1232, and L1278, for example, one, two, three, four, five, six, seven, eight, or nine. For example, the receptor binding domain may comprise at least one sequence of amino acid variations selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, and L1278F, for example, one, two, three, four, five, six, seven, eight, or nine.

[0088] For example, the receptor binding domain may include N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, and L1278F, and may include at least one or two amino acid variations selected from the group consisting of T1063P, H1064R, and Y1117F. For example, the receptor binding domain may include amino acid variations of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, and L1278F, and may include amino acid variations of 'T1063P and H1064R' or 'Y1117F'.

[0089] The receptor binding domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but may include one or more amino acid variations at positions other than those having different amino acids at the corresponding positions of botulinum toxin type A1. For example, the receptor binding domain may include an amino acid variation at the position S1142. For example, the amino acid variation may include S1142Y, S1142R, S1142I, S1142F, or S1142H.

[0090] Additionally, the positions of the 9 mutable amino acids were also selected from sequences that are common to botulinum toxin type A2 and A6, while being different from botulinum toxin type A1.

[0091] In one embodiment, the receptor binding domain may include all variations of the amino acid sequences of N880, W1068, and N1090, and N954, M968, T990, Q991, N1025, N1026, N1052, T1232, and L1278.

[0092] In one embodiment, the receptor binding domain may include all variations in the amino acid sequences of N880S, W1068M, and N1090S, and N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, and L1278F.

[0093] In one embodiment, the receptor binding domain may include one of SEQ ID NOs 1 to 7 and 33 to 42.

[0094] Amino acid variations can each be independently selected from deletions, modifications, and substitutions. For example, an amino acid variation can be a substitution.

[0095] A recombinant polypeptide according to one embodiment of the present invention may include a translocation domain of recombinant botulinum toxin type A1. The recombinant botulinum toxin type A1 translocation domain may include amino acid variations at one or more locations among positions that have amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but different amino acids at corresponding locations of said botulinum toxin type A1. For example, the locations of the amino acid variations of the recombinant translocation domain of one embodiment may include at least one selected from the group consisting of L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818. For example, the recombination potential domain of one embodiment may include at least one amino acid variant selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V.

[0096] Alternatively, the recombinant translocation domain of one embodiment may comprise a translocation domain of botulinum toxin type A1 and may comprise at least one amino acid variation at a position selected from the group consisting of D589, E734, and E809. For example, the recombinant translocation domain of one embodiment may comprise a translocation domain of botulinum toxin type A1 and may comprise at least one amino acid variation at a position selected from the group consisting of D589K, E734K, and E809K.

[0097] In one embodiment, the translocation domain may comprise or be composed of a translocation domain of wild-type botulinum toxin type A1, or may comprise or be composed of a translocation domain of botulinum toxin type A1 comprising at least one amino acid variant selected from the group consisting of D589K, L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, A818V, E734K and E809K.

[0098] In one embodiment of the present invention, the recombinant polypeptide (e.g., a recombinant receptor binding domain) may further comprise a translocation domain. The translocation domain may comprise a polypeptide selected from the group consisting of a translocation domain of botulinum toxin type A1, a translocation domain of botulinum toxin type A2, a translocation domain of botulinum toxin type A6, and variants thereof (e.g., the aforementioned recombinant translocation domain of botulinum toxin type A1). For example, the translocation domain may comprise a polypeptide selected from the group consisting of a translocation domain of botulinum toxin type A2, a translocation domain of botulinum toxin type A6, and variants thereof. For example, the translocation domain may comprise a translocation domain of botulinum toxin type A2 or a translocation domain of botulinum toxin type A6.

[0099] A recombinant polypeptide of one embodiment may include the aforementioned recombinant receptor binding domain and / or the aforementioned recombinant potential domain. In one embodiment, the recombinant polypeptide includes the aforementioned receptor binding domain of botulinum toxin type A1 and

[0100] The recombinant polypeptide of one embodiment may further comprise a light chain domain. The light chain domain may be a recombinant polypeptide. For example, the light chain domain may comprise a polypeptide selected from the group consisting of botulinum toxin type A1, botulinum toxin type A4, and variants thereof. For example, the light chain domain may comprise a subdomain of botulinum toxin type A1 or a variant thereof, a subdomain of botulinum toxin type A4 or a variant thereof. A variant of each subdomain of botulinum toxin type A1 or A4 may have homology of 99.8% or more, 99.5% or more, 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 95% or more, 93% or more, 92% or more, 91% or more, or 90% or more with the sequence of each subdomain of wild-type botulinum toxin type A1 or A4. For example, variants of each subdomain of botulinum toxin type A1 or A4 may include one, two, three, four, five, six, seven, eight, or nine amino acid variations in the sequence of each subdomain of wild-type botulinum toxin type A1 or A4.

[0101] In one embodiment, the sequences of botulinum toxin type A1 and A4 light chains were divided into 1st, 2nd, 3rd, and 4th domains according to the following criteria.

[0102] The first domain of the botulinum toxin type A1 light chain and the first domain of the A4 light chain are defined as a region containing an α-exosite that primarily interacts with the substrate SNAP25. The first domain of the botulinum toxin type A1 and A4 light chains may include amino acids at positions 1 to 20 and 90 to 150, for example, amino acids at positions 1, 5, 10, 15, or 20 and amino acids at positions 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150.

[0103] In one embodiment, the first domain of the light chain of botulinum toxin type A1 and the first domain of the light chain of botulinum toxin type A4 comprise a portion containing an α-exosite that includes a site for interbinding with the substrate SNAP25, and amino acids at positions 1, 5, 10, 15, or 20 to amino acids at positions 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150, for example, amino acids at positions 20 to 90, amino acids at positions 20 to 100, amino acids at positions 20 to 110, amino acids at positions 20 to 120, amino acids at positions 20 to 130, amino acids at positions 20 to 140, 10 It may include amino acids at positions 10 to 90, amino acids at positions 10 to 100, amino acids at positions 10 to 110, amino acids at positions 10 to 120, amino acids at positions 10 to 130, or amino acids at positions 10 to 140. For example, the first domain of the light chain of botulinum toxin type A1 and the first domain of the light chain of botulinum toxin type A4 may include amino acids at positions 1 to 120.

[0104] The second domain of the botulinum toxin type A1 light chain and the second domain of the A4 light chain are defined as parts primarily involved in the enzymatic activity of the light chain, containing 170 loops and binding to the cofactor zinc. The second domain of the botulinum toxin type A1 and A4 light chain may include amino acids at positions 91 to 151, for example, amino acids at positions 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, and 151, and amino acids at positions 210 to 270, for example, amino acids at positions 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, and 270.

[0105] In one embodiment, the second domain of the botulinum toxin type A1 light chain and the second domain of the A4 light chain are involved in the enzymatic activity of the light chain, include a 170-loop, and include a site that binds to zinc as a cofactor, and amino acids at positions 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, and 151 to amino acids at positions 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, and 270, for example, amino acids at positions 91 to 270, 101 It may include amino acids at positions 111 to 250, amino acids at positions 121 to 240, amino acids at positions 131 to 230, amino acids at positions 141 to 220, or amino acids at positions 151 to 210. For example, the second domain of the light chain of botulinum toxin type A1 and the second domain of the light chain of botulinum toxin type A4 may include amino acids at positions 121 to 240.

[0106] The third domain of the botulinum toxin type A1 light chain and the third domain of the A4 light chain are defined as domains that include 250 loops and 370 loops that stabilize substrate binding, and include sites involved in both the α-exosite and β-exosite, which are substrate binding sites. The third domain of the botulinum toxin type A1 and A4 light chain may include amino acids 211 to 271 (e.g., amino acids 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, 261, 266, and 271) to amino acids 386 to 446 (e.g., amino acids 386, 391, 396, 401, 406, 411, 416, 421, 426, 431, 436, 441, and 446).

[0107] The third domain of the botulinum toxin type A1 light chain and the third domain of the A4 light chain include 250 loops and 370 loops that stabilize substrate binding, and include a site involved in both the α-exosite and β-exosite substrate binding sites, and amino acids 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, 261, 266 and 271 to amino acids 386, 391, 396, 401, 406, 411, 416, 421, 426, 431, 436, 441 and 446, for example, It may include amino acids 211 to 446, amino acids 221 to 436, amino acids 231 to 426, amino acids 241 to 416, amino acids 251 to 406, or amino acids 261 to 396. For example, the third domain of the light chain of botulinum toxin type A1 and the third domain of the light chain of botulinum toxin type A4 may include amino acids at positions 241 to 396.

[0108] The fourth domain of the botulinum toxin type A1 light chain (SEQ No. 4), and the fourth domain of the A4 light chain (SEQ No. 8) can be defined as domains including a site 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 chain may include amino acids 362-425 (e.g., amino acids 362, 367, 372, 377, 382, ​​387, 392, 397, 402, 407, 412, 417, 422 and 425) to amino acids 410-448 (e.g., amino acids 410, 414, 417, 420, 425, 430, 435, 440, 445, and 448).

[0109] In one embodiment, the fourth domain of the botulinum toxin type A1 light chain (SEQ No. 4) and the fourth domain of the A4 light chain (SEQ No. 8) comprise a site involved in the structural flexibility of the botulinum toxin type A light chain, and amino acids 362, 367, 372, 377, 382, ​​387, 392, 397, 402, 407, 412, 417, 422 and 425 to 410, 414, 417, 420, 425, 430, 435, 440, 445, and 448, for example, amino acids 362 to 448, amino acids 372 to 438 or 382 to 428 It may include amino acids. For example, the fourth domain of the light chain of botulinum toxin type A1 and the fourth domain of the light chain of botulinum toxin type A4 may include amino acids at positions 397 to 425.

[0110] The light chain domain may be such that the sequence of the third domain among the first, second, third, and fourth domains of a botulinum toxin type A light chain other than botulinum toxin type A4 is substituted with the sequence of the third domain of botulinum toxin type A4 or a variant thereof. The variant of the third domain of botulinum toxin type A4 may have 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 95% or more, 93% or more, 92% or more, 91% or more, or 90% or more homology with the sequence of the third domain of wild-type botulinum toxin type A4. For example, the variant of the third domain of botulinum toxin type A4 may include one, two, three, four, five, six, seven, eight, or nine amino acid variations in the sequence of the third domain of wild-type botulinum toxin type A4. Here, amino acid variations may each be independently selected from deletions, modifications, and substitutions, and, for example, the amino acid variation may be an amino acid substitution. The same content as described for the variant of the third domain of botulinum toxin type A4 may also apply to the variant of the fourth domain of botulinum toxin type A4, the variant of the first domain of botulinum toxin type A1, the variant of the second domain of botulinum toxin type A1, and the variant of the fourth domain of the light chain of botulinum toxin type A1 described below. For example, these variants may have 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 95% or more, 93% or more, 92% or more, 91% or more, or 90% or more homology with the sequence of the wild-type domain, and may contain one, two, three, four, five, six, seven, eight, or nine amino acid variations in the sequence of the wild-type domain.

[0111] The light chain domain may be such that the sequence of the fourth domain among the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain is further substituted with the sequence of the fourth domain of botulinum toxin type A4 or a variant thereof. The light chain domain may be such that the sequence of the second domain among the first, second, third, and fourth domains of the botulinum toxin type A light chain other than botulinum toxin type A1 is substituted with the sequence of the second domain of botulinum toxin type A1 or a variant thereof.

