Clostridium neurotoxins including exogenous activation loops

Abstract

This invention provides a method for producing Clostridium neurotoxins containing an exogenous activation loop, modified Clostridium neurotoxins, and pharmaceutical compositions. [Solution] A method for proteolytically processing a single-chain Clostridium neurotoxin into a corresponding double-chain Clostridium neurotoxin, comprising: providing a single-chain Clostridium neurotoxin; and contacting the single-chain Clostridium neurotoxin with enterokinase or factor Xa, wherein the single-chain Clostridium neurotoxin has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - A method in which an activation loop containing Cys (SEQ ID NO: 1) is present, a=1 to 10, b=4 to 15, and enterokinase or factor Xa hydrolyzes the peptide bond of the activation loop, thereby producing a double-stranded Clostridium neurotoxin.

Description

[Technical Field]

[0001] This invention relates to Clostridium neurotoxins and methods for activating and using them. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent, specific protein toxins that can poison neurons and other cells they reach. Examples of such Clostridium neurotoxins include the neurotoxin produced by C. tetani (TeNT) and the neurotoxin produced by C. botulinum (BoNT) serotypes A-G and X (see WO2018 / 009903A2), as well as those produced by C. baratii and C. butyricum.

[0003] Clostridium neurotoxins are among the most potent toxins known. For example, botulinum neurotoxin has a median lethal dose (LD50) in mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50 They have a value of ) . Both tetanus and botulinum toxin act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0004] Clostridium neurotoxins are expressed as single-chain polypeptides in Clostridium. Each Clostridium neurotoxin has a catalytic light chain separated from the heavy chain (containing the N-terminal transposition domain and the C-terminal receptor-binding domain) by an exposed region called the activation loop. During protein maturation, the light and heavy chains of the Clostridium neurotoxin are separated by proteolytic cleavage of the activation loop, and these are joined by disulfide bridges to create a fully active double-chain toxin. This process must be replicated during recombinant toxin production. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Exogenous proteases such as trypsin or Lys-C are used to proteolytically activate single-chain Clostridium neurotoxins. However, for some Clostridium neurotoxins, incubation with Lys-C or trypsin results in partial or inadequate cleavage of the single-chain polypeptide, resulting in the production of contaminated single-chain and / or inactive cleavage / degradation products (e.g., BoNT / E), requiring purification of the full-length double-chain polypeptide. Therefore, there is currently no universal exogenous protease for the activation of Clostridium neurotoxins. This is particularly problematic when identifying novel Clostridium neurotoxins or generating modified (e.g., chimeric or hybrid) neurotoxins, requiring screening of multiple proteases to determine correct activation.

[0006] Botulinum neurotoxin serotype X (BoNT / X) was recently identified (WO2018 / 009903 A2). BoNT / X is particularly problematic to activate, as its polypeptide is known to be completely degraded by cleavage using trypsin or Lys-C.

[0007] The present invention overcomes one or more of the above problems.

[0008] The protease enterokinase exhibits significantly higher substrate specificity compared to conventionally used trypsin and Lys-C. This protease recognizes and cleaves the immediate C-terminus of the DDDDK peptide sequence (SEQ ID NO: 72). Notably, this sequence is not present in all of the Clostridium neurotoxin activation loops (see Figure 1), and therefore, enterokinase has been previously excluded as a protease for use in Clostridium neurotoxin activation. [Means for solving the problem]

[0009] The inventors have surprisingly found that enterokinase recognizes and cleaves the immediate C-terminal portion of the IDGR sequence present in the BoNT / C1 activation loop (see Figure 1). Advantageously, this sequence can also be recognized and cleaved by factor Xa, another protease exhibiting high substrate specificity (e.g., compared to trypsin and Lys-C). Furthermore, the BoNT / C1 activation loop also contains lysine and arginine residues, allowing for cleavage by either lysine or trypsin. Thus, the inventors have surprisingly found that the BoNT / C1 loop constitutes a universal activation loop for Clostridium neurotoxins, thus providing flexibility for the use of four different proteases.

[0010] In one aspect, the present invention relates to a method for proteolytically processing a single-chain Clostridium neurotoxin (e.g., a modified Clostridium neurotoxin as described herein) into a corresponding double-chain Clostridium neurotoxin, a. To provide a single-chain Clostridium neurotoxin. b. Contacting single-chain Clostridium neurotoxin with enterokinase Includes, Single-chain Clostridium neurotoxins have polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Has an activation loop including Cys (sequence number 1), The present invention provides a method in which an enterokinase hydrolyzes the peptide bond of the activation loop, thereby generating a double-stranded Clostridium neurotoxin (e.g., a modified double-stranded Clostridium neurotoxin as described herein).

[0011] In related embodiments, the present invention relates to a method for proteolytically processing a single-chain Clostridium neurotoxin (e.g., a modified Clostridium neurotoxin as described herein) into a corresponding double-chain Clostridium neurotoxin, a. To provide a single-chain Clostridium neurotoxin. b. Contacting single-chain Clostridium neurotoxin with factor Xa Includes, Single-chain Clostridium neurotoxins have polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Has an activation loop including Cys (sequence number 1), The present invention provides a method in which factor Xa hydrolyzes the peptide bonds of the activation loop, thereby generating a double-stranded Clostridium neurotoxin (e.g., a modified double-stranded Clostridium neurotoxin as described herein).

[0012] The single-chain Clostridium neurotoxin is preferably the modified single-chain Clostridium neurotoxin of the present invention, and the activation loop is the exogenous activation loop. Advantageously, the inventors have found that replacing the endogenous Clostridium neurotoxin activation loop with the exogenous activation loop shown as SEQ ID NO: 1 (which contains a protease cleavage site in its natural context) overcomes the problems associated with modifying the endogenous activation loop to insert a protease cleavage site (e.g., factor Xa cleavage site, e.g., Ile-Asp-Gly-Arg [SEQ ID NO: 18] or Ile-Glu-Gly-Arg [SEQ ID NO: 19]). In particular, modifying the endogenous activation loop to insert a protease cleavage site can lead to conformational changes, which may then have a negative effect on cleavage efficiency (see Example 7 herein).

[0013] In a particularly preferred embodiment, the method of the present invention includes the use of enterokinase.

[0014] In one aspect, the present invention is directed to the use of enterokinase for hydrolyzing peptide bonds of a polypeptide (e.g., Clostridium neurotoxin) comprising the sequence shown as SEQ ID NO: 18 or SEQ ID NO: 19 (preferably SEQ ID NO: 18). Preferably, enterokinase hydrolyzes the peptide bond immediately C-terminal to SEQ ID NO: 18 or SEQ ID NO: 19 (more preferably SEQ ID NO: 18) contained within the polypeptide sequence. In one embodiment, the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to the polypeptide sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

[0015] The present invention also provides a method for producing a modified Clostridium neurotoxin, comprising: a. identifying the endogenous activation loop of a Clostridium neurotoxin, wherein the Clostridium neurotoxin is characterized in that the peptide bonds outside the endogenous activation loop of the Clostridium neurotoxin are hydrolyzed by trypsin or Lys-C; b. replacing the endogenous activation loop with an exogenous activation loop, thereby providing a modified Clostridium neurotoxin, wherein the exogenous activation loop comprises the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1); and the method comprises the above steps.

[0016] The present invention also provides a method for producing a modified Clostridium neurotoxin, comprising: a. identifying the endogenous activation loop of a Clostridium neurotoxin, wherein the Clostridium neurotoxin is characterized in that the endogenous activation loop is inefficiently proteolytically processed by trypsin or Lys-C; b. replacing the endogenous activation loop with an exogenous activation loop, thereby providing a modified Clostridium neurotoxin, wherein the exogenous activation loop comprises the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b-Includes Cys (sequence number 1) This provides a method that includes [something].

[0017] In one embodiment, the Clostridium neurotoxin is characterized in that the peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C, and the endogenous activation loop is inefficiently processed in a proteolytic manner by trypsin or Lys-C.

[0018] In embodiments where the endogenous activation loop is inefficiently proteolytically processed by trypsin (preferably, the peptide bonds outside the endogenous activation loop of the Clostridium neurotoxin are not hydrolyzed by trypsin), the method may further include contacting the modified Clostridium neurotoxin with trypsin capable of hydrolyzing the peptide bonds in the exogenous activation loop of the modified Clostridium neurotoxin. Similarly, in embodiments where the endogenous activation loop is inefficiently proteolytically processed by Lys-C (preferably, the peptide bonds outside the endogenous activation loop of the Clostridium neurotoxin are not hydrolyzed by Lys-C), the method may further include contacting the modified Clostridium neurotoxin with Lys-C capable of hydrolyzing the peptide bonds in the exogenous activation loop of the modified Clostridium neurotoxin.

[0019] In one embodiment, the method includes a step of screening a Clostridium neurotoxin for its suitability for use in the method of the present invention. The screening step may include determining whether the peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C. Alternatively, the screening step may further include determining whether the endogenous activation loop of the Clostridium neurotoxin is inefficiently proteolytically processed by trypsin or Lys-C.

[0020] In contrast to the original Clostridium neurotoxin, in one embodiment, the modified Clostridium neurotoxin of the present invention is not inefficiently processed proteolytically by enterokinase or factor Xa, and / or the peptide bonds outside the exogenous activation loop of the modified Clostridium neurotoxin are not hydrolyzed by enterokinase or factor Xa. Therefore, the original Clostridium neurotoxin is preferably resistant to proteolytic processing by enterokinase and / or factor Xa.

[0021] Clostridium neurotoxins can be identified as suitable for modification in the methods of the present invention by an assay comprising contacting 1 mg of Clostridium neurotoxin with at least 0.25 μg of trypsin at ≥3350 units / mg or with Lys-C at ≥200 units / mg in 50 mM Tris-HCl pH 8.0, 50 mM NaCl reaction buffer, at at least 4°C for at least 5 hours.

[0022] In one embodiment, the assay involves contacting 1 mg of Clostridium neurotoxin with 0.25 μg of trypsin at a ratio of ≥3350 units / mg (approximately 1:611 molar ratio of Clostridium neurotoxin to trypsin) or with Lys-C at a ratio of ≥200 units / mg (approximately 1:734 molar ratio of Clostridium neurotoxin to Lys-C) in 50 mM Tris-HCl pH 8.0, 50 mM NaCl reaction buffer, at 4°C for 18 hours.

[0023] In another embodiment, the assay involves contacting 1 mg of Clostridium neurotoxin with 0.40 μg of trypsin at a ratio of ≥3350 units / mg (approximately 1:978 molar ratio of Clostridium neurotoxin to trypsin) or with Lys-C at a ratio of ≥200 units / mg (approximately 1:1174 molar ratio of Clostridium neurotoxin to Lys-C) in 50 mM Tris-HCl pH 8.0, 50 mM NaCl reaction buffer, at 20°C for 5 hours.

[0024] The trypsin used is preferably commercially available TrypZean (Sigma number T3568). The trypsin may have a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 47. In one embodiment, the trypsin may have a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 47. Preferably, the trypsin may have the polypeptide sequence shown as SEQ ID NO: 47. One unit of the trypsin (Trypzean) is defined as the amount of enzyme that, in a reaction volume of 3.2 mL, produces a change in absorbance at 253 nm of 0.003 per minute at pH 7.6 and 25°C using a 0.23 mM solution of Na-benzoyl-L-arginine ethyl ester (BAEE) as the substrate.

[0025] The Lys-C used is preferably commercially available Lys-C (Sigma number 000000011047825001). Lys-C may have a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 48. In one embodiment, Lys-C may have a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 48. Preferably, Lys-C may have the polypeptide sequence shown as SEQ ID NO: 48. One unit of Lys-C is defined as the amount of enzyme that hydrolyzes 1.0 μmol of Tos-Gly-Pro-Lys-pNA per minute at 25°C and pH 7.

[0026] If, by SDS-PAGE (preferably stained with Coomassie or a dye of equivalent sensitivity), one or more cleavage products are observed in addition to the cleavage products of the H and L chains of Clostridium neurotoxin, it is confirmed that the peptide bonds outside the endogenous activation loop of Clostridium neurotoxin are hydrolyzed by trypsin or Lys-C. Preferably, after performing the above assay, if, by SDS-PAGE, at least 3, 4, 5, 6, 7, 8, 9, or 10 cleavage products are observed in addition to the cleavage products of the chains and L chains of Clostridium neurotoxin, it is confirmed that the peptide bonds outside the endogenous activation loop of Clostridium neurotoxin are hydrolyzed by trypsin or Lys-C.

[0027] Furthermore, if less than 70% of the endogenous activation loop is proteolytically processed by trypsin or Lys-C, resulting in a double-stranded Clostridium neurotoxin (evaluated by SDS-PAGE after performing the above assay, preferably stained with Coomassie or a dye of equivalent sensitivity), it is confirmed that the endogenous activation loop is inefficiently proteolytically cleaved by trypsin or Lys-C. Preferably, if less than 60%, 50%, 40%, 30%, 10%, or 5% of the endogenous activation loop is proteolytically processed by trypsin or Lys-C (evaluated by SDS-PAGE after performing the above assay), the Clostridium neurotoxin may be characterized by the inefficient proteolytic cleavage of the endogenous activation loop by trypsin or Lys-C. More preferably, if less than 30% of the endogenous activation loop is proteolytically processed by trypsin or Lys-C (as assessed by SDS-PAGE after performing the above assay), Clostridium neurotoxins may be characterized by the inefficient proteolytic cleavage of the endogenous activation loop by trypsin or Lys-C.

[0028] The Clostridium neurotoxin (before modification) is preferably one in which the peptide bond (either inside or outside the activation loop) is not hydrolyzed or substantially not hydrolyzed by enterokinase or factor Xa. The term "substantially not hydrolyzed" means that less than 10%, 5%, 4%, 3%, 2%, or 1% of the Clostridium neurotoxin present in the reactant contains peptide bonds that are hydrolyzed by enterokinase or factor Xa in the method of the present invention.

[0029] In one embodiment, the method of the present invention further comprises contacting a modified Clostridium neurotoxin with enterokinase or factor Xa (more preferably enterokinase) to generate a corresponding double-stranded modified Clostridium neurotoxin.

[0030] In one embodiment, the present invention provides a modified Clostridium neurotoxin (which can be obtained, for example, by the method of the present invention), wherein the endogenous activation loop of the Clostridium neurotoxin is replaced by an exogenous activation loop, thereby generating a modified Clostridium neurotoxin, the exogenous activation loop being a polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Includes Cys (sequence number 1).

[0031] In one embodiment, the Clostridium neurotoxin (unmodified) is characterized in that the peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C. In another embodiment, the Clostridium neurotoxin (unmodified) is characterized in that the endogenous activation loop is inefficiently processed proteolytically by trypsin or Lys-C. In yet another embodiment, the Clostridium neurotoxin (unmodified) is characterized in that the peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C, and the endogenous activation loop is inefficiently processed proteolytically by trypsin or Lys-C. Confirmation of these characteristics of the Clostridium neurotoxin (unmodified) is preferably by the assay described above.

[0032] The present invention may include replacing the endogenous activation loop of any Clostridium neurotoxin with the exogenous activation loop described herein. Preferably, the Clostridium neurotoxin is not BoNT / C1. The Clostridium neurotoxin may be botulinum neurotoxin or tetanus neurotoxin. Preferably, the Clostridium neurotoxin is botulinum neurotoxin (BoNT), for example, BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X.

[0033] In one embodiment, the Clostridium neurotoxin for use in the present invention is BoNT / X, BoNT / E, or a BoNT / A1C1 hybrid. Preferably, the Clostridium neurotoxin is BoNT / X or BoNT / E, both of which are characterized in that trypsin and / or Lys-C hydrolyze the peptide bonds outside their endogenous activation loop, and / or both Clostridium neurotoxins contain an endogenous activation loop that is inefficiently proteolytically processed by trypsin and / or Lys-C. Most preferably, the Clostridium neurotoxin for use in the present invention is BoNT / X.

[0034] When used herein, the term “endogenous activation loop” means the activation loop present in the Clostridium neurotoxin of interest, e.g., the Clostridium neurotoxin of the indicated serotype. For example, BoNT / A1 comprises the BoNT / A1 heavy and light chains, and therefore the endogenous activation loop of BoNT / A1 is the A1 activation loop. For Clostridium neurotoxin chimeras or hybrids, those skilled in the art will know, for example, the L chain and H chain. N The "endogenous activation loop" can be identified by determining the serotype(s) from which the domain originates. In some embodiments, chimeric or hybrid Clostridium neurotoxins may have an endogenous activation loop that is a fusion of activation loops derived from two different serotypes. For example, a chimeric Clostridium neurotoxin, e.g., BoNT / A1C1, has a BoNT / A1 light chain and a transposition domain, and therefore the endogenous BoNT / A1C1 activation loop is the A1 activation loop. An example of an activation loop is provided in Figure 1.

[0035] Preferably, the “endogenous activation loop” is any activation loop other than SEQ ID NO: 1. In one embodiment, the “endogenous activation loop” is any activation loop other than SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0036] In contrast, "exogenous activation loop," as used herein, means an activation loop different from the endogenous activation loop present in the Clostridium neurotoxin of interest, e.g., the Clostridium neurotoxin of the indicated serotype. For example, the BoNT / C1 activation loop has a different polypeptide sequence from the wild-type BoNT / A1 activation loop, and therefore the BoNT / C1 activation loop is exogenous to BoNT / A1. Regarding Clostridium neurotoxin chimeras or hybrids, those skilled in the art will know, for example, the L chain and H chain. N By determining the serotype(s) from which the domain originates, it is possible to determine whether the activation loop is an "exogenous activation loop". The L chain is a BoNT / BL chain, and H NIf the domain originates from BoNT / D, the endogenous activation loop may contain portions of both the BoNT / B and BoNT / D sequences. If the activation loop (e.g., the C1 activation loop) differs from this, it is considered an "exogenous activation loop."

[0037] Determining whether an activation loop is an "exogenous activation loop" can be done by aligning the sequence of the target Clostridium neurotoxin with the activation loop and verifying whether the activation loop is present in the target Clostridium neurotoxin sequence. If it is not present, the activation loop can be identified as an exogenous activation loop.

