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 polypeptides, compositions comprising the same, and uses and methods thereof

[0001] The present invention relates to a recombinant polypeptide, and more particularly, to a recombinant polypeptide comprising a receptor binding domain of botulinum toxin type A1 comprising one or more amino acid mutations 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 the neurotransmitter acetylcholine from axon terminals at 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), which facilitates translocation of the light chain into the cytosol, and a C-terminal domain (Hc), which recognizes and binds to cell-surface receptors on nerve cells. Once inside the cell, the light chain specifically proteolytically cleaves a portion of the soluble NSF-attachment protein receptor (SNARE), thereby inactivating neurotransmitter release. Botulinum toxins are divided 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 variations in their amino acid sequences. Among these, botulinum toxin type A1 has been extensively studied at the molecular level and in preclinical and clinical trials and is currently widely used in the pharmaceutical industry. In contrast, the pharmacological properties of other botulinum toxin type A subtypes have been less studied or reported due to the difficulty in isolating purified toxins. Currently, only some of the botulinum toxin type A subtypes have been characterized biochemically, cellularly, and in vivo. Reported studies have revealed that botulinum toxin type A subtypes exhibit distinct properties from botulinum toxin type A1 in terms of mechanisms of potency, intracellular transport, and persistence, through various in vitro and in vivo studies. Recently, in vitro characterization of botulinum toxin types A7 and A8 has been reported.

[0004] One object of the present invention is to provide a recombinant polypeptide comprising at least one 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 the recombinant botulinum toxin comprising the same.

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

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

[0008] A recombinant polypeptide according to one embodiment of the present invention may comprise a receptor binding domain of botulinum toxin type A1. The receptor binding domain is conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but may comprise amino acid mutations at one or more positions among positions having different amino acids at the corresponding positions in botulinum toxin type A1.

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

[0010] The receptor binding domain may further comprise an amino acid mutation at at least one position selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, Y1117, T1232, and L1278. As used herein, for example, comprising an amino acid mutation at position N954 means that amino acid 954, asparagine, is substituted with another amino acid, deleted, or modified.

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

[0014] In one embodiment, the recombinant polypeptide further comprises 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. For example, the translocation domain may comprise or consist of a translocation domain of botulinum toxin type A1 or a variant thereof.

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

[0016] In one embodiment, the recombinant polypeptide comprises a translocation domain of botulinum toxin type A1, wherein the position of the amino acid mutation of the translocation domain may include 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 comprise at least one amino acid mutation selected from the group consisting of D589K, E734K and E809K.

[0018] In one embodiment, the recombinant polypeptide 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 A4, and variants thereof.

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

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

[0021] In one embodiment, the receptor binding domain may have a lower half-maximal inhibitory concentration value in a botulinum toxin heavy chain competition assay compared to 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 potency, 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 NO: 8 to SEQ ID NO: 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, wherein the receptor binding domain may comprise an amino acid mutation at two or more positions selected from among N954, Y1117, T1232, R1273, and L1278. The receptor binding domain may comprise two or more amino acid mutations selected from among N954S, Y1117F, T1232R, R1273K, and L1278F. The receptor binding domain may comprise an amino acid mutation at two or more positions selected from among N954, T1232, and L1278. The receptor binding domain may comprise an amino acid mutation at two or more positions selected from among N954S, T1232R, and L1278F. The amino acid mutations of the receptor binding domain may be 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, a recombinant polypeptide comprises a receptor binding domain of a botulinum toxin type A1, and a half-maximal inhibitory concentration value of a variant of the receptor binding domain according to a botulinum toxin heavy chain competition assay may be lower than that of a wild-type receptor binding domain. The variant of the receptor binding domain may comprise an amino acid mutation 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 comprise at least one amino acid mutation 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 comprise an amino acid mutation at position S1142. The amino acid mutation may comprise S1142Y, S1142R, S1142I, S1142F or S1142H.

[0025] According to one embodiment of the present invention, the recombinant botulinum toxin comprises a mutant in which one or more amino acids among the amino acids of the receptor binding domain sequence of botulinum toxin type A1 are mutated, wherein the amino acid mutations include 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 in need of 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 recombinant botulinum toxin may comprise a light chain domain selected from the group consisting of the aforementioned botulinum toxin type A1 light chain, the botulinum toxin type A4 light chain, and variants thereof. In addition, the recombinant botulinum toxin may further comprise the aforementioned translocation domain. The recombinant botulinum toxin has a safety margin when administered to a subject compared to wild-type botulinum toxin type A1; and DAS. AUC　 or CMAP AAC ; at least one of which exhibits superior values, and the remainder may be at least equivalent to the wild-type toxin. For example, the recombinant botulinum toxin may have a safety margin when administered to a subject compared to wild-type botulinum toxin type A1; and DAS AUC　 or CMAP AAC ; at least one of which exhibits 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 may comprise a receptor binding domain comprising a variant of a botulinum toxin type A light chain and comprising at least one amino acid mutation 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 a recombinant polypeptide of SEQ ID NOs: 1 to 7, 33 to 42 and 45 to 49.

[0027] The recombinant botulinum toxin may comprise 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. The recombinant botulinum toxin may have at least one of safety and duration of action superior to wild-type botulinum toxin type A1 when administered to a subject in need of treatment, and the remaining aspects may be at least equivalent to the wild-type toxin. The recombinant botulinum toxin may have a safety margin when administered to a subject compared to wild-type botulinum toxin type A1; and DAS. AUC　 or CMAP AAC ; one or more of them may show a higher value.

[0028] According to one embodiment of the present invention, the recombinant polypeptide may comprise a botulinum toxin type A1 translocation domain. The translocation domain may comprise an amino acid mutation at one or more positions among positions that are conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but have different amino acids at the corresponding positions in botulinum toxin type A1.

[0029] The position of the amino acid mutation of the above 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 translocation domain may include at least one amino acid mutation selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V. A recombinant botulinum toxin including the above translocation domain may exhibit superior specific potency compared to a 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 thermal denaturation temperature (Tm) of 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 changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of sequence number 10 to the right calf muscle at doses of 1.2, 4, and 12 U / kg in mice. 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 changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of sequence number 11 to the right calf muscle at doses of 1.2, 4, and 12 U / kg in mice. The DAS values ​​on each measurement day were analyzed using the Mann-Whitney test ( a p = 0.065, bp = 0.093).

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

[0035] Figure 5 is a graph showing the temporal changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 22 and 23 to the right calf muscle at doses of 1.2, 4, and 12 U / kg in mice. 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 the CMAP values ​​at the injection site (ipsilateral) and non-injection site (contralateral) 7 days after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 22 and 23 at a dose of 12 U / kg to the right calf muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxin according to the injection site were analyzed using a two-tailed t-test (*p<0.05, **p<0.01).

[0037] Figure 7 is a graph showing the temporal changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 14 and 15 at doses of 1.2, 4, and 12 U / kg to the right calf muscle in mice. 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 the CMAP values ​​at the injection site (ipsilateral) and non-injection site (contralateral) 7 days after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 14 and 15 at a dose of 12 U / kg to the right calf muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxin according to the injection site were analyzed using a two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001).

[0039] Figure 9 is a graph showing the temporal changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 12 and 13 at doses of 1.2, 4, and 12 U / kg to the right calf muscle in mice. 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 the CMAP values ​​at the injection site (ipsilateral) and non-injection site (contralateral) 1 week (day 7) after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxins of sequence numbers 12 and 13 at a dose of 12 U / kg to the right calf muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxin according to the injection site were analyzed using a two-tailed t-test (*p<0.05).

[0041] Figure 11 is a graph showing the temporal changes in DAS values ​​after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of sequence number 16 to the right calf muscle at doses of 1.2, 4, and 12 U / kg in mice. 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 CMAP values ​​at the injection site (ipsilateral) and non-injection site (contralateral) 1 week (day 6) after administration of botulinum toxin type A product (BoNT / A) and recombinant botulinum toxin of sequence number 16 at a dose of 12 U / kg to the right calf muscle in mice. The CMAP values ​​of botulinum toxin type A product and recombinant botulinum toxin according to the injection site were analyzed using a two-tailed t-test (**p<0.01).

[0043] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly used in the art. To facilitate understanding of the present invention, the following definitions will apply, and singular expressions include plural expressions, and vice versa, unless the context clearly dictates otherwise.

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

[0045] When interpreting components, even if not explicitly stated otherwise, they are interpreted as including a tolerance. In this specification, the term "about" is used to encompass a typical tolerance in the relevant technical field. For example, the term "about" may mean that a tolerance of up to 5%, 10%, 15%, or 20% exists within a given value or range of values.

[0046] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. These terms apply to naturally occurring amino acid polymers, including non-naturally occurring amino acids in which one or more amino acid residues are synthetically formed, such as chemical analogs of related naturally occurring amino acids. These terms also encompass post-translational modifications of polypeptides, such as 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 neurons and execute a global cellular mechanism to inhibit neurotransmitter release.

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

[0049] For example, the botulinum toxin can be selected from the group consisting of types 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 sp. 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 can 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. BoNT subtypes include chimeric BoNTs, e.g., BoNT / DC, BoNT / CD, BoNT / FA, etc.

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

[0052] In one embodiment, the term “variant” of a botulinum toxin (including both wild type toxin and recombinant toxin) may mean a botulinum toxin that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identity with the sequence of a reference botulinum toxin. For example, the term “variant” of a botulinum toxin (including both wild type toxin and recombinant toxin) can mean a botulinum toxin having 1 to 10, 1 to 7, 1 to 5, or 1 to 3, or 1 amino acid mutation from 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 mutations. In one embodiment, the amino acid mutations include both conservative and non-conservative mutations, for example, conservative mutations in which an amino acid is mutated but the properties (hydrophilicity, hydrophobicity, etc.) of the mutated amino acid are maintained.

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

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

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

[0056] In one embodiment, the recombinant botulinum toxin composition may be a composition that does not contain animal proteins. For example, in one embodiment, the recombinant botulinum toxin composition may not contain a protein stabilizer derived from an animal. In one embodiment, the recombinant botulinum toxin composition may not include albumin, such as human serum albumin or recombinant human albumin.