[0112] The light chain domain may be such that the sequence of the fourth domain among the first, second, third, and fourth domains of the recombinant botulinum toxin type A light chain is further substituted with the sequence of the fourth domain of botulinum toxin type A1 or a variant thereof. In one embodiment, the light chain domain may comprise the first and second domains of the botulinum toxin type A1 light chain; or variants thereof. The light chain domain may also further comprise the third and fourth domains of the botulinum toxin type A4 light chain; or variants thereof.

[0113] In one embodiment, each variant of the first, second, third, or fourth domain of the botulinum toxin type A light chain may mean a subdomain of the botulinum toxin type A light chain having 98% or more homology or 99% or more homology with each of the first, second, third, or fourth domains of the wild-type botulinum toxin type A light chain. In one embodiment, each variant of the first, second, third, or fourth domain of the botulinum toxin type A light chain may mean a subdomain of the botulinum toxin type A light chain having 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acid variations, for example, one, two, three, four, five, six, seven, eight, nine, or 10 amino acid variations, in the first, second, third, or fourth domain of the wild-type botulinum toxin type A light chain.

[0114] The light chain domain of one embodiment may include a recombinant light chain disclosed in PCT-KR2022-008793 or PCT-KR2022-008798, which is incorporated herein by reference in its entirety.

[0115] The light chain domain of one embodiment may include a first domain and a second domain of a wild-type botulinum toxin type A1 light chain; or variants thereof and a third domain and a fourth domain of a wild-type botulinum toxin type A4 light chain; or variants thereof.

[0116] In the case where the light chain domain of one embodiment is a variant, the variant of said subdomain may have 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, or 90% or more homology with the sequence of the light chain subdomain of wild-type botulinum toxin. For example, the variant of said subdomain may include one, two, three, four, five, six, seven, eight, or nine amino acid variations in the sequence of the light chain subdomain of wild-type botulinum toxin.

[0117] In the case where the light chain domain of one embodiment is a variant, the third domain of the botulinum toxin type A4 light chain may be mutated. For example, the variant of the third domain of botulinum toxin type A4 may have 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, or 90% or more homology with the sequence of the third domain of wild-type botulinum toxin type A4. For example, the variant of the third domain of botulinum toxin type A4 may include one, two, three, four, five, six, seven, eight, or nine amino acid variations in the sequence of the third domain of wild-type botulinum toxin type A4. For example, a variant of the third domain of botulinum toxin type A4 may have a variant of an amino acid at position 260 and / or position 264 relative to the full-length light chain domain. For example, a variant of the third domain of botulinum toxin type A4 may have leucine at position 260 substituted with phenylalanine and isoleucine at position 264 substituted with arginine relative to the full-length light chain domain.

[0118] The description of the light chain domain described above may be applied in the same way to the light chain domain to be described below, to the extent that there are no contradictions.

[0119] The recombinant receptor binding domain of one embodiment may include one or more sequences selected from SEQ ID NOs. 1 to 7, 33 to 42 and 45 to 49.

[0120] The recombinant polypeptide of one embodiment may further comprise a cell penetrating peptide. In one embodiment, the cell penetrating peptide may be IMTP-8. The cell penetrating peptide of one embodiment may be linked to the recombinant polypeptide through a GS linker. The cell penetrating peptide may be linked to the recombinant polypeptide directly or indirectly through a GS linker.

[0121] The GS linker is an amino acid linker rich in glycine and containing glycine and serine, and may include a conventional GS linker used in the field, and may be a linker containing four or more or five or more Gs and Ss. For example, the GS linker may be GGGGS.

[0122] In one embodiment, the receptor binding domain may have a half-maximum inhibitory concentration value based on botulinum toxin heavy chain competition analysis that is lower than that of the wild-type receptor binding domain. For example, in one embodiment, the receptor binding domain may have a half-maximum inhibitory concentration value based on ELISA-based botulinum toxin heavy chain competition analysis that is lower than that of the wild-type receptor binding domain.

[0123] A recombinant polypeptide of one embodiment further comprises a light chain domain, wherein the light chain domain comprises a polypeptide selected from the group consisting of botulinum toxin type A1, botulinum toxin type A2, and variants thereof; and further comprises a translocation domain, wherein the translocation domain may comprise a polypeptide selected from the group consisting of a translocation domain of botulinum toxin type A1, a translocation domain of botulinum toxin type A2, a translocation domain of botulinum toxin type A6, and variants thereof.

[0124] In one embodiment, if the translocation domain is a variant, the variant of the translocation domain may have 99.5% or more, 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, 90% or more, 89% or more, 88% or more, 87% or more, 86% or more, or 85% or more homology with the sequence of the translocation domain of a wild-type botulinum toxin (e.g., botulinum toxin type A1, A2, or A6). For example, a variant of the said subdomain may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acid variations in the sequence of the light chain subdomain of wild-type botulinum toxin, or thirty, twenty-five, twenty, eighteen, fifteen or fewer amino acid variations.

[0125] The translocation domain of one embodiment may include at least one amino acid variant selected from the group consisting of L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818.

[0126] The potential domain of one embodiment may include at least one amino acid variant selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V.

[0127] The translocation domain of one embodiment may include at least one amino acid variant selected from the group consisting of D589, E734, and E809.

[0128] The translocation domain of one embodiment may include at least one amino acid variant selected from the group consisting of D589K, E734K, and E809K.

[0129] The recombinant polypeptide of one embodiment may have increased potency, safety, or half-life compared to wild-type botulinum toxin type A.

[0130] The recombinant polypeptide of one embodiment may include one or more sequences selected from SEQ ID NOs 1 to 7, 33 to 42 and 45 to 49 of Table 1 to be described later; SEQ ID NOs 8 to 32, 43 and 44, or SEQ ID NOs 1 to 49.

[0131] A recombinant polypeptide according to one embodiment of the present invention comprises a receptor binding domain of botulinum toxin type A1, and the receptor binding domain may comprise amino acid variations at two or more positions selected from N954, Y1117, T1232, R1273, and L1278. For example, the receptor binding domain may comprise two or more amino acid variations selected from N954S, Y1117F, T1232R, R1273K, and L1278F. The receptor binding domain of one embodiment may comprise amino acid variations at two or more positions selected from N954, T1232, and L1278. For example, the receptor binding domain may comprise amino acid variations at two or more positions selected from N954S, T1232R, and L1278F.

[0132] For example, amino acid variations of the receptor binding domain may include N954S and T1232R; N954S and L1278F; T1232R and L1278F; T1232R and R1273K; R1273K and L1278F; N954S, T1232R and L1278F; Y1117F, R1273K and L1278F; or T1232R, R1273K and L1278F.

[0133] A recombinant polypeptide according to one embodiment of the present invention may include a receptor binding domain of botulinum toxin type A1. Here, the half-maximum inhibitory concentration value according to the botulinum toxin heavy chain competitive analysis of a variant of the receptor binding domain may be lower than that of the wild-type receptor binding domain. Here, the variant of the receptor binding domain may include an amino acid variation at at least one position selected from the group consisting of N880, N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, W1068, N1090, Y1117, S1142, T1232, and L1278.

[0134] For example, the receptor binding domain may include an amino acid variation at the S1142 position. For example, the amino acid variation may include S1142Y, S1142R, S1142I, S1142F, or S1142H.

[0135] A recombinant botulinum toxin according to one embodiment of the present invention may include a variant in which two or more amino acids among the amino acids of the receptor binding domain sequence of botulinum toxin type A1 are mutated. The amino acid mutations may include two or more selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, Y1117F, S1142Y, S1142R, S1142I, S1142F, S1142H, T1232R, and L1278F. When administered to a subject requiring treatment, the recombinant botulinum toxin of one embodiment may be superior in one or more of efficacy and safety compared to wild-type botulinum toxin type A1, and the remainder may be at least equivalent to the wild-type toxin. For example, the recombinant botulinum toxin of one embodiment is CMAP or DAS (e.g., CMAP) compared to wild-type botulinum toxin type A1. AUCor DAS AAC ); and at least one of weight loss or safety margin may be superior, and the remainder may be at least equivalent to the wild-type toxin. In this specification, “equivalent or superior” may mean that the corresponding value of the example substance is not statistically significantly less effective than the corresponding value of the substance to be compared.

[0136] A recombinant botulinum toxin according to one embodiment may comprise a light chain domain selected from the group consisting of botulinum toxin type A1 light chain, botulinum toxin type A2 light chain, and variants thereof. Here, the description of the light chain domain described above may be applied in the same way.

[0137] For example, the light chain domain of a recombinant botulinum toxin of one embodiment may include a first domain and a second domain of a botulinum toxin type A1 light chain; or variants thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or variants thereof. For example, the first domain of the light chain domain of the recombinant botulinum toxin may include a first domain of a botulinum toxin type A1 light chain or a variant thereof, the second domain may include a second domain of a botulinum toxin type A1 light chain or a variant thereof, the third domain may include a third domain of a botulinum toxin type A4 light chain or a variant thereof, and the fourth domain may include a fourth domain of a botulinum toxin type A4 light chain or a variant thereof.

[0138] For example, a variant of the third domain of botulinum toxin type A4 may have a variant of an amino acid at position 260 and / or position 264 relative to the full-length light chain domain. For example, a variant of the third domain of botulinum toxin type A4 may have leucine at position 260 substituted with phenylalanine and isoleucine at position 264 substituted with arginine relative to the full-length light chain domain.

[0139] The recombinant botulinum toxin of one embodiment, compared to wild-type botulinum toxin type A1, has a safety margin when administered to an individual; and DAS AUC　 or CMAP AAC ; At least one of them may show a superior value, and the remainder may be at least equivalent to the wild-type toxin. In the recombinant botulinum toxin of one embodiment, the translocation domain may comprise a polypeptide selected from the group consisting of the translocation domain of botulinum toxin type A1, the translocation domain of botulinum toxin type A2, the translocation domain of botulinum toxin type A6, and variants thereof. The translocation domain of one embodiment may comprise at least one amino acid variant selected from the group consisting of D589, E734, and E809. The translocation domain of one embodiment may comprise at least one amino acid variant selected from the group consisting of D589K, E734K, and E809K.

[0140] The recombinant botulinum toxin of one embodiment may include the aforementioned botulinum toxin type A light chain variant (e.g., variants of the A1-1, A1-2, A4-3, and A4-4 forms) and may include a receptor binding domain comprising one or more amino acid variants selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, S1142Y, S1142R, S1142I, S1142F, S1142H, Y1117F, T1232R, and L1278F. The receptor binding domain of one embodiment may include the recombinant polypeptides of SEQ ID NOs 1 to 7, 33 to 42 and 45 to 49. The recombinant botulinum toxin of one embodiment may include a translocation domain comprising one or more amino acid variations among L260F and I264R.