[0038] Preferably, the entire endogenous activation loop is replaced by the exogenous activation loop described herein. However, in some embodiments, only a portion of the endogenous activation loop is replaced, for example, at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the endogenous activation loop are replaced.

[0039] Replacement of the endogenous activation loop can be achieved by any method known in the art. For example, replacement can be achieved by amino acid modification. In one embodiment, the endogenous activation loop can be replaced by deleting one or more amino acid residues of the endogenous activation loop. The endogenous activation loop can be replaced by substituting one or more amino acid residues of the endogenous activation loop with amino acid residues of the exogenous activation loop. In some embodiments, the endogenous activation loop (or a portion thereof) may be deleted, and the exogenous activation loop may be inserted, preferably at a position originally occupied by the endogenous activation loop. Alternatively, the endogenous activation loop may be retained in the modified Clostridium neurotoxin of the present invention and preferably inactivated (e.g., by mutation). It is preferable that the endogenous activation loop (or a portion thereof, more preferably the entire endogenous activation loop) is not present in the modified Clostridium neurotoxin of the present invention. It is preferable that the exogenous activation loop occupies a position in the Clostridium neurotoxin originally occupied by the endogenous activation loop.

[0040] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art and may be used in carrying out the present invention. For example, amino acid modification can be introduced by modifying the DNA sequence encoding Clostridium neurotoxin. This can be achieved using standard molecular cloning techniques, for example, by site-directed mutagenesis in which a short chain (oligonucleotide) of DNA encoding the desired amino acid(s) is used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting a portion of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence may be chemically synthesized.

[0041] In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 23, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71. In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71. Preferably, the endogenous activation loop includes a polypeptide sequence represented as SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71.

[0042] In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. Preferably, the endogenous activation loop includes a polypeptide sequence represented as SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69.

[0043] In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 24. In another embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 24. Preferably, the endogenous activation loop includes a polypeptide sequence represented as SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 24.

[0044] Preferably, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with respect to SEQ ID NO: 20. In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with respect to SEQ ID NO: 20. More preferably, the endogenous activation loop includes the polypeptide sequence shown as SEQ ID NO: 20.

[0045] Preferably, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with SEQ ID NO: 21. In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 21. More preferably, the endogenous activation loop includes the polypeptide sequence shown as SEQ ID NO: 21.

[0046] Preferably, the endogenous activation loop includes a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity with SEQ ID NO: 24. In one embodiment, the endogenous activation loop includes a polypeptide sequence having at least 95% sequence identity with SEQ ID NO: 24. More preferably, the endogenous activation loop includes the polypeptide sequence shown as SEQ ID NO: 24.

[0047] This invention relates to a system where the endogenous activation loop is replaced by an exogenous activation loop, such as Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b The method and Clostridium neurotoxin are comprised of an exogenous activation loop containing a polypeptide represented as -Cys (SEQ ID NO: 1). Xaa or Yaa can be any amino acid. The number of amino acids at positions Xaa and Yaa is indicated by the letters "a" and "b", respectively. In one embodiment, "a" and "b" can be any integers that allow proteolytic cleavage of the activation loop and result in an active double-stranded Clostridium neurotoxin. In one embodiment, "a" is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, "b" is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, "a" is ≤12, ≤11, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, or ≤4. In one embodiment, "b" is ≤20, ≤19, ≤18, ≤17, ≤16, ≤15, ≤14, ≤13, ≤12, ≤11, ≤10, or ≤9.

[0048] In one embodiment, "a" is 1 to 12, for example, 1 to 10. Preferably, "a" is 1 to 7, for example, 2 to 4. More preferably, "a" is 3. In one embodiment, "b" is 1 to 20, for example, 4 to 15. Preferably, "b" is 6 to 10. More preferably, "b" is 8.

[0049] Xaa or Yaa is not intended to be limited to only one type of amino acid. Therefore, one or more residues present at position Xaa can be independently selected from the standard amino acids, namely aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. One or more residues present at position Yaa can be independently selected from the standard amino acids, namely aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. Preferably, the amino acid at position Yaa (more preferably the amino acid immediately C-terminal to the Arg residue in SEQ ID NO: 1) is not proline.

[0050] Alternatively, one or more residues located at position Xaa or Yaa may be independently selected from non-standard amino acids (amino acids that are not part of the 20 standard sets mentioned above). For example, non-standard amino acids may include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, L-ornithine, L-2-amino-3-guanidinopropionic acid or lysine, arginine and / or ornithine D-isomers, and 4-fluorophenylalanine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using the Escherichia coli (E. coli) nutrient-dependent expression host.

[0051] The properties of standard amino acids are shown in the table below: [Table A]

[0052] The following amino acids are considered to be charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0053] The sequence Ile-Asp / Glu-Gly-Arg contained in Sequence ID No. 1 refers to a site that was surprisingly discovered by the inventors to be recognized by enterokinase (and factor Xa). Preferably, the sequence is Ile-Asp-Gly-Arg, for example, Cys-(Xaa) a -Ile-Asp-Gly-Arg-(Yaa) b-Cys. Enterokinase and factor Xa are thought to hydrolyze the peptide bond immediately C-terminal to Arg in SEQ ID NO: 1 (i.e., the peptide bond between Arg and Yaa).

[0054] In one embodiment, the amino acid residue of Xaa immediately N-terminal to Ile in SEQ ID NO: 1 is an uncharged hydrophobic amino acid, preferably alanine. In some embodiments, "a" is at least 2, and Xaa includes at least a C-terminal uncharged polar amino acid and a charged basic amino acid immediately N-terminal to it. The charged basic amino acid is preferably lysine. Therefore, in embodiments where "a" is at least 2, Xaa may include at least Lys-Ala, where Ala is immediately N-terminal to Ile in SEQ ID NO: 1.

[0055] In one embodiment, Xaa includes or consists of the sequence HKA.

[0056] In one embodiment, the amino acid residue of Yaa immediately C-terminal to Arg in SEQ ID NO: 1 is an uncharged polar amino acid, preferably serine. In some embodiments, "b" is at least 2, and Yaa includes at least an N-terminal uncharged polar amino acid and an uncharged hydrophobic amino acid immediately C-terminal thereto. The uncharged hydrophobic amino acid is preferably leucine. Therefore, in embodiments where "b" is at least 2, Yaa may include at least Ser-Leu, where Ser is immediately C-terminal to Arg in SEQ ID NO: 1.

[0057] In one embodiment, Yaa comprises or consists of the sequence SLYNKTLDC.

[0058] In some embodiments, the exogenous activation loop has at least 70% sequence identity with respect to SEQ ID NO: 2. In one embodiment, the exogenous activation loop has at least 80%, 85%, or 90% sequence identity with respect to SEQ ID NO: 2. Preferably, the exogenous activation loop has at least 95% sequence identity with respect to SEQ ID NO: 2. More preferably, the exogenous activation loop has at least 99% sequence identity with respect to SEQ ID NO: 2.

[0059] In a particularly preferred embodiment, the extrinsic loop includes SEQ ID NO: 2. More preferably, the extrinsic loop consists of SEQ ID NO: 2.

[0060] The exogenous loop may also be a variant of SEQ ID NO: 2, for example, SEQ ID NO: 3, or a sequence having at least 70% sequence identity to it. SEQ ID NO: 3 is a variant of SEQ ID NO: 2 in which the enterokinase recognition site IDGR is mutated to IEGR. In one embodiment, the exogenous activation loop has at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 3. Preferably, the exogenous activation loop has at least 95% sequence identity to SEQ ID NO: 3. More preferably, the exogenous activation loop has at least 99% sequence identity to SEQ ID NO: 3.

[0061] In a particularly preferred embodiment, the extrinsic loop includes SEQ ID NO: 3. More preferably, the extrinsic loop consists of SEQ ID NO: 3.

[0062] The Clostridium neurotoxin of the present invention (e.g., modified Clostridium neurotoxin) can be encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In one embodiment, the Clostridium neurotoxin of the present invention can be encoded by a nucleotide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. Preferably, the Clostridium neurotoxin of the present invention can be encoded by a nucleotide sequence comprising (more preferably comprising) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0063] The Clostridium neurotoxin of the present invention (e.g., modified Clostridium neurotoxin) may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. In one embodiment, the Clostridium neurotoxin of the present invention may contain a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. Preferably, the Clostridium neurotoxin of the present invention may contain (more preferably consist of) the polypeptide sequence represented as SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

[0064] The Clostridium neurotoxin of the present invention (e.g., modified Clostridium neurotoxin) is preferably BoNT / X, and the Clostridium neurotoxin is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, the Clostridium neurotoxin is encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 4. Preferably, the Clostridium neurotoxin is encoded by a nucleotide sequence containing (or consisting of) SEQ ID NO: 4. The Clostridium neurotoxin of the present invention is preferably BoNT / X, and the Clostridium neurotoxin contains a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, the Clostridium neurotoxin contains a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 5. Preferably, the Clostridium neurotoxin contains (or consists of) a polypeptide sequence shown as SEQ ID NO: 5.

[0065] The Clostridium neurotoxin of the present invention (e.g., modified Clostridium neurotoxin) is preferably BoNT / E, and the Clostridium neurotoxin is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 10. In one embodiment, the Clostridium neurotoxin is encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 10. Preferably, the Clostridium neurotoxin is encoded by a nucleotide sequence containing (or consisting of) SEQ ID NO: 10. The Clostridium neurotoxin of the present invention is preferably BoNT / E, and the Clostridium neurotoxin contains a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 11. In one embodiment, the Clostridium neurotoxin contains a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 11. Preferably, the Clostridium neurotoxin contains (or consists of) a polypeptide sequence shown as SEQ ID NO: 11.

[0066] In some embodiments, the polypeptide sequence (or the nucleotide sequence encoding it) of the present invention may include a purified tag, such as a His- tag. The present invention is also intended to include polypeptide sequences (and the nucleotide sequences encoding them) from which the purified tag has been removed.

[0067] The present invention comprises contacting a single-chain Clostridium neurotoxin (e.g., a modified Clostridium neurotoxin of the present invention) with a protease capable of hydrolyzing the peptide bond in the activation loop of the single-chain Clostridium neurotoxin, thereby generating a double-chain Clostridium neurotoxin. The protease may be an endopeptidase. The protease may be an enterokinase, factor Xa, Lys-C, or trypsin. Preferably, the protease may be an enterokinase or factor Xa, more preferably an enterokinase.

[0068] The terms “enterokinase” or “EK” encompass the enterokinases described herein and any proteases having structural and / or functional similarity (preferably structural and functional similarity) to hydrolyze the peptide bond of SEQ ID NO: 1. A suitable enterokinase is the enterokinase light chain, commercially available from NEB (P8070). One unit can be defined as the amount of enzyme required to cleave 25 μg of MBP-EK-paramyosin-ΔSal substrate to 95% completion in a total reaction volume of 25 μl (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 (pH 8.0 at 25°C)) at 25°C for 16 hours.

[0069] In one embodiment, the enterokinase comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 49. In some embodiments, the enterokinase comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 49. Preferably, the enterokinase comprises SEQ ID NO: 49 (more preferably, comprises SEQ ID NO: 49).

[0070] In some embodiments, the enterokinase may further contain a heavy chain, and the heavy and light chains are connected by disulfide bridges. Such enterokinases are commercially available (e.g., from R&D Systems).

[0071] The term “Factor Xa” encompasses any protease having structural and / or functional similarity (preferably structural and functional similarity) to the Factor Xa described herein and the peptide bond of Sequence ID No. 1. A suitable Factor Xa is commercially available from NEB (P8010). One unit can be defined as the amount of Factor Xa required to cleave 50 μg of MBP fusion protein test substrate, MBP-ΔSal (the substrate MBP-ΔSal is a maltose-binding protein fused to a truncated form of paramyosin, with the amino acid Ile-Glu-Gly-Arg at the fusion junction) to 95% completion in a reaction dose of 50 μl (20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2 (pH 8.0)) at 23°C for 6 hours or less.

[0072] In one embodiment, factor Xa comprises a polypeptide sequence having a heavy chain having at least 70% sequence identity with SEQ ID NO: 50 and a light chain having at least 70% sequence identity with SEQ ID NO: 51, with the heavy and light chains connected by disulfide bridges. In some embodiments, factor Xa comprises a polypeptide sequence having a heavy chain having at least 80% or 90% sequence identity with SEQ ID NO: 50 and a light chain having at least 80% or 90% sequence identity with SEQ ID NO: 51, with the heavy and light chains connected by disulfide bridges. Preferably, factor Xa comprises SEQ ID NO: 50 and SEQ ID NO: 51 (more preferably, comprising SEQ ID NO: 50 and SEQ ID NO: 51), with the heavy and light chains connected by disulfide bridges.

[0073] Contact may occur under any suitable conditions that result in the formation of the corresponding double-stranded Clostridium neurotoxin from more than 30%, 40%, 50%, or 60% (preferably more than 70%) of the proteolytically processed single-stranded Clostridium neurotoxin, with or without substantial hydrolysis of the peptide bond outside the activation loop of the Clostridium neurotoxin. "Without substantial hydrolysis" may mean that less than 5%, 4%, 3%, 2%, or 1% of the contacted Clostridium neurotoxin is hydrolyzed by the protease in the manner of the present invention at the peptide bond outside the activation loop.

[0074] Those skilled in the art can select appropriate reaction times, temperatures, buffers, and molar ratios of protease to single-chain Clostridium neurotoxin to achieve the above. Optimization of such conditions can be empirically determined using routine techniques, such as SDS-PAGE (stained with, for example, Coomassie or a similarly sensitive dye) visual analysis or spectroscopic techniques (e.g., mass spectrometry) of the reaction products after contact.

[0075] When evaluated by SDS-PAGE (stained with, for example, Coomassie or a similarly sensitive dye), the method of the present invention preferably results in the production of only the L and H chains of Clostridium neurotoxin.

[0076] In one embodiment, the protease-mediated proteolytic processing in the method of the present invention results in the production of fewer than five degradation products of Clostridium neurotoxin L chains or H chains, more preferably four, three, two, or fewer than one degradation product, and preferably the L chains and H chains produced by the method of the present invention are full-length L chains and H chains.

[0077] Therefore, in a particularly preferred embodiment, the protease used in the method of the present invention (e.g., enterokinase or factor Xa) hydrolyzes only the peptide bond of SEQ ID NO: 1, and more preferably only the peptide bond between Arg and Yaa of SEQ ID NO: 1.

[0078] In one embodiment, contact occurs for at least one hour, for example, at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 hours.

[0079] In one embodiment, contact occurs at a temperature of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

[0080] In one embodiment, contact occurs at a temperature of 1 to 10°C (preferably about 4°C). Preferably, contact occurs at a temperature of 1 to 10°C (more preferably about 4°C) for 10 to 25 hours (preferably 15 to 20 hours).

[0081] In one embodiment, contact occurs at a temperature of 15-25°C (preferably about 20°C). Preferably, contact occurs at a temperature of 15-25°C (more preferably about 20°C) for 10-25 hours (preferably 15-20 hours).

[0082] In one embodiment, contact occurs at a temperature of 20-30°C (preferably about 25°C). Preferably, contact occurs at a temperature of 20-30°C (more preferably about 25°C) for 10-25 hours (preferably 15-20 hours).

[0083] The contact step of the method of the present invention may include the use of at least 1 μg of protease per 1 mg of Clostridium neurotoxin. In one embodiment, the contact step of the method of the present invention includes the use of at least 0.1, 0.2, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 μg of protease per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention includes the use of at least 2 μg (more preferably at least 4 μg) of protease per 1 mg of Clostridium neurotoxin.

[0084] In one embodiment, the contact step of the method of the present invention includes the use of ≤20 μg of protease per 1 mg of Clostridium neurotoxin. In one embodiment, the contact step of the method of the present invention includes the use of ≤15 μg of protease per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention includes the use of ≤10 μg of protease per 1 mg of Clostridium neurotoxin. More preferably, the contact step of the method of the present invention includes the use of ≤7 μg of protease per 1 mg of Clostridium neurotoxin.

[0085] The contact step of the method of the present invention may include the use of 0.1 to 20 μg of protease per 1 mg of Clostridium neurotoxin. In one embodiment, the method of the present invention may include the use of 1 to 10 μg of protease per 1 mg of Clostridium neurotoxin, preferably 4 to 7 μg of protease per 1 mg of Clostridium neurotoxin.

[0086] The contact step of the method of the present invention may include the use of at least 10, 20, 30, 40, 50, 60, or 70 units of enterokinase per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of at least 60 units (more preferably at least 70 units) of enterokinase per 1 mg of Clostridium neurotoxin. In some embodiments, the contact step of the method of the present invention may include the use of ≤150, ≤140, ≤130, ≤120, ≤110, ≤100, or ≤90 units of enterokinase per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of ≤100 units (more preferably ≤90 units) of enterokinase per 1 mg of Clostridium neurotoxin. The contact step of the method of the present invention may include the use of 50 to 110 units of enterokinase per 1 mg of Clostridium neurotoxin. In one embodiment, the method of the present invention may involve the use of 70 to 90 units of enterokinase per 1 mg of Clostridium neurotoxin, for example, about 80 units of enterokinase per 1 mg of Clostridium neurotoxin.

[0087] The contact step of the method of the present invention may include the use of at least 0.5, 1, 2, 3, 4, or 5 units of factor Xa per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of at least 3 units (more preferably at least 4 units) of factor Xa per 1 mg of Clostridium neurotoxin. In some embodiments, the contact step of the method of the present invention may include the use of ≤15, ≤14, ≤13, ≤12, ≤11, ≤10, ≤9, ≤8, or ≤7 units of factor Xa per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of ≤8 units (more preferably ≤7 units) of factor Xa per 1 mg of Clostridium neurotoxin. The contact step of the method of the present invention may include the use of 0.5 to 15 units of factor Xa per 1 mg of Clostridium neurotoxin. In one embodiment, the method of the present invention may involve the use of 1 to 10 units (preferably 4 to 7 units) of factor Xa per 1 mg of Clostridium neurotoxin, for example, about 5 or 6 units of factor Xa per 1 mg of Clostridium neurotoxin.