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

[0058] A "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, such as albumin (e.g., human serum albumin or recombinant human albumin) and / or sodium chloride, is present in the pharmaceutical composition. A pharmaceutical composition is a preparation suitable for administration to a subject, such as a human patient. The pharmaceutical composition may be in a lyophilized form, for example, a solution formed after reconstitution of a lyophilized pharmaceutical composition with saline or water, or in the form of a solution that does not require reconstitution. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and / or albumin.

[0059] The term "active ingredient" refers to a biologically or physiologically effective substance in a formulation or composition. Specifically, in the context of a pharmaceutical formulation or composition, the term "active ingredient" refers to a constituent substance that exhibits the desired pharmaceutical effect. For example, the active ingredient of the present invention may be uncomplexed botulinum toxin, free of complexing proteins.

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

[0061] In one embodiment, the pharmaceutical composition may be administered transdermally, subcutaneously, or intramuscularly. In a specific embodiment, the composition may be administered locally to a muscle or group of muscles. For example, reduction of forehead wrinkles or skin wrinkles may be achieved by administering the composition transdermally or subcutaneously to the wrinkle. 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 prefilled syringe. The composition of one embodiment may be administered transdermally using a transdermal patch or microneedles.

[0062] "Pharmaceutical composition" refers to 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, such as albumin (human serum albumin or recombinant human albumin) and / or sodium chloride. The pharmaceutical composition is a preparation suitable for administration to a subject, such as a mammal including a human. The subject to which the composition of the present invention is administered may include, without limitation, a human or an animal, such as a human, pig, dog, cat, cow, horse, or rat.

[0063] The pharmaceutical composition may be in liquid or lyophilized form. If the pharmaceutical composition is a lyophilized formulation, the lyophilized pharmaceutical composition may be reconstituted using, for example, saline solution or water. The pharmaceutical composition may be in solution form. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and / or albumin.

[0064] "Treat," "treating," or "treatment" means the alleviation or reduction (including partial reduction, substantial reduction, almost complete reduction, and complete reduction), resolution, or prevention (whether temporary or permanent) of a disease, disorder, or condition, so as to achieve a desired therapeutic result, for example, by healing injured or damaged tissue, or by altering, changing, strengthening, improving, ameliorating, and / or beautifying an existing or recognized disease, disorder, or condition. As used herein, "treatment" includes prevention. "Prevention" means delaying the onset of a disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

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

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

[0067] (b) ameliorating the disease, disorder, or medical condition, i.e., eliminating or causing regression of the disease, disorder, or medical condition in a patient, including by antagonizing the effect of other treatments;

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

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

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

[0071] In one embodiment, a therapeutically effective amount of botulinum toxin is from about 0.01 U / kg to about 100 U / kg, from about 0.1 U / kg to about 100 U / kg, from about 0.2 U / kg to about 100 U / kg, from about 0.2 U / kg to about 50 U / kg, from about 0.2 U / kg to about 30 U / kg, from about 0.2 U / kg to about 10 U / kg, from about 0.2 U / kg to about 1 U / kg, from about 1.2 U / kg, from about 4 U / kg, or from about 12 U / kg. In another embodiment, the dose 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 for an adult weighing 60 kg.

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

[0073] The composition of one embodiment may be administered in a therapeutically effective amount, and the term "effective amount" or "therapeutically effective amount" as used herein refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to any medical treatment or prevention. The effective dosage level may be determined according to the severity of the disease, the activity of the drug, the patient's age, weight, health, and sex, sensitivity to the drug, the timing of administration, the route of administration, and the rate of excretion of the composition of the present disclosure, the duration of treatment, drugs used concurrently or in combination with the composition of the present disclosure, and other factors known in the medical arts.

[0074] In one embodiment, the botulinum toxin may be administered in single or multiple treatment sessions. The dosage may be administered as a single or divided injection at the injection site. In multiple treatment sessions, the botulinum toxin may be administered at intervals of no more than six months, four months, or three months. In multiple treatment sessions, the interval between botulinum toxin administrations includes the first treatment and the second treatment, and the dosage of the second treatment may be less than, more than, or the same as the dosage of the first treatment.

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

[0076] Botulinum toxin includes botulinum toxin derivatives. The botulinum toxin derivatives may be derivatives of native botulinum toxin or recombinant native botulinum toxin having botulinum toxin activity, which contain one or more chemical or functional modifications on a portion or a portion of the chain. For example, the botulinum toxin derivatives may be modified toxins having one or more amino acids deleted, modified, or substituted. The botulinum toxin may be a recombinant peptide, a fusion protein, or a hybrid neurotoxin prepared, for example, from subunits or domains of different toxin serotypes. The botulinum toxin may also be part of a whole molecule having toxic activity, or may be used as part of a combination or conjugated molecule, for example, a fusion protein.

[0077] The term 'domain' refers to a tertiary structure in a given protein sequence and a conserved portion that can evolve, function, and exist independently of the rest of the protein chain.

[0078] Because domains are independently stable, domains can be swapped between one protein and another by genetic engineering to create chimera proteins.

[0079] The term 'homology' refers to the degree of amino acid sequence identity between polypeptides. For example, if a first amino acid sequence is identical to a second amino acid sequence, the first and second amino acid sequences are 100% homologous.

[0080] The term "recombination" refers to the process by which genetic elements, such as DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid), are disassembled and reassembled, resulting in a sequence that deviates from the original sequence. Molecular biology experiments allow for the artificial recombining of DNA fragments. DNA thus artificially recombined is called recombinant DNA.

[0081] A recombinant polypeptide according to one embodiment of the present invention may comprise a receptor binding domain (RBD) of botulinum toxin type A1. The receptor binding domain may comprise amino acid mutations. The receptor binding domain may be conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but may comprise amino acid mutations at one or more positions among positions having different amino acids at the corresponding positions in botulinum toxin type A1.

[0082] For example, the amino acid mutation may be a substitution of at least a portion of the sequence of botulinum toxin type A1 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 amino acid mutation may be a substitution of at least a portion of the amino acid sequence of botulinum toxin type A1 with an amino acid other than the amino acid sequence common to botulinum toxin types A2 and A6. For example, asparagine, which is amino acid at position 880 of the receptor binding domain of botulinum toxin type A1, may be a substitution of an amino acid other than serine, which is amino acid at position 880 of botulinum toxin types A2 and 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 another amino acid, such as threonine or glutamine.

[0085] In one embodiment, the recombinant polypeptide may comprise at least one, for example, one, two, or three, amino acid mutations 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, amino acid mutations selected from the group consisting of N880S, W1068M, and N1090S. The amino acid mutations may each independently be selected from among deletions, modifications, and substitutions. For example, the amino acid mutations may be substitutions.

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

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

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

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

[0090] Additionally, the positions of the 9 amino acids that can be mutated were selected by selecting a sequence that is common among the sequences of botulinum toxin types A2 and A6, but different from that of botulinum toxin type A1.

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

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

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

[0094] Amino acid mutations can be independently selected from among deletions, modifications, and substitutions. For example, an amino acid mutation can be a substitution.

[0095] A recombinant polypeptide according to one embodiment of the present invention may comprise a translocation domain of a recombinant botulinum toxin type A1. The recombinant botulinum toxin type A1 translocation domain may comprise an amino acid mutation at one or more positions among positions that are conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but have different amino acids at the corresponding positions in the botulinum toxin type A1. For example, the positions of the amino acid mutations in the recombinant translocation domain of one embodiment may include at least one selected from the group consisting of L681, D696, 1713, K730, E734, K779, N789, G804, E809, L815, K816, and A818. For example, the recombinant translocation domain of one embodiment may comprise at least one amino acid mutation 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 a botulinum toxin type A1, and may comprise at least one amino acid mutation 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 a botulinum toxin type A1, and may comprise at least one amino acid mutation at a position selected from the group consisting of D589K, E734K, and E809K.

[0097] In one embodiment, the translocation domain comprises or consists of a translocation domain of a wild-type botulinum toxin type A1, or comprises or consists of a translocation domain of a botulinum toxin type A1 comprising at least one amino acid mutation 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., the 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., a recombinant translocation domain of the recombinant botulinum toxin type A1 described above). 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] In one embodiment, the recombinant polypeptide may comprise the recombinant receptor binding domain described above and / or the recombinant translocation domain described above. In one embodiment, the recombinant polypeptide comprises the receptor binding domain of the botulinum toxin type A1 described above and

[0100] In one embodiment, the recombinant polypeptide 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. The variant of each subdomain of botulinum toxin type A1 or A4 may have a sequence identity of at least 99.8%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 95%, at least 93%, at least 92%, at least 91%, or at least 90% to a sequence identity of each subdomain of wild-type botulinum toxin type A1 or A4. For example, a variant of each subdomain of botulinum toxin type A1 or A4 may comprise one, two, three, four, five, six, seven, eight or nine amino acid mutations 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 domains 1, 2, 3, and 4 according to the criteria below.