[0141] The variant light chain of the recombinant botulinum toxin of one embodiment may comprise a first domain and a second domain of the botulinum toxin type A1 light chain; or variants thereof, and a third domain and a fourth domain of the botulinum toxin type A4 light chain; or variants thereof. The light chain domain of one embodiment may comprise a variant of the second domain of the botulinum toxin type A1 light chain, wherein the leucine at position 260 of the variant of the second domain of the botulinum toxin type A1 light chain is substituted with phenylalanine, and the isoleucine at position 264 is substituted with arginine. The recombinant botulinum toxin of one embodiment may exhibit inhibited diffusion, increased duration, and rapid onset of effect when administered to a patient compared to wild-type botulinum toxin. Compared to wild-type botulinum toxin type A1, the recombinant botulinum toxin of one embodiment exhibits safety when administered to an individual; One or more of the duration of action and the remainder may show a higher value, while the remainder may be at least equivalent to the wild-type toxin. For example, the recombinant botulinum toxin of one embodiment has a higher safety margin when administered to an individual compared to wild-type botulinum toxin type A1, and DAS AUC　 or CMAP AAC This may be equivalent to or better than the wild-type toxin. For example, the recombinant botulinum toxin of one embodiment has a safety margin equivalent to or better than that of wild-type botulinum toxin type A1 when administered to an individual, and DAS AUC　 or CMAP AAC This may be higher than the wild-type toxin.

[0142] Botulinum toxins comprising various recombinant polypeptides included in the present invention described above may have increased efficacy, safety, or half-life compared to wild-type botulinum toxin type A (e.g., wild-type botulinum toxin type A1). For example, a botulinum toxin comprising the recombinant polypeptide of one embodiment may exhibit inhibited diffusion, increased duration, and rapid onset of effect when administered to a patient compared to wild-type botulinum toxin.

[0143] A botulinum toxin comprising a recombinant polypeptide of one embodiment may include pharmaceutically acceptable excipients or additives. Pharmaceutically acceptable excipients or additives may be stabilizers, ionic compounds, surfactants, buffers, lyophilizing protective agents, or combinations thereof, for example, amino acids (e.g., methionine), salts (e.g., NaCl), buffers, nonionic surfactants (e.g., polysorbates, e.g., polysorbate 20, poloxamer), sugars (e.g., disaccharides such as monosaccharides, sucrose, trehalose, etc.), sugar alcohols (e.g., sorbitol), or combinations thereof.

[0144] The botulinum toxin composition comprising the recombinant polypeptide of the present invention can be formulated in any form, such as a solid or liquid preparation, for example, a lyophilized powder, a liquid, or a pre-filled syringe preparation.

[0145] In one embodiment of the present invention, a composition for improving, preventing, or treating a disease is provided, comprising a botulinum toxin containing a recombinant polypeptide as an active ingredient.

[0146] In one embodiment of the present invention, a use is provided for improving or treating a disease by administering a botulinum toxin composition having a botulinum toxin containing a recombinant polypeptide as an active ingredient to an individual.

[0147] In one embodiment of the present invention, a method is provided for improving, preventing, or treating a disease by administering a botulinum toxin composition having a botulinum toxin containing a recombinant polypeptide as an active ingredient to an individual.

[0148] In one embodiment of the present invention, the use of a botulinum toxin comprising a recombinant polypeptide is provided in the manufacture of a drug for improving, preventing, or treating a disease.

[0149] In one embodiment, the condition or disease may include wrinkles, square jaw, pointed jaw, wound, skin softening, scar, acne, pores, elasticity, reduced sebum secretion and / or keloid; and / or facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, central pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus and / or hyperhidrosis.

[0150] In one embodiment of the present invention, a botulinum toxin composition comprising a recombinant polypeptide may include or not include an animal protein (e.g., albumin such as human serum albumin). If the composition of one embodiment does not include an animal protein, it may include an amino acid as a stabilizer. In one embodiment, the amino acid may be any of the 20 standard amino acids found in nature, but is not limited thereto. The amino acid of one embodiment may include any known as a stabilizer in the art. For example, in one embodiment, the amino acid may include nonpolar amino acids, e.g., methionine, isoleucine, and / or tryptophan.

[0151] The present invention will be described in more detail below with reference to examples, but this is merely for the purpose of explaining the invention and is not intended to limit its scope in any way.

[0152] Example 1: Purification of domain-substituted botulinum toxin type A heavy chain receptor binding domain

[0153] A recombinant receptor binding domain expression vector (cloned in the pET28a vector) was introduced into BL21(DE3) cells, a type of E. coli, to induce the expression of a recombinant botulinum toxin type A heavy chain receptor binding domain (RBD) with a substituted domain, i.e., a recombinant polypeptide corresponding to each embodiment of the present invention. Expression was induced with IPTG (Isopropyl β-D-1-thiogalactopyranoside). Specifically, IPTG was added to the liquid medium, and BL21(DE3) cells cultured at an appropriate temperature for an appropriate time were centrifuged to precipitate the cells. The medium was removed from the supernatant, and the precipitated cells were resuspended in buffer A (20 mM sodium phosphate, 150 mM NaCl, 10 mM Imidazole, pH 7.3). The cells were then lysed using an ultrasonic disperser on ice. After centrifuging the lysed cells to remove cell debris, the recombinant polypeptide was isolated from the supernatant by binding the His tag to the Ni-NTA resin using Ni-NTA resin. The amino acid sequence of the isolated recombinant polypeptide is listed in Table 1.

[0154] Sequence Number Receptor Binding Domain Sequence 1A1(N880S)2A1(W1068M)3A1(N1090S)4A1(N880S, W1068M)5A1(N880S, N1090S)6A1(W1068M, N1090S)7A1(N880S, W1068M, N1090S)33A1(T1232R, L1278F)34A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)35A1 (N954S, T1232R, L1278F)36A1 (Y1117F, R1273K, L1278F)37A1 (T1232R, R1273K, L1278F)38A1 (S1142Y)39A1 (S1142R)40A1 (S1142I)41A1 (S1142F)42A1 (S1142H)45A1 (H1064Y)46A1 (H1064W)47A1 (T1063P,H1064F)48A1 (T1063P,H1064W)49A1 (T1063P,H1064Y)

[0155] The parentheses in Table 1 indicate the location where amino acid mutations occurred in wild-type botulinum toxin and the information on the mutated amino acids. For example, SEQ ID NO. 1 refers to a recombinant polypeptide in which the asparagine at position 880 of the receptor binding domain of wild-type botulinum toxin type A1 is substituted with serine.

[0156] Example 2: ELISA-based BoNT Hc competition assay

[0157] The purified recombinant polypeptides of sequence numbers 2, 6, 33, 34 and 35 to 43, 45 to 49 were analyzed through a competition assay.

[0158] Differentiated Ntera-2 cells (CRL-1973, ATCC) were seeded into 96-well plates and cultured for 4 days at 37 °C and 5% CO2 in DMEM / F12 (WelGene) supplemented with 5% FBS (Gibco), B27 (Gibco), and Antibiotic-Antimycotic (Gibco). After 4 days, the cells were simultaneously treated with 300 U / well BoNT / A (150 kDa, Medytox, Batch No.: RBTS2301) and BoNT HC (botulinum toxin heavy chain) at various concentrations for 48 hours. Treatment concentrations of BoNT / A1 WT HC (botulinum toxin type A1 wild-type heavy chain, hereinafter A1 WT HC) and recombinant polypeptides were applied at 5-fold intervals, ranging from 5 to a maximum of 15,625 times the molar concentration of BoNT / A (in this specification, BoNT / A is defined as including both wild-type botulinum toxin serotype A and recombinant botulinum toxin serotype A. It is obvious to those skilled in the art that BoNT / A used in the embodiments herein refers to wild-type BoNT / A unless otherwise noted). After treatment with BoNT / A and HC, the medium was replaced with fresh medium and cultured for an additional 2 days; subsequently, the cells were treated with lysis buffer to perform an ELISA test. The ELISA test was performed on anti-SNAP25 197After coating the immunoplate with an antibody (Medytox) and reacting it with cell lysate, BoNT / A-cleaved SNAP25 was captured and detected using biotinylated-anti-SNAP25 antibody (LS Bio) and streptavidin-HRP (Thermo Scientific). A chromogenic solution (TMB substrate, Sigma-Aldrich) was added, and the absorbance of the chromogenic reaction was measured at 450 nm using a microplate reader (SpectraMax i3, Molecular Devices). Graphpad Prism 7.05 (GraphPad Software Inc., CA, USA) was used for graph presentation and analysis. The response to HC treatment concentration was determined using a 4-parameter logistic (4PL) non-linear regression method to calculate the half-maximum inhibition concentration (IC10). 50 The IC50(uM) value of the calculated variant HC was divided by the IC50(uM) value of A1 WT HC to obtain the relative inhibitory activity value compared to the wild-type A1 botulinum toxin heavy chain, as shown in the table below.

[0159]

[0160] As a result of ELISA-based botulinum toxin heavy chain competitive analysis, the recombinant polypeptide according to one embodiment showed a high relative inhibitory activity value compared to the wild-type receptor binding domain, confirming an increase in binding affinity to the receptor. Through this, it was confirmed that the recombinant polypeptide of one embodiment can exhibit superior efficacy and improved safety compared to the wild type.

[0161] Example 3: Preparation and Purification of Domain-Substituted Recombinant Botulinum Toxin

[0162] After confirming the effects of the receptor binding domains of SEQ ID NOs. 1 to 7, which were confirmed in Examples 1 and 2, a recombinant receptor binding domain was designed that further includes additional amino acid substitutions in the sequences of SEQ ID NOs. 1 to 7. For example, the receptor binding domain included in the recombinant botulinum toxin of SEQ ID NO. 8 includes all amino acid variations of the recombinant polypeptide of SEQ ID NO. 7, and also includes additional variations. Here, the additional variations were selected at a position that is conserved with amino acids common to wild-type botulinum toxin type A2 and botulinum toxin type A6, but has different amino acids at the corresponding position in botulinum toxin type A1. At that position, the amino acid of botulinum toxin type A1 was substituted with an amino acid conserved in botulinum toxin types A2 and A6.

[0163] Subsequently, a recombinant botulinum toxin containing a designed recombinant receptor binding domain was prepared.

[0164] Specifically, a recombinant full-length botulinum toxin gene containing the light and heavy chains of recombinant botulinum toxin type A was cloned using the pMTL80000 vector system via site-directed mutagenesis. The cloned products are shown in Table 2 below. The recombinant botulinum toxin was prepared using a detoxified Hall A-hyper strain in which the toxin gene was inactivated using the Clostron system.