[0088] The contact step of the method of the present invention may include the use of at least 0.02, 0.04, 0.06, or 0.08 units of Lys-C per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of at least 0.04 units of Lys-C per 1 mg of Clostridium neurotoxin. In some embodiments, the contact step of the method of the present invention may include the use of ≤0.5, ≤0.4, or ≤0.2 units of Lys-C per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of ≤0.2 units of Lys-C per 1 mg of Clostridium neurotoxin. The contact step of the method of the present invention may include the use of 0.02 to 0.5 units of Lys-C per 1 mg of Clostridium neurotoxin. Preferably, the method of the present invention may include the use of 0.04 to 0.2 units of Lys-C per 1 mg of Clostridium neurotoxin.

[0089] The contact step of the method of the present invention may include the use of at least 0.1, 0.2, 0.3, or 0.4 units of trypsin per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of at least 0.4 units of trypsin per 1 mg of Clostridium neurotoxin. In some embodiments, the contact step of the method of the present invention may include the use of ≤2.5, ≤2.3, ≤2.1, or ≤1.9 units of trypsin per 1 mg of Clostridium neurotoxin. Preferably, the contact step of the method of the present invention may include the use of ≤1.8 units of trypsin per 1 mg of Clostridium neurotoxin. The contact step of the method of the present invention may include the use of 0.1 to 2.5 units of trypsin per 1 mg of Clostridium neurotoxin. Preferably, the method of the present invention may include the use of 0.3 to 2 units (more preferably 0.4 to 1.8 units) of trypsin per 1 mg of Clostridium neurotoxin.

[0090] In one embodiment, Clostridium neurotoxin (e.g., unmodified) may be BoNT / X. The reference BoNT / X sequence is shown as SEQ ID NO: 33. A histidine-tagged version of BoNT / X is presented as SEQ ID NO: 34. The reference nucleotide sequence encoding BoNT / X is shown as SEQ ID NO: 32.

[0091] In one embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / A. The reference BoNT / A sequence is shown as Sequence ID No. 35.

[0092] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / B. The reference BoNT / B sequence is shown as Sequence ID No. 36.

[0093] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / C. The reference BoNT / C1 sequence is shown as Sequence ID No. 37.

[0094] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / D. The reference BoNT / D sequence is shown as Sequence ID No. 38.

[0095] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / E. The reference BoNT / E sequence is shown as Sequence ID No. 39.

[0096] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / F. The reference BoNT / F sequence is shown as Sequence ID No. 40.

[0097] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be BoNT / G. The reference BoNT / G sequence is shown as Sequence ID No. 41.

[0098] In another embodiment, the Clostridium neurotoxin (e.g., before modification) may be TeNT. The reference TeNT sequence is shown as Sequence ID No. 42.

[0099] As discussed above, Clostridium neurotoxins are composed of two polypeptide chains: a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain has a C-terminal targeting component (receptor-binding domain or H). C Domain) and N-terminal transposition component (H N Includes domains.

[0100] Examples of light chain reference sequences include the following: Botulinum toxin type A neurotoxin: amino acid residues 1-448 Botulinum toxin type B neurotoxin: amino acid residues 1-440 Botulinum C1 neurotoxin: amino acid residues 1-441 Botulinum toxin type D neurotoxin: amino acid residues 1-445 Botulinum toxin type E neurotoxin: amino acid residues 1-422 Botulinum toxin type F neurotoxin: amino acid residues 1-439 Botulinum toxin type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: Amino acid residues 1-457.

[0101] For recently identified BoNT / X molecules, it has been reported that the light chain corresponds to amino acids 1-439, and the light chain boundary can vary by approximately 25 amino acids (e.g., 1-414 or 1-464).

[0102] Because slight variations may occur depending on the serotype, the reference sequences identified above should be considered as guides. For example, US2007 / 0166332 (whose full text is incorporated herein by reference) cites a slightly different Clostridium sequence. Botulinum toxin type A neurotoxin: amino acid residues M1-K448 Botulinum toxin type B neurotoxin: amino acid residues M1-K441 Botulinum C1 neurotoxin: amino acid residues M1-K449 Botulinum toxin type D neurotoxin: amino acid residues M1-R445 Botulinum toxin type E neurotoxin: amino acid residues M1-R422 Botulinum toxin type F neurotoxin: amino acid residues M1-K439 Botulinum toxin type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: Amino acid residues M1-A457.

[0103] The translocation domain is a molecule that enables the translocation of a protease to a target cell, resulting in the functional expression of protease activity within the target cell's cytosol. Whether any molecule (e.g., a protein or peptide) possesses the necessary translocation function of the present invention can be confirmed by any one of several conventional assays.

[0104] For example, Shone C. (1987) describes an in vitro assay using liposomes exposed to a test molecule. The presence of the required transposition function can be easily monitored. +This is confirmed by the release of labeled NAD from liposomes [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

[0105] Further examples are provided by Blaustain R. (1987), who describes a simple in vitro assay using a planar phospholipid bilayer membrane. The membrane is exposed to the test molecule, and the required transposition function is confirmed by an increase in conductance across the membrane [see Blaustain (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120].

[0106] Further methodologies for evaluating membrane fusions and, consequently, identifying transposition domains suitable for use in the present invention are provided in Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

[0107] The present invention also encompasses mutant transposition domains insofar as the mutant domain still demonstrates the required transposition activity. For example, the mutant may have at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, or at least 98% amino acid sequence homology with the reference transposition domain. When the term "fragment" is used in relation to a transposition domain, it means a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference transposition domain. In the case of a Clostridium transposition domain, the fragment is preferably a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference transposition domain (e.g., H NThe domain has at least 100, preferably at least 150, more preferably at least 200, and most preferably at least 250 amino acid residues. The transposition "fragment" of the present invention comprises a fragment of a mutant transposition domain based on a reference sequence.

[0108] The transposition domains are preferably capable of forming ion-permeable pores in the lipid membrane under low pH conditions. Preferably, it has been found that only those portions of the protein molecule capable of forming pores within the endosomal membrane are used.

[0109] Transposition domains can be obtained from microbial protein sources, particularly from bacterial or viral protein sources. Therefore, in one embodiment, the transposition domain is a transposition domain of an enzyme, such as a bacterial toxin or viral protein.

[0110] It has been well demonstrated that certain domains of bacterial toxin molecules can form such pores. It is also known that certain transposition domains of membrane fusion proteins expressed by viruses can form such pores. Such domains may be used in the present invention.

[0111] The transposition domain originates from Clostridium, for example, H N It can be a domain (or its functional component). H N This refers to a portion or fragment of the Clostridium neurotoxin's H chain that is approximately equivalent to the amino-terminus half of the H chain, or the domain in the intact H chain that corresponds to that fragment. In one embodiment, the H of the H chain C The function is H C It can be removed by deletion of the amino acid sequence (either at the DNA synthesis level by nuclease or protease treatment, or at the post-synthesis level). Alternatively, H C The function can be inactivated by chemical or biochemical treatment. Therefore, in some embodiments, the H chain may be unable to bind to the binding site on the target cell to which the natural Clostridium neurotoxin (i.e., holotoxin) binds.

[0112] Examples of suitable (reference) inversion domains include the following: Botulinum toxin type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum toxin type F neurotoxin - amino acid residues (440-864) Botulinum toxin type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879).

[0113] Because slight variations may occur depending on the serotype, the reference sequences identified above should be considered as guides. For example, US2007 / 0166332 (whose full text is incorporated herein by reference) cites a slightly different Clostridium sequence. Botulinum toxin type A neurotoxin - amino acid residues (A449~K871) Botulinum toxin type B neurotoxin - amino acid residues (A442~S858) Botulinum type C neurotoxin - amino acid residues (T450~N866) Botulinum type D neurotoxin - amino acid residues (D446~N862) Botulinum toxin type E neurotoxin - amino acid residues (K423~K845) Botulinum toxin type F neurotoxin - amino acid residues (A440~K864) Botulinum toxin type G neurotoxin - amino acid residues (S447~S863) Tetanus neurotoxin - amino acid residues (S458~V879).

[0114] In relation to the present invention, various Clostridium neurotoxins H2, including transposition domains, are available in embodiments of the present invention. NThe region may be useful insofar as these active fragments promote the release of non-cytotoxic proteases (e.g., the L chain of Clostridium) from intracellular vesicles into the cytoplasm of target cells, thereby participating in the execution of the overall cellular mechanism by which Clostridium neurotoxins proteolytically cleave substrates. H derived from the heavy chain of Clostridium neurotoxins. N The region is approximately 410-430 amino acids long and includes a transposition domain. Research has shown that H2 is derived from the heavy chain of Clostridium neurotoxin. N It has been shown that the entire length of the region is not necessary for the transposition activity of the transposition domain. Therefore, aspects of this embodiment include, for example, Clostridium neurotoxin H2 containing a transposition domain having a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. N It may include a region. Other embodiments of this embodiment include, for example, a Clostridium neurotoxin H comprising a transposition domain having a length of at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, and at most 425 amino acids. N It may include a region.

[0115] For further details regarding the genetic basis of toxin production in Clostridium botulinum and C. tetani, the inventors refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0116] H N The term refers to the naturally occurring neurotoxin H N Parts, as well as modified H N Modified H having amino acid sequences and / or synthetic amino acid residues that do not occur naturally, as long as the portion still demonstrates the above transposition function. N To include a part.

[0117] Alternatively, the transposition domain may be of non-Clostridian origin. Examples of non-Clostridian (see reference) transposition domains include the transposition domain of diphtheria toxin [O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the transposition domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-3559], and the transposition domain of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, Examples of transposition domains include, but are not limited to, transposition domains [8437-8442], various membrane-fusionable or hydrophobic peptides with transposition function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp.7934-7938] and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp.1986-1992]. The transposition domain may closely resemble transposition domains present in naturally occurring proteins, or may contain amino acid mutations, provided that the mutations do not disrupt the transposition ability of the transposition domain.

[0118] Specific examples of viral (reference) transposition domains suitable for use in the present invention include specific transposition domains of membrane fusion proteins expressed by viruses. For example, Wagner et al. (1992) and Murata et al. (1992) describe the transposition (i.e., membrane fusion and vesicle formation) functions of several membrane-fusion and amphiphilic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other viral membrane fusion proteins known to possess desired transposition activity include the transposition domain of the membrane fusion peptide of Semryki Forest Virus (SFV), the transposition domain of the vesicular stomatitis virus (VSV) glycoprotein G, the transposition domain of the SER virus F protein, and the transposition domain of the foamy viral envelope glycoprotein. Virus-encoded aspike proteins have specific applications in connection with the present invention, for example, the E1 protein of SFV and the G protein of VSV.

[0119] The use of transposition domains listed in the table below (see reference) includes the use of their sequence variants. A variant may include one or more conserved nucleic acid substitutions and / or deletions or insertions, insofar as the variant has the required transposition function. A variant may also include one or more amino acid substitutions and / or deletions or insertions, insofar as the variant has the required transposition function. [Table B]

[0120] Clostridium neurotoxin H C Examples of domain reference sequences include the following:

[0121] BoNT / A-N872~L1296 BoNT / B-E859~E1291 BoNT / C1-N867~E1291 BoNT / D-S863~E1276 BoNT / E-R846~K1252 BoNT / F-K865~E1274 BoNT / G-N864~E1297 TeNT-I880~D1315.

[0122] Regarding the recently identified BoNT / X, H C The domain corresponds to amino acids 893–1306, and it has been reported that the domain boundary can change by approximately 25 amino acids (for example, 868–1306 or 918–1306).

[0123] Clostridium neurotoxins described herein may further comprise a translocation-promoting domain. This domain facilitates the delivery of non-cytotoxic proteases to the cytosol of target cells, as described, for example, in WO08 / 008803 and WO08 / 008805, which are incorporated herein by reference.

[0124] For example, suitable transposition-promoting domains include enveloped viral membrane fusion peptide domains, such as influenza virus membrane fusion peptide domains (e.g., influenza A virus membrane fusion peptide domain with 23 amino acids), alphavirus membrane fusion peptide domains (e.g., semliki forest virus membrane fusion peptide domain with 26 amino acids), vediclovirus membrane fusion peptide domains (e.g., vesicular stomatitis virus membrane fusion peptide domain with 21 amino acids), respirovirus membrane fusion peptide domains (e.g., Sendai virus membrane fusion peptide domain with 25 amino acids), and mo Examples include rubirivirus membrane fusion peptide domains (e.g., a 25-amino acid canine distempervirus membrane fusion peptide domain), abrasive virus membrane fusion peptide domains (e.g., a 25-amino acid Newcastle disease virus membrane fusion peptide domain), henipavirus membrane fusion peptide domains (e.g., a 25-amino acid Hendravirus membrane fusion peptide domain), metapneumovirus membrane fusion peptide domains (e.g., a 25-amino acid human metapneumovirus membrane fusion peptide domain), or spumavirus membrane fusion peptide domains, such as a monkey foam virus membrane fusion peptide domain, or fragments or variants thereof.

[0125] As a further example, the relocation-promoting domain is Clostridium neurotoxin H CN It may include the domain or fragments thereof or variants. More specifically, Clostridium neurotoxin H CN The relocation-promoting domain may have lengths of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. In this regard, Clostridium neurotoxin H CN The transposition-promoting domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include the following: Botulinum toxin type A neurotoxin - amino acid residues (872-1110) Botulinum toxin type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum toxin type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum toxin type F neurotoxin - amino acid residues (865-1105) Botulinum toxin type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127).

[0126] The above sequence positions may vary slightly depending on the serotype / subtype, and the appropriate (reference) Clostridium neurotoxin H CN Further examples of domains include the following: Botulinum toxin type A neurotoxin - amino acid residues (874-1110) Botulinum toxin type B neurotoxin - amino acid residues (861-1097) Botulinum type C neurotoxin - amino acid residues (869-1111) Botulinum type D neurotoxin - amino acid residues (865-1098) Botulinum toxin type E neurotoxin - amino acid residues (848-1085) Botulinum toxin type F neurotoxin - amino acid residues (867-1105) Botulinum toxin type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127).

[0127] Any of the above-mentioned promotion domains can be combined with any of the previously described transposition domain peptides suitable for use in the present invention. For example, a non-Clostridium promotion domain can be combined with a non-Clostridium transposition domain peptide, or with a Clostridium transposition domain peptide. Alternatively, Clostridium neurotoxin H CN The relocation-promoting domain can be combined with a non-Clostridium relocation domain peptide. Alternatively, Clostridium neurotoxin HCN The promotion domain can be combined with Clostridium transposition domain peptides, and examples include the following: Botulinum toxin type A neurotoxin - amino acid residues (449-1110) Botulinum toxin type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum toxin type F neurotoxin - amino acid residues (440-1105) Botulinum toxin type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127).

[0128] In some embodiments, the Clostridium neurotoxin of the present invention is functional H of Clostridium neurotoxin C The domain may be missing. Therefore, the Clostridium neurotoxin, in the binding assay described in Shone et al. (1985) Eur. J. Biochem. 151, 75-82, binds to the rat synaptosome membrane (Clostridium H C They cannot bind (via their constituent components). In one embodiment, the Clostridium neurotoxin preferably lacks the last 50 C-terminal amino acids of the Clostridium neurotoxin holotoxin. In another embodiment, the Clostridium neurotoxin preferably lacks the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of the Clostridium neurotoxin holotoxin. Alternatively, H C Binding activity can be neutralized / reduced by mutagenesis—for example, referring to BoNT / A for convenience, modification of one or two amino acid residues in the ganglioside binding pocket (W1266 to L and Y1267 to F) can be neutralized by mutagenesis. CThis causes the region to lose its receptor binding function. Similar mutations can be made in peptide components of non-serotype A Clostridium, for example, in constructs based on botulinum B (mutations to W1262 to L and Y1263 to F) or botulinum E (mutations to W1224 to L and Y1225 to F). Other mutations to the active site (e.g., Y1267S in botulinum toxin A and the corresponding highly conserved residue in other Clostridium neurotoxins) are H C The same loss of receptor binding activity is achieved. Details of this mutation and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which are incorporated herein by reference.

[0129] H of the natural Clostridium neurotoxin C A peptide contains approximately 400-440 amino acid residues, each comprising two functionally distinct domains of approximately 25 kDa, namely the N-terminal region (H CN (commonly called peptide or domain) and C-terminal region (H CCIt consists of peptides (commonly called domains). This fact is confirmed by the following publications, each of which is incorporated herein by reference in full: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Furthermore, the C-terminal region (H) comprising 160-200 amino acid residues at the C-terminus. CC It has been well demonstrated, and this fact has also been confirmed by the publications mentioned above, that Clostridium neurotoxins are involved in binding to their innate cell receptors, namely nerve endings at the neuromuscular junction. Therefore, throughout this specification, Clostridium heavy chains are involved in the functional heavy chain H C Lacking a peptide (or domain), the heavy chain cannot bind to the cell surface receptor to which the natural Clostridium neurotoxin binds, meaning that the Clostridium heavy chain is simply functionally ineffective. CC This means that it lacks a peptide. In other words, H CC The peptide region may be partially or completely deleted, or otherwise modified (e.g., by conventional chemical or proteolytic treatment) to inactivate its intrinsic binding ability to nerve terminals at the neuromuscular junction.