[0102] The first domain of the botulinum toxin type A1 light chain and the first domain of the A4 light chain are defined as the portion containing the α-exosite, which primarily interacts with the substrate SNAP25. The first domain of the botulinum toxin type A1 and the A4 light chain may comprise an amino acid at positions 1 to 20 or an amino acid at positions 90 to 150, for example, an amino acid at positions 1, 5, 10, 15 or 20 or an amino acid 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 that interacts with the substrate SNAP25, which is an α-exosite-containing portion, and comprises an amino acid at position 1, 5, 10, 15 or 20 to an amino acid at position 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150, for example, an amino acid at position 20 to 90, an amino acid at position 20 to 100, an amino acid at position 20 to 110, an amino acid at position 20 to 120, an amino acid at position 20 to 130, an amino acid at position 20 to 140, It may include an amino acid at positions 10 to 90, an amino acid at positions 10 to 100, an amino acid at positions 10 to 110, an amino acid at positions 10 to 120, an amino acid at positions 10 to 130, or an amino acid 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 the part that is mainly involved in the enzymatic activity of the light chain, contains a 170 loop, and is the part that binds to the cofactor zinc. The second domain of the botulinum toxin type A1 and A4 light chain can comprise 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, comprise a 170 loop, comprise a site for binding to a cofactor zinc, and comprise 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, It may include an amino acid at positions 101 to 260, an amino acid at positions 111 to 250, an amino acid at positions 121 to 240, an amino acid at positions 131 to 230, an amino acid at positions 141 to 220, or an amino acid 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 an amino acid 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 a 250 loop and a 370 loop that stabilize substrate binding and include sites involved in both the α-exosite and β-exosite substrate binding sites. The third domain of botulinum toxin type A1 and A4 light chains can comprise 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 a 250 loop and a 370 loop that stabilize substrate binding, and include sites 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. For example, it may include amino acids at positions 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 (SEQ ID NO: 4) of the botulinum toxin type A1 light chain and the fourth domain (SEQ ID NO: 8) of the A4 light chain can be defined as domains that include a region involved in the structural flexibility of the botulinum toxin type A light chain. The fourth domain of a botulinum toxin type A1 and A4 light chain can comprise 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 (SEQ ID NO: 4) of the botulinum toxin type A1 light chain and the fourth domain (SEQ ID NO: 8) of the A4 light chain comprise a region 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 amino acids 410, 414, 417, 420, 425, 430, 435, 440, 445, and 448, for example, amino acids 362 to 448, amino acids 372 to 438, or amino acids 382 to It may contain amino acids at positions 428 to 425. 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 contain amino acids at positions 397 to 425.

[0110] The light chain domain may be one in which 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 replaced 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 a homology of 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 to the sequence of the third domain of wild-type botulinum toxin type A4. For example, a variant of the third domain of a botulinum toxin type A4 may comprise one, two, three, four, five, six, seven, eight or nine amino acid mutations in the sequence of the third domain of a wild-type botulinum toxin type A4. Here, the amino acid mutations may each independently be selected from among deletions, modifications and substitutions, and for example, the amino acid mutation may be an amino acid substitution. Substantially the same as described for the variant of the third domain of a botulinum toxin type A4 may also be applied to the variant of the fourth domain of a botulinum toxin type A4, the variant of the first domain of a botulinum toxin type A1, the variant of the second domain of a botulinum toxin type A1, and the variant of the fourth domain of a botulinum toxin type A1 light chain described below. For example, these variants may be at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 95%, at least 93%, at least 92%, at least 91% or at least 90% homologous to the sequence of the wild-type domain, and may comprise one, two, three, four, five, six, seven, eight or nine amino acid mutations from the sequence of the wild-type domain.

[0111] The light chain domain may be one in which the sequence of the fourth domain among the first, second, third, and fourth domains of a 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 one in which the sequence of the second domain among the first, second, third, and fourth domains of a 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 further comprise a sequence of the fourth domain among the first, second, third, and fourth domains of a recombinant botulinum toxin type A light chain, substituted with the sequence of the fourth domain of a botulinum toxin type A1 or a variant thereof. In one embodiment, the light chain domain may comprise the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof. The light chain domain may further comprise the third domain and the fourth domain of a botulinum toxin type A4 light chain; or a variant thereof.

[0113] In one embodiment, each of the variants of the first, second, third or fourth domains of a botulinum toxin type A light chain may refer to a subdomain of a botulinum toxin type A light chain that has at least 98% homology or at least 99% homology with the first, second, third or fourth domains of a wild-type botulinum toxin type A light chain, respectively. In one embodiment, each of the variants of the first, second, third or fourth domains of a botulinum toxin type A light chain can mean a subdomain of a botulinum toxin type A light chain having from 1 to 10, from 1 to 7, from 1 to 5 or from 1 to 3 amino acid mutations, for example, from 1, 2, 3, or 4 domains of a wild-type botulinum toxin type A light chain.

[0114] In one embodiment, the light chain domain may comprise a recombinant light chain as disclosed in PCT-KR2022-008793 or PCT-KR2022-008798, which are incorporated herein by reference in their entirety.

[0115] In one embodiment, the light chain domain may comprise the first domain and the second domain of a wild-type botulinum toxin type A1 light chain; or a variant thereof; and the third domain and the fourth domain of a wild-type botulinum toxin type A4 light chain; or a variant thereof.

[0116] When the light chain domain of one embodiment is a variant, the variant of the subdomain may have at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, or at least 90% identity to the sequence of a light chain subdomain of a wild-type botulinum toxin. For example, the variant of the subdomain may comprise one, two, three, four, five, six, seven, eight, or nine amino acid mutations in the sequence of a light chain subdomain of a wild-type botulinum toxin.

[0117] In one embodiment, when the light chain domain is a variant, the third domain of the botulinum toxin type A4 light chain may be a variant. For example, the variant of the third domain of the botulinum toxin type A4 may have at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, or at least 90% identity to the sequence of the third domain of the wild-type botulinum toxin type A4. For example, the variant of the third domain of the botulinum toxin type A4 may comprise one, two, three, four, five, six, seven, eight, or nine amino acid mutations in the sequence of the third domain of the wild-type botulinum toxin type A4. For example, a variant of the third domain of botulinum toxin type A4 may have a mutation in the 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 a mutation in the leucine at position 260 replaced by phenylalanine and a mutation in the isoleucine at position 264 replaced by arginine relative to the full-length light chain domain.

[0118] The description of the light chain domain described above can be equally applied to the light chain domain described below, to the extent that there is no contradiction.

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

[0120] In one embodiment, the recombinant polypeptide may further comprise a cell-penetrating peptide. In one embodiment, the cell-penetrating peptide may be IMTP-8. In one embodiment, the cell-penetrating peptide may be linked to the recombinant polypeptide via a GS linker. The cell-penetrating peptide may be linked to the recombinant polypeptide directly or indirectly via the 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 art, 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 lower half-maximal inhibitory concentration (HMIC) value in a botulinum toxin heavy chain competition assay compared to a wild-type receptor binding domain. For example, in one embodiment, the receptor binding domain may have a lower HMIC value in an ELISA-based botulinum toxin heavy chain competition assay compared to a wild-type receptor binding domain.

[0123] In one embodiment, the recombinant polypeptide 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 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.

[0124] In one embodiment, when the translocation domain is a variant, the variant of the translocation domain may have a sequence identity of at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, or at least 85% to 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 subdomain may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acid mutations in the sequence of the light chain subdomain of a wild-type botulinum toxin, or may comprise thirty, twenty-five, twenty-eighteen, fifteen or fewer amino acid mutations.

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

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

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

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

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

[0130] In one embodiment, the recombinant polypeptide may comprise one or more sequences selected from SEQ ID NOs: 1 to 7, 33 to 42 and 45 to 49; SEQ ID NOs: 8 to 32, 43 and 44, or SEQ ID NOs: 1 to 49 of Table 1 described below.

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

[0132] For example, amino acid mutations in 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 comprise a receptor binding domain of botulinum toxin type A1. Here, the half-maximal inhibitory concentration value of a variant of the receptor binding domain according to a botulinum toxin heavy chain competition assay may be lower than that of a wild-type receptor binding domain. Here, the variant of the receptor binding domain may comprise an amino acid mutation 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 comprise an amino acid mutation at position S1142. For example, the amino acid mutation may comprise S1142Y, S1142R, S1142I, S1142F, or S1142H.

[0135] A recombinant botulinum toxin according to one embodiment of the present invention may include a mutant 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. In one embodiment, the recombinant botulinum toxin may have at least one of superior efficacy and safety compared to a wild-type botulinum toxin type A1 when administered to a subject in need of treatment, and the remainder may be at least equivalent to the wild-type toxin. For example, the recombinant botulinum toxin of one embodiment has a higher CMAP or DAS (e.g., CMAP) than the wild type botulinum toxin type A1. AUCor DAS AAC ); and at least one of weight loss or safety margin is superior, and the remainder may be at least equivalent to the wild-type toxin. In the present specification, “equivalent or greater” may mean that the corresponding value of the embodiment substance is not statistically significantly inferior in effect to the corresponding value of the comparative substance.

[0136] In one embodiment, the recombinant botulinum toxin may comprise a light chain domain selected from the group consisting of a botulinum toxin type A1 light chain, a botulinum toxin type A2 light chain, and variants thereof. Here, the light chain domain may be identically described with respect to the light chain domain described above.

[0137] For example, the light chain domain of the recombinant botulinum toxin of one embodiment may comprise 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. For example, the first domain of the light chain domain of the recombinant botulinum toxin may comprise a first domain of a botulinum toxin type A1 light chain or a variant thereof, the second domain may comprise a second domain of a botulinum toxin type A1 light chain or a variant thereof, the third domain may comprise a third domain of a botulinum toxin type A4 light chain or a variant thereof, and the fourth domain may comprise 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 mutation in the 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 a mutation in the leucine at position 260 replaced by phenylalanine and a mutation in the isoleucine at position 264 replaced by arginine relative to the full-length light chain domain.

[0139] In one embodiment, the recombinant botulinum toxin has a safety margin when administered to a subject compared to wild-type botulinum toxin type A1; and DAS AUC　 or CMAP AAC ; at least one of which exhibits a superior value, and the remainder may be at least equivalent to the wild-type toxin. In one embodiment of the recombinant botulinum toxin, 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. In one embodiment, the translocation domain may comprise at least one amino acid mutation selected from the group consisting of D589, E734, and E809. In one embodiment, the translocation domain may comprise at least one amino acid mutation selected from the group consisting of D589K, E734K, and E809K.

[0140] In one embodiment, the recombinant botulinum toxin comprises a receptor binding domain comprising a botulinum toxin type A light chain variant as described above (e.g., variants of the A1-1, A1-2, A4-3, A4-4 forms) and comprising one or more amino acid mutations 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 one embodiment, the receptor binding domain may comprise a recombinant polypeptide of SEQ ID NOs: 1 to 7, 33 to 42, and 45 to 49. In one embodiment, the recombinant botulinum toxin may comprise a translocation domain comprising one or more amino acid mutations of L260F and I264R.