[0165] 시이번벨경창전위 대녀수용체 정다대8A1A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)9A1A1(L681I / D696N / I713T / K730E / E734K / K779S / N789D / G804A / E809K / L815V / K816R / A818V)A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)10A1-1, A1-2, A4-3, A4-4A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)11A1-1, A1-2, A4-3, A4-4 (L260F, I264R)A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)12A1-1, A1-2, A4-3, A4-4, (L260F, I264R)A1(L681I / D696N / I713T / K730E / E734K / K779S / N789D / G804A / E809K / L815V / K816R / A818V)A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)13A1-1, A1-2, A4-3, A4-4A1(L681I / D696N / I713T / K730E / E734K / K779S / N789D / G804A / E809K / L815V / K816R / A818V)A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)14A1-1, A1-2, A4-3, A4-4, (L260F, I264R)A1A1(N880S,W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, Y1117F, T1232R, L1278F)15A1-1, A1-2, A4-3, A4-4, (L260F, I264R)A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R T1232R, L1278F)16A1-1, A1-2, A4-3, A4-4A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, Y1117F, T1232R, L1278F)17A1-1, A1-2, A4-3, A4-4A1A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R T1232R, L1278F),18A1-1, A1-2, A4-3, A4-4A1(D589K, E734K, E809K)A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)19A1-1, A1-2, A4-3, A4-4(L260F, I264R)A1(D589K, E734K, E809K)A1(N880S, W1068M, N1090S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1232R, L1278F)22A1-1, A1-2, A4-3, A4-4A1A1(Y1117F, R1273K, L1278F)23A1-1, A1-2, A4-3, A4-4A1A1(T1232R, R1273K, L1278F)24A1-1, A1-2, A4-3, A4-4A1A1(N954S)25A1-1, A1-2, A4-3, A4-4A1A1(L1278F)26A1-1, A1-2, A4-3,A4-4A1A1(T1232R)27A1-1, A1-2, A4-3, A4-4A1A1(N1025T)28A1-1, A1-2, A4-3, A4-4A1A1(N1026K)29A1-1, A1-2, A4-3, A4-4A1A1(Y1117F)30A1-1, A1-2, A4-3, A4-4A1A1(W1068M, N1090S)31A1-1, A1-2, A4-3, A4-4(L260F, I264R)A1A1(Y1117F, R1273K, L1278F)32A1-1, A1-2, A4-3, A4-4(L260F, I264R)A1A1(T1232R, R1273K, L1278F)43A1-1, A1-2, A4-3, A4-4A1R1273K44A1A1(L681I / D696N / I713T / K730E / E734K / K779S / N789D / G804A / E809K / L815V / K816R / A818V)A1,

[0166] Detoxified Clostridium botulinum strains, including strains in which the toxin gene has been inactivated or knocked out, can be used in a method for producing purified recombinant botulinum toxin or purified recombinant botulinum toxin complexes. Methods commonly used in the art may be used to produce strains in which the toxin gene has been inactivated or knocked out. The recombinant botulinum toxins of SEQ ID NOs 8 to 21, produced by the Clostron method, were purified into a 150 kDa form that does not contain complex components. Additionally, two recombinant polypeptides (SEQ ID NOs 20 and 21, respectively) were further produced by additionally linking the cell-permeating peptide IMTP8 to each of the recombinant botulinum toxins of SEQ ID NOs 10 and 11 using a GS linker. All production of the above recombinant botulinum toxins was carried out in a facility authorized for toxin production in accordance with regulatory authorities.

[0167] Example 4: Evaluation of Thermal Stability of Recombinant Botulinum Toxin

[0168] The thermal stability of the recombinant botulinum toxins SEQ ID NOs. 10 and 11 prepared in Example 3 was compared. As a stability evaluation method, the intermediate temperature of thermal denaturation (Tm) was evaluated, and generally, a high intermediate temperature of thermal denaturation is desirable. The thermal stability of each domain-substituted recombinant botulinum toxin type A was evaluated using the Thermal Shift Typology (PTS) assay. The PTS assay was performed according to the manufacturer's method using the Protein Thermal Shift Dye Kit (Catalog No. 4461146, Applied Biosystems). Specifically, 10 μg of each recombinant botulinum toxin was mixed with a protein thermal shift die and buffer to make a total volume of 20 μl, and then the ROX signal was checked in RT-PCR (C1000 thermal cycler equipped with a CFX96 optical reaction module, Bio-Rad) by increasing the temperature from 20°C to 95°C at a rate of 1°C every 60 seconds. Based on the melting curve, the melting temperature (Tm) of each recombinant botulinum toxin was determined. The melting points of each recombinant botulinum toxin are shown in Figure 1 and Table 3.

[0169] ConstructTm (°C)ΔTmTm_std Wild type(7s)52.80.00.3 Sequence No. 10(7s)54.21.30.3 Sequence No. 11(7s)54.31.50.3 Wild type(7s)52.70.00.3 Sequence No. 12(7s)55.52.80.0 Sequence No. 13(7s)55.52.80.0 Sequence No. 14(7s)54.51.80.0 Sequence No. 16(7s)54.01.30.0 Sequence No. 20(7s)56.03.30.0

[0170] The results confirmed that the thermal stability of the recombinant botulinum toxins of SEQ ID NOs 10, 11 and SEQ ID NOs 12, 13, 14, 16, and 20 increased compared to the wild type. Specifically, while the melting point of wild-type botulinum toxin type A1 was 52.8°C, the melting points of SEQ ID NOs 10 and 11 increased to 54.2°C and 54.3°C, respectively. While the melting point of wild-type botulinum toxin type A1 was 52.7°C, the melting points of SEQ ID NOs 12, 13, 14, 16, and 20 increased to 55.5°C, 55.5°C, 54.5°C, 54.0°C, and 56.0°C, respectively. Through this, it is confirmed that the thermal stability of the recombinant botulinum toxin according to the present invention is significantly improved compared to the wild type.

[0171] Example 5: Evaluation of the efficacy of recombinant botulinum toxin in mice

[0172] (1) Recombinant botulinum toxin of SEQ ID NO. 10

[0173] Materials and Methods

[0174] Animals: Female 5–6 week old CD1 (ICR) mice were purchased from Orient Bio Co., Ltd. After a 1-week acclimatization and quarantine period, 6–7 week old mice were used in the experiment. Sterilized laboratory solid feed (R40-10, SAFE, France) was provided free of charge, and tap water sterilized at high temperature and high pressure was provided free of charge. During acclimatization, quarantine, and the experimental period, mice were housed under specific-pathogen-free conditions set at a temperature of 23 ± 3℃, relative humidity of 55 ± 15%, lighting time of 12 hours (8:00 AM–8:00 PM), ventilation rate of 15 times / hour, and illuminance of 150–300 Lux. This study was conducted following review and approval (A-2022-007, A-2024-005) by the Meditox Animal Ethics Committee.

[0175] Test substance: Botulinum toxin type A product (BoNT / A) was used as 100 units of Coretox (Lot No.: E623001, Meditox). Recombinant botulinum toxin of SEQ ID NO. 10 was prepared at 163 U / mL and used in the experiment. The potency of the recombinant botulinum toxin was measured using the mouse potency assay method prior to the efficacy test, and the test was conducted based on the potency values ​​derived. In the efficacy test, the dosage of each test substance was administered identically based on the unit (U: a value representing the biological activity of botulinum toxin, where 1 U is the median lethal dose of a mouse via intraperitoneal administration).

[0176] Both the botulinum toxin type A product and the recombinant botulinum toxin of the examples were diluted to concentrations of 6, 20, and 60 U / mL, respectively, before administration.

[0177] The day the test substance was administered was designated as day 0. After anesthetizing mice with an injectable anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), each test substance was administered at a dose of 0.2 mL / kg into the right calf muscle of the mice using a Hamilton syringe according to the group composition in Table 4.

[0178] Group Animals Administered Substance Dosage (U / kg) Route of Administration Evaluation Index 16 BoNT / A 1.2 Right calf muscle DAS 26 BoNT / A4 Right calf muscle DAS 36 BoNT / A 12 Right calf muscle DAS, body weight 46 Recombinant botulinum toxin 1.2 Right calf muscle DAS 56 Recombinant botulinum toxin 4 Right calf muscle DAS 66 Recombinant botulinum toxin 12 Right calf muscle DAS, body weight

[0179] The evaluation was performed using the digit abduction score (DAS) method, which visually assesses the degree of muscle paralysis in mice. The DAS values ​​indicated in Table 5 (Mouse DAS Evaluation Criteria) represent the degree of muscle paralysis based on the shape of the toes on the side administered to the mouse.

[0180] Scoring Criteria 0 Normal, no difference in shape from the uninjected foot 1 The space between the toes is narrowed, or two toes are joined together and the rest are fully extended 2 All toes are significantly narrowed, or three toes are joined together 3 The foot is bent and four toes are joined together 4 The foot is bent and all toes are joined together

[0181] According to the composition of the study groups in Table 4, DAS measurements were evaluated from day 1 after administration until the DAS score reached 0 for doses administered at 1.2, 4, and 12 U / kg. The time to reach a DAS score of 0 was measured for each individual to calculate the average DAS recovery period. Regarding body weight, to compare the effect on safety, the change in body weight at week 1 after administration relative to pre-administration weight in the 12 U / kg high-dose group was compared between the botulinum toxin type A product group and the recombinant botulinum toxin group. Statistics: Graphpad Prism 7.05 (GraphPad Software Inc., CA, USA) was used for graph presentation and DAS ED 50It was used for calculation, and SPSS software 25.0 (SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. Normality was tested using the Kolmogorov-Smirnov test. Non-parametric data were statistically analyzed using the Mann-Whitney test, and for parametric data, a two-tailed t-test was performed, and a p-value less than 0.05 was considered statistically significant. Results: Referring to Figure 2, after administering a single dose of botulinum toxin type A product (BoNT / A: Coretox) and the recombinant botulinum toxin of our institute to the right calf muscle of mice at doses of 1.2, 4, and 12 U / kg, a DAS evaluation result showed that a significant increase in DAS scores compared to BoNT / A was observed in the 1.2 U / kg recombinant botulinum toxin group on days 3, 4, and 7 after administration, and in the 4 U / kg recombinant botulinum toxin group on days 7, 14, and 21 after administration. In the BoNT / A 1.2, 4, and 12 U / kg administration groups, recovery was observed in all individuals at 2, 4, and 6 weeks after administration, respectively, while in the recombinant botulinum toxin administration groups at the same dose, recovery was observed in all individuals at 3, 5, and 8 weeks, which was an increased period (Fig. 2). When the DAS recovery time was calculated for each individual and the duration of action was compared, the average durations were confirmed to be 9.0, 19.8, and 33.8 days for the BoNT / A 1.2, 4, and 12 U / kg administration groups, respectively, and 17.5, 29.2, and 42.8 days for the recombinant botulinum toxin 1.2, 4, and 12 U / kg administration groups, respectively; a 1.9-fold, 1.5-fold, and 1.3-fold increase in the duration of action was observed for each dose, and the duration of action was significantly increased in the 1.2 and 4 U / kg recombinant botulinum toxin administration groups compared to the BoNT / A administration groups (Table 6). In the 12 U / kg administration group, the change in body weight at week 1 compared to before administration was 97% on average in the BoNT / A administration group and 95% on average in the SEQ ID NO. 10 administration group, and no significant difference was observed.

[0182] Average DAS duration of action (days) Substance 1.2 U / kg 4 U / kg 12 U / kg BoNT / A9.019.833.8 Sequence number 1017.5**29.2**42.8 † Growth rate (times) 1.9 1.5 1.3

[0183] **p<0.01, †p=0.081, compared to the group administered the same dose of BoNT / A, two-tailed t-tests or Mann-Whitney tests performed in mice. From the above results, the potent efficacy and increased duration of action of the recombinant botulinum toxin of the present invention compared to the botulinum toxin type A product (BoNT / A) were confirmed.

[0184] (2) Recombinant botulinum toxin of SEQ ID NO. 11

[0185] Except for the fact that the recombinant botulinum toxin of SEQ ID NO. 11 was prepared at 384 U / mL and used in the experiment, the remaining content was evaluated for efficacy in the same manner as the example with the recombinant botulinum toxin of SEQ ID NO. 10.