[0130] Therefore, in one embodiment, the Clostridium neurotoxin H of the present invention NThe peptide is the C-terminal peptide portion of Clostridium neurotoxin (H CC ) is missing part of the natural Clostridium neurotoxin H C It lacks binding function. For example, in one embodiment, Clostridium H is extended towards the C-terminus. N The peptide lacks the 40 amino acid residues at the C-terminus of the Clostridium neurotoxin heavy chain, or the 60 amino acid residues at the C-terminus, or the 80 amino acid residues at the C-terminus, or the 100 amino acid residues at the C-terminus, or the 120 amino acid residues at the C-terminus, or the 140 amino acid residues at the C-terminus, or the 150 amino acid residues at the C-terminus, or the 160 amino acid residues at the C-terminus. In another embodiment, Clostridium H of the present invention N The peptide is the C-terminal peptide portion of Clostridium neurotoxin (H CC ) is missing the entirety, and therefore the H of the natural Clostridium neurotoxin C It lacks binding function. For example, in one embodiment, Clostridium H N The peptide lacks the 165 amino acid residues at the C-terminus of the Clostridium neurotoxin heavy chain, or the 170 amino acid residues at the C-terminus, or the 175 amino acid residues at the C-terminus, or the 180 amino acid residues at the C-terminus, or the 185 amino acid residues at the C-terminus, or the 190 amino acid residues at the C-terminus, or the 195 amino acid residues at the C-terminus. As a further example, Clostridium H of the present invention N The peptide is selected from the group consisting of the following Clostridium H CC The reference array is missing. Botulinum toxin type A neurotoxin - amino acid residues (Y1111~L1296) Botulinum toxin type B neurotoxin - amino acid residues (Y1098~E1291) Botulinum type C neurotoxin - amino acid residues (Y1112~E1291) Botulinum type D neurotoxin - amino acid residues (Y1099~E1276) Botulinum type E neurotoxin - amino acid residues (Y1086~K1252) Botulinum toxin type F neurotoxin - amino acid residues (Y1106~E1274) Botulinum type G neurotoxin - amino acid residues (Y1106~E1297) Tetanus neurotoxin - amino acid residues (Y1128~D1315).

[0131] Since slight variations may occur depending on the serotype, the reference sequences identified above should be considered as guides.

[0132] The present invention is suitable for application to numerous different varieties of Clostridium neurotoxins. Therefore, in relation to the present invention, the term “Clostridium neurotoxin” encompasses toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified Clostridium neurotoxins or derivatives derived from any of the above. The term “Clostridium neurotoxin” also encompasses botulinum neurotoxin serotype H. Preferably, the Clostridium neurotoxin is not BoNT / C1.

[0133] Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself and several accessory proteins that form complexes with it. Currently, there are nine different classes of botulinum neurotoxin, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such serotyping classification correlates with the percentage of sequence identity at the amino acid level. The BoNT protein of a given serotype can be further divided into different subtypes based on the percentage of amino acid sequence identity.

[0134] BoNT is absorbed in the gastrointestinal tract and enters the systemic circulation, where it binds to the presynaptic membrane of cholinergic nerve endings, inhibiting the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP), BoNT / C1, BoNT / A, and BoNT / E cleave 25kDa synaptosome-associated protein (SNAP-25), and BoNT / C1 cleaves syntaxin. BoNT / X is known to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0135] Tetanus toxin is produced in a single serotype by C. tetani. C. butyricum produces BoNT / E, and C. baratii produces BoNT / F.

[0136] The term “Clostridium neurotoxin” also includes, but is not limited to, modified Clostridium neurotoxins and their derivatives, as described below. A modified Clostridium neurotoxin or derivative may contain one or more modified amino acids compared to the natural (unmodified) form of the Clostridium neurotoxin, or may contain one or more inserted amino acids that are not present in the natural (unmodified) form of the Clostridium neurotoxin. For example, a modified Clostridium neurotoxin may have a modified amino acid sequence in one or more domains compared to the natural (unmodified) Clostridium neurotoxin sequence. Such modifications may modify the functional aspects of the toxin, such as bioactivity or persistence. Thus, in one embodiment, the modified Clostridium neurotoxin of the present invention is a modified Clostridium neurotoxin or a modified Clostridium neurotoxin derivative or a modified Clostridium neurotoxin derivative.

[0137] Modified Clostridium neurotoxins have modified heavy chain amino acid sequences (e.g., modified H CThe domain may have one or more modifications, and the modified heavy chains bind to target nerve cells with higher or lower affinity than natural (unmodified) Clostridium neurotoxins. C Such modifications in a domain are H C This may include modifying residues in the protein (SV2 or synaptotagmin) binding site that alters the binding of the domain to ganglioside binding sites or to the binding of ganglioside receptors and / or protein receptors on target neurons. Examples of such modified Clostridium neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0138] Modified Clostridium neurotoxins may have one or more modifications in the amino acid sequence of the light chain, for example, modifications in the substrate-binding or catalytic domain that can alter or modify the SNARE protein specificity of the modified L chain. Examples of such modified Clostridium neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0139] Modified Clostridium neurotoxins may contain one or more modifications that increase or decrease the bioactivity and / or biological persistence of the modified Clostridium neurotoxin. For example, modified Clostridium neurotoxins may contain leucine- or tyrosine-based motifs that increase or decrease the bioactivity and / or biological persistence of the modified Clostridium neurotoxin. Suitable leucine-based motifs include xDxxxLL (SEQ ID NO: 74), xExxxLL (SEQ ID NO: 75), xExxxIL (SEQ ID NO: 76), and xExxxLM (SEQ ID NO: 77) (wherein x is any amino acid). A suitable tyrosine-based motif is Yxx-Hy (SEQ ID NO: 78) (wherein Hy is a hydrophobic amino acid). Examples of modified Clostridium neurotoxins containing leucine- and tyrosine-based motifs are described in WO2002 / 08268, the full text of which is incorporated herein by reference.

[0140] The term "Clostridium neurotoxin" encompasses hybrid and chimeric Clostridium neurotoxins. A hybrid Clostridium neurotoxin comprises at least a portion of the light chain derived from one Clostridium neurotoxin or its subtype, and at least a portion of the heavy chain derived from another Clostridium neurotoxin or Clostridium neurotoxin subtype. In one embodiment, a hybrid Clostridium neurotoxin may contain a whole light chain derived from one Clostridium neurotoxin subtype and a heavy chain derived from another Clostridium neurotoxin subtype. In another embodiment, a chimeric Clostridium neurotoxin may contain a portion of the heavy chain (e.g., a binding domain) derived from one Clostridium neurotoxin subtype, and another portion of the heavy chain may be derived from another Clostridium neurotoxin subtype. Similarly, or or otherwise, a therapeutic element may contain light chain portions derived from different Clostridium neurotoxins. Such hybrid or chimeric Clostridium neurotoxins are useful, for example, as a means of delivering the therapeutic utility of such Clostridium neurotoxins to patients who are immunologically resistant to a given Clostridium neurotoxin subtype, to patients who may have receptors for a given Clostridium neurotoxin heavy chain binding domain at lower-than-average concentrations, or to patients who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric Clostridium neurotoxins are described in US8,071,110, the full text of which is incorporated herein by reference. Thus, in one embodiment, the modified Clostridium neurotoxin of the present invention is a modified hybrid Clostridium neurotoxin or a modified chimeric Clostridium neurotoxin.

[0141] In a particularly preferred embodiment, the Clostridium neurotoxin is BoNT / X containing at least one domain derived from a non-BoNT / X Clostridium neurotoxin (e.g., a BoNT / X hybrid or chimera).

[0142] For example, in one embodiment, the Clostridium neurotoxin (including the exogenous activation loop) of the present invention may include the following: i. BoNT / XL chain and non-BoNT / XH chain N and H C domain, ii. BoNT / XH N Domain and non-BoNT / XL strands and H C domain, iiiBoNT / XH C Domain and non-BoNT / XL strands and H N domain, iv. BoNT / XL strand and H N Domain and non-BoNT / XH C domain, v.BoNT / XL chain and H C Domain and non-BoNT / XH N domain or vi.BoNT / XH N Domain and H C Domain and non-BoNT / XL strands.

[0143] In one embodiment, the modified Clostridium neurotoxin of the present invention comprises a BoNT / XL chain and H N Domain and BoNT / AH C Includes domains. In one embodiment, BoNT / XL chain and H N Domain and BoNT / AH C The modified Clostridium neurotoxin of the present invention, including the domain, is encoded by a nucleotide sequence having at least 70% sequence identity with respect to SEQ ID NO: 6. In one embodiment, the BoNT / XL chain and H N Domain and BoNT / AH C The modified Clostridium neurotoxin of the present invention, including the domain, is encoded by a nucleotide sequence having at least 80% or 90% sequence identity with respect to SEQ ID NO: 6. Preferably, the BoNT / XL chain and H N Domain and BoNT / AH C The modified Clostridium neurotoxin of the present invention, comprising the domain, is encoded by a nucleotide sequence comprising (more preferably) SEQ ID NO: 6. In one embodiment, the BoNT / XL chain and HN Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 7. In one embodiment, BoNT / X L-chain and H N Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains comprises a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 7. Preferably, BoNT / X L-chain and H N Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains comprises (more preferably, consists of) the polypeptide sequence shown as SEQ ID NO: 7.

[0144] In one embodiment, the modified Clostridium neurotoxin of the present invention is BoNT / X L-chain and H N Domain and BoNT / B H C Domain. In one embodiment, BoNT / X L-chain and H N Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 8. In one embodiment, BoNT / X L-chain and H N Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains is encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 8. Preferably, BoNT / X L-chain and H N Domain and BoNT / A H C The modified Clostridium neurotoxin of the present invention comprising the Domain and BoNT / A H domains is encoded by a nucleotide sequence comprising (more preferably, consisting of) SEQ ID NO: 8. In one embodiment, BoNT / X L-chain and H N Domain and BoNT / A H CThe modified Clostridium neurotoxin of the present invention, including the domain, contains a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 9. In one embodiment, the BoNT / XL chain and H N Domain and BoNT / AH C The modified Clostridium neurotoxin of the present invention, including the domain, contains a polypeptide sequence with at least 80% or 90% sequence identity to SEQ ID NO: 9. Preferably, the BoNT / XL chain and H N Domain and BoNT / AH C The modified Clostridium neurotoxin of the present invention, comprising the domain, comprises (more preferably) the polypeptide sequence shown as Sequence ID No. 9.

[0145] In one embodiment, the modified Clostridium neurotoxin of the present invention comprises a BoNT / XL chain and H N Domain and BoNT / CH C Includes domains. In one embodiment, the modified Clostridium neurotoxin of the present invention includes BoNT / XL chains and H N Domain and BoNT / DH C Includes domains. In one embodiment, the modified Clostridium neurotoxin of the present invention includes BoNT / XL chains and H N Domain and BoNT / EH C Includes domains. In one embodiment, the modified Clostridium neurotoxin of the present invention includes BoNT / XL chains and H N Domain and BoNT / FH C Includes domains. In one embodiment, the modified Clostridium neurotoxin of the present invention includes BoNT / XL chains and H N Domain and BoNT / GH C Includes domains. In one embodiment, the modified Clostridium neurotoxin of the present invention includes BoNT / XL chains and H N Domain and TeNT H C Includes the domain.

[0146] In one embodiment, the Clostridium neurotoxin is BoNT / A, which includes at least one domain derived from a non-BoNT / A Clostridium neurotoxin.

[0147] For example, in one embodiment, the Clostridium neurotoxin (including the exogenous activation loop) of the present invention may include the following: i. BoNT / AL chains and non-BoNT / AH chains N and H C domain, ii. BoNT / AH N Domains and non-BoNT / AL chains and H C domain, iii. BoNT / AH C Domains and non-BoNT / AL chains and H N domain, iv. BoNT / AL chain and H N Domain and non-BoNT / AH C domain, v.BoNT / AL chain and H C Domain and non-BoNT / AH N domain, or vi.BoNT / AH N Domain and H C Domains and non-BoNT / AL strands.

[0148] In one embodiment, the modified Clostridium neurotoxin of the present invention comprises a BoNT / AL chain and H N Domain and BoNT / C1 H C Includes domains. In one embodiment, BoNT / AL chain and H N Domain and BoNT / C1 H C The modified Clostridium neurotoxin of the present invention, including the domain, is encoded by a nucleotide sequence having at least 70% sequence identity with respect to SEQ ID NO: 12. In one embodiment, the BoNT / AL chain and H N Domain and BoNT / C1 H CThe modified Clostridium neurotoxin of the present invention, including the domain, is encoded by a nucleotide sequence having at least 80% or 90% sequence identity with respect to SEQ ID NO: 12. Preferably, the BoNT / AL chain and H N Domain and BoNT / C1 H C The modified Clostridium neurotoxin of the present invention, comprising the domain, is encoded by a nucleotide sequence comprising (more preferably) SEQ ID NO: 12. In one embodiment, the BoNT / AL chain and H N Domain and BoNT / C1 H C The modified Clostridium neurotoxin of the present invention, including the domain, contains a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 13. In one embodiment, the BoNT / AL chain and H N Domain and BoNT / C1 H C The modified Clostridium neurotoxin of the present invention, including the domain, comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 13. Preferably, the BoNT / AL chain and H N Domain and BoNT / C1 H C The modified Clostridium neurotoxin of the present invention, comprising the domain, comprises (more preferably) the polypeptide sequence shown as Sequence ID No. 13.

[0149] In one embodiment, the modified Clostridium neurotoxin of the present invention comprises a BoNT / AL chain and H N Domain and BoNT / BH C Includes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and BoNT / DH C Includes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and BoNT / EH C Includes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and BoNT / FH CIncludes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and BoNT / GH C Includes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and BoNT / XH C Includes a domain. In one embodiment, the modified Clostridium neurotoxin of the present invention includes a BoNT / AL chain and H N Domain and TeNT H C Includes the domain.

[0150] For example, in one embodiment, the Clostridium neurotoxin (including the exogenous activation loop) of the present invention may include the following: i.BoNT / BL chains and non-BoNT / BH chains N and H C domain, ii. BoNT / BH N Domains and non-BoNT / BL strands and H C domain, iii. BoNT / BH C Domains and non-BoNT / BL strands and H N domain, iv. BoNT / BL chain and H N Domain and non-BoNT / BH C domain, v.BoNT / BL chain and H C Domain and non-BoNT / BH N domain, or vi.BoNT / BH N Domain and H C Domains and non-BoNT / BL strands.

[0151] For example, in one embodiment, the Clostridium neurotoxin (including the exogenous activation loop) of the present invention may include the following: i. BoNT / DL chain and non-BoNT / XD H N and H C domain, ii. BoNT / DH N Domains and non-BoNT / DL strands and H C domain, iii. BoNT / DH C Domains and non-BoNT / DL strands and H N domain, iv. BoNT / DL chain and H N Domain and non-BoNT / DH C domain, v.BoNT / DL chain and H C Domain and non-BoNT / DH N domain, or vi.BoNT / DH N Domain and H C Domains and non-BoNT / DL strands.

[0152] For example, in one embodiment, the Clostridium neurotoxin (including the exogenous activation loop) of the present invention may include the following: i. BoNT / EL chains and non-BoNT / EH chains N and H C domain, ii. BoNT / EH N Domains and non-BoNT / EL strands and H C domain, iii. BoNT / EH C Domains and non-BoNT / EL strands and H N domain, iv. BoNT / EL chain and H N Domain and non-BoNT / EH C domain, v.BoNT / EL chain and H C Domain and non-BoNT / EH N domain, or vi.BoNT / EH N Domain and H C Domains and non-BoNT / EL strands.

[0153] For example, in one embodiment, the Clostridium neurotoxin of the present invention (including an exogenous activation loop) may include the following. i. BoNT / F L chain and non-BoNT / F H N and H C domain, ii. BoNT / F H N domain and non-BoNT / F L chain and H C domain, iii. BoNT / F H C domain and non-BoNT / F L chain and H N domain, iv. BoNT / F L chain and H N domain and non-BoNT / F H C domain, v. BoNT / F L chain and H C domain and non-BoNT / F H N domain, or vi. BoNT / F H N domain and H C domain and non-BoNT / F L chain.

[0154] For example, in one embodiment, the Clostridium neurotoxin of the present invention (including an exogenous activation loop) may include the following. i. BoNT / G L chain and non-BoNT / G H N and H C domain, ii. BoNT / G H N domain and non-BoNT / G L chain and H C domain, iii. BoNT / G H C domain and non-BoNT / G L chain and H N domain, iv. BoNT / G L chain and H N domain and non-BoNT / G H C domain, v. BoNT / G L chain and H C domain and non-BoNT / G H N domain, or vi. BoNT / G H N domain and H C domain and non-BoNT / G L chain.

[0155] For example, in one embodiment, the Clostridium neurotoxin (including an exogenous activation loop) of the present invention may include the following. i. TeNT L chain and non-TeNT H N and H C domain, ii. TeNT H N domain and non-TeNT L chain and H C domain, iii. TeNT H C domain and non-TeNT L chain and H N domain, iv. TeNT L chain and H N domain and non-TeNT H C domain, v. TeNT L chain and H C domain and non-TeNT H N domain, or vi. TeNT H N domain and H C domain and non-TeNT L chain.

[0156] The term "Clostridium neurotoxin" may also encompass newly discovered botulinum neurotoxin protein family members expressed by non-Clostridium microorganisms, such as the toxin encoded by the genus Enterococcus, which has the closest sequence identity to BoNT / X; the toxin encoded by Weissella oryzae, which cleaves VAMP2 at W89-W90 and is called BoNT / Wo (NCBI reference sequence: WP_027699549.1); the toxin encoded by Enterococcus faecium, which cleaves VAMP2 and SNAP25 (GenBank: OTO22244.1); and the toxin encoded by Chryseobacterium pipero (NCBI reference sequence: WP_034687872.1).

[0157] The term “Clostridium neurotoxin” shall encompass retargeted Clostridium neurotoxins. In retargeted Clostridium neurotoxins, the Clostridium neurotoxin is modified to include an exogenous ligand known as the targeting moiety (TM). The TM is selected to provide binding specificity to desired target cells and, as part of the retargeting process, the native binding moiety of the Clostridium neurotoxin (e.g., H) is modified. C Domain or H CCThe domain may be removed. Retargeting techniques include, for example, EP-B-0689459, WO1994 / 021300, EP-B-0939818, US6,461,617, US7,192,596, WO1998 / 007864, EP-B-0826051, US5,989,545, US6,395,513, US6,962, 703, WO1996 / 033273, EP-B-0996468, US7,052,702, WO1999 / 017806, EP-B-1107794, US6,632,440, WO2000 / 010598, WO2001 / 21213, WO2006 / 059093, WO2000 / 62814, WO2000 / 04926, WO1993 / 15766, WO2000 / 61192 and WO1999 / 58571, all of which are incorporated herein by reference in their entirety. Accordingly, in one embodiment, the modified Clostridium neurotoxin of the present invention is a modified, retargeted Clostridium neurotoxin. The modified Clostridium neurotoxin of the present invention has the functional H of the Clostridium neurotoxin C The polypeptide may lack a domain and may also lack any functionally equivalent TM. Therefore, the polypeptide lacks the intrinsic binding function for Clostridium neurotoxin, and in the binding assay described in Shone et al. (1985) Eur. J. Biochem. 151, 75-82, it does not bind to rat synaptosome membrane (Clostridium H). C It cannot be bound via its constituent components or via any functionally equivalent TM. In one embodiment, TM is preferably not a wheat germ agglutinin (WGA) peptide.