[0141] In one embodiment, the variant light chain of the recombinant botulinum toxin may comprise 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. In one embodiment, the light chain domain may comprise a variant of a second domain of a 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 may be substituted with phenylalanine, and the isoleucine at position 264 may be substituted with arginine. In one embodiment, the recombinant botulinum toxin may exhibit reduced diffusion, increased duration of action, and rapid onset of action when administered to a subject compared to wild-type botulinum toxin. In one embodiment, the recombinant botulinum toxin may exhibit improved safety when administered to a subject compared to wild-type botulinum toxin type A1; and at least one of the durations of action may exhibit higher values, and the remainder may be at least equivalent to the wild-type toxin. For example, the recombinant botulinum toxin of one embodiment may have a higher safety margin when administered to a subject, and a DAS, compared to wild-type botulinum toxin type A1. 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 the wild type botulinum toxin type A1 when administered to a subject, and the DAS AUC　 or CMAP AAC This may be higher than the wild type toxin.

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

[0143] A botulinum toxin comprising a recombinant polypeptide of one embodiment may comprise a pharmaceutically acceptable excipient or additive. A pharmaceutically acceptable excipient or additive may be a stabilizer, an ionic compound, a surfactant, a buffer, a lyoprotectant, or a combination thereof, such as an amino acid (e.g., methionine), a salt (e.g., NaCl), a buffer, a non-ionic surfactant (e.g., a polysorbate, e.g., polysorbate 20, a poloxamer), a sugar (e.g., a monosaccharide, a disaccharide such as sucrose, trehalose, etc.), a sugar alcohol (e.g., sorbitol), or a combination thereof.

[0144] The botulinum toxin composition comprising the recombinant polypeptide of the present invention may 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, which comprises a botulinum toxin comprising a recombinant polypeptide as an active ingredient.

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

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

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

[0149] In one embodiment, the condition or disease may include wrinkles, square jaw, jowls, scars, laxity, acne, enlarged pores, decreased elasticity, sebum secretion, and / or keloids; and / or facial spasms, blepharospasm, torticollis, blepharospasm, cervical dystonia, mid-pharyngeal dystonia, spasmodic dysphonia, migraines, pruritus ani, and / or hyperhidrosis.

[0150] In one embodiment of the present invention, a botulinum toxin composition comprising a recombinant polypeptide may or may 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, but is not limited to, the 20 standard amino acids found in nature. The amino acid of one embodiment may include any amino acid known in the art as a stabilizer. For example, in one embodiment, the amino acid may include a non-polar amino acid, such as methionine, isoleucine, and / or tryptophan.

[0151] Hereinafter, the present invention will be described in more detail with examples, but these are only for the purpose of explaining the present invention and are not intended to limit the scope in any way.

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

[0153] A recombinant receptor binding domain expression vector (cloned into pET28a vector) was introduced into BL21(DE3) cells, a type of Escherichia coli (E. coli), to induce 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 example of the present invention. Expression was induced with IPTG ((Isopropyl β-D-1-thiogalactopyranoside). That is, after IPTG was added to the liquid medium, BL21 (DE3) cells cultured at an appropriate temperature for an appropriate time were centrifuged to precipitate the cells. The medium in the supernatant was removed, and the precipitated cells were resuspended in A buffer (20 mM sodium phosphate, 150 mM NaCl, 10 mM Imidazole, pH 7.3), and then the cells were disrupted using an ultrasonic disperser on ice. After centrifuging the disrupted cells, the supernatant from which cell debris had been removed was used to separate the recombinant polypeptide from the supernatant through binding of the His tag to the Ni-NTA resin. The amino acid sequence of the separated recombinant polypeptide is shown 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 positions of amino acid mutations in wild-type botulinum toxin and the information on the mutated amino acid. For example, SEQ ID NO: 1 refers to a recombinant polypeptide in which 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 SEQ ID NOs: 2, 6, 33, 34 and 35 to 43, 45 to 49 were analyzed through competition assay.

[0158] Differentiated Ntera-2 cells (CRL-1973, ATCC) were seeded in 96-well plates and cultured in DMEM / F12 (WelGene) supplemented with 5% FBS (Gibco), B27 (Gibco), and Antibiotic-antimycotic (Gibco) for 4 days at 37 °C in 5% CO2. After 4 days, the cells were treated simultaneously with 300 U / well BoNT / A (150 kDa, Medytox, Batch No.: RBTS2301) and various concentrations of BoNT HC (botulinum toxin heavy chain) for 48 h. The treatment concentrations of BoNT / A1 WT HC (botulinum toxin type A1 wild-type heavy chain, hereinafter A1 WT HC) and recombinant polypeptides were treated at 5-fold intervals from 5 to 15625 times the molar concentration of BoNT / A (in this specification, BoNT / A is defined to include 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 examples herein refers to wild-type BoNT / A unless otherwise stated). After treatment with BoNT / A and HC, the medium was replaced with fresh medium, and the cells were cultured for an additional 2 days, and then treated with lysis buffer to perform an ELISA test. The ELISA test was performed on anti-SNAP25 197After coating the immunoplate with antibody (Medytox), the cell lysate was reacted to capture BoNT / A-cleaved SNAP25, which was then detected using biotinylated-anti-SNAP25 antibody (LS Bio) and streptavidin-HRP (Thermo Scientific). After treating with a chromogenic solution (TMB substrate, Sigma-Aldrich), the color reaction was measured at a wavelength of 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, and the half-maximal inhibitory concentration (IC) was calculated using a four-parameter logistic (4PL) non-linear regression method according to the HC treatment concentration. 50 ) was calculated, and the IC50 (uM) value of the A1 WT HC was divided by the IC50 (uM) value of the calculated mutant HC, and the relative inhibitory activity value compared to the wild type A1 botulinum toxin heavy chain was recorded in the table below.

[0159]

[0160] Results from an ELISA-based botulinum toxin heavy chain competition assay revealed that the recombinant polypeptide according to one embodiment exhibited a higher relative inhibitory activity value compared to the wild-type receptor binding domain, confirming increased binding affinity to the receptor. This confirmed that the recombinant polypeptide according to one embodiment exhibits superior efficacy and improved safety compared to the wild-type polypeptide.

[0161] Example 3: Preparation and purification of domain-substituted recombinant botulinum toxin

[0162] After confirming the effectiveness of the receptor binding domains of SEQ ID NOS: 1 to 7, which were effective in Examples 1 and 2, a recombinant receptor binding domain further comprising additional amino acid substitutions in the sequences of SEQ ID NOS: 1 to 7 was designed. For example, the receptor binding domain included in the recombinant botulinum toxin of SEQ ID NO: 8 includes all the amino acid mutations of the recombinant polypeptide of SEQ ID NO: 7, as well as an additional mutation. Here, the additional mutation was selected at a position that is conserved as a common amino acid in wild-type botulinum toxin type A2 and botulinum toxin type A6, but has a different amino acid at the corresponding position in botulinum toxin type A1. The amino acid of botulinum toxin type A1 at that position was substituted with an amino acid that is conserved in botulinum toxin types A2 and A6.

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

[0164] Specifically, a recombinant full-length botulinum toxin gene, including recombinant botulinum toxin type A light chain and heavy chain, was cloned using the pMTL80000 vector system by site-directed mutagenesis. The cloned products are shown in Table 2 below. The recombinant botulinum toxin was used by producing a detoxified hall A-hyper strain in which the toxin gene was inactivated using the ClosTron system.

[0165] 시이벨벨경창전위 대녀수용체 보드대8A1A1A1(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] A detoxified Clostridium botulinum strain, including a strain in which the toxin gene is inactivated or knocked out, can be used in a method for producing a purified recombinant botulinum toxin or a purified recombinant botulinum toxin complex. A method for producing a strain in which the toxin gene is inactivated or knocked out can be used a method commonly used in the art. The recombinant botulinum toxins of SEQ ID NOs: 8 to 21 produced by the clostron method were purified into a 150 kDa form free of complex components. In addition, two recombinant polypeptides (SEQ ID NOs: 20 and 21, respectively) were additionally produced by additionally linking the cell-penetrating peptide IMTP8 to each of the recombinant botulinum toxins of SEQ ID NOs: 10 and 11 using a GS linker. All production of the recombinant botulinum toxins was performed in a facility permitted for toxin production in accordance with regulations of the authorities.

[0167] Example 4: Evaluation of thermal stability of recombinant botulinum toxin

[0168] Among the recombinant botulinum toxins manufactured in Example 3, the thermal stabilities of the recombinant toxins of SEQ ID NOs. 10 and 11 were compared. The thermal denaturation midpoint (Tm) was evaluated as a stability evaluation method, and a high Tm is generally desirable. The thermal stability of each domain-substituted recombinant botulinum toxin type A was evaluated using the thermal shift assay (PTS). The thermal shift assay was performed using the Protein Thermal Shift Dye Kit (Catalog No. 4461146, Applied Biosystems) according to the manufacturer's method. Specifically, 10 μg of each recombinant botulinum toxin was mixed with a protein thermal shift dye and buffer to make 20 μl, and the ROX signal was confirmed by increasing the temperature by 1 °C per 60 s from 20 °C to 95 °C using RT-PCR (C1000 thermal cycler equipped with a CFX96 optical reaction module, Bio-Rad). The melting temperature (Tm) of each recombinant botulinum toxin was determined based on the melting curve. The melting points of each recombinant botulinum toxin are shown in Fig. 1 and Table 3.

[0169] ConstructTm (°C)ΔTmTm_stdWild type (7s)52.80.00.3SEQ ID NO: 10(7s)54.21.30.3SEQ ID NO: 11(7s)54.31.50.3Wild type (7s)52.70.00.3SEQ ID NO: 12(7s)55.52.80.0SEQ ID NO: 13(7s)55.52.80.0SEQ ID NO: 14(7s)54.51.80.0SEQ ID NO: 16(7s)54.01.30.0SEQ ID NO: 20(7s)56.03.30.0

[0170] The results show that the thermal stability of the recombinant botulinum toxins of SEQ ID NOs: 10, 11 and 12, 13, 14, 16 and 20 increased compared to the wild type. Specifically, while the melting point of the 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 the 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 sequence number 10

[0173] Materials and Methods

[0174] Animals: Female CD1 (ICR) mice, 5-6 weeks old, were purchased from Orient Bio Co., Ltd., and after one week of acclimatization and quarantine, 6-7-week-old mice were used in the experiment. Sterilized solid food for laboratory animals (R40-10, SAFE, France) was fed ad libitum, and tap water was sterilized by high temperature and high pressure autoclaving and provided ad libitum. During the acclimatization, quarantine, and experimental periods, mice were raised under specific pathogen-free conditions set at a temperature of 23 ± 3℃, a relative humidity of 55 ± 15%, a lighting period of 12 h (8:00 AM to 8:00 PM), ventilation rate of 15 times / h, and illumination intensity of 150-300 Lux. This study was conducted after review and approval (A-2022-007, A-2024-005) by the Animal Experiment Ethics Committee of Medytox Co., Ltd.

