Clostridial neurotoxins containing an extrinsic activation loop

Abstract

To provide a method for producing clostridial neurotoxin comprising an exogenous activation loop.SOLUTION: The present invention relates to a method for proteolytically processing a single-chain clostridial neurotoxin into a corresponding di-chain clostridial neurotoxin, the method comprising: providing a single-chain clostridial neurotoxin; and contacting the single-chain clostridial neurotoxin with enterokinase or a factor Xa, wherein the single-chain clostridial neurotoxin has an activation loop comprising a polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 1), wherein a=1-10 and b=4-15, and wherein the enterokinase or the factor Xa hydrolyses a peptide bond of the activation loop, thereby producing a di-chain clostridial neurotoxin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to clostridial neurotoxins and methods of activating and using same. [Background technology]

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

[0003] Clostridial neurotoxins are among the most potent toxins known. For example, botulinum neurotoxins have a median lethal dose (LD) in mice ranging from 0.5 to 5 ng / kg, depending on the serotype. 50 ) value. Both tetanus and botulinum toxins 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] Clostridial neurotoxins are expressed in Clostridium as single-chain polypeptides. Each clostridial neurotoxin has a catalytic light chain separated from a heavy chain (including an N-terminal cartwheel domain and a C-terminal receptor-binding domain) by an exposed region called the activation loop. During protein maturation, proteolytic cleavage of the activation loop separates the light and heavy chains of the clostridial neurotoxin, which join together via disulfide bridges to create a fully active dichain toxin. This process must be reproduced during recombinant toxin production. Summary of the Invention [Problem to be solved by the invention]

[0005] Exogenous proteases, such as trypsin or Lys-C, are used to proteolytically activate single-chain clostridial neurotoxins. However, for some clostridial neurotoxins, incubation with Lys-C or trypsin results in partial or inappropriate cleavage of the single-chain polypeptide, resulting in the generation of contaminating single-chain and / or inactive cleavage / degradation products (e.g., in the case of BoNT / E), necessitating the purification of the full-length double-chain polypeptide. Therefore, there is currently no universal exogenous protease for activating clostridial neurotoxins. This is particularly problematic when identifying novel clostridial neurotoxins or generating modified (e.g., chimeric or hybrid) neurotoxins, requiring the 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, and cleavage with trypsin or Lys-C has been shown to completely degrade the polypeptide.

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

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

[0009] The present inventors surprisingly found that enterokinase recognizes and cleaves the IDGR sequence immediately C-terminal to the sequence present in the BoNT / C1 activation loop (see FIG. 1). Advantageously, this sequence can also be recognized and cleaved by factor Xa, another protease that exhibits 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 present inventors surprisingly found that the BoNT / C1 loop constitutes a universal activation loop for clostridial neurotoxins, thus providing flexibility for using four different proteases.

[0010] In one aspect, the present invention provides a method for proteolytically processing a single-chain clostridial neurotoxin (e.g., of a modified clostridial neurotoxin described herein) into a corresponding two-chain clostridial neurotoxin, comprising: a. providing a single-chain clostridial neurotoxin; b. contacting a single-chain clostridial neurotoxin with enterokinase; Including, Single-chain clostridial neurotoxins have the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - has an activation loop containing Cys (SEQ ID NO: 1), Methods are provided in which enterokinase hydrolyzes the peptide bond of the activation loop, thereby generating a di-chain clostridial neurotoxin (eg, a modified di-chain clostridial neurotoxin described herein).

[0011] In a related aspect, the invention provides a method for proteolytically processing a single-chain clostridial neurotoxin (e.g., of a modified clostridial neurotoxin described herein) into a corresponding two-chain clostridial neurotoxin, comprising: a. providing a single-chain clostridial neurotoxin; b. contacting a single-chain clostridial neurotoxin with factor Xa; Including, Single-chain clostridial neurotoxins have the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - has an activation loop containing Cys (SEQ ID NO: 1), Methods are provided in which Factor Xa hydrolyzes the peptide bond of the activation loop, thereby generating a di-chain clostridial neurotoxin (eg, a modified di-chain clostridial neurotoxin described herein).

[0012] The single-chain clostridial neurotoxin is preferably a modified single-chain clostridial neurotoxin of the present invention, and the activation loop is an exogenous activation loop. Advantageously, the present inventors have found that replacing the endogenous clostridial neurotoxin activation loop with the exogenous activation loop shown as SEQ ID NO: 1 (containing 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., a 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 may 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 methods of the present invention involve the use of enterokinase.

[0014] In one aspect, the invention is directed to the use of enterokinase to hydrolyze peptide bonds of a polypeptide (e.g., a Clostridial neurotoxin) comprising the sequence set forth 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 the polypeptide sequence set forth as SEQ ID NO: 1, or a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0015] The present invention also provides a method for making a modified Clostridial neurotoxin, comprising the steps of: a. Identifying the intrinsic activation loop of a clostridial neurotoxin, wherein the clostridial neurotoxin is characterized in that a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin is hydrolyzed by trypsin or Lys-C; b. Replacing the endogenous activation loop with an exogenous activation loop, thereby providing a modified Clostridial neurotoxin, wherein the exogenous activation loop has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - containing Cys (SEQ ID NO: 1); The present invention provides a method comprising:

[0016] The present invention also provides a method for making a modified Clostridial neurotoxin, comprising the steps of: a. Identifying the intrinsic activation loop of a clostridial neurotoxin, which is characterized in that the intrinsic 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 Clostridial neurotoxin, wherein the exogenous activation loop has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b- containing Cys (SEQ ID NO: 1); The present invention provides a method comprising:

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

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

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

[0020] In contrast to clostridial neurotoxins (before modification), in one embodiment, the modified clostridial neurotoxins of the present invention are not inefficiently proteolytically processed by enterokinase or factor Xa, and / or peptide bonds outside the extrinsic activation loop of the modified clostridial neurotoxins are not hydrolyzed by enterokinase or factor Xa. Thus, the clostridial neurotoxins (before modification) are preferably resistant to proteolytic processing by enterokinase and / or factor Xa.

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

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

[0023] In another embodiment, the assay comprises contacting 1 mg of clostridial neurotoxin with 0.40 μg of trypsin at ≧3350 units / mg