[0158] In one embodiment, the modified Clostridium neurotoxin of the present invention is the modified LH described herein. N It may contain polypeptides.

[0159] In one embodiment, the modified Clostridium neurotoxin is the modified LH described herein. N It may include polypeptides and targeting moieties (TM).

[0160] Reference Modified LHN Polypeptide sequences are presented herein as SEQ ID NOs. 53-60, but modified LH N The polypeptide sequence may have at least 70% sequence identity with any of sequence numbers 53-60. In one embodiment, modified LH N The polypeptide sequence may have at least 80% or 90% sequence identity with any of sequence numbers 53 to 60. Preferably, modified LH N The polypeptide sequence comprises (more preferably consists of) any of sequence numbers 53 to 60.

[0161] In one embodiment, TM may comprise an anthrax toxin protective antigen (PA) or a fragment thereof. The reference sequence of PA is shown as SEQ ID NO: 52. In some embodiments, PA comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 52 or a fragment thereof. In some embodiments, PA comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 52 or a fragment thereof. In other embodiments, PA comprises (or comprises) a polypeptide sequence shown as SEQ ID NO: 52 or a fragment thereof.

[0162] Therefore, in one embodiment, the modified Clostridium neurotoxin of the present invention comprises a Clostridium neurotoxin non-cytotoxic protease domain and a Clostridium neurotoxin translocation domain (e.g., the LH of Clostridium neurotoxin). N ) and TM containing PA or a fragment thereof. The modified Clostridium neurotoxin contains the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Includes an exogenous activation loop containing Cys (sequence number 1).

[0163] Therefore, in one embodiment, the modified Clostridium neurotoxin is PA or a fragment thereof and LH N / A, LH N / B, LH N / D, LH N / E, LH N / F, LHN / G, LH N / X or LH N The endogenous Clostridium neurotoxin activation loop contains the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -It is replaced by an exogenous activation loop containing Cys (sequence number 1).

[0164] Lys-C hydrolyzes one or more peptide bonds outside the endogenous activation loop of the Clostridium neurotoxin. For example, it has been shown that Lys-C hydrolyzes one or more peptide bonds in PA™, so Lys-C hydrolyzes LH N It is not suitable for use with conventional Clostridium neurotoxins, including PA™.

[0165] Therefore, in some embodiments, the modified Clostridium neurotoxin is PA (or a fragment thereof) and i. Amino acid residues 1-871 of sequence number 35, ii. Amino acid residues 1-858 of SEQ ID NO: 36, iii. Amino acid residues 1-862 of SEQ ID NO: 38, iv. Amino acid residues 1-845 of sequence number 39, v. Amino acid residues 1-864 of sequence number 40, vi. Amino acid residues 1-863 of sequence number 41, vii. Amino acid residues 1-879 of sequence number 42 or viii. Amino acid residues 1-924 of sequence number 33 Includes, The endogenous Clostridium neurotoxin activation loop is the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -It replaces the exogenous activation loop containing Cys (sequence number 1).

[0166] In one embodiment, the modified Clostridium neurotoxin has a polypeptide sequence having at least 70% sequence identity to a polypeptide comprising: a. SEQ ID NO: 52, and b. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60.

[0167] In one embodiment, the modified Clostridium neurotoxin has a polypeptide sequence having at least 80% or 90% sequence identity to a polypeptide comprising: a. SEQ ID NO: 52, and b. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60.

[0168] Preferably, the modified Clostridium neurotoxin has a polypeptide sequence comprising: a. SEQ ID NO: 52, and b. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60.

[0169] In one embodiment, the modified Clostridium neurotoxin has a polypeptide sequence having at least 70% sequence identity to a polypeptide comprising: a. a fragment of SEQ ID NO: 52, and b. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60.

[0170] In one embodiment, the modified Clostridium neurotoxin has a polypeptide sequence having at least 80% or 90% sequence identity to a polypeptide comprising: a. a fragment of SEQ ID NO: 52, and b. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60.

[0171] Preferably, the modified Clostridium neurotoxin has a polypeptide sequence comprising the following: a. Fragment of sequence number 52, and b. Sequence ID 53, Sequence ID 54, Sequence ID 55, Sequence ID 56, Sequence ID 57, Sequence ID 58, Sequence ID 59, or Sequence ID 60.

[0172] In one embodiment, the PA fragment may be PAd1 located at residues 1-258 of SEQ ID NO: 52. In one embodiment, the PA fragment may be PAd2 located at residues 259-487 of SEQ ID NO: 52. In one embodiment, the PA fragment may be PAd3 located at residues 488-594 of SEQ ID NO: 52. In one embodiment, the PA fragment may be PAd4 located at residues 595-735 of SEQ ID NO: 52. In other embodiments, the PA fragment may contain any combination of PAd1, PAd2, PAd3, or PAd4.

[0173] The 83 kDa PA (PA83) is proteolytically processed by furin or other furin-like proteases, and thus the N-terminal fragment can be removed (PA20). The 63 kDa processed form known as PA63 is an oligomer-capable form of PA.

[0174] In one embodiment, the PA fragment may include (or consist of) one or more of PA63, PAd3-d4, PAd2-d4, and PAd4.

[0175] In one embodiment, the PA fragment may be the PA fragment (or a variant thereof) that retains the C-terminal receptor-binding domain of PA or its binding activity to ANTXR2 or nociceptor neuron-binding proteins.

[0176] The present invention also encompasses Clostridium neurotoxins having non-natural protease cleavage sites. In such Clostridium neurotoxins, the natural protease cleavage site (also known as the activation site, as described above) is modified or replaced with a protease cleavage site that is not natural for the Clostridium neurotoxin (i.e., an exogenous cleavage site). Such a site requires an exogenous protease for cleavage, thereby allowing for improved control over the timing and location of the cleavage event. Examples of non-natural protease cleavage sites that may be used in Clostridium neurotoxins include: TEV (Tobacco Sex Virus) (ENLYFQ↓G) (Sequence ID 79) Thrombin (LVPR↓GS) (Sequence ID 80) PreScission (LEVLFQ↓GP) (Sequence ID 81).

[0177] Further protease cleavage sites include recognition sequences cleaved by non-cytotoxic proteases, such as the light chains of Clostridium neurotoxins. These include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences cleaved by non-cytotoxic proteases, such as the light chains of Clostridium neurotoxins. Clostridium neurotoxins containing non-natural protease cleavage sites are described in US7,132,259, EP1206554-B2 and US2007 / 0166332, all of which are incorporated herein by reference in their entirety. Inteins, which are self-cleaving sequences, are also included under the term protease cleavage site. Self-splicing reactions can be controlled, for example, by changing the concentration of the reducing agent present.

[0178] The present invention also encompasses Clostridium neurotoxins containing “destructive cleavage sites.” In such Clostridium neurotoxins, the non-natural protease cleavage sites are incorporated into the Clostridium neurotoxin at a location such that cleavage at the site reduces or inactivates the activity of the Clostridium neurotoxin. Destructive protease cleavage sites may be susceptible to cleavage by local proteases in the event that the Clostridium neurotoxin migrates to a non-target site after administration. Suitable non-natural protease cleavage sites include those mentioned above. Clostridium neurotoxins containing destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, both of which are incorporated herein by reference in their entirety.

[0179] The modified Clostridium neurotoxins of the present invention, particularly their light chain components, can be PEGylated—this may help increase the stability of the light chain components, e.g., the duration of action. Pegylation is particularly preferred when the light chain contains a BoNT / A, B, or C1 protease. Pegylation preferably involves the addition of PEG to the N-terminus of the light chain components. For example, the N-terminus of the light chain can be extended using one or more amino acid residues (e.g., cysteine) that may be identical or different. One or more of the amino acid residues may have their own PEG molecule attached to it (e.g., by covalent bond). An example of this technique is described in WO2007 / 104567, the full text of which is incorporated herein by reference.

[0180] The modified Clostridium neurotoxin of the present invention may not contain the complex-forming proteins present in naturally occurring Clostridium neurotoxin complexes.

[0181] The modified Clostridium neurotoxin of the present invention can be produced using recombinant nucleic acid technology. Therefore, in one embodiment, the modified Clostridium neurotoxin (as described above) is a recombinant modified Clostridium neurotoxin.

[0182] In another embodiment, the present invention provides a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding the modified Clostridium neurotoxin described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.

[0183] In a preferred embodiment, the vector has a promoter selected from the following: Promoter / Inducer / Normal induction conditions Tac (Hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM).

[0184] In another preferred embodiment, the vector has a promoter selected from the following: Promoter, Inducer, Normal induction conditions Tac (Hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM) T5-lac operator / IPTG / 0.2mM (0.05~2.0mM).

[0185] The nucleic acid molecules of the present invention can be prepared using any suitable process known in the art. Therefore, the nucleic acid molecules can be prepared using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be prepared using molecular biology techniques.

[0186] The DNA constructs of the present invention are preferably designed by computer and then synthesized by conventional DNA synthesis techniques.

[0187] The nucleic acid sequence information described above is optionally modified for codon bias according to the final host cell expression system to be used (e.g., Escherichia coli).

[0188] In one embodiment, the present invention provides a nucleotide sequence encoding a modified Clostridium neurotoxin of the present invention. The nucleotide sequence includes a sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In one embodiment, the nucleotide sequence includes a sequence having at least 80% or 90% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. Preferably, the nucleotide sequence includes (more preferably) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0189] The nucleotide sequence of the present invention encodes a polypeptide containing Sequence ID No. 1.

[0190] The terms “nucleotide sequence” and “nucleic acid” are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0191] The present invention provides a method for producing a single-chain (modified) Clostridium neurotoxin protein having a light chain and a heavy chain, comprising expressing the nucleic acid described herein in a suitable host cell, lysing the host cell to provide a host cell homogenate containing the single-chain (modified) Clostridium neurotoxin protein, and isolating the single-chain (modified) Clostridium neurotoxin protein. In one aspect, the present invention provides a method for proteolytically processing the (modified) Clostridium neurotoxin of the present invention into a corresponding double-chain Clostridium neurotoxin, comprising contacting the (modified) Clostridium neurotoxin with a protease (preferably an endopeptidase, e.g., enterokinase or factor Xa) to thereby produce a double-chain Clostridium neurotoxin (e.g., the light chain and heavy chain are joined together by a disulfide bond).

[0192] The present invention therefore provides a double-stranded Clostridium neurotoxin that can be obtained by the method of the present invention.

[0193] The term “can be obtained” also encompasses the term “obtained” in this specification. In one embodiment, the term “can be obtained” means “obtained.”

[0194] The Clostridium neurotoxin of the present invention may be found to be useful in pharmaceuticals or cosmetics as appropriate. In use, the Clostridium neurotoxin is preferably in a double-stranded form.

[0195] The (modified) Clostridium neurotoxins of the present invention may be used to prevent or treat certain medical or cosmetic diseases and conditions. Accordingly, in a further embodiment, the present invention provides the above-described (modified) Clostridium neurotoxins for use in pharmaceuticals.

[0196] In related embodiments, the present invention provides the above-described (modified) Clostridium neurotoxin for use in the prevention or treatment of diseases or conditions selected from: conditions associated with unwanted immunosecretion; strabismus, blepharospasm, esotropia; dystonia (e.g., spasmodic dystonia, mangooral dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia); torticollis (e.g., spasmodic torticollis); cosmetic (cosmetic) applications benefiting from cellular / muscle incapacitation (due to SNARE downregulation or inactivation); ocular motor neuromuscular disorders or conditions (e.g., concomitant strabismus, hypertropia, lateral rectus paralysis, nystagmus, thyroid abnormalities); writer's cramp; blepharospasm; bruxism; Wilson's disease; tremor; tics; segmental myoclonus; spasms; and spasms due to chronic multiple sclerosis. Contraction, spasticity resulting from abnormal bladder management, animus, back spasms, muscle cramps, tension headaches, levator pelvis syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain from muscle spasms), headache pain (e.g., tension headaches), wrinkled forehead, wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological disorders, chronic neurogenic inflammation, and smooth muscle disorders.

[0197] If the (modified) Clostridium neurotoxin of the present invention includes a BoNT / X sequence (or a portion thereof), the Clostridium neurotoxin may be able to target other types of secretory cells other than neurons due to its ability to cleave VAMP4, VAMP5 and / or Ykt6. In some embodiments, the secretory cells to be targeted are secretory immune cells. "Secretory immune cells," as used herein, refers to immune cells that secrete cytokines, chemokines, or antibodies. Such secretory immune cells may include, but are not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Secretory immune cells that secrete antibodies (e.g., leukocytes) may also be targeted by the Clostridium neurotoxin of this disclosure. Examples of antibody-secreting cells, but are not limited to, plasma B cells, plasma cells, plasma cells, and effector B cells. In some embodiments, the Clostridium neurotoxin can modulate the immune response. Therefore, the therapeutic use of Clostridium neurotoxins of the present invention for treating unwanted secretions, preferably conditions associated with unwanted immunosecretion, is further considered herein. Conditions associated with unwanted immunosecretion include, but are not limited to, inflammation, psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic diseases.

[0198] In one embodiment, the present invention provides a pharmaceutical composition comprising the (modified) Clostridium neurotoxin or double-stranded Clostridium neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt.

[0199] The (modified) Clostridium neurotoxin of the present invention may be formulated for oral, parenteral, serial infusion, inhalation, or topical application. Compositions suitable for injection may be in the form of a solution, suspension or emulsion, or a dry powder dissolved or suspended in a suitable vehicle before use.

[0200] In the case of (modified) Clostridium neurotoxins intended for local delivery, the (modified) Clostridium neurotoxin may be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0201] Local delivery methods include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of (modified) Clostridium neurotoxins enable delivery to the lungs and / or other nasal cavities and / or bronchi or airways.

[0202] The (modified) Clostridium neurotoxin of the present invention can be administered to a patient by intraarachnoid or epidural injection into the spinal column at the level of the spinal cord segments involved in the innervation of the affected organ.

[0203] The preferred route of administration is laparoscopic and / or localized, particularly by intramuscular injection.

[0204] The dosage range for administering the (modified) Clostridium neurotoxin of this invention is intended to produce the desired therapeutic effect. It will be acknowledged that the required dosage range will vary depending on the exact nature of the (modified) Clostridium neurotoxin or composition, the route of administration, the nature of the formulation, the patient's age, the nature, degree, or severity of the patient's condition, and, if any, the judgment of the attending physician. These variations in dosage levels can be adjusted using standard empirical routines for optimization.

[0205] The appropriate daily dose (per kg of patient body weight) is in the range of 0.0001 to 1 ng / kg, preferably 0.0001 to 0.5 ng / kg, more preferably 0.002 to 0.5 ng / kg, and particularly preferably 0.004 to 0.5 ng / kg. The unit dose can vary from less than 1 picogram to 30 ng, but is usually 0.01 to 1 ng per dose, which can be administered daily, or preferably less frequently, for example, weekly or monthly.

[0206] A particularly preferred dosing regimen is based on 0.05 ng of (modified) Clostridium neurotoxin as a 1× dose. In this regard, preferred doses are in the range of 1× to 100× (i.e., 0.05 to 5 ng).

[0207] Fluid dosage forms are typically prepared using (modified) Clostridium neurotoxin and a pyrogen-free sterile vehicle. The (modified) Clostridium neurotoxin may be dissolved or suspended in the vehicle, depending on the vehicle and concentration used. In solution preparation, the (modified) Clostridium neurotoxin is dissolved in the vehicle, the solution is made isotonic by the addition of sodium chloride if necessary, sterilized by filtration through a sterile filter using aseptic techniques, and then filled and sealed in suitable sterile vials or ampoules. Alternatively, if the solution stability is adequate, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives, such as buffers, lubricants, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics, may be dissolved in the vehicle.

[0208] The dry powder, which is dissolved or suspended in a suitable vehicle before use, can be prepared by filling a sterile container with pre-sterilized components using aseptic techniques in a sterile area. Alternatively, the components can be dissolved in a suitable container using aseptic techniques in a sterile area. The product is then freeze-dried, and the container is aseptically sealed.

[0209] Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved, and sterilization cannot be achieved by filtration. The components may be isolated in a sterile state, or, after isolation, may be sterilized, for example, by gamma irradiation.

[0210] Advantageously, to promote the uniform distribution of the constituent components, the composition(s) may contain a suspending agent, such as polyvinylpyrrolidone.

[0211] Administration according to the present invention can utilize various delivery technologies, including particulate capsule encapsulation, virus delivery systems, or high-pressure aerosol impact.

[0212] Various methods and related embodiments of the present invention shall be equally applicable to other methods, Clostridium neurotoxins, for example, modified Clostridium neurotoxins (whether in single-chain or double-chain form), uses, or pharmaceutical compositions, and vice versa.

[0213] sequence homology

[0214] While not limited to these, various sequence alignment methods, including global, local, and hybrid methods, such as the segment approach, can be used to determine the identity percentage. The protocols for determining the identity percentage are commonplace within the scope of the art. The global method determines the best alignment by aligning the sequence from the beginning to the end of the molecule, summing the scores of individual residue pairs, and imposing a gap penalty. Examples of lesser-known methods include CLUSTAL W, see Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement, see Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). The local method aligns sequences by identifying one or more conserved motifs shared by all of the input sequences.Examples of lesser-known methods include Match-box sampling, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); and Align-M, see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0215] Therefore, the sequence identity percentage is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap start penalty of 10, a gap extension penalty of 1, and the Henikoff and Henikoff "blosum 62" scoring matrix (amino acids are indicated by standard single-letter codes) as shown below.