[0186] Results: After single administration of botulinum toxin type A product and recombinant botulinum toxin SEQ NO. 11 at doses of 1.2, 4, and 12 U / kg to the right calf muscle of mice, DAS evaluation results showed an increase in DAS scores compared to BoNT / A in the groups administered 1.2 and 4 U / kg of recombinant botulinum toxin SEQ NO. 11 (Fig. 3). In the BoNT / A 1.2, 4, and 12 U / kg administration groups, recovery was observed in all individuals at 2, 4, and 6 weeks after administration, respectively, while in the groups administered the same dose of recombinant botulinum toxin, recovery was observed in all individuals at 3, 5, and 7 weeks, which was higher than in the groups administered the same dose (Fig. 3). As a result of calculating the DAS recovery time for each individual and comparing the duration of action, the average durations for the BoNT / A 1.2, 4, and 12 U / kg administration groups were confirmed to be 7.2, 16.7, and 31.5 days, respectively, and for the recombinant botulinum toxin 1.2, 4, and 12 U / kg administration groups were 14.0, 25.3, and 36.2 days, respectively, showing a 1.9-fold, 1.5-fold, and 1.1-fold increase in duration of action for each dose, respectively, and the duration of action was significantly increased in the 1.2 U / kg recombinant botulinum toxin administration group compared to the BoNT / A administration group (Table 7). Regarding the change in body weight at week 1 compared to before administration in the 12 U / kg administration group, no decrease in body weight was observed, with an average of 99% in the BoNT / A administration group and an average of 104% in the SEQ ID No. 11 administration group.

[0187] Average DAS duration of action (days) DAS ED 50 (U / kg)Substance 1.2 U / kg 4 U / kg 12 U / kgBoNT / A7.216.731.52.9 Sequence No. 1114.0*25.3 † 36.21.9 Growth rate (times) 1.91.51.11.5

[0188] *p<0.05, †p=0.089, compared to the group administered BoNT / A at the same dose, two-tailed t-test or Mann-Whitney test. From the results performed in mice, the strong efficacy and increased duration of action of the recombinant botulinum toxin of SEQ ID NO. 11 compared to the botulinum toxin type A product (BoNT / A) were confirmed.

[0189] Example 6: Evaluation of the efficacy of recombinant botulinum toxin in rats

[0190] Materials and Methods

[0191] Animals: Female 6-week-old SD (Sprague-Dawley) rats were purchased from Orient Bio Co., Ltd., and after acclimatization and quarantine for 1 week, 7-week-old rats were used in the experiment. The rearing environment was the same as in Example 5. This study was conducted after review and approval (A-2023-001) by the Meditox Animal Ethics Committee.

[0192] Test substance: Botulinum toxin type A product (BoNT / A) was used as 100 units of Coretox (Lot No.: E623001, Meditox). Recombinant botulinum toxin of SEQ ID NO. 10 was prepared at 444 U / mL, and recombinant botulinum toxin of SEQ ID NO. 11 was prepared at 366 U / mL and used in the experiment. For the placebo (placebo group), only the botulinum toxin was excluded from the recombinant botulinum toxin, while the remaining excipient components were used identically.

[0193] Botulinum toxin type A products and recombinant botulinum toxins were each diluted to a concentration of 120 U / mL before administration.

[0194] The day the test substance was administered was designated as day 0. Mice were anesthetized using an injectable anesthetic (60 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), and each test substance was administered at a dose of 0.1 mL / kg into the right calf muscle of rats using a Hamilton syringe according to the group composition in Table 8.

[0195] Group Animal Administered Substance Dose (U / kg) Route of Administration Evaluation Index 16 Placebo 0 Right calf muscle DAS, CMAP 26 BoNT / A 12 Right calf muscle DAS, CMAP, Body weight 36 Recombinant botulinum toxin (SEQ No. 10) 12 Right calf muscle DAS, CMAP, Body weight 46 Recombinant botulinum toxin (SEQ No. 11) 12 Right calf muscle DAS, CMAP, Body weight

[0196] The evaluation utilized the digit abduction score (DAS) 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 responding to external electrical stimulation. The DAS values ​​indicated in Table 9 (Rat DAS Evaluation Criteria) represent the degree of muscle paralysis based on the toe shape of the administered side, while the CMAP values ​​represent the degree of direct inhibition of muscle contraction caused by nerve block.

[0197] Scoring Criteria: 0 Normal 1 Loss of abduction of the fifth toe 2 Three toes fused together 3 Four toes fused together 4 Loss of abduction of all toes and fused together

[0198] CMAP was measured using an UltraPro S100 device on the left (toxin-free) gastrocnemius muscle of each rat. After anesthetizing with an injectable anesthetic, the hair at the measurement site was shaved, and the rats were positioned in a prone position. The negative electrode was placed on the sciatic nerve of the leg to be measured, and the positive electrode was placed approximately 0.5 cm away from that point relative to the spine. The recording electrode and reference electrode were placed on the gastrocnemius muscle and Achilles tendon, respectively, and the ground electrode was placed on the rectus femoris muscle. The stimulation level and duration were set to 25–30 mA and 0.2 ms, respectively, and the amplifier filter range was set to 2–10 K at 60 Hz. Under these conditions, the height from the base to the peak of the waveform was recorded as the CMAP measurement data. Based on the test group composition in Table 8, DAS measurements were evaluated from day 3 until the DAS score reached 0. The average DAS recovery period was calculated by measuring the time it took for the DAS score to reach zero for each individual. CMAP was measured in the left calf muscle 7 days after administration to evaluate diffusion to the non-administration site. In addition, regarding CMAP measurement, CMAP AAC (CMAP baselineAUC - CMAP AUC For comparison, CMAP values ​​were measured at 2–3 week intervals until recovery was observed and calculated based on reported literature (Dermatologic Surgery 2020; 46(12): e132). Statistics: Same as Example 1.

[0199] Results: After administering a single dose of botulinum toxin type A and recombinant botulinum toxins at a dose of 12 U / kg to the right gastrocnemius muscle of rats, a DAS evaluation was performed. In the group administered with the 12 U / kg recombinant botulinum toxin SEQ No. 10, a significant increase in DAS scores compared to BoNT / A was observed on days 49 and 56 after administration (Fig. 4). An increase in DAS scores compared to BoNT / A was also observed in the group administered with the recombinant botulinum toxin SEQ No. 11 (Fig. 4). In the BoNT / A group, recovery was observed in all individuals at 10 weeks after administration, while in the group administered with the same dose of recombinant botulinum toxin SEQ No. 10, recovery was observed at 14 weeks, which was higher than in the group administered with the same dose of recombinant botulinum toxin SEQ No. 11, and at 11 weeks after administration (Fig. 4). As a result of comparing the duration of action by calculating the DAS recovery time for each individual, the average for the BoNT / A administration group was confirmed to be 50.2 days, while the recombinant botulinum toxin administration group of SEQ ID NO. 10 was 72.3 days and the recombinant botulinum toxin administration group of SEQ ID NO. 11 was 65.3 days, showing a significant increase in the duration of action of 1.4 times and 1.3 times, respectively (Table 10).

[0200] Average DAS duration of action (days) Growth rate (times) Substance 12 U / kg BoNT / A 50.2n / a Sequence number 1072.3*1.4 Sequence number 1165.3*1.3

[0201] *p<0.05, comparison with the BoNT / A administration group, two-tailed t-test. As a result of measuring CMAP in the calf muscle on the contralateral side on day 7 after botulinum toxin administration, no significant difference was observed between the group administered recombinant botulinum toxin SEQ No. 10 and the BoNT / A administration group, while significantly increased CMAP values ​​were observed in the group administered recombinant botulinum toxin SEQ No. 11 (Fig. 4). CMAP AACAs a result of calculating and comparing, the average value of the BoNT / A administration group was 3558.5, and the average value of the botulinum toxin administration group of SEQ ID NO. 10 was 3883.3, showing a significant increase of 1.1 times compared to the BoNT / A administration group, while the average value of the botulinum toxin administration group of SEQ ID NO. 11 was 3592.5, showing an equivalent or greater value. In the 12 U / kg administration group, regarding the change in body weight at week 1 compared to before administration, the average change was 107% in the botulinum toxin type A product administration group, 108% in the botulinum toxin administration group of SEQ ID NO. 10, and 108% in the botulinum toxin administration group of SEQ ID NO. 11, showing an equivalent effect compared to the BoNT / A administration group.

[0202] From the above results performed in rats, the strong efficacy and increased duration of action of recombinant botulinum toxins compared to botulinum toxin type A product (BoNT / A) were confirmed, and in the case of the recombinant botulinum toxin of SEQ ID NO. 11, a reduction in diffusion compared to the BoNT / A administration group was confirmed.

[0203] Example 7: DAS ED of recombinant botulinum toxin in mice 50 , IM LD 50 test

[0204] Materials and Methods

[0205] The animals and rearing environment are the same as in Example 5, and this study was conducted after review and approval (A-2022-007, A-2022-008) by the Meditox Animal Ethics Committee.

[0206] Test substance: Botulinum toxin type A product (BoNT / A) was used as 100 units of Coretox (Lot No.: E623001, Meditox). Recombinant botulinum toxin of SEQ ID NO. 10 was prepared at 1306 U / mL and recombinant botulinum toxin of SEQ ID NO. 11 was prepared at 1056 U / mL and used in the experiment. For the placebo (placebo group), only the botulinum toxin was excluded from the recombinant botulinum toxin, while the remaining excipient components were used identically.

[0207] Botulinum toxin type A products and recombinant botulinum toxin are DAS ED 50 Prior to test administration, it was diluted to concentrations of 6, 20, 60, and 200 U / mL, respectively, and IM LD50 50 Prior to test administration, the samples were diluted to concentrations of 300, 450, and 675 U / mL, respectively. A total of two replicate tests were performed.

[0208] The day the test substance was administered was designated as Day 0, and after anesthetizing mice using an injectable anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), the group composition (DAS ED in Table 11) 50 Test group, IM LD in Table 12 50 Each test substance was administered to the right calf muscle of mice at a dose of 0.2 mL / kg using a Hamilton syringe according to the test group.

[0209] Group Animals Administered Substance Dosage (U / kg) Route of Administration Evaluation Index 16 Placebo 0 Right calf muscle DAS2 6BoNT / A1.2 Right calf muscle DAS3 6BoNT / A4 Right calf muscle DAS4 6BoNT / A12 Right calf muscle DAS5 6BoNT / A40 Right calf muscle DAS6 6 Sequence No. 101.2 Right calf muscle DAS7 6 Sequence No. 104 Right calf muscle DAS8 6 Sequence No. 1012 Right calf muscle DAS9 6 Sequence No. 1040 Right calf muscle DAS10 6 Sequence No. 111.2 Right calf muscle DAS11 6 Sequence No. 114 Right calf muscle DAS12 6 Sequence No. 1112 Right calf muscle DAS13 6 Sequence No. 1140 Right calf muscle DAS

[0210] Group Animals Administered Substance Dosage (U / kg) Route of Administration Evaluation Index 1 10BoNT / A60 Right calf muscle Survival 2 10BoNT / A90 Right calf muscle Survival 3 10BoNT / A135 Right calf muscle Survival 4 10 Sequence No. 1060 Right calf muscle Survival 5 10 Sequence No. 1090 Right calf muscle Survival 6 10 Sequence No. 10135 Right calf muscle Survival 7 10 Sequence No. 1160 Right calf muscle Survival 8 10 Sequence No. 1190 Right calf muscle Survival 9 10 Sequence No. 11135 Right calf muscle Survival

[0211] The evaluation was performed using the digit abduction score (DAS) method, which visually assesses the degree of muscle paralysis in mice. The DAS values ​​listed in Table 11 indicate the degree of muscle paralysis based on the shape of the toes on the side administered to the mouse. According to the test group composition in Table 11, DAS measurements were evaluated from day 1 to day 14 after administration at doses of 1.2, 4, 12, and 40 U / kg, and according to the test group composition in Table 12, the occurrence of deaths at each dose was checked for 14 days after administration.