[0175] Test material: 100 units of botulinum toxin type A product (BoNT / A) Coretox (Lot No.: E623001, Medytox) were used. Recombinant botulinum toxin of sequence number 10 was manufactured at 163 U / mL and used in the experiment. The potency of the recombinant botulinum toxin was measured using a mouse potency test method before the efficacy test, and the test was performed based on the derived potency value. In the efficacy test, the administered dose of each test material was administered identically based on the unit (U: a value indicating the biological activity of botulinum toxin, where 1 U is the half-lethal dose of mice by intraperitoneal administration).

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

[0177] The day on which the test substance was administered was set as day 0, and the mice were anesthetized using an injection anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine). Then, each test substance was administered to the right calf muscle of the mice at 0.2 mL / kg using a Hamilton syringe according to group composition Table 4.

[0178] Military animal number Administered substance dose (U / kg) Administration route Evaluation index 16 BoNT / A 1.2 Right calf muscle DAS 26 BoNT / A 4 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 used the digit abduction score (DAS), which visually assesses the degree of muscle paralysis in mice. The DAS values ​​shown in Table 5 (Mouse DAS Evaluation Criteria) indicate the degree of muscle paralysis based on the shape of the toe on the side where the drug was administered.

[0180] Score Criteria 0 Normal, no different from the shape of the foot on the non-injected side 1 Narrowing of the space between the toes, or two toes close together and the rest fully extended 2 Significant narrowing of the space between all toes, or three toes close together 3 Curved foot with four toes close together 4 Curved foot with all toes close together

[0181] Table 4 DAS measurements were performed at doses of 1.2, 4, and 12 U / kg according to the composition of the test group from 1 day after administration until the DAS score became 0. The average DAS recovery period was calculated by measuring the time when the DAS score became 0 for each individual. In the case of body weight, in order to compare the effect on safety, the change in body weight at 1 week after administration compared to the body weight before administration in the 12 U / kg high-dose administration group was compared between the botulinum toxin type A product administration group and the recombinant botulinum toxin administration group. Statistics: Graphpad Prism 7.05 (GraphPad Software Inc., CA, USA) was used for graph presentation and DAS ED. 50SPSS software 25.0 (SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. Normality test was performed using the Kolmogorov-Smirnov test. Nonparametric data were statistically analyzed using the Mann-Whitney test, and for parametric data, a two-tailed t-test was performed, and statistical significance was determined when the p-value was less than 0.05. Results: Referring to Figure 2, after a single administration of botulinum toxin type A product (BoNT / A: Coretox) and our recombinant botulinum toxin at doses of 1.2, 4, and 12 U / kg to the right calf muscle of the mouse, a significant increase in the DAS score was observed in the 1.2 U / kg recombinant botulinum toxin administration group on days 3, 4, and 7 after administration, and in the 4 U / kg recombinant botulinum toxin administration group on days 7, 14, and 21 after administration compared to BoNT / A. 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, respectively, after administration. In the recombinant botulinum toxin administration group of the same dose, recovery was observed in all individuals at 3, 5, and 8 weeks, which was an increase (Fig. 2). The DAS recovery time for each individual was calculated and the duration of action was compared. As a result, the average duration of action was 9.0, 19.8, and 33.8 days in the BoNT / A 1.2, 4, and 12 U / kg administration groups, respectively, and the average duration of action was 17.5, 29.2, and 42.8 days in 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 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 group (Table 6). In the 12 U / kg administration group, no significant difference was observed in the 1st week body weight change compared to before administration, with an average of 97% in the BoNT / A administration group and an average of 95% in the sequence number 10 administration group.

[0182] Average DAS duration of action (days) Substance 1.2 U / kg 4 U / kg 12 U / kg BoNT / A9.0 19.8 33.8 Sequence number 1017.5**29.2**42.8 † Growth rate (x) 1.91.51.3

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

[0184] (2) Recombinant botulinum toxin of sequence number 11

[0185] Except that the recombinant botulinum toxin of sequence number 11 was manufactured at 384 U / mL and used in the experiment, the efficacy evaluation was conducted in the same manner as in the example for the recombinant botulinum toxin of sequence number 10.

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

[0187] Average DAS duration of action (days)DAS ED 50 (U / kg) Substance 1.2 U / kg 4 U / kg 12 U / kg BoNT / A7.2 16.73 1.5 2.9 Sequence number 1114.0*25.3 † 36.21.9 Growth rate (x) 1.91.51.11.5

[0188] *p<0.05, †p=0.089, compared to the same dose of BoNT / A administered group, two-tailed t-test or Mann-Whitney test. From the above results performed on mice, the strong efficacy and increased duration of action of the recombinant botulinum toxin of sequence number 11 were confirmed compared to the botulinum toxin type A product (BoNT / A).

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

[0190] Materials and Methods

[0191] Animals: Six-week-old female SD (Sprague-Dawley) rats were purchased from Orient Bio Co., Ltd. After one week of acclimatization and quarantine, seven-week-old rats were used for 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 Animal Experiment Ethics Committee of Medytox Co., Ltd.

[0192] Test Material: Botulinum toxin type A product (BoNT / A) was used at 100 units of Coretox (Lot No.: E623001, Medytox). Recombinant botulinum toxin of sequence number 10 was manufactured at 444 U / mL, and recombinant botulinum toxin of sequence number 11 was manufactured at 366 U / mL and used in the experiment. The placebo group used the same excipient components as the recombinant botulinum toxin except for botulinum toxin.

[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 on which the test substance was administered was set as day 0, and the mice were anesthetized using an injection anesthetic (60 mg / kg ketamine hydrochloride + 10 mg / kg xylazine). Then, each test substance was administered to the right calf muscle of the rats at 0.1 mL / kg using a Hamilton syringe according to group composition Table 8.

[0195] Military animal number Administered substance dose (U / kg) Administration route 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 ID NO: 10) 12 Right calf muscle DAS, CMAP, body weight 46 Recombinant botulinum toxin (SEQ ID NO: 11) 12 Right calf muscle DAS, CMAP, body weight

[0196] The evaluation used the digit abduction score (DAS) method, which visually evaluates the degree of muscle paralysis in rats, and the compound muscle action potential (CMAP) test method, which measures the action potential of muscles in response to external electrical stimulation. The DAS value shown in Table 9 (rat DAS evaluation criteria) indicates the degree of muscle paralysis by the shape of the toe on the administered side in rats, and the CMAP value indicates the degree of direct muscle contraction inhibition caused by nerve block.

[0197] Score 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 and fusion of all toes

[0198] CMAP was measured in the gastrocnemius muscle of each rat (the non-toxin-treated site) using an UltraPro S100 device. After anesthesia with an intravenous anesthetic, the hair at the measurement site was shaved, and the rats were positioned in the prone position. The cathode was placed over the sciatic nerve on the leg to be measured, and the anode was placed approximately 0.5 cm from the sciatic nerve. The recording and reference electrodes were placed over the gastrocnemius and Achilles tendon, respectively, and the ground electrode was placed over the rectus femoris. The stimulation level and duration were 25–30 mA and 0.2 ms, and the amplifier filter range was set to 2–10 K at 60 Hz. The height from the base to the peak of the waveform was recorded as the CMAP measurement value. According to the composition of the test groups in Table 8, the DAS was measured from day 3 until the DAS score reached 0. The average DAS recovery period was calculated by measuring the time when the DAS score became 0 for each individual. CMAP measurements were taken in the left calf muscle 7 days after administration to assess the spread of the toxin to non-administered sites. In addition, for CMAP measurements, CMAP AAC (CMAP baselineAUC - CMAP AUC ) For comparison, measurements were taken at 2-3 week intervals until recovery of CMAP values ​​was observed, and the results were calculated based on the reported literature (Dermatologic Surgery 2020; 46(12): e132). Statistics: Same as Example 1.

[0199] Results: After a single administration of botulinum toxin type A and recombinant botulinum toxins at a dose of 12 U / kg to the right calf muscle of rats, the DAS evaluation showed that the 12 U / kg recombinant botulinum toxin group with a sequence number 10 showed a significant increase in DAS scores compared to BoNT / A on days 49 and 56 after administration (Fig. 4). The recombinant botulinum toxin group with a sequence number 11 also showed an increase in DAS scores compared to BoNT / A (Fig. 4). Recovery was observed in all individuals in the BoNT / A group at 10 weeks after administration, and in the recombinant botulinum toxin group with the same dose of SEQ ID NO: 10, recovery was observed in all individuals at 14 weeks, and in the botulinum toxin group with a sequence number 11, recovery was observed in all individuals at 11 weeks (Fig. 4). The DAS recovery time for each individual was calculated and the duration of action was compared. As a result, the average duration of action was confirmed to be 50.2 days in the BoNT / A administration group, 72.3 days in the recombinant botulinum toxin administration group with sequence number 10, and 65.3 days in the recombinant botulinum toxin administration group with sequence number 11, 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) Increase rate (fold) Substance 12 U / kg BoNT / A 50.2 n / a Sequence number 1072.3*1.4 Sequence number 1165.3*1.3

[0201] *p<0.05, compared to the BoNT / A administration group, two-tailed t-test. 7 days after botulinum toxin administration, no significant difference was observed in the CMAP measurement results of the calf muscle on the contralateral side in the recombinant botulinum toxin administration group of sequence number 10 compared to the BoNT / A administration group, while a significantly increased CMAP value was observed in the recombinant botulinum toxin administration group of sequence number 11 (Fig. 4). CMAP AACAs a result of calculating and comparing, the BoNT / A administration group had an average of 3558.5, the botulinum toxin administration group of sequence number 10 had an average of 3883.3, which was a significant increase of 1.1 times compared to the BoNT / A administration group, and the botulinum toxin administration group of sequence number 11 had an average of 3592.5, which was an equivalent or higher value. In the 12 U / kg administration group, the change in body weight at 1 week compared to before administration was an average of 107% in the botulinum toxin type A product administration group, an average of 108% in the botulinum toxin administration group of sequence number 10, and an average of 108% in the botulinum toxin administration group of sequence number 11, showing an effect at a level equivalent 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 were confirmed compared to botulinum toxin type A product (BoNT / A), and in the case of the recombinant botulinum toxin of sequence number 11, a decrease in proliferation was confirmed compared to the BoNT / A administration group.