[0216] The "sequence identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, the identity percentage can be calculated as the number of identical nucleotides / amino acids, divided by the total number of nucleotides / amino acids and multiplied by 100. The calculation of sequence identity percentage may also take into account the number of gaps that need to be introduced to optimize the alignment of the two or more sequences, and the length of each gap. Sequence comparison of two or more sequences and determination of the identity percentage can be performed using specific mathematical algorithms, such as BLAST, which will be familiar to those skilled in the art.

number

[0217] Next, the identity percentage is calculated as follows:

number

[0218] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, such as conservative amino acid substitutions, other substitutions that do not significantly affect polypeptide folding or activity, small deletions of typically 1 to about 30 amino acids, small amino- or carboxyl-terminal extensions, e.g., amino-terminal methionine residues, or small linker peptides or affinity tags of up to about 20 to 25 residues (see below). Conservative amino acid substitutions Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobic: Leucine Isoleucine Valine Aromatic: Phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine Methionine

[0219] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptide of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptide of the present invention may also contain amino acid residues that do not exist in nature.

[0220] Examples of amino acids that do not exist in nature, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating amino acid residues that do not exist in nature into proteins are known in the art. For example, in vitro systems in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs may be used. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing Escherichia coli (E. coli) S30 extract and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In the second method, translation is carried out in Xenopus laevis oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996).In the third method, *E. coli* cells are cultured in the absence of the native amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-native amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-native amino acid is incorporated into the polypeptide in place of its native counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Native amino acid residues can be converted to non-native species by in vitro chemical modification. To further expand the range of substitutions, chemical modification may be combined with site-directed mutagenesis (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0221] A limited number of non-conserved amino acids, amino acids not coded by the genetic code, naturally occurring amino acids, and unnatural amino acids can be substituted for amino acid residues in the polypeptide of the present invention.

[0222] The essential amino acids in the polypeptide of the present invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Along with mutations of putative contact site amino acids, the sites of biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of the essential amino acids can also be inferred from homology analysis with relevant components of the polypeptide of the present invention (e.g., transposition or protease components).

[0223] Multiple amino acid substitutions can be performed and tested using known methods of mutagenesis and screening, e.g., those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutagenerated polypeptide to determine the range of acceptable substitutions at each position. Other methods that may be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0224] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with many common dictionaries of the terms used herein.

[0225] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. Numerical ranges include numerical values ​​that define the range. Unless otherwise specified, any nucleic acid sequence is written from left to right in a 5' to 3' orientation, and amino acid sequences are written from left to right in an amino to carboxyl orientation.

[0226] The headings provided herein are not limitations on any particular aspect or embodiment of this disclosure.

[0227] Amino acids are referred to herein by their names, three-letter abbreviations, or one-letter abbreviations. The term “protein,” as used herein, includes proteins, polypeptides, and peptides. In this specification, the term “amino acid sequence” is synonymous with the terms “polypeptide” and / or “protein.” In some instances, the term “amino acid sequence” is synonymous with the term “peptide.” In some instances, the term “amino acid sequence” is synonymous with the term “enzyme.” The terms “protein” and “polypeptide” are used synonymously herein. Conventional one-letter and three-letter codes for amino acid residues may be used in this disclosure and claims. Three-letter codes for amino acids as defined in accordance with the Joint Committee on Biochemical Nomenclature of IUPACIUB (JCBN). It is also understood that polypeptides may be encoded by a sequence of more than one nucleotide due to the degeneracy of the genetic code.

[0228] Other definitions of terms may appear throughout this specification. It should be understood that this disclosure is not limited to the specific embodiments described, and may be modified to do so, before the exemplary embodiments are described in more detail. It should also be understood that the scope of this disclosure is defined solely by the appended claims, and that the technical terms used herein are merely for the purpose of describing specific embodiments and are not intended to be limiting.

[0229] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, is also specifically disclosed unless the context explicitly indicates otherwise. Each smaller range between a stated value or intervening value within a stated range and any other stated value or intervening value within that range is included in this disclosure. The upper and lower limits of these smaller ranges may be included in or excluded from the range independently, and each range in which one of the limits is included in a smaller range, neither is included, or both are included is also included in this disclosure, and if the limits are specifically excluded within the stated range, that shall apply. If a stated range includes one or both limits, the range that excludes one or both of the included limits is also included in this disclosure.

[0230] In this specification and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to “Clostridium neurotoxins” includes multiple such candidate substances, and a reference to one or more Clostridium neurotoxins and their equivalents known to those skilled in the art includes references to one or more Clostridium neurotoxins and their equivalents.

[0231] The publications discussed herein were provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as constituting prior art of the claims attached herein.

[0232] Embodiments of the present invention are described herein simply as examples with reference to the following figures and examples. [Brief explanation of the drawing]

[0233] [Figure 1]Figure 1 shows a comparison of the protein sequences of the activation loops of all BoNT serotypes and tetanus toxins, indicating two adjacent cysteines that form a disulfide bridge connecting the light and heavy chains of the toxin molecule. The factor Xa cleavage sites (IDGRs) in BoNT / C1 and BoNT / CD are underlined. [Figure 2] Figure 2 shows that all four tested proteases—trypsin (TrypZean), Lys-C, factor Xa (FXa), and enterokinase (EK)—have the ability to cleave the BoNT / C1 activation loop and produce double-stranded molecules compared to an untreated C-protease control. (A,B) BoNT / C1(0) (SEQ ID NO: 15) treated with factor Xa (FXa), enterokinase (EK), and trypsin (time course from 1 to 16 hours) as shown, tested by SDS-PAGE under non-reducing (A) and reducing conditions (B). Similarly, proteolytic digestion with Lys-C produces double-stranded molecules of BoNT / C1 (C). Both trypsin and Lys-C exhibit off-target cleavage within the heavy chain of BoNT / C1. 1-Benchmark (5 μl), 2-Control sample (-LysC)-DTT, 3-Control sample (-LysC)+DTT, 4-Activated-DTT, 5-Activated+DTT. [Figure 3]Figure 3(A) shows the BoNT / X cleavage by Lys-C. Samples were tested under non-reducing and reducing (+DTT) conditions. 1-Benchmark (5 μl), 2-Protease-free control, 3-LysC 0.125 μg / ml, 4-LysC 0.25 μg / ml, 5-LysC 0.5 μg / ml, 6-LysC 1 μg / ml, 7-LysC 2 μg / ml, 8-LysC 4 μg / ml, 9-Protease-free control + DTT, 10-LysC 0.125 μg / ml + DTT, 11-LysC 0.25 μg / ml + DTT, 12-LysC 0.5 μg / ml + DTT, 13-LysC 1 μg / ml + DTT, 14-LysC 2 μg / ml + DTT, 15-LysC 4 μg / ml + DTT, and 16-Benchmark (5 μl). Figure 3(B) shows the BoNT / X cleavage by trypsin (TrypZean), factor Xa, and enterokinase. Samples were tested under non-reducing and reducing (+DTT) conditions. 1-Benchmark (5 μl), 2-Protease-free control, 3-TrypZean 0.125 μg / ml, 4-TrypZean 0.25 μg / ml, 5-Protease-free control, 6-TrypZean 1 μg / ml, 7-TrypZean 2 μg / ml, 8-TrypZean 4 μg / ml, 9-Factor Xa 5 μg / ml, 10-Enterokinase 0.01 μg / ml, 11-Protease-free control + DTT, 12-TrypZean 0.125 μg / ml + DTT, 13-TrypZean 0.25 μg / ml + DTT, 14-Protease-free control + DTT, 15-TrypZean 1 μg / ml + DTT, 16-TrypZean 2 μg / ml + DTT, 17-TrypZean 4 μg / ml + DTT, 18-factor Xa 5 μg / ml, 19-enterokinase 0.01 μg / ml, and 20-benchmark (5 μl). [Figure 4]Figure 4 shows modified BoNT / X (SEQ ID NO: 5) treated with the indicated protease and successful BoNT double-strand formation events, comparing non-reducing (-DTT) and reducing (+DTT) conditions. Lanes 4-7 show samples during the process. Lanes 8-11 show the final samples after the final polishing step. 1-Captured HisHP load, 2-Control-DTT-protease, 3-Control+DTT-protease, 4-Activated-DTT+EK, 5-Activated+DTT+EK, 6-Activated-DTT+FXa, 7-Activated+DTT+FXa, 8-Final-DTT EK activity, 9-Final+DTT EK activity, 10-Final-DTT FXa activity, 11-Final+DTT FXa activity. [Figure 5] Figure 5(A) shows the BoNT / E values ​​obtained after testing with 10 μg / ml Lysobacter enzymogenes-derived ("Lys-C") and Pseudomonas aeruginosa-derived ("rLys-C") endoproteases Lys-C at 37°C for 2 hours. Samples were tested under non-reducing and reducing (+TCEP) conditions. Figure 5(B) shows the BoNT / E values ​​obtained after treatment with the indicated amount of trypsin at 20°C for 7 hours. (C) Samples stored at -20°C. (T) Protease-free control at 20°C. Samples were tested under reducing (+DTT) conditions. [Figure 6] Figure 6 shows the modified BoNT / E (SEQ ID NO: 11) treated with the indicated protease and the successful BoNT double-strand formation event, comparing non-reducing (-DTT) and reducing (+DTT) conditions. 1-Benchmark (5 μl), 2-Control sample (-EK)-DTT, 3-Control sample (-EK)+DTT, 4-Activated (+EK)-DTT, 5-Activated (+EK)+DTT, 11-Benchmark (5 μl), 6-Control sample (-FXa)-DTT, 7-Control sample (-FXa)+DTT, 8-Activated (+FXa)-DTT, 9-Activated (+FXa)+DTT. [Figure 7]Figure 7(A) shows modified BoNT / A1C1 (SEQ ID NO: 13) treated with factor Xa protease and successful BoNT double-strand formation events, comparing non-reducing (-DTT) and reducing (+DTT) conditions. 1-Benchmark ladder, 2-Control (-FXa-DTT), 3-Control (-FXa+DTT), 4-Activated (+FXa-DTT), 5-Activated (+FXa+DTT). Figure 7(B) shows cleavage of BoNT / A1 with FXa or EK after 2 hours, compared to a positive control (double-stranded BoNT / A1). [Figure 8] Figure 8 shows BoNT / A1C1 and BoNT / C1 dose-dependent inhibition of glutamate release from primary rat neurons. [Figure 9] Figure 9 shows the intact mass spectrometry of enterokinase-activated non-reducible modified BoNT / E (SEQ ID NO: 11), which has the indicated mass of 143853 Da. [Figure 10] Figure 10 shows the intact mass spectrometry of enterokinase-activated reduced-modified BoNT / E (SEQ ID NO: 11), with the indicated masses of 47518Da and 96338Da. [Figure 11] Figure 11 shows the intact mass spectrometry of non-reducible modified BoNT / E (SEQ ID NO: 11) activated by factor Xa, with a indicated mass of 143850 Da. [Figure 12] Figure 12 shows the intact mass spectrometry of reduced modified BoNT / E (SEQ ID NO: 11) activated by factor Xa, with the indicated masses of 47518 Da and 96335 Da. [Figure 13]Figure 13 shows LHN / A1 treated with EK, containing an EK cleavage site inserted into the activated loop (SEQ ID NO: 44), compared with a natural A1 loop (SEQ ID NO: 46) treated with Lys-C. 1-Benchmark (5 μl), 2-Empty, 3-SEQ ID NO: 44+EK-DTT, 4-SEQ ID NO: 44+EK+DTT, 5-SEQ ID NO: 44-EK-DTT, 6-SEQ ID NO: 44-EK+DTT, 7-Benchmark, 8-SEQ ID NO: 46-LysC-DTT, 9-SEQ ID NO: 46-LysC-DTT, 10-SEQ ID NO: 46+LysC-DTT, 11-SEQ ID NO: 46-LysC+DTT, 12-SEQ ID NO: 46+LysC+DTT, and 13-SEQ ID NO: 46-LysC+DTT. [Figure 14] Figure 14 shows the activation of a modified BoNT / XA (SEQ ID NO: 7) using FXa. [Figure 15] Figure 15 shows the activation of a modified BoNT / XB (SEQ ID NO: 9) using FXa.

[0234] Sequence List

[0235] The presence of the first Met amino acid residue or the corresponding first codon is optional if it is shown in any of the following sequence numbers.

[0236] Sequence ID 1 (C1 activation loop consensus sequence) Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys

[0237] Sequence ID 2 (C1 activation loop) CHKAIDGRSLYNKTLDC

[0238] Sequence ID No. 3 (C1 activating loop mutant) CHKAIEGRSLYNKTLDC

[0239] Sequence ID 4 (Nucleic acid sequence, BoNT / X, with C1 activation loop)

[0240] Sequence ID 5 (polypeptide sequence, BoNT / X, with C1 activation loop)

[0241] Sequence ID 6 (Nucleic acid sequence, BoNT / XA [LH N XH C A], with C1 activation loop)

[0242] Sequence ID 7 (polypeptide sequence, BoNT / XA [LH N XH C A], with C1 activation loop)

[0243] Sequence ID 8 (Nucleic acid sequence, BoNT / XB [LH N XH C B], with C1 activation loop)

[0244] Sequence ID 9 (polypeptide sequence, BoNT / XB [LH N XH C B], with C1 activation loop)

[0245] Sequence ID 10 (Nucleic acid sequence, BoNT / E, with C1 activation loop)

[0246] Sequence ID 11 (polypeptide sequence, BoNT / E, with C1 activation loop) [ka]

[0247] Sequence ID 12 (Nucleic acid sequence, BoNT / A1C1, with C1 activation loop)

[0248] Sequence ID 13 (polypeptide sequence, BoNT / A1C1, with C1 activation loop) [ka]

[0249] Sequence ID 14 (Nucleic acid sequence, BoNT / C1(0) (Endonegative))

[0250] Sequence ID 15 (polypeptide sequence, BoNT / C1(0) (Endonegative))

[0251] Sequence ID 16 (Nucleic acid sequence, BoNT / C1)

[0252] Sequence ID 17 (polypeptide sequence, BoNT / C1)

[0253] Sequence ID No. 18 (Enterokinase and Factor Xa cleavage sites) IDGR

[0254] Sequence ID No. 19 (Enterokinase and Factor Xa cleavage site mutation) IEGR

[0255] Sequence ID 20 (BoNT / X activation loop) CPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGC

[0256] Sequence ID 21 (BoNT / A1 & A6 activation loop) CVRGIITSKTKSLDKGYNKALNDLC

[0257] Sequence ID 22 (BoNT / B2, B3 & B6 activation loop) CKSVRAPGIC

[0258] Sequence ID 23 (BoNT / D activation loop) CLRLTKNSRDDSTC

[0259] Sequence ID 24 (BoNT / E1 to E5, E9 & E12 activation loop) CKNIVSVKGIRKSIC

[0260] Sequence ID 25 (BoNT / F1 and F6 activation loop) CKSVIPRKGTKAPPRLC

[0261] Sequence ID 26 (BoNT / F2 and F3 activation loop) CKSIIPRKGTKQSPSLC

[0262] Sequence ID 27 (BoNT / F4 activation loop) CKSIIPRKGTKAPPRLC

[0263] Sequence ID 28 (BoNT / F5 activation loop) CLNSSFKKNTKKPLC

[0264] Sequence ID 29 (BoNT / F7 activation loop) CKSIVSKKGTKNSLC

[0265] Sequence ID 30 (TeNT activation loop) CKKIIPPTNIRENLYNRTASLTDLGGELC

[0266] Sequence ID 31 (BoNT / G activation loop) CKPVMYKNTGKSEQC

[0267] Sequence ID 32 (nucleic acid sequence, wild-type BoNT / X-10HT)

[0268] Sequence ID 33 (polypeptide sequence, BoNT / X) MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNNALDKRD QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

[0269] Sequence ID 34 (polypeptide sequence, wild-type BoNT / X-10HT)

[0270] sequence number 35 (BoNT / A - UniProt P10845) MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINMNFTKLKNFT GLFEFYKLCCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPANLIGNMLYKDDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRWVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRWYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0271] sequence number 36 (BoNT / B - UniProt P10844)

[0272] Sequence ID 37 (BoNT / C - UniProt P18640)

[0273] Sequence ID 38 (BoNT / D - UniProt P19321)

[0274] Sequence ID 39 (BoNT / E - UniProt Q00496)

[0275] Sequence ID 40 (BoNT / F - UniProt A7GBG3)

[0276] Sequence ID 41 (BoNT / G - UniProt Q60393)

[0277] Sequence ID 42 (TeNT - UniProt P04958)

[0278] Sequence ID 43 (Nucleic acid sequence, LH N (A1, EK cutting section included)

[0279] Sequence ID 44 (Polypeptide sequence, LH N (A1, EK cutting section included) MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKT KSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKY TMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIF SGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEAHHHHHHHHHH

[0280] Sequence ID 45 (Nucleic acid sequence, BoNT, with natural A1 loop)

[0281] Sequence ID 46 (polypeptide sequence, BoNT, with natural A1 loop)

[0282] Liquid 47 (Liquid Liquid, Trypsin) MHPLLILAFVGAAVAFPSDDDDKIVGGYTCAENSVPYQVSLNAGYHFCGGSLINDQWVVS AAHCYQYHIQVRLGEYNIDVLEGGEQFIDASKIIRHPKYSSWTLDNDILLIKLSTPAVIN ARVSTLALPSACASGSTECLISGWGNTLSSGVNYPDLLQCLEAPLLSHADCEASYPGEIT NNMICAGFLEGGKDSCQGDSGGPVACNGQLQGIVSWGYGCAQKGKPGVYTKVCNYVDWIQ ETIAANS