[0212] Statistics: DAS ED for the highest DAS score for each individual in the DAS exam 50 It was calculated using a 3-metric logistic model after fixing the minimum score of 0 and the maximum score of 4 using Graphpad Prism. IM LD 50 The values ​​were calculated for the number of deceased subjects for each administered dose using probit analysis with SPSS software (SPSS Inc., IL, USA).

[0213] Results: As shown in Table 11, after single dose-wise administration of the botulinum toxin type A product and the recombinant botulinum toxins of the example to the right gastrocnemius muscle of mice, DAS ED 50As a result of calculating [the values], the average values ​​from the two repeated tests were 1.7 U / kg for the BoNT / A administration group, 1.2 U / kg for the recombinant botulinum toxin administration group of SEQ ID NO. 10, and 1.9 U / kg for the recombinant botulinum toxin administration group of SEQ ID NO. 11, showing an approximately 1.4-fold increase in the recombinant botulinum toxin administration group of SEQ ID NO. 10 compared to the BoNT / A administration group (Table 13). As shown in Table 12, the occurrence of mortality was observed for 14 days after administering the botulinum toxin type A product and the recombinant botulinum toxins of the examples to the right calf muscle of mice, and the median lethal dose (IM LD50) was determined. 50 As a result of calculating the values, the average of the two tests for the BoNT / A administration group was observed to be 61.7 U / kg, for the recombinant botulinum toxin administration group of SEQ ID NO. 10 it was 78.0 U / kg, and for the recombinant botulinum toxin administration group of SEQ ID NO. 11 it was 97.1 U / kg, showing an increase of approximately 1.3 times for the recombinant botulinum toxin administration group of SEQ ID NO. 10 and approximately 1.6 times for the recombinant botulinum toxin administration group of SEQ ID NO. 11 compared to the BoNT / A administration group (Table 13).

[0214] Article Average DAS ED 50 (U / kg) Average IM LD 50 (U / kg)Safety Margin*BoNT / A1.761.736.3 Sequence No. 101.278.065.0 Sequence No. 111.997.151.1

[0215] *Safety margin: IM LD 50 (U / kg) / DAS ED 50 (U / kg)DAS ED 50 Wow IM LD 50The safety margin was calculated from the results and found to be 36.3 for the BoNT / A administration group, 65.0 for the recombinant botulinum toxin administration group of SEQ ID NO. 10, and 51.1 for the recombinant botulinum toxin administration group of SEQ ID NO. 11. Compared to the BoNT / A administration group, a 1.8-fold increase was observed in the recombinant botulinum toxin administration group of SEQ ID NO. 10 and a 1.4-fold increase in the recombinant botulinum toxin administration group of SEQ ID NO. 11. These results confirmed the strong efficacy and improved safety effects of the recombinant botulinum toxin.

[0216] Example 8: Evaluation of the efficacy of recombinant botulinum toxin in mice

[0217] (1) Recombinant botulinum toxin of sequence numbers 22 and 23

[0218] The recombinant botulinum toxin of SEQ ID NO. 22 was prepared at 416 U / mL and the recombinant botulinum toxin of SEQ ID NO. 23 was prepared at 424 U / mL for the experiment, and the composition of the test groups is as shown in Table 14 below. The test substances or measurement methods used for the efficacy evaluation were conducted in the same manner as in the example of the recombinant botulinum toxin of SEQ ID NO. 10 described above. For CMAP measurement, the method used was the same as in Example 6. AAC The value was calculated.

[0219] Group Animals Administered Substance Dosage (U / kg) Route of Administration Evaluation Index 16 Placebo Right calf muscle DAS, CMAP 26 BoNT / A1.2 Right calf muscle DAS 36 BoNT / A4 Right calf muscle DAS 46 BoNT / A12 Right calf muscle DAS, CMAP, Body weight 56 ​​Sequence No. 221.2 Right calf muscle DAS 66 Sequence No. 224 Right calf muscle DAS 76 Sequence No. 2212 Right calf muscle DAS, CMAP, Body weight 86 Sequence No. 231.2 Right calf muscle DAS 96 Sequence No. 234 Right calf muscle DAS 106 Sequence No. 2312 Right calf muscle DAS, CMAP, Body weight

[0220] After administering a single dose of botulinum toxin type A product and botulinum toxin SEQ NO. 22 at doses of 1.2, 4, and 12 U / kg to the right calf muscle of a mouse, the results of the DAS evaluation showed that in the group administered with botulinum toxin SEQ NO. 22 at 1.2 U / kg, a significant increase in DAS scores compared to BoNT / A was observed on days 1, 3, 4, and 7 after administration, and in the group administered with botulinum toxin SEQ NO. 23 at 1.2 and 4 U / kg, an increase in DAS scores compared to BoNT / A was observed (Fig. 5). As a result of comparing the duration of action by calculating the DAS recovery time for each individual, the average times for the BoNT / A 1.2, 4, and 12 U / kg administration groups were confirmed to be 9.8, 21.0, and 25.3 days, respectively, while for the botulinum toxin administration groups of SEQ ID NO. 22, the average times were 15.2, 23.0, and 37.4 days, respectively. In other words, compared to wild-type BoNT / A, the duration of action of the recombinant polypeptide of the present invention was observed to increase by 1.6, 1.1, and 1.5 times for each dose, and the DAS recovery times for the botulinum toxin administration groups of SEQ ID NO. 23 were 16.3, 27.3, and 35.5 days, respectively, showing an increase in the duration of action by 1.7, 1.3, and 1.4 times for each dose (Table 15). In addition, in the 12 U / kg administration groups of botulinum toxin SEQ NO. 22 and botulinum toxin SEQ NO. 23, the duration of action was significantly increased compared to the BoNT / A administration group (Table 15). DAS ED 50 The results showed that the BoNT / A administration group had a value of 3.2 U / kg, the SEQ ID NO. 22 administration group had 1.1 U / kg, and the SEQ ID NO. 23 administration group had 2.1 U / kg, indicating 2.9 times and 1.5 times stronger efficacy, respectively, compared to the BoNT / A administration group. On the 7th day after administration, CMAP measurements confirmed the strong efficacy, with a significant decrease in injection site CMAP values ​​observed in the botulinum toxin groups of SEQ ID NO. 22 and SEQ ID NO. 23 compared to the BoNT / A administration group; however, no significant difference was observed in the contralateral injection site CMAP values ​​compared to the BoNT / A administration group (Fig. 6). CMAPAAC As a result of calculating and comparing [values], the average value for the BoNT / A administration group was 2077.1, the average value for the botulinum toxin administration group of SEQ ID NO. 22 was 2680.3, and the average value for the botulinum toxin administration group of SEQ ID NO. 23 was 2612.9, showing a significant 1.3-fold increase compared to the BoNT / A administration group. In the 12 U / kg administration group, the change in body weight at week 1 compared to pre-administration was 96% for the BoNT / A administration group, 96% for the botulinum toxin administration group of SEQ ID NO. 22, and 97% for the botulinum toxin administration group of SEQ ID NO. 23, showing an effect equivalent to that of the BoNT / A administration group. From the above results performed in mice, the strong efficacy and extended duration of action of the recombinant polypeptides botulinum toxin of SEQ ID NO. 22 and botulinum toxin of SEQ ID NO. 23 were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0221] Average DAS duration of action (days) Growth rate (times) Substance 1.2 U / kg 4 U / kg 12 U / kg 1.2 U / kg 4 U / kg 12 U / kg BoNT / A 9.8 21.0 25.3 N / AN / AN / A Sequence No. 2215.2b 23.0 37.4 *1.6 1.1 1.5 Sequence No. 2316.3a 27.3a 35.5 *1.7 1.3 1.4

[0222] *p<0.05, a p=0.05, b p=0.08, comparison with the group administered equal doses of BoNT / A, two-tailed t-test or Mann-Whitney test

[0223] (2) Recombinant botulinum toxin of sequence numbers 14 and 15

[0224] The botulinum toxin type A product (BoNT / A) is the same as in Example 1, and the recombinant botulinum toxin of SEQ ID NO. 14 was prepared at 394 U / mL and the recombinant botulinum toxin of SEQ ID NO. 15 was prepared at 419 U / mL for use in the experiment. The composition of the test group is as shown in Table 16, and the remaining details are the same as those described in (1) of Example 8 above. However, CMAP AAC ...was not produced.

[0225] Group Animals Administered Substance Dose (U / kg) Route of Administration Evaluation Index 16 Placebo-Right calf muscle DAS, CMAP 26 BoNT / A1.2 Right calf muscle DAS 36 BoNT / A4 Right calf muscle DAS 46 BoNT / A12 Right calf muscle DAS, CMAP 56 Sequence No. 141.2 Right calf muscle DAS 66 Sequence No. 144 Right calf muscle DAS 76 Sequence No. 1412 Right calf muscle DAS, CMAP 86 Sequence No. 151.2 Right calf muscle DAS 96 Sequence No. 154 Right calf muscle DAS 106 Sequence No. 1512 Right calf muscle DAS, CMAP

[0226] After administering a single dose of botulinum toxin type A and recombinant botulinum toxin at doses of 1.2, 4, and 12 U / kg to the right calf muscle of mice, the results of the DAS evaluation showed that for the botulinum toxin of SEQ ID NO. 14, an increase in the DAS score was observed in the 4 U / kg administration group compared to BoNT / A (Fig. 7). When the DAS recovery time was calculated for each individual and the duration of action was compared, an equivalent or greater duration of action was observed in the recombinant botulinum toxin administration group compared to BoNT / A (Table 17). The DAS ED50 calculation results showed that the BoNT / A administration group was 5.8 U / kg, the SEQ ID NO. 14 administration group was 3.3 U / kg, and the SEQ ID NO. 15 administration group was 3.0 U / kg, indicating that the efficacy was 1.8 times and 1.9 times stronger, respectively, compared to the BoNT / A administration group. As a result of measuring CMAP on day 7 after administration, a significant decrease in injection site CMAP values ​​was observed in the groups administered botulinum toxin SEQ NO. 14 and SEQ NO. 15 compared to the BoNT / A group, confirming a strong efficacy, and a significant increase in non-injection site CMAP was observed in the group administered botulinum toxin SEQ NO. 15 compared to the BoNT / A group, confirming a diffusion inhibitory effect (Fig. 8). Regarding the change in body weight at week 1 compared to pre-administration in the 12 U / kg administration group, the average change was 95% in the BoNT / A group and 97% in the SEQ NO. 14 group, with no significant difference observed compared to the BoNT / A group, while the average change was 111% in the SEQ NO. 15 group, showing a significant increase in body weight compared to the BoNT / A group (p < 0.001). From the above results performed in mice, the strong efficacy, increased duration of action, and improved diffusion effect of the recombinant botulinum toxin of the present invention compared to botulinum toxin type A product (BoNT / A) were confirmed.