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

[0204] Materials and Methods

[0205] The animals and breeding environment were the same as in Example 5, and this study was conducted after review and approval (A-2022-007, A-2022-008) by the Animal Experiment Ethics Committee of Medytox Co., Ltd.

[0206] Test Material: 100 units of Coretox (Lot No.: E623001, Medytox) were used for the botulinum toxin type A product (BoNT / A). The recombinant botulinum toxin of sequence number 10 was manufactured at 1306 U / mL, and the recombinant botulinum toxin of sequence number 11 was manufactured at 1056 U / mL and used in the experiment. The placebo (placebo group) was used with the same excipient components as the recombinant botulinum toxin except for the botulinum toxin.

[0207] Botulinum toxin type A products and recombinant botulinum toxin are DAS ED 50 Before test administration, it was diluted to concentrations of 6, 20, 60, and 200 U / mL, respectively, and IM LD 50 The solution was diluted to concentrations of 300, 450, and 675 U / mL before test administration. The test was repeated twice in total.

[0208] The day on which the test substance was administered was set as day 0, and the mice were anesthetized using an injection anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine) and then divided into groups (DAS ED in Table 11). 50 Test group, IM LD in Table 12 50 According to the test group, each test substance was administered at 0.2 mL / kg into the right calf muscle of the mouse using a Hamilton syringe.

[0209] Military animal number administered substance dose (U / kg) route of administration evaluation index 16 Placebo 0 Right calf muscle DAS 2 6 BoNT / A 1.2 Right calf muscle DAS 3 6 BoNT / A 4 Right calf muscle DAS 4 6 BoNT / A 12 Right calf muscle DAS 5 6 BoNT / A 40 Right calf muscle DAS 66 Sequence number 101.2 Right calf muscle DAS 76 Sequence number 104 Right calf muscle DAS 86 Sequence number 1012 Right calf muscle DAS 96 Sequence number 1040 Right calf muscle DAS 106 Sequence number 111.2 Right calf muscle DAS 116 Sequence number 114 Right calf muscle DAS 126 Sequence number 1112 Right Calf muscle DAS136 Sequence number 1140 Right calf muscle DAS

[0210] Military animal number Administered substance dose (U / kg) Administration route Evaluation index 110 BoNT / A60 Right calf muscle Survival 210 BoNT / A90 Right calf muscle Survival 310 BoNT / A135 Right calf muscle Survival 410 Sequence number 1060 Right calf muscle Survival 510 Sequence number 1090 Right calf muscle Survival 610 Sequence number 10135 Right calf muscle Survival 710 Sequence number 1160 Right calf muscle Survival 810 Sequence number 1190 Right calf muscle Survival 910 Sequence number 11135 Right calf muscle Survival

[0211] The evaluation used the digit abduction score (DAS) method, which visually assesses the degree of muscle paralysis in mice. The DAS values ​​shown in Table 11 indicate the degree of muscle paralysis based on the shape of the toes on the administered side in mice. According to the composition of the test groups 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. The occurrence of deaths was checked for 14 days after administration at each administered dose according to the composition of the test groups in Table 12.

[0212] Statistics: DAS ED for highest DAS score for each subject in the DAS test 50 The minimum score was 0 and the maximum score was 4, and then the 3-indicator logistic model was used to calculate the IM LD. 50 The values ​​were calculated through probit analysis using SPSS software (SPSS Inc., IL, USA) for each dose of death.

[0213] Results: As shown in Table 11, after single administration of botulinum toxin type A product and recombinant botulinum toxin of the example at different doses to the right calf muscle of the mouse, DAS ED 50As a result of calculating the average of two repeated tests, the BoNT / A administration group was 1.7 U / kg, the recombinant botulinum toxin administration group of sequence number 10 was 1.2 U / kg, and the recombinant botulinum toxin administration group of sequence number 11 was 1.9 U / kg, and an increase of approximately 1.4 times was observed in the recombinant botulinum toxin administration group of sequence number 10 compared to the BoNT / A administration group (Table 13). As shown in Table 12, after administering the botulinum toxin type A product and the recombinant botulinum toxins of the examples to the right calf muscle of mice, the occurrence of deaths was observed for 14 days, and the median lethal dose (IM LD) was calculated. 50 ) was calculated, the BoNT / A administration group was observed to have an average of 61.7 U / kg in two tests, the recombinant botulinum toxin administration group of sequence number 10 was observed to have 78.0 U / kg, and the recombinant botulinum toxin administration group of sequence number 11 was observed to have 97.1 U / kg, indicating that the recombinant botulinum toxin administration group of sequence number 10 showed an increase of approximately 1.3 times and the recombinant botulinum toxin administration group of sequence number 11 showed an increase of approximately 1.6 times compared to the BoNT / A administration group (Table 13).

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

[0215] *Safety margin: IM LD 50 (U / kg) / DAS ED 50 (U / kg)DAS ED 50 Wow IM LD 50As a result of calculating the safety margin from the results, the BoNT / A administration group was 36.3, the recombinant botulinum toxin administration group with sequence number 10 was 65.0, and the recombinant botulinum toxin 1 administration group with sequence number 11 was 51.1. Compared to the BoNT / A administration group, the recombinant botulinum toxin administration group with sequence number 10 increased 1.8-fold and the recombinant botulinum toxin administration group with sequence number 11 increased 1.4-fold. From these results, the strong efficacy and safety improvement effect of recombinant botulinum toxin were confirmed.

[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 sequence number 22 was manufactured at 416 U / mL, and the recombinant botulinum toxin of sequence number 23 was manufactured at 424 U / mL, and the test group composition is as shown in Table 14 below. The test substances or measurement methods used for efficacy evaluation were carried out in the same manner as in the example of the recombinant botulinum toxin of sequence number 10 described above. In the case of CMAP measurement, CMAP was measured in the same manner as in Example 6. AAC The value was calculated.

[0219] Military animal number Administered substance dose (U / kg) Administration route 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 number 221.2 Right calf muscle DAS 66 Sequence number 224 Right calf muscle DAS 76 Sequence number 2212 Right calf muscle DAS, CMAP, body weight 86 Sequence number 231.2 Right calf muscle DAS 96 Sequence number 234 Right calf muscle DAS 106 Sequence number 2312 Right calf muscle DAS, CMAP, body weight

[0220] After single administration of botulinum toxin type A and botulinum toxin of sequence number 22 at doses of 1.2, 4, and 12 U / kg to the right calf muscle of the mouse, the DAS evaluation results showed that in the 1.2 U / kg botulinum toxin type A group with sequence number 22, a significant increase in DAS score was observed compared to BoNT / A on days 1, 3, 4, and 7 after administration, and in the botulinum toxin type A group with sequence number 23, an increase in DAS score compared to BoNT / A was observed in the 1.2 and 4 U / kg groups (Fig. 5). The DAS recovery time for each individual was calculated and the duration of action was compared. As a result, the average durations were 9.8, 21.0, and 25.3 days for the BoNT / A 1.2, 4, and 12 U / kg administration groups, respectively, and the average durations for the botulinum toxin administration group of SEQ ID NO: 22 were 15.2, 23.0, and 37.4 days, respectively. That is, compared to the wild-type BoNT / A, the duration of action of the recombinant polypeptide of the present invention was observed to increase by 1.6-fold, 1.1-fold, and 1.5-fold for each dose, and the DAS recovery times for the botulinum toxin administration group of SEQ ID NO: 23 were 16.3, 27.3, and 35.5 days on average, respectively, indicating that the duration of action increased by 1.7-fold, 1.3-fold, and 1.4-fold for each dose (Table 15). In addition, in the groups administered 12 U / kg of botulinum toxin of sequence number 22 and botulinum toxin of sequence number 23, the duration of action was significantly increased compared to the BoNT / A group (Table 15). DAS ED 50 The results showed that the BoNT / A administration group had 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, which were 2.9 and 1.5 times stronger than the BoNT / A administration group, respectively. As a result of measuring CMAP on the 7th day after administration, a significant decrease in the CMAP value at the injection site was observed in the botulinum toxin administration groups of SEQ ID NO: 22 and SEQ ID NO: 23 compared to the BoNT / A administration group, confirming the strong efficacy. In the case of the CMAP value at the contralateral injection site, no significant difference was observed compared to the BoNT / A administration group (Fig. 6). CMAPAAC As a result of calculating and comparing, the BoNT / A administration group had an average of 2077.1, the botulinum toxin administration group with sequence number 22 had an average of 2680.3, and the botulinum toxin administration group with sequence number 23 had an average of 2612.9, which showed a significant increase of 1.3 times compared to the BoNT / A administration group. In the 12 U / kg administration group, the botulinum toxin administration group with sequence number 22 had an average of 96%, and the botulinum toxin administration group with sequence number 23 had an average of 97% compared to before administration, showing an effect at a similar level to the BoNT / A administration group. From the above results performed in mice, the strong efficacy and increased duration of action of the botulinum toxin with sequence number 22 and the botulinum toxin with sequence number 23, which are recombinant polypeptides, were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0221] Average DAS duration of action (days) Increase rate (fold) 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 number 2215.2b 23.03 7.4*1.6 1.11.5 Sequence number 2316.3a 27.3a 35.5*1.7 1.3 1.4

[0222] *p<0.05, a p=0.05, b p=0.08, compared to the same dose of BoNT / A administered group, 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 sequence number 14 was manufactured at 394 U / mL and the recombinant botulinum toxin of sequence number 15 was manufactured at 419 U / mL and used 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 the above-mentioned Example 8. However, CMAP AAC was not produced.