[0283] Liquid Liquid 48 (Liquid Liquid Liquid, Lys-C) GVSGSCNIDVVCPEGNGHRDV IRSVAAYSKQGTMWCTGSLVN NSANDKKMYFLTANHCGMTTA AIASSMVVYWNYQNSTCRAPG SSSSGANGDGSLAQSQTGAVV RATNAASDFTLLELNTAANPA YNLFWAGWDRRDQNFAGATAI HHPNVAEKRISHSTVATEISG YNGATGTSHLHVFWQASGGVT EPGSSGSPIYSPEKRVLGQLH 211 GGPSSCSATGADRSDYYGRVF TSWTGGGTSATRLSDWLDAAG TGAQFIDGLDSTGTPPV

[0284] Sequence ID 49 (Polypeptide sequence, enterokinase light chain) IVGGSDSREGAWPWVVALYFDDQQVCGASLVSRDWLVSAAHCVYGRNMEPSKWKAVLGLHMASNLTSPQIETRLIDQIVINRHYNKRRKNNDIAMMHLEMKVNYTDYIQPICLPEEN QVFPPGRICSIAGWGALIYQGSTADVLQEADVPLLSNEKCQQQMPEYNITENMVCAGYDAGGVDSCQGDSGGPLMCQENNRWLLAGVTSFGYQCALPNRPGVYARVPRFTEWIQSFLH

[0285] Sequence ID 50 (Polypeptide sequence, Factor Xa heavy chain) IVGGRDCAEGECPWQALLVNEENEGFCGGTILNEFYVLTAAHCLHQAKRFTVRVGDRNTEQEEGNEMAHEVEMTVKHSRFVKETYDFDIAVLRLKTPIRFRRNVAPACLPEKDWAEATLMTQKTGIVSG FGRTHEKGRLSSTLKMLEVPYVDRSTCKLSSSFTITPNNMFCAGYDTQPEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKFGVYTKVSNFLKWIDKIMKARAGAAGSRGHSEAPATWTVPPPLPL

[0286] Sequence ID 51 (polypeptide sequence, factor Xa light chain) ANSFLEEVKQGNLERECLEEACSLEEAREVFEDAEQTDEFWSKYKDGDQCEGHPCLNQGHCKDGIGDYTCTCAEGFEGKNCEFSTREICSLDNGGCDQFCREERSEVRCSCAHGYVLGDDSKSCVSTERFPCGKFTQGRS

[0287] Sequence ID 52 (Potential sequence, Anthrax Toxin Protective Antigen - NCBI Ref Seq: NP_052806) 1 mkkrkvlipl malstilvss tgnleviqae vkqenrllne sesssqgllg yyfsdlnfqa 61 pmvvtssttg dlsipssele nipsenqyfq saiwsgfikv kksdeytfat sadnhvtmwv 121 ddqevinkas nsnkirlekg rlyqikiqyq renptekgld fklywtdsqn kkevissdnl 181 qlpelkqkss nsrkkrstsa gptvpdrdnd gipdsleveg ytvdvknkrt flspwisnih 241 ekkgltkyks spekwstasd pysdfekvtg ridknvspea rhplvaaypi vhvdmeniil 301 sknedqstqn tdsqtrtisk ntstsrthts evhgnaevha sffdiggsvs agfsnsnsst 361 vaidhslsla gertwaetmg lntadtarln aniryvntgt apiynvlptt slvlgknqtl 421 atikakenql sqilapnnyy psknlapial naqddfsstp itmnynqfle lektkqlrld 481 tdqvygniat ynfengrvrv dtgsnwsevl pqiqettari ifngkdlnlv erriaavnps 541 dplettkpdm tlkealkiaf gfnepngnlq yqgkditefd fnfdqqtsqn iknqlaelna 601 tniytvldki klnakmnili rdkrfhydrn niavgadesv vkeahrevin ssteglllni 661 dkdirkilsg yiveiedteg lkevindryd mlnisslrqd gktfidfkky ndklplyisn 721 pnykvnvyav tkentiinps engdtstngi kkilifskkg yeig

[0288] Sequence ID 53 (LH N (with A, C1 activation loop) [ka]

[0289] Sequence ID 54 (LH N (with B, C1 activation loop) [ka]

[0290] Sequence ID 55 (LH N (with D, C1 activation loop) [ka]

[0291] Sequence ID 56 (LH N (with E, C1 activation loop) [ka]

[0292] Sequence ID 57 (LH N (with F, C1 activation loop) [ka]

[0293] Sequence ID 58 (LH N (with G, C1 activation loop) [ka]

[0294] Sequence ID 59 (LH N(TeNT, with C1 activation loop) [ka]

[0295] Sequence ID 60 (LH N (with / X, C1 activation loop) [ka] [Examples]

[0296] material and method

[0297] material

[0298] • 5ml HiTrap Butyl HP (GE code: 28411005) • 5ml HiTrap Q HP (GE number: 17-1154-01) • 5ml HiTrap phenyl HP column (GE code 17-5195-01) • CHT Type II column (Biorad number 7324756) TrypZean (Sigma serial number T3568) ·Lys-C (Sigma number 000000011047825001) Enterokinase, light chain (NEB number P8070) ·Factor Xa (NEB number P8010) • ACQUITY UPLC Protein BEH C4 Column (Waters No. 186004495)

[0299] Protein purification

[0300] Escherichia coli (E. coli) BL21(DE3) or NiCo(DE3)(NEB) was used for protein expression. Generally, the bacteria were cultured at 37°C until induction, then the temperature was reduced to 16°C, and protein expression was induced overnight using 1 mM IPTG.

[0301] BoNT / AC with C1 loop (SEQ ID NO: 13)

[0302] Bacterial pellets were pulverized by sonication in lysis buffer (50 mM Tris-HCl pH=8) and clarified by centrifugation. The ammonium sulfate concentration was adjusted to 1.3 M, and the target protein was captured using butyl HP resin (GE). The fraction containing the target protein was desalted and loaded onto Q HP resin (GE). The purified protein was activated overnight at 4°C with 6 μg / 1 mg BoNT factor Xa (NEB), followed by polishing with phenyl HP resin (GE).

[0303] BoNT / E (SEQ ID NO: 11) has a C1 loop.

[0304] Bacterial pellets were pulverized by sonication in lysis buffer (100 mM sodium phosphate, pH=7.8, 100 mM NaCl) and clarified by centrifugation. The ammonium sulfate concentration was adjusted to 1.25 M, and the target protein was captured using butyl HP resin (GE). The fraction containing the target protein was desalted and loaded onto Q HP resin (GE). The purified protein was activated overnight at 4°C with 5 μg / 1 mg BoNT factor Xa (NEB) or with 80 U / ml enterokinase (NEB), and subsequently polished using CHT type II resin (Biorad).

[0305] BoNT / X(Sequence ID 5)

[0306] Bacterial pellets were pulverized by sonication in lysis buffer (50 mM Tris-HCl pH=8) and clarified by centrifugation. Target proteins were captured using a HisTrap HP column (GE). The fraction containing the target proteins was desalted and loaded onto Q HP resin (GE). The purified proteins were activated overnight at 4°C with 5 μg / 1 mg BoNT factor Xa (NEB) or with 80 U / ml enterokinase (NEB), and then polished using a 1 ml HisTrap column (GE).

[0307] LC / MS

[0308] Prior to analysis, samples were buffered with 50 mM ammonium bicarbonate. Samples were either intact proteins or reduced by incubation with 10 mM DTT at 37°C for 30 minutes. Samples were tested using a Waters Acquity H-Class UPLC system combined with a Waters Xevo G2-XS QToF mass spectrometer. Mobile phase A: Water, 0.1% formic acid Mobile phase B: 0.1% formic acid in acetonitrile • Column: ACQUITY UPLC Protein BEH C4 (Waters)

[0309] [Example 1]

[0310] The BoNT / C1 activation loop can be cleaved by multiple proteases.

[0311] Inactive BoNT / C1(0) (SEQ ID NO: 15) was incubated with a series of proteases: trypsin, Lys-C, enterokinase, and factor Xa. All proteases showed cleavage within the activation loop. BoNT / C1(0) (SEQ ID NO: 15) was digested overnight at 4°C and 25°C with enterokinase (EK) or factor Xa (FXa). Furthermore, BoNT / C1(0) was digested with trypsin over a 16-hour period at 20°C (Figures 2A, B). All three proteases were able to cleave the BoNT / C1 activation loop and produce double-stranded molecules compared to an untreated control. However, further cleavage products were observed after trypsin and Lys-C digestion.

[0312] [Example 2]

[0313] Characterization and Improvement of BoNT / X Proteolytic Activation

[0314] Partially purified wild-type BoNT / X-10HT (SEQ ID NO: 34) was incubated overnight at 4°C with gradually increasing doses of trypsin (TrypZean) and Lys-C, as well as factor Xa (FXa) and enterokinase (EK).

[0315] Figure 3 shows that wild-type BoNT / X was completely degraded by both Lys-C (Figure 3A) and trypsin (Figure 3B, lanes 12-13 and 15-17). Notably, FXa and EK were unable to cleave the protein into double-stranded form (Figure 3B, lanes 18 and 19, respectively).

[0316] To improve BoNT / X activation, the BoNT / X activation loop was replaced with the BoNT / C1 activation loop (SEQ ID NO: 2), creating a modified BoNT protein, SEQ ID NO: 5. The modified BoNT was purified using several chromatographic steps and treated with enterokinase (EK) or factor Xa (FXa) to confirm that the presence of the BoNT / C loop enables the generation of double-stranded molecules. Surprisingly, Figure 4 shows that EK and FXa specifically cleave the modified BoNT / X into a double-stranded form.

[0317] [Example 3]

[0318] Characterization and Improvement of BoNT / E Proteolytic Activation

[0319] Wild-type BoNT / E was cleaved using Lys-C and trypsin (TryZean). Figure 5A shows that Lys-C inaccurately cleaved / degraded BoNT / E. Treatment with trypsin resulted in shortening of BoNT / E, which meant that a further purification step was required to separate the full-length protein from the shortened product (Figure 5B).

[0320] To improve BoNT / E activation, the BoNT / E activation loop was replaced with the BoNT / C1 activation loop (SEQ ID NO: 2), creating a modified BoNT protein, SEQ ID NO: 11. The modified BoNT was purified using several chromatographic steps and treated with enterokinase (EK) or factor Xa (FXa) to confirm that the presence of the BoNT / C loop enables the generation of double-stranded molecules. Surprisingly, Figure 6 shows that EK and FXa specifically cleave the modified BoNT / E into a double-stranded form.

[0321] [Example 4]

[0322] Proteolytic activation of BoNT / A1C1 chimeras

[0323] To facilitate the activation of proteolytic proteins, the BoNT / C loop is used (LH N / A1 and C1 H C The modified BoNT protein, sequence number 13, was introduced into a BoNT / A1C1 chimera (with the domain). The BoNT / A1 activation loop of BoNT / A1C1 was replaced with the BoNT / C1 loop to create modified BoNT protein sequence number 13. The modified BoNT was purified using several chromatographic steps and treated with factor Xa (FXa) to confirm that the presence of the BoNT / C loop enables the generation of a double-stranded molecule. Figure 7A shows that FXa specifically cleaves modified BoNT / A1C1 into a double-stranded form. For comparison, wild-type Met BoNT / A1 (commercially available from Metabiologics A1080116) containing the A1 activation loop was incubated with FXa and EK. Figure 7B shows that FXa does not cleave the A1 activation loop, EK cleaves it only with minimal activity, and both FXa and EK result in the formation of additional inappropriate cleavage products (degradation products).

[0324] [Example 5]

[0325] BoNT containing a C1 loop retains SNARE cleavage activity.

[0326] Rat primary cortical neurons were treated for 24 hours with BoNT / A1C1 (SEQ ID NO: 13) from Example 4, which contains a BoNT / C1 activation loop, and recombinant BoNT / C1 (SEQ ID NO: 17) was purified. After incubation, SNARE-dependent glutamate release from cells stimulated with potassium chloride was measured (Figure 8). These data confirm the activity of Clostridium neurotoxin modified to include a BoNT / C1 activation loop.

[0327] [Example 6]

[0328] Factor Xa and enterokinase cleave the BoNT / C activation loop at the same site IDGR↓SL.

[0329] Purified BoNT / E containing the BoNT / C1 loop (SEQ ID NO: 11) was activated with either enterokinase or factor Xa protease and incubated with 10 mM DTT to reduce the disulfide bridge and separate the light and heavy chains. Liquid chromatography-mass spectrometry analysis of intact protein mass was performed on reduced and non-reduced modified BoNT / E (SEQ ID NO: 11) samples to map the cleavage sites of both proteases. Both proteases cleaved BoNT / E to produce light and heavy chains of identical size, indicating that both enterokinase and factor Xa cleave at the same site (Table 1). [Table 1]

[0330] Comparative Example 7

[0331] Insertion of protease recognition site into endogenous loop

[0332] The BoNT / C1 activation loop is the only BoNT activation loop that contains the naturally occurring cleavage sites of the site-specific protease FXa (and surprisingly, EK) (see Figure 1). All other loops are cleaved by non-specific proteases, such as trypsin or Lys-C. Cleavage by Lys-C and trypsin often leads to unwanted protein shortening because the cleavage site is determined more by protease reachability than by a specific recognition sequence.

[0333] The innate activation loops derived from each serotype have evolved to enable protease access and process toxins into a double-stranded form by Clostridium. While we don't want to be constrained by theory, it is thought that mutating these loops to create protease recognition sites could lead to conformational changes, which could negatively impact cleavage efficiency.

[0334] To test this hypothesis, a polypeptide (LH) having a BoNT / A1 light chain and transposition domain was used. N / A1) was modified to include the EK protease recognition sequence DDDDK (SEQ ID NO: 44). Modified LH N The efficiency of proteolytic cleavage of / A1 using EK was evaluated and compared with cleavage using Lys-C in the wild-type A1 activation loop (note that a direct comparison using EK is not possible because there is no EK recognition site in the wild-type loop). Figure 13 shows that cleavage of the modified loop is considerably less efficient than that of the wild-type loop.

[0335] [Example 8]

[0336] Proteolytic activation of BoNT / XA chimeras

[0337] A BoNT / XA chimera containing the BoNT / X light chain and transposition domain, the BoNT / A1 binding domain, and the BoNT / C1 activation loop was constructed (SEQ ID NO: 7). Figure 14 shows that the double-stranded form of the modified BoNT / XA chimera was generated after activation using FXa.

[0338] [Example 9]

[0339] Activation of proteolytic activity of BoNT / XB chimeras

[0340] A BoNT / XB chimera was constructed containing the light chain and transposition domain of BoNT / X, the binding domain of BoNT / B, and the BoNT / C1 activation loop (SEQ ID NO: 9). Figure 15 shows that the double-stranded form of the modified BoNT / XB chimera was generated after activation using FXa.

[0341] All publications listed in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the present invention, which will be apparent to those skilled in the art in biochemistry and biotechnology or related fields, are intended to fall within the scope of the following claims.