[0227] Average DAS duration of action (days) Growth rate (times) Substance 1.2 U / kg 4 U / kg 12 U / kg 1.2 U / kg 4 U / kg 12 U / kg BoNT / A 10.5 20.8 26.3 N / AN / AN / A Sequence No. 1413.8 20.5 33.8 1.3 1.01.3 Sequence No. 1514.7 22.8 31.5 1.4 1.11.2

[0228] (3) Recombinant botulinum toxin of sequence numbers 12 and 13

[0229] The recombinant botulinum toxin of SEQ ID NO. 12 was prepared at 559 U / mL, and the recombinant botulinum toxin of SEQ ID NO. 13 was prepared at 440 U / mL, and the composition of the test group is as shown in Table 18 below. The test substance or measurement method used for the efficacy evaluation was carried out in the same manner as in the examples of the recombinant botulinum toxins of SEQ ID NO. 22 and 23 described above.

[0230] Group Animal Administered Substance Dose (U / kg) Route of Administration Evaluation Index 16 Placebo 0 Right calf muscle DAS, CMAP 26 BoNT / A 1.2 Right calf muscle DAS 36 BoNT / A 4 Right calf muscle DAS 46 BoNT / A 12 Right calf muscle DAS, CMAP, Body weight 56 ​​Sequence No. 12 1.2 Right calf muscle DAS 66 Sequence No. 12 4 Right calf muscle DAS 76 Sequence No. 12 12 Right calf muscle DAS, CMAP, Body weight 86 Sequence No. 13 1.2 Right calf muscle DAS 96 Sequence No. 13 4 Right calf muscle DAS 106 Sequence No. 13 12 Right calf muscle DAS, CMAP, Body weight

[0231] After single-dose administration of botulinum toxin type A and botulinum toxins SEQ ID NOs. 12 and 13 at doses of 1.2, 4, and 12 U / kg to the right gastrocnemius muscle of mice, DAS evaluation results showed that the 1.2 and 4 U / kg groups demonstrated an increase in DAS scores compared to BoNT / A, indicating an equivalent or superior effect, while the 12 U / kg group showed an equivalent effect (Fig. 9). When the duration of action was compared by calculating the DAS recovery time for each individual, the duration of action was significantly increased in the 1.2 U / kg group of botulinum toxin SEQ ID NO. 12 and the 4 U / kg group of botulinum toxin SEQ ID NO. 13 compared to the BoNT / A group (Table 19). DAS ED 50 The results showed that the BoNT / A administration group had a value of 3.0 U / kg, the botulinum toxin administration group of SEQ ID NO. 12 had 2.5 U / kg, and the botulinum toxin administration group of SEQ ID NO. 13 had 2.8 U / kg, indicating 1.2 and 1.1 times stronger efficacy, respectively, compared to the BoNT / A administration group. On the 7th day after administration, CMAP measurements showed equivalent efficacy in the injection site CMAP value, while a significant increase in the contralateral injection site CMAP value was observed in the SEQ ID NO. 12 administration group (Fig. 10). CMAP AAC As a result of calculating and comparing [values], the average value for the BoNT / A administration group was 2502.2, and the average value for the botulinum toxin administration group of SEQ ID NO. 13 was 3041.9, showing a significant 1.2-fold increase compared to the BoNT / A administration group. Regarding the change in body weight at week 1 compared to pre-administration in the 12 U / kg administration group, the average change was 96% for the BoNT / A administration group, 95% for the botulinum toxin administration group of SEQ ID NO. 12, and 97% for the botulinum toxin administration group of SEQ ID NO. 13, indicating an equivalent level of efficacy compared to the BoNT / A administration group. From the above results performed in mice, the strong efficacy, increased duration of action, and improved diffusion effects of the recombinant botulinum toxins SEQ ID NO. 12 and 13 were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0232] Average DAS duration of action (days) Growth rate (times) Substance 1.2 U / kg 4 U / kg 12 U / kg 1.2 U / kg 4 U / kg 12 U / kg BoNT / A 5.5 16.3 38.7 N / AN / AN / A Sequence Number 12 11.2 **21.3 38.0 2.0 1.3 1.0 Sequence Number 138.8 25.0 * 29.3 1.6 1.5 0.8

[0233] *p<0.05, **p<0.01, comparison with the group administered the same dose of BoNT / A, two-tailed t-test

[0234] (4) Recombinant botulinum toxin of sequence number 16

[0235] The recombinant botulinum toxin of SEQ ID NO. 16 was prepared at 444 U / mL, and the composition of the test group is as shown in Table 20 below. The test substance or measurement method used for the efficacy evaluation was carried out in the same manner as in the examples of the recombinant botulinum toxins of SEQ ID NOs 22 and 23 described above.

[0236] Group Animal Administered Substance Dosage (U / kg) Route of Administration Evaluation Index 16 Placebo 0 Right calf muscle DAS, CMAP 26 BoNT / A 1.2 Right calf muscle DAS 36 BoNT / A 4 Right calf muscle DAS 46 BoNT / A 12 Right calf muscle DAS, CMAP, Body weight 56 ​​Sequence No. 16 1.2 Right calf muscle DAS 66 Sequence No. 16 4 Right calf muscle DAS 76 Sequence No. 16 12 Right calf muscle DAS, CMAP, Body weight

[0237] After single-dose administration of botulinum toxin type A and the recombinant botulinum toxin SEQ NO. 16 at doses of 1.2, 4, and 12 U / kg to the right gastrocnemius muscle of mice, the results of the DAS evaluation showed that a significant increase in DAS scores compared to BoNT / A was observed in the group administered with the 12 U / kg recombinant botulinum toxin SEQ NO. 16 on day 21 after administration, and increases in DAS scores compared to BoNT / A were also observed at other measurement points (Fig. 11). When the duration of action was compared by calculating the DAS recovery time for each individual, the duration of action was significantly increased in the group administered with the 4 U / kg recombinant botulinum toxin SEQ NO. 16 compared to the BoNT / A group (Table 21). DAS ED 50 The results showed that the BoNT / A administration group had a value of 2.0 U / kg, while the recombinant botulinum toxin administration group of SEQ ID NO. 16 had a value of 1.8 U / kg, indicating a 1.1-fold stronger efficacy compared to the BoNT / A group. On day 7 after administration, CMAP measurements confirmed the stronger efficacy of the recombinant botulinum toxin administration group of SEQ ID NO. 16, as a significant decrease in the injection site CMAP value was observed compared to the BoNT / A group; however, no significant difference was observed in the contralateral injection site CMAP value (Fig. 12). CMAP AAC As a result of calculating and comparing [values], the average value for the BoNT / A administration group was 2061.9, and the average value for the botulinum toxin administration group of SEQ ID NO. 16 was 2659.7, showing a significant 1.3-fold increase compared to the BoNT / A administration group. Regarding the change in body weight at week 1 compared to pre-administration in the 12 U / kg administration group, the average was 94% for the BoNT / A administration group and 93% for the recombinant botulinum toxin administration group of SEQ ID NO. 16, with no significant difference observed compared to the BoNT / A administration group. From the above results performed in mice, the strong efficacy and extended duration of action of the recombinant botulinum toxin of SEQ ID NO. 16 were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0238] Average DAS duration of action (days) Increase rate (times) Substance 1.2 U / kg 4 U / kg 12 U / kg 1.2 U / kg 4 U / kg 12 U / kg BoNT / A 11.218.732.7N / AN / AN / A Sequence number 1613.826.8*45.5 a 1.21.41.4

[0239] *p<0.05, a p=0.05, comparison with the group administered equal doses of BoNT / A, two-tailed t-test or Mann-Whitney test

[0240] Example 9: Evaluation of the efficacy of recombinant botulinum toxins SEQ ID NOs. 24 to 32 and 43 in mice

[0241] The efficacy evaluation was conducted under the same conditions as in Example 5 for the animals and rearing environment.

[0242] Test substance: Botulinum toxin type A product (BoNT / A) 100 units of Coretox (Lot No.: E623001, Meditox) was used. Recombinant botulinum toxin was prepared in liquid form for the experiment and diluted to concentrations of 6, 20, and 60 U / mL prior to administration. Ten types of recombinant botulinum toxin were used in the experiment, and a total of four tests were performed. After administering botulinum toxin type A product and recombinant botulinum toxin to the right calf muscle of mice at doses of 1.2, 4, and 12 U / kg, the duration of action was compared via DAS evaluation, and body weight at week 1 after administration at the 12 U / kg dose was compared.

[0243] Results: After administering a single dose of botulinum toxin type A (BoNT / A: Coretox) and 10 types of recombinant botulinum toxins at doses of 1.2, 4, and 12 U / kg to the right calf muscle of mice, the DAS evaluation results showed that for the recombinant botulinum toxin of SEQ ID NO. 24, a 1.6-fold and 1.1-fold increase in DAS duration compared to BoNT / A was observed at doses of 1.2 and 4 U / kg, respectively, while an equivalent duration of action was observed at 12 U / kg. For the recombinant botulinum toxin of SEQ ID NO. 25, a 1.3-fold, 1.4-fold, and 1.2-fold increase in the duration of DAS compared to BoNT / A was observed at doses of 1.2, 4, and 12 U / kg; for the recombinant botulinum toxin of SEQ ID NO. 26, a 1.2-fold, 1.1-fold, and 1.3-fold increase in the duration of DAS compared to BoNT / A was observed at doses of 1.2, 4, and 12 U / kg; and for the recombinant botulinum toxin of SEQ ID NO. 27, a 1.2-fold, 1.1-fold, and 1.1-fold increase in the duration of DAS compared to BoNT / A was observed at doses of 1.2, 4, and 12 U / kg. In the case of the recombinant botulinum toxin of SEQ ID NO. 28, a 1.2-fold and 1.3-fold increase in the duration of DAS compared to BoNT / A was observed at doses of 1.2 and 4 U / kg, and a duration of DAS equivalent to that of BoNT / A was observed at a dose of 12 U / kg. In the case of the recombinant botulinum toxins of SEQ ID NO. 29 to 31, a duration of DAS equivalent to that of BoNT / A was observed at doses of 1.2, 4, and 12 U / kg. In the case of the recombinant botulinum toxin of SEQ ID NO. 32, a 1.6-fold and 1.3-fold increase in the duration of DAS compared to BoNT / A was observed at doses of 1.2 and 4 U / kg, and a duration of DAS equivalent to that of BoNT / A was observed at a dose of 12 U / kg. In the case of sequence number 43, a 1.4-fold increase in the duration of action of DAS compared to BoNT / A was observed at a dose of 1.2 U / kg, and a duration of action equivalent to that of BoNT / A was observed at doses of 4 U / kg and 12 U / kg.