[0225] Military animal number 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 number 141.2 right calf muscle DAS 66 Sequence number 144 right calf muscle DAS 76 Sequence number 1412 right calf muscle DAS, CMAP 86 Sequence number 151.2 right calf muscle DAS 96 Sequence number 154 right calf muscle DAS 106 Sequence number 1512 right calf muscle DAS, CMAP

[0226] After single administration 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 a mouse, the DAS evaluation results showed that the botulinum toxin of sequence number 14 showed an increase in the DAS score compared to BoNT / A in the 4 U / kg administration group (Fig. 7). The DAS recovery time for each individual was calculated and the duration of action was compared, and the duration of action was observed to be equally or more extended in the recombinant botulinum toxin administration group compared to BoNT / A (Table 17). The DAS ED50 results showed that the BoNT / A administration group had an effect of 5.8 U / kg, the SEQ ID NO: 14 administration group had an effect of 3.3 U / kg, and the SEQ ID NO: 15 administration group had an effect of 3.0 U / kg, which were 1.8 and 1.9 times stronger than the BoNT / A administration group, respectively. On the 7th day after administration, the CMAP measurement results showed that the botulinum toxin of sequence number 14 and the botulinum toxin of sequence number 15 showed a significant decrease in the CMAP value at the injection site compared to the BoNT / A administration group, confirming the strong efficacy, and the botulinum toxin of sequence number 15 showed a significant increase in the CMAP at the non-injection site compared to the BoNT / A administration group, confirming the diffusion inhibition effect (Fig. 8). In the 12 U / kg administration group, the change in body weight at 1 week compared to before administration was 95% on average in the BoNT / A administration group and 97% on average in the SEQ ID NO: 14 administration group. No significant difference was observed compared to the BoNT / A administration group, and in the SEQ ID NO: 15 administration group, a significant weight gain was observed compared to the BoNT / A administration group with an average of 111% (p < 0.001). From the above results performed in mice, we confirmed the strong efficacy, increased duration of action, and improved diffusion of the recombinant botulinum toxin of this invention compared to the botulinum toxin type A product (BoNT / A).

[0227] Average DAS duration of action (days) Increase rate (fold) 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 20.826.3 N / AN / AN / A Sequence number 1413.820.5 33.81.3 1.01.3 Sequence number 1514.722.83 1.51.4 1.11.2

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

[0229] A recombinant botulinum toxin of sequence number 12 was prepared at 559 U / mL, and a recombinant botulinum toxin of sequence number 13 was prepared at 440 U / mL, and the composition of the test groups is as shown in Table 18 below. The test substances or measurement methods used for efficacy evaluation were the same as those in the examples of the recombinant botulinum toxins of sequence numbers 22 and 23 described above.

[0230] Military animal number Administered substance dose (U / kg) Administration route Evaluation index 16 Placebo 0 Right calf muscle DAS, CMAP 2 6 BoNT / A 1.2 Right calf muscle DAS 3 6 BoNT / A 4 Right calf muscle DAS 4 6 BoNT / A 12 Right calf muscle DAS, CMAP, body weight 5 6 Sequence number 12 1.2 Right calf muscle DAS 6 6 Sequence number 12 4 Right calf muscle DAS 7 6 Sequence number 12 12 Right calf muscle DAS, CMAP, body weight 8 6 Sequence number 13 1.2 Right calf muscle DAS 9 6 Sequence number 13 4 Right calf muscle DAS 10 6 Sequence number 13 12 Right calf muscle DAS, CMAP, body weight

[0231] After single administration of botulinum toxin type A and botulinum toxins of sequence numbers 12 and 13 at doses of 1.2, 4, and 12 U / kg to the right calf muscle of the mouse, the DAS evaluation results showed that the 1.2 and 4 U / kg administration groups showed an increase in DAS scores compared to BoNT / A, demonstrating an effect equivalent to or greater than that of BoNT / A, and the 12 U / kg administration group showed an equivalent effect (Fig. 9). The DAS recovery time for each individual was calculated and the duration of action was compared, and the duration of action was significantly increased in the 1.2 U / kg administration group of botulinum toxin of sequence number 12 and the 4 U / kg administration group of botulinum toxin of sequence number 13 compared to the BoNT / A administration group (Table 19). DAS ED 50 The results showed that the BoNT / A group had an efficacy of 3.0 U / kg, the botulinum toxin group with sequence number 12 had an efficacy of 2.5 U / kg, and the botulinum toxin group with sequence number 13 had an efficacy of 2.8 U / kg, which were 1.2 and 1.1 times stronger than the BoNT / A group, respectively. As a result of measuring CMAP on the 7th day after administration, an equivalent level of effect was observed in the CMAP value at the injection site, and a significant increase in the CMAP value at the contralateral injection site was observed in the group administered with sequence number 12 (Fig. 10). CMAP AAC As a result of calculating and comparing, the BoNT / A administration group had an average of 2502.2, and the botulinum toxin administration group of SEQ ID NO: 13 had an average of 3041.9, showing a significant increase of 1.2 times compared to the BoNT / A administration group. In the 12 U / kg administration group, the 1-week body weight change compared to before administration was 96% on average in the BoNT / A administration group, 95% on average in the botulinum toxin administration group of SEQ ID NO: 12, and 97% on average in the botulinum toxin administration group of SEQ ID NO: 13, showing an effect at a similar level to the BoNT / A administration group. From the above results performed in mice, the strong efficacy, increased duration of action, and improved diffusion effect of the botulinum toxin of SEQ ID NO: 12 and 13, which are recombinant botulinum toxins, were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0232] Average DAS duration of action (days) Increase rate (fold) 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 16.338.7 N / AN / AN / ASeq. No. 1211.2**21.338.02.01.31.0 Seq. No. 138.825.0*29.31.61.50.8

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

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

[0235] A recombinant botulinum toxin of sequence number 16 was prepared at 444 U / mL, and the composition of the test group is as shown in Table 20 below. The test substances or measurement methods used for efficacy evaluation were carried out in the same manner as in the examples of the recombinant botulinum toxins of sequence numbers 22 and 23 described above.

[0236] Military animal number Administered substance dose (U / kg) Administration route Evaluation index 16 Placebo 0 Right calf muscle DAS, CMAP 2 6 BoNT / A 1.2 Right calf muscle DAS 3 6 BoNT / A 4 Right calf muscle DAS 4 6 BoNT / A 12 Right calf muscle DAS, CMAP, body weight 5 6 Sequence number 16 1.2 Right calf muscle DAS 6 6 Sequence number 16 4 Right calf muscle DAS 7 6 Sequence number 16 12 Right calf muscle DAS, CMAP, body weight

[0237] After single administration of botulinum toxin type A and recombinant botulinum toxin of sequence number 16 at doses of 1.2, 4, and 12 U / kg to the right calf muscle of the mouse, the DAS evaluation results showed that a significant increase in the DAS score was observed in the 12 U / kg recombinant botulinum toxin of sequence number 16 group compared to BoNT / A on day 21 after administration, and an increase in the DAS score compared to BoNT / A was also observed at other measurement time points (Fig. 11). The DAS recovery time for each individual was calculated and the duration of action was compared, and the duration of action was significantly increased in the 4 U / kg recombinant botulinum toxin of sequence number 16 group compared to the BoNT / A group (Table 21). DAS ED 50 The results showed that the BoNT / A administration group had an effect 1.1 times stronger than the BoNT / A administration group, with 2.0 U / kg and the recombinant botulinum toxin administration group with sequence number 16 having an effect 1.8 U / kg. The CMAP measurement result on the 7th day after administration confirmed the strong effect by showing a significant decrease in the CMAP value at the injection site in the recombinant botulinum toxin administration group with sequence number 16 compared to the BoNT / A administration group, and no significant difference was observed in the CMAP value at the contralateral injection site compared to the BoNT / A administration group (Fig. 12). CMAP AAC As a result of calculating and comparing, the BoNT / A administration group had an average of 2061.9, and the botulinum toxin administration group with sequence number 16 had an average of 2659.7, showing a significant increase of 1.3 times compared to the BoNT / A administration group. In the 12 U / kg administration group, the change in body weight at 1 week compared to before administration was an average of 94% in the BoNT / A administration group and an average of 93% in the recombinant botulinum toxin administration group with sequence number 16, showing no significant difference compared to the BoNT / A administration group. From the above results performed in mice, the strong efficacy and increased duration of action of the recombinant botulinum toxin with sequence number 16, a recombinant botulinum toxin, were confirmed compared to the botulinum toxin type A product (BoNT / A).

[0238] Average DAS duration of action (days) Increase rate (fold) Substance 1.2 U / kg 4 U / kg 12 U / kg 1.2 U / kg 4 U / kg 12 U / kg BoNT / A 11.2 18.732.7 N / AN / AN / A Sequence number 1613.826.8*45.5 a 1.21.41.4

[0239] *p<0.05, a p=0.05, compared to the same dose of BoNT / A administered group, two-tailed t-test or Mann-Whitney test

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

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

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

[0243] Results: After single administration of botulinum toxin type A product (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 the mouse, the DAS evaluation results showed that the recombinant botulinum toxin of sequence number 24 showed a 1.6-fold and 1.1-fold increase in DAS duration compared to BoNT / A at doses of 1.2 and 4 U / kg, respectively, and an equivalent duration of action was observed at 12 U / kg. For the recombinant botulinum toxin of sequence number 25, the DAS duration of action was increased by 1.3-fold, 1.4-fold, and 1.2-fold compared to BoNT / A at doses of 1.2, 4, and 12 U / kg, for the recombinant botulinum toxin of sequence number 26, the DAS duration of action was increased by 1.2-fold, 1.1-fold, and 1.3-fold compared to BoNT / A at doses of 1.2, 4, and 12 U / kg, and for the recombinant botulinum toxin of sequence number 27, the DAS duration of action was increased by 1.2-fold, 1.1-fold, and 1.1-fold compared to BoNT / A at doses of 1.2, 4, and 12 U / kg. For the recombinant botulinum toxin of sequence number 28, a 1.2-fold and 1.3-fold increase in DAS duration of action compared to BoNT / A was observed at doses of 1.2 and 4 U / kg, and a DAS duration equivalent to BoNT / A was observed at doses of 12 U / kg. For the recombinant botulinum toxins of sequence numbers 29 to 31, a DAS duration equivalent to BoNT / A was observed at doses of 1.2, 4, and 12 U / kg. For the recombinant botulinum toxin of sequence number 32, a 1.6-fold and 1.3-fold increase in DAS duration of action compared to BoNT / A was observed at doses of 1.2 and 4 U / kg, and a DAS duration equivalent to BoNT / A was observed at doses of 12 U / kg. For sequence number 43, a 1.4-fold increase in DAS duration of action compared to BoNT / A was observed at a dose of 1.2 U / kg, and a duration of action equivalent to BoNT / A was observed at doses of 4 U / kg and 12 U / Kg.