[0342] item [Item 1] A method for proteolytically processing single-chain Clostridium neurotoxins into their corresponding double-chain Clostridium neurotoxins, a. To provide a single-chain Clostridium neurotoxin. b. Contacting single-chain Clostridium neurotoxin with enterokinase or factor Xa Includes, Single-chain Clostridium neurotoxins have the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -It has an activation loop containing Cys (sequence number 1), where a=1~10 and b=4~15. A method by which enterokinase or factor Xa hydrolyzes the peptide bonds of the activation loop, thereby producing a double-stranded Clostridium neurotoxin. [Item 2] The method described in item 1, where a = 2 to 4. [Item 3] The method described in item 1 or 2, wherein a=3. [Item 4] The method described in any one of the above items, wherein b = 6 to 10. [Item 5] The method described in any one of the above items, wherein b=8. [Item 6] The method according to any one of the above items, wherein the activation loop comprises a polypeptide sequence having at least 70% sequence identity with respect to sequence number 2 or sequence number 3. [Item 7] The method according to any one of the above items, wherein the activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity with respect to SEQ ID NO: 2 or SEQ ID NO: 3. [Item 8] The method according to any one of the above items, wherein the activation loop comprises a polypeptide sequence having sequence number 2 or sequence number 3. [Item 9] The method according to any one of the above items, wherein the activation loop includes sequence number 2. [Item 10] The method according to any one of the above items, wherein the activation loop consists of sequence number 2. [Item 11] The method according to any one of the above items, wherein a single-chain Clostridium neurotoxin is brought into contact with enterokinase. [Item 12] Clostridium neurotoxins, Clostridium neurotoxins functional H C A method according to any one of the above items, lacking a domain. [Item 13] The method according to any one of the preceding items, wherein the Clostridium neurotoxin is a retargeted Clostridium neurotoxin containing a non-Clostridium targeting moiety (TM). [Item 14] The method according to any one of the preceding items, wherein the Clostridium neurotoxin is a retargeted Clostridium neurotoxin containing TM, and TM contains anthrax toxin protective antigen (PA) or a fragment thereof. [Item 15] Clostridium neurotoxin, a. Sequence ID No. 52 or a fragment thereof, and ☐ Sequence IDs 53, 54, 55, 56, 57, 58, 59, or 60 The method according to item 14, having a polypeptide sequence that has at least 70% sequence identity with respect to a polypeptide containing [Item 16] The method described in any one of the above items, wherein the Clostridium neurotoxin is not BoNT / C1. [Item 17] The method according to any one of the above items, wherein the Clostridium neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT. [Item 18] The method according to any one of the preceding items, wherein the Clostridium neurotoxin is BoNT / X, BoNT / E, chimeric BoNT, or hybrid BoNT. [Item 19] Single-chain Clostridium neurotoxin, a. Encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or b. The method according to any one of the above items, comprising a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. [Item 20] A method for producing a modified Clostridium neurotoxin, including the following steps: a. A step to identify the endogenous activation loop of Clostridium neurotoxin, where the Clostridium neurotoxin is i. The peptide bond outside the endogenous activation loop of Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C. ii. The endogenous activation loop is inefficiently processed into a proteolytic state by trypsin or Lys-C. Characterized by; b. A step of replacing an endogenous activation loop with an exogenous activation loop to provide a modified Clostridium neurotoxin, wherein the exogenous activation loop is a polypeptide sequence Cys-(Xaa) a-Ile-Asp / Glu-Gly-Arg-(Yaa) b -It includes Cys (sequence number 1), where a=1 to 10 and b=4 to 15. [Item 21] The method according to item 20, wherein the endogenous activation loop is one or more selected from SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. [Item 22] The method according to item 20 or 21, wherein Clostridium neurotoxins are identified as suitable for use in a method that involves contacting Clostridium neurotoxins with trypsin or Lys-C and confirming the hydrolysis of peptide bonds outside the endogenous activation loop of Clostridium neurotoxins. [Item 23] The method according to any one of items 20 to 22, further comprising contacting a modified Clostridium neurotoxin with an enterokinase or factor Xa to produce a corresponding double-stranded modified Clostridium neurotoxin. [Item 24] The method described in any one of items 20 to 23, where a = 2 to 4. [Item 25] The method described in any one of items 20 to 24, where a=3. [Item 26] The method described in any one of items 20 to 25, where b = 6 to 10. [Item 27] The method described in any one of items 20 to 26, where b=8. [Item 28] The method according to any one of items 20 to 27, wherein the exogenous activation loop comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3. [Item 29] The method according to any one of items 20 to 28, wherein the exogenous activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3. [Item 30] The method according to any one of items 20 to 29, wherein the exogenous activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3. [Item 31] The method described in any one of items 20 to 30, including SEQ ID NO: 2, wherein the exogenous activation loop is an exogenous activation loop. [Item 32] The method described in any one of items 20 to 31, wherein the exogenous activation loop is sequence number 2. [Item 33] The method according to any one of items 23 to 32, wherein a modified Clostridium neurotoxin is brought into contact with enterokinase. [Item 34] Clostridium neurotoxins, Clostridium neurotoxins functional H C A method described in any one of items 20 to 33, lacking a domain. [Item 35] The method according to any one of items 20 to 34, wherein the Clostridium neurotoxin is a retargeted Clostridium neurotoxin containing a non-Clostridium targeting moiety (TM). [Item 36] The method according to any one of items 20 to 35, wherein the Clostridium neurotoxin is a retargeted Clostridium neurotoxin containing TM, and TM contains anthrax toxin protective antigen (PA) or a fragment thereof. [Item 37] Clostridium neurotoxin, a. Sequence ID No. 52 or a fragment thereof, and ☐ Sequence IDs 53, 54, 55, 56, 57, 58, 59, or 60 The method according to item 36, wherein the polypeptide sequence has at least 70% sequence identity with respect to the polypeptide containing. [Item 38] Clostridium neurotoxin is not BoNT / C1, as described in any one of items 20 to 37. [Item 39] The method according to any one of items 20 to 38, wherein the Clostridium neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT. [Item 40] The method according to any one of items 20 to 39, wherein the Clostridium neurotoxin is BoNT / X, BoNT / E, chimeric BoNT, or hybrid BoNT. [Item 41] Modified Clostridium neurotoxin, a. Encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or b. The method according to any one of items 20 to 40, comprising a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. [Item 42] The endogenous activation loop of Clostridium neurotoxin is replaced by an exogenous activation loop, thereby providing a modified Clostridium neurotoxin. The exogenous activation loop is the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Includes Cys (sequence number 1), a=1 to 10, and b=4 to 15. A modified Clostridium neurotoxin (which can be obtained, for example, by the method described in any one of items 20 to 41), characterized in that the peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C and / or the endogenous activation loop is inefficiently proteolytically processed by trypsin or Lys-C. [Item 43] Modified Clostridium neurotoxin as described in item 42, wherein the endogenous activation loop is one or more selected from SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. [Item 44] Modified Clostridium neurotoxins as described in item 42 or 43, where a=2 to 4. [Item 45] A modified Clostridium neurotoxin described in any one of items 42 to 44, wherein a=3. [Item 46] A modified Clostridium neurotoxin described in any one of items 42 to 45, where b = 6 to 10. [Item 47] A modified Clostridium neurotoxin described in any one of items 42 to 46, wherein b=8. [Item 48] A modified Clostridium neurotoxin according to any one of items 42 to 47, wherein the exogenous activation loop comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3. [Item 49] A modified Clostridium neurotoxin according to any one of items 42 to 48, wherein the exogenous activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3. [Item 50] A modified Clostridium neurotoxin according to any one of items 42 to 49, wherein the exogenous activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3. [Item 51] An exogenous activation loop is a modified Clostridium neurotoxin as described in any one of items 42 to 50, including Sequence ID No. 2. [Item 52] An exogenous activation loop comprising a modified Clostridium neurotoxin as described in any one of items 42 to 51, comprising Sequence ID No. 2. [Item 53] Clostridium neurotoxins, Clostridium neurotoxins functional H C A modified Clostridium neurotoxin lacking a domain, as described in any one of items 42 to 52. [Item 54] A modified Clostridium neurotoxin as described in any one of items 42 to 53, wherein the Clostridium neurotoxin is a retargeted Clostridium neurotoxin containing a non-Clostridium targeting moiety (TM). [Item 55] A modified Clostridium neurotoxin described in any one of items 42 to 54, which is not BoNT / C1. [Item 56] A modified Clostridium neurotoxin as described in any one of items 42 to 55, wherein the Clostridium neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT. [Item 57] Modified Clostridium neurotoxins as described in any one of items 42 to 56, wherein the Clostridium neurotoxin is BoNT / X, BoNT / E, chimeric BoNT, or hybrid BoNT. [Item 58] Single-chain Clostridium neurotoxin, a. Encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or b. A modified Clostridium neurotoxin according to any one of items 42 to 57, comprising a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. [Item 59] The modified Clostridium neurotoxin is a retargeted Clostridium neurotoxin comprising a targeting moiety (TM), wherein the TM comprises an anthrax toxin protective antigen (PA) or a fragment thereof, as described in any one of items 42 to 58. [Item 60] Modified Clostridium neurotoxin, a. Sequence ID No. 52 or a fragment thereof, and ☐ Sequence IDs 53, 54, 55, 56, 57, 58, 59, or 60 A modified Clostridium neurotoxin as described in item 59, having a polypeptide sequence having at least 70% sequence identity with the polypeptide containing [the specified substance]. [Item 61] A method for proteolytically processing a modified Clostridium neurotoxin described in any one of items 42 to 60 into a corresponding double-stranded Clostridium neurotoxin, comprising contacting the modified Clostridium neurotoxin with enterokinase or factor Xa to generate a double-stranded Clostridium neurotoxin. [Item 62] A double-stranded Clostridium neurotoxin that can be obtained by the method described in any one of items 1 to 19, 23 to 41, or 61. [Item 63] A pharmaceutical composition comprising a modified Clostridium neurotoxin as described in any one of items 42 to 60 or a double-stranded Clostridium neurotoxin as described in item 62 and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt. [Item 64] Conditions associated with unwanted immunosecretion, strabismus, blepharospasm, esotropia, dystonia (e.g., spasmodic dystonia, mangooral dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic (cosmetic) applications benefiting from cellular / muscle incapacitation (due to SNARE downregulation or inactivation), ocular motor neuromuscular disorders or conditions (e.g., concomitant strabismus, hypertropia, lateral rectus palsy, nystagmus, thyroid abnormal muscle disease), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back cramps, calf cramps, tension headaches, levator pelvis Modified Clostridium neurotoxins as described in any one of items 42 to 60, double-stranded Clostridium neurotoxin as described in item 62, or pharmaceutical compositions as described in item 63, for use in the treatment of one or more of the following: syndromes, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissures, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, myalgia (e.g., pain from muscle spasms), headache pain (e.g., tension headaches), wrinkled forehead, wrinkles of the skin, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological disorders, chronic neurogenic inflammation, and smooth muscle disorders. [Item 65] Use of enterokinase for hydrolysis of peptide bonds in polypeptides containing polypeptide sequences indicated as SEQ ID NO: 18 or SEQ ID NO: 19. [Item 66] A nucleotide sequence encoding a modified Clostridium neurotoxin, comprising a sequence having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. [Explanation of Symbols]

[0343] [Figure 1] Protein ID: Protein number aa start: aa start Sequence: Array aa end: aa finished SEQ ID NO: Sequence ID [Figure 2] 20℃ Time course (hr): 20℃ time elapsed (hours) intact: intact [Figure 4] intact: intact [Figure 5] Benchmark: Benchmark -ve control: Negative control +ve control: Positive control Trypsine: Trypsine intact: intact [Figure 6] intact: intact [Figure 7] intact: intact [Figure 8] Glutamate: Glutamic acid Stimulated: stimulated Basal: Basal [Figures 9-12] Intensity [Counts]: Mass [Da]: Mass [Da] [Figure 14] Bench Mark ladder: Benchmark ladder HisTrap load: HisTrap Road Control: Contrast Control+DTT: Control + DTT Activated: Activated Activated+DTT: Activated+DTT [Figure 15] Bench Mark ladder: Benchmark ladder Final product 1μg: Final product 1μg Final product 1μg + DTT: Final product 1μg + DTT intact: intact

Claims

1. A method for processing single-strand modified Clostridium neurotoxins into corresponding double-strand modified Clostridium neurotoxins in a proteolytic manner, a) To provide a single-chain modified Clostridium neurotoxin in which the endogenous activation loop is replaced by an exogenous activation loop, b) The modified single-chain Clostridium neurotoxin is brought into contact with factor Xa, The aforementioned single-chain Clostridium neurotoxin has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -It has an activation loop containing Cys (sequence number 1), where a=1 to 10 and b=4 to 20. The aforementioned modified Clostridium neurotoxin is not botulinum neurotoxin serotype C1 (BoNT / C1), and A method comprising hydrolyzing the peptide bond of the activation loop with factor Xa, thereby generating the double-stranded modified Clostridium neurotoxin.

2. A method for proteolytically processing single-chain Clostridium neurotoxins into their corresponding double-chain Clostridium neurotoxins, a) To provide a single-chain Clostridium neurotoxin, b) The single-chain Clostridium neurotoxin is brought into contact with enterokinase, The aforementioned single-chain Clostridium neurotoxin has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Has an activation loop containing Cys (sequence number 1), where a is at least 1 and b is at least 1, and A method comprising hydrolyzing the peptide bond of the activation loop with enterokinase, thereby generating the double-stranded Clostridium neurotoxin.

3. A method for producing a modified Clostridium neurotoxin, wherein the method is a) A step to identify the endogenous activation loop of Clostridium neurotoxin, where the Clostridium neurotoxin is i. The peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C. ii. The endogenous activation loop is inefficiently processed in a proteolytic manner by trypsin or Lys-C, or iii. It must be a combination of i. and ii. Characterized by, b) A step of replacing the endogenous activation loop with an exogenous activation loop, thereby providing the modified Clostridium neurotoxin, wherein the exogenous activation loop is the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Includes Cys (sequence number 1), where a=1 to 10 and b=4 to 20, The modified Clostridium neurotoxin is BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G or TeNT, or a chimera of BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G or TeNT, or a retargeted Clostridium neurotoxin containing a non-Clostridium targeting moiety (TM), in a method.

4. The method according to claim 3, wherein the Clostridium neurotoxin is identified as suitable for use in a method comprising contacting the Clostridium neurotoxin with trypsin or Lys-C and confirming the hydrolysis of peptide bonds outside the endogenous activation loop of the Clostridium neurotoxin.

5. The method according to claim 3 or 4, further comprising contacting the modified Clostridium neurotoxin with factor Xa to produce a corresponding double-stranded modified Clostridium neurotoxin.

6. The method according to any one of claims 1 or 3 to 5, wherein the endogenous activation loop is one or more of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71.

7. a) i. a = 1 to 10 ii. a = 2 to 4, or iii. a=3, b) i. b = 4 to 20. ii. b = 6 to 10, or iii. b=8, c) i. The exogenous activation loop includes a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

3. ii. The exogenous activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3, iii. The exogenous activation loop includes SEQ ID NO: 2, or iv. The exogenous activation loop consists of SEQ ID NO: 2, or d) any combination of at least two of a) to c), The method according to any one of claims 1 to 6.

8. a) The Clostridium neurotoxin has a functional H C Missing domain, b) The Clostridium neurotoxin is a retargeted Clostridium neurotoxin comprising a targeting moiety (TM), where TM comprises an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the fragment comprises PAd1, PAd2, PAd3, PAd4, or a combination thereof. c) The Clostridium neurotoxin, i. Sequence ID 52 or a fragment thereof, where the fragment is PAd1, PAd2, PAd3, PAd4, or a combination thereof, and ii. Sequence IDs 53, 54, 55, 56, 57, 58, 59, or 60, A polypeptide sequence having at least 70% sequence identity with a polypeptide containing, or d) any combination of at least two of a) to c), The method according to any one of claims 1 to 7.

9. The Clostridium neurotoxin mentioned above i. Encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12, or ii. A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 13, The method according to any one of claims 1 to 7.

10. The endogenous activation loop of Clostridium neurotoxin is replaced by an exogenous activation loop, thereby providing a modified Clostridium neurotoxin. The exogenous activation loop is a polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1), where a = 1 to 10 and b = 4 to 20, Modified Clostridium neurotoxins, which are BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G or TeNT, or chimeras of BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G or TeNT, or retargeted Clostridium neurotoxins containing non-Clostridium targeting moieties (TM).

11. The modified Clostridium neurotoxin according to claim 10, wherein the endogenous activation loop is one or more of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71.

12. a) i) a = 1 to 10, ii) a = 2 to 4, or iii) a=3, b) i) b = 4 to 20, ii) b = 6 to 10, or iii) b=8, c) i) The exogenous activation loop includes a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, ii) The exogenous activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3, iii) The exogenous activation loop includes SEQ ID NO: 2, or iv) The exogenous activation loop consists of Sequence ID No. 2, d) The Clostridium neurotoxin is (i) The peptide bond outside the endogenous activation loop of the Clostridium neurotoxin is hydrolyzed by trypsin or Lys-C. (ii) The endogenous activation loop is inefficiently processed in a proteolytic manner by trypsin or Lys-C, or (iii) Any combination of (i) and (ii), Characterized by, e) any combination of at least two of a) to d), The modified Clostridium neurotoxin according to claim 10 or 11.

13. a. The Clostridium neurotoxin has a functional H C Missing domain, b. The Clostridium neurotoxin is a retargeted Clostridium neurotoxin comprising a targeting moiety (TM), where TM comprises an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the fragment comprises PAd1, PAd2, PAd3, PAd4, or a combination thereof. c. The Clostridium neurotoxin mentioned above i) Sequence ID 52 or a fragment thereof, where the fragment is PAd1, PAd2, PAd3, PAd4, or a combination thereof, and ii) Sequence ID 53, Sequence ID 54, Sequence ID 55, Sequence ID 56, Sequence ID 57, Sequence ID 58, Sequence ID 59 or Sequence ID 60, A polypeptide sequence having at least 70% sequence identity with a polypeptide containing, or at least two arbitrary combinations of da to c. A modified Clostridium neurotoxin according to any one of claims 10 to 12.

14. i. Encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12, or ii. A polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 13, A modified Clostridium neurotoxin according to any one of claims 10 to 12.

15. A modified Clostridium neurotoxin comprising a polypeptide sequence having at least 80% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 13, In its single-chain form, the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b A modified Clostridium neurotoxin containing an activation loop with -Cys (sequence number 1), where a=1 to 10 and b=4 to 20.

16. The modified Clostridium neurotoxin according to claim 15, which is a double-stranded modified Clostridium neurotoxin.

17. A method for proteolytically processing a modified Clostridium neurotoxin according to any one of claims 10 to 16 into a corresponding double-stranded modified Clostridium neurotoxin, A method comprising contacting the modified Clostridium neurotoxin with a protease to produce the double-stranded modified Clostridium neurotoxin.

18. A double-stranded modified Clostridium neurotoxin that can be obtained by the method described in any one of claims 1, 6 to 11, or 17.

19. A double-stranded Clostridium neurotoxin that can be obtained by the method described in any one of claims 2 or 7 to 11.

20. a) A modified Clostridium neurotoxin according to any one of claims 10 to 16, or b) A modified Clostridium neurotoxin encoded by a nucleic acid encoding a modified Clostridium neurotoxin having at least 80% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12, wherein in single-chain form, the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b A modified Clostridium neurotoxin containing an activation loop including -Cys (sequence number 1), where a=1 to 10 and b=4 to 20. A double-strand modified Clostridium neurotoxin obtained by contacting it with a protease.

21. (a) (i) Modified Clostridium neurotoxin as defined in any one of claims 10 to 16 (ii) A double-stranded modified Clostridium neurotoxin as defined in claim 18 or 20, or (iii) a double-stranded Clostridium neurotoxin as defined in claim 19, and (b) pharmaceutically acceptable carriers, excipients, adjuvants, propellants, salts or any combination of at least two thereof A pharmaceutical composition containing the following:

22. Conditions associated with unwanted immunosecretion, strabismus, blepharospasm, esotropia, dystonia, torticollis, cosmetic therapy applications benefiting from cellular / muscular incapacitation, ocular motor neuromuscular disorders or conditions, writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, back spasms, calf cramps, tension headaches, levator pelvis syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limbs A modified Clostridium neurotoxin according to any one of claims 10 to 16, a double-stranded modified Clostridium neurotoxin according to claim 18 or 20, or a double-stranded Clostridium neurotoxin according to claim 19, for use in the treatment of spasticity, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretion, muscle pain, headache, wrinkled forehead, wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological disorders, chronic neurogenic inflammation, smooth muscle disorders, or any combination thereof.

23. In vitro or in vitro, the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b The use of enterokinase for hydrolyzing peptide bonds of a polypeptide comprising -Cys (SEQ ID NO: 1), wherein a is at least 1 and b is at least 1, Enterokinase is used to hydrolyze the peptide bond between Arg and Yaa in SEQ ID NO: 1 contained within the polypeptide.

24. A nucleic acid encoding a modified Clostridium neurotoxin, comprising a sequence having at least 80% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12, When in single-chain form, the modified Clostridium neurotoxin has the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa) b A nucleic acid containing an activation loop including -Cys (sequence number 1), where a=1 to 10 and b=4 to 20.

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