[0244] In addition, safety evaluations were conducted by observing changes in body weight after administering doses of 1.2, 4, and 12 U / kg of the recombinant botulinum toxins of SEQ ID NOs 28 to 32 and 43. As a result, all showed a level of body weight reduction equivalent to that of wild-type BoNT / A, confirming that they exhibit at least equivalent safety to wild-type botulinum toxins.

[0245] From the above results performed in mice, the increased duration of action or equivalent or greater safety of recombinant botulinum toxin compared to wild-type botulinum toxin type A product (BoNT / A) is confirmed.

[0246] Example 10: Evaluation of the efficacy of recombinant botulinum toxins of SEQ ID NOs. 18 and 19 in mice

[0247] The efficacy evaluation was conducted under the same conditions as in Example 5 for the animals and rearing environment.

[0248] Test substance: 100 units of botulinum toxin type A product (BoNT / A) Coretox (Lot No.: E623001, Meditox) were used. Recombinant botulinum toxin was prepared in liquid form for the experiment and diluted to a concentration of 60 U / mL prior to administration. Two types of recombinant botulinum toxin were used in the experiment, and a total of two tests were performed. After administering botulinum toxin type A product and recombinant botulinum toxin at a dose of 12 U / kg into the right calf muscle of mice, efficacy was compared via CMAP evaluation, and body weight was compared at 1 week after administration.

[0249] Results: When the CMAP values ​​at the injection site were compared at week 1 after a single dose of 12 U / kg of botulinum toxin type A product (BoNT / A: Coretox) and recombinant botulinum toxin SEQ No. 18 were administered to the right calf muscle of mice, a significant decrease was observed in the SEQ No. 18 group with an average CMAP value of 0.1 mV compared to the BoNT / A group with an average CMAP value of 0.9 mV (p<0.001). Regarding body weight change at week 1, 97% of the BoNT / A group and 93% of the SEQ No. 18 group were observed, so no significant difference was observed. When comparing the CMAP values ​​at the injection site at week 1 after a single dose of 12 U / kg of the recombinant botulinum toxin of SEQ ID NO. 19, a significant decrease was observed in the group administered with SEQ ID NO. 19 (average 0.3 mV) compared to the group administered with BoNT / A (average 1.3 mV) (p<0.01). Regarding body weight change at week 1, a 93% change in body weight was observed in both the BoNT / A and SEQ ID NO. 19 botulinum toxin groups, indicating equivalent efficacy. From the above results performed in mice, the strong efficacy and equivalent safety of the recombinant botulinum toxins SEQ ID NO. 18 and 19 compared to the botulinum toxin type A product (BoNT / A) were confirmed.

[0250] Example 11: Cell-based activity of recombinant botulinum toxin of SEQ ID NO. 44

[0251] Materials and Methods

[0252] The cell line used in the experiment was the same as in Example 2, and the cells were treated with BoNT / A (150 kDa, Medytox, Batch No.: RBTS2301) and recombinant botulinum toxin of SEQ No. 44 (150 kDa, Medytox) serially diluted to 300, 60, 12, 2.4, and 0.48 U / well for 48 hours. Afterward, the cell culture medium was replaced and the cells were cultured for an additional 48 hours, after which the cells were treated with lysis buffer to perform an ELISA test. The method of performing the ELISA test was the same as in Example 2.

[0253] result

[0254] As a result of analyzing the activity of cell-based wild-type and recombinant botulinum toxins using the ELISA method, an 11-fold increase in activity was confirmed in the recombinant botulinum toxin-treated group compared to wild-type botulinum toxin.

[0255] SEQ ID No. EC50 (U / well) Relative increase in activity compared to wild type BoNT / A 0.866 - SEQ ID No. 440.07811-fold increase

[0256] Through this, it was confirmed that the recombinant polypeptide can exhibit superior efficacy compared to the wild type.

[0257] A recombinant polypeptide comprising a receptor binding domain having an amino acid variation according to one embodiment exhibits superior efficacy compared to a wild-type receptor binding domain. A botulinum toxin comprising a recombinant polypeptide according to one embodiment exhibits increased efficacy, safety, or half-life compared to wild-type botulinum toxin.

Claims

It includes the receptor binding domain of botulinum toxin type A1, and A recombinant polypeptide in which the receptor binding domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but includes amino acid variations at one or more positions having different amino acids at the corresponding positions of botulinum toxin type A1. In Article 1, A recombinant polypeptide in which the position of the amino acid variation comprises at least one selected from the group consisting of N880, W1068, and N1090. In Article 1, A recombinant polypeptide in which the receptor binding domain comprises at least one amino acid variant selected from the group consisting of N880S, W1068M, and N1090S. In Article 2, A recombinant polypeptide wherein the receptor binding domain further comprises an amino acid variation at at least one position selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, Y1117, T1232, and L1278. In Paragraph 4, A recombinant polypeptide wherein the receptor binding domain further comprises at least one amino acid variant selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, Y1117F, T1232R, and L1278F. In Article 1, A recombinant polypeptide further comprising a translocation domain, wherein the translocation domain comprises a polypeptide selected from the group consisting of a translocation domain of botulinum toxin type A1, a translocation domain of botulinum toxin type A2, a translocation domain of botulinum toxin type A6, and variants thereof. In Article 6, It comprises a translocation domain of botulinum toxin type A1, wherein the translocation domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but comprises one or more amino acid variations at a position having different amino acids at a corresponding position of botulinum toxin type A1, or A recombinant polypeptide comprising the translocation domain of botulinum toxin type A1 and comprising an amino acid variation at position 589 In Article 6, A recombinant polypeptide comprising a translocation domain of botulinum toxin type A1, wherein the position of the amino acid variant of the translocation domain comprises at least one selected from the group consisting of D589, L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818. In Article 1, A recombinant polypeptide further comprising a light chain domain, wherein the light chain domain comprises a polypeptide selected from the group consisting of botulinum toxin type A1, botulinum toxin type A4, and variants thereof. In Article 9, The above light chain domain is, A first domain and a second domain of a botulinum toxin type A1 light chain; or a variant thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, comprising, or A recombinant polypeptide comprising a first domain and a second domain of a botulinum toxin type A1 light chain; or a variant thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, wherein leucine at position 260 of the light chain domain is substituted with phenylalanine and isoleucine at position 264 is substituted with arginine. In Article 1, The above receptor binding domain is a recombinant polypeptide in which the half-maximum inhibitory concentration value according to botulinum toxin heavy chain competitive analysis is lower than that of the wild-type receptor binding domain. In Article 1, It further includes light chain domains and translocation domains, A recombinant polypeptide having increased potency, safety, or half-life compared to wild-type botulinum toxin type A. In Article 12, The above recombinant polypeptide comprises at least one selected from the recombinant botulinum toxins of SEQ ID NOs. 8 to 32, SEQ ID NO. 43, and SEQ ID NO.

44. It includes the receptor binding domain of botulinum toxin type A1, and A recombinant polypeptide in which the receptor binding domain comprises amino acid variations at two or more positions selected from N954, Y1117, T1232, R1273, and L1278. In Article 14, A recombinant polypeptide in which the receptor binding domain comprises two or more amino acid variations selected from N954S, Y1117F, T1232R, R1273K, and L1278F. In Article 14, A recombinant polypeptide in which the receptor binding domain comprises amino acid variations at two or more positions selected from N954S, T1232R, and L1278F. In Article 14, A recombinant polypeptide in which the amino acid variants of the receptor binding domain include N954S and T1232R; N954S and L1278F; T1232R and L1278F; T1232R and R1273K; R1273K and L1278F; N954S, T1232R and L1278F; Y1117F, R1273K and L1278F; or T1232R, R1273K and L1278F. Includes a variant of the receptor binding domain of botulinum toxin type A1, and A recombinant polypeptide in which the half-maximum inhibitory concentration value according to the botulinum toxin heavy chain competitive analysis of the variant of the receptor binding domain is lower than that of the wild-type receptor binding domain. In Article 18, A recombinant polypeptide wherein the variant of the receptor binding domain comprises an amino acid variant at at least one position selected from the group consisting of N880, N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, W1068, N1090, Y1117, S1142, T1232, and L1278. It includes a variant in which one or more amino acids of the receptor binding domain sequence of botulinum toxin type A1 are modified, and The above amino acid variant comprises one or more selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, Y1117F, S1142Y, S1142R, S1142I, S1142F, S1142H, T1232R, and L1278F, and when administered to a subject requiring treatment, at least one of efficacy and safety is superior compared to wild-type botulinum toxin type A1, and the remainder is at least equivalent to the wild-type toxin, a recombinant botulinum toxin. In Article 20, A recombinant botulinum toxin comprising a light chain domain selected from the group consisting of botulinum toxin type A1 light chain, botulinum toxin type A4 light chain, and variants thereof. In Article 21, The above light chain domain is, A first domain and a second domain of a botulinum toxin type A1 light chain; or a variant thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, comprising, or A recombinant botulinum toxin comprising a first domain and a second domain of a botulinum toxin type A1 light chain; or a variant thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, wherein the leucine at position 260 of the light chain domain is substituted with phenylalanine and the isoleucine at position 264 is substituted with arginine. In Article 20, It includes a prepositional domain, The above-mentioned translocation domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but includes amino acid variations at one or more positions having different amino acids at the corresponding positions of botulinum toxin type A1, or A recombinant botulinum toxin in which the above-mentioned translocation domain comprises a translocation domain of botulinum toxin type A1 and comprises at least one amino acid variant selected from the group consisting of D589, E734, and E809. A recombinant botulinum toxin comprising a variant of the botulinum toxin type A light chain and a receptor binding domain comprising one or more selected amino acid variants selected from the group consisting of N880S, N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, W1068M, N1090S, S1142Y, S1142R, S1142I, S1142F, S1142H, Y1117F, T1232R, and L1278F. In Article 24, The receptor binding domain is a recombinant botulinum toxin comprising the recombinant polypeptides of SEQ ID NOs. 1 to 7, 33 to 42 and 45 to 49. In Article 24, The above variant of the botulinum toxin type A light chain is, A first domain and a second domain of a botulinum toxin type A1 light chain; or variants thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or variants thereof, comprising, or A recombinant botulinum toxin comprising a first domain and a second domain of a botulinum toxin type A1 light chain; or a variant thereof, and a third domain and a fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, wherein leucine at position 260 is substituted with phenylalanine and isoleucine at position 264 is substituted with arginine. In Article 24, The above-mentioned recombinant botulinum toxin is a recombinant botulinum toxin that, compared to wild-type botulinum toxin type A1, exhibits one or more higher values ​​for safety and duration of action when administered to an individual. It includes a botulinum toxin type A1 translocation domain, and A recombinant polypeptide in which the above-mentioned translocation domain is conserved with amino acids common to botulinum toxin type A2 and botulinum toxin type A6, but includes amino acid variations at one or more positions having different amino acids at the corresponding positions of botulinum toxin type A1. In Article 28, A recombinant polypeptide wherein the position of the amino acid variant of the above-mentioned translocation domain comprises at least one selected from the group consisting of L681, D696, I713, K730, E734, K779, N789, G804, E809, L815, K816, and A818. In Article 28, A recombinant polypeptide in which the above-mentioned translocation domain comprises at least one amino acid variation selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V.

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