[0244] In addition, a safety evaluation was conducted by observing changes in body weight after administration of 1.2, 4, and 12 U / kg doses of recombinant botulinum toxins of sequence numbers 28 to 32 and 43, and as a result, all of them showed a level of weight loss equivalent to that of wild-type BoNT / A, confirming that they exhibit safety at least equivalent to that of wild-type botulinum toxin.

[0245] The above results obtained in mice confirm the increased duration of action or equivalent or higher safety of the recombinant botulinum toxin compared to the wild-type botulinum toxin type A product (BoNT / A).

[0246] Example 10: Evaluation of the efficacy of recombinant botulinum toxins of sequence numbers 18 and 19 in mice.

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

[0248] Test material: 100 units of botulinum toxin type A (BoNT / A) Coretox (Lot No.: E623001, Medytox) were used. Recombinant botulinum toxin was prepared in liquid form and used in the experiment, and was diluted to a concentration of 60 U / mL before administration. Two types of recombinant botulinum toxins were used in the experiment, and a total of two tests were conducted. Botulinum toxin type A product and recombinant botulinum toxin were administered to the right calf muscle of mice at a dose of 12 U / kg, and the efficacy was compared through CMAP evaluation and body weight comparison at 1 week after administration was performed.

[0249] Results: After a single injection of 12 U / kg of botulinum toxin type A (BoNT / A: Coretox) and recombinant botulinum toxin of sequence number 18 into the right calf muscle of mice, the CMAP values ​​at the injection site were compared at 1 week. A significant decrease was observed in the group administered with sequence number 18, with an average of 0.1 mV, compared to the average CMAP value of 0.9 mV in the BoNT / A group (p<0.001). In the case of body weight change at 1 week, the BoNT / A group showed a 97% weight change, while the group administered with sequence number 18 showed a 93% weight change, showing no significant difference. After a single administration of 12 U / kg of the recombinant botulinum toxin of sequence number 19, the CMAP values ​​at the injection site were compared at 1 week. A significant decrease was observed in the botulinum toxin group of sequence number 19, with an average of 0.3 mV, compared to the average CMAP value of 1.3 mV in the BoNT / A group (p<0.01). In terms of body weight change at 1 week, both the BoNT / A and botulinum toxin groups of sequence number 19 showed a 93% change in body weight, indicating a comparable level of efficacy. From the above results performed in mice, the strong efficacy and equivalent level of safety of the recombinant botulinum toxins of sequence numbers 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 recombinant botulinum toxin sequence number 44 (150 kDa, Medytox) at stepwise dilutions of 300, 60, 12, 2.4, and 0.48 U / well for 48 hours. After replacing the cell culture medium, the cells were cultured for an additional 48 hours, and then treated with lysis buffer to perform an ELISA test. The ELISA test performing method was the same as in Example 2.

[0253] result

[0254] The activity of cell-based wild-type and recombinant botulinum toxin was analyzed using ELISA, and an 11-fold increase in activity was confirmed in the recombinant botulinum toxin-treated group compared to the wild-type botulinum toxin.

[0255] Sequence number EC50 (U / well) Relative activity increase compared to wild type BoNT / A0.866-SEQ ID 440.078 11-fold increase

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

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

Claims

1. Contains a receptor binding domain of botulinum toxin type A1, A recombinant polypeptide wherein the receptor binding domain is conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but contains an amino acid mutation at one or more positions among positions having different amino acids in the corresponding positions of botulinum toxin type A1.

2. In paragraph 1, A recombinant polypeptide, wherein the position of the above amino acid mutation comprises at least one selected from the group consisting of N880, W1068 and N1090.

3. In paragraph 1, A recombinant polypeptide, wherein the receptor binding domain comprises at least one amino acid mutation selected from the group consisting of N880S, W1068M and N1090S.

4. In paragraph 2, A recombinant polypeptide, wherein the receptor binding domain further comprises an amino acid mutation at at least one position selected from the group consisting of N954, M968, T990, Q991, N1025, N1026, N1052, T1063, H1064, Y1117, T1232, and L1278.

5. In paragraph 4, A recombinant polypeptide, wherein the receptor binding domain further comprises at least one amino acid mutation selected from the group consisting of N954S, M968I, T990N, Q991K, N1025T, N1026K, N1052K, T1063P, H1064R, Y1117F, T1232R and L1278F.

6. In paragraph 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.

7. In paragraph 6, A translocation domain of botulinum toxin type A1, wherein the translocation domain is conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but includes an amino acid mutation at one or more positions among positions having different amino acids at the corresponding positions in botulinum toxin type A1, or A recombinant polypeptide comprising a translocation domain of botulinum toxin type A1 and comprising an amino acid mutation at position 589 8. In paragraph 6, A recombinant polypeptide comprising a translocation domain of botulinum toxin type A1, wherein the amino acid mutation position 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.

9. In paragraph 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.

10. In paragraph 9, The above light chain domain is, comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, or A recombinant polypeptide comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the 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 replaced with phenylalanine, and the isoleucine at position 264 is replaced with arginine.

11. In paragraph 1, A recombinant polypeptide wherein the receptor binding domain has a lower half-maximal inhibitory concentration value in a botulinum toxin heavy chain competition assay compared to the wild-type receptor binding domain.

12. In paragraph 1, It further comprises a light chain domain and a translocation domain, A recombinant polypeptide having increased potency, safety or half-life compared to wild-type botulinum toxin type A.

13. In paragraph 12, A recombinant polypeptide comprising at least one selected from the recombinant botulinum toxins of SEQ ID NO: 8 to SEQ ID NO: 32, SEQ ID NO: 43, and SEQ ID NO:

44.

14. Contains a receptor binding domain of botulinum toxin type A1, A recombinant polypeptide, wherein the receptor binding domain comprises an amino acid mutation at two or more positions selected from N954, Y1117, T1232, R1273, and L1278.

15. In paragraph 14, A recombinant polypeptide, wherein the receptor binding domain comprises two or more amino acid mutations selected from N954S, Y1117F, T1232R, R1273K and L1278F.

16. In paragraph 14, A recombinant polypeptide, wherein the receptor binding domain comprises an amino acid mutation at two or more positions selected from N954S, T1232R and L1278F.

17. In paragraph 14, A recombinant polypeptide comprising amino acid mutations of the receptor binding domain, wherein the amino acid mutations 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.

18. Contains a variant of the receptor binding domain of botulinum toxin type A1, A recombinant polypeptide wherein the half-maximal inhibitory concentration value of a mutant of the receptor binding domain according to a botulinum toxin heavy chain competition assay is lower than that of the wild-type receptor binding domain.

19. In paragraph 18, A recombinant polypeptide, wherein the variant of the receptor binding domain comprises an amino acid mutation 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.

20. A variant in which at least one amino acid in the receptor binding domain sequence of botulinum toxin type A1 is mutated, A recombinant botulinum toxin, wherein the amino acid mutation comprises at least one 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 wherein at least one of efficacy and safety is superior compared to wild-type botulinum toxin type A1 when administered to a subject in need of treatment, and the remainder is at least equivalent to the wild-type toxin.

21. In paragraph 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.

22. In paragraph 21, The above light chain domain is, comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, or A recombinant botulinum toxin comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the 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.

23. In paragraph 20, Contains a translocation domain, The above translocation domain is conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but contains an amino acid mutation at one or more positions among the positions having different amino acids at the corresponding positions of the botulinum toxin type A1, or A recombinant botulinum toxin, wherein the translocation domain comprises a translocation domain of botulinum toxin type A1 and comprises at least one amino acid mutation selected from the group consisting of D589, E734, and E809.

24. A recombinant botulinum toxin comprising a receptor binding domain comprising a variant of a botulinum toxin type A light chain, wherein the receptor binding domain comprises at least one amino acid mutation 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.

25. In paragraph 24, A recombinant botulinum toxin comprising a recombinant polypeptide of SEQ ID NO: 1 to 7, 33 to 42 and 45 to 49, wherein the receptor binding domain is 26. In paragraph 24, The above variant of botulinum toxin type A light chain is, comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the fourth domain of a botulinum toxin type A4 light chain; or a variant thereof, or A recombinant botulinum toxin comprising the first domain and the second domain of a botulinum toxin type A1 light chain; or a variant thereof, and the third domain and the 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.

27. In paragraph 24, The recombinant botulinum toxin above is a recombinant botulinum toxin that, compared to wild-type botulinum toxin type A1, exhibits at least one higher value among safety and duration of action when administered to an individual.

28. Contains a botulinum toxin type A1 translocation domain, A recombinant polypeptide wherein the above translocation domain is conserved with common amino acids in botulinum toxin type A2 and botulinum toxin type A6, but contains an amino acid mutation at one or more positions among positions having different amino acids in the corresponding positions of botulinum toxin type A1.

29. In paragraph 28, A recombinant polypeptide, wherein the amino acid mutation position 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.

30. In paragraph 28, A recombinant polypeptide, wherein the translocation domain comprises at least one amino acid mutation selected from the group consisting of L681I, D696N, I713T, K730E, E734K, K779S, N789D, G804A, E809K, L815V, K816R, and A818V.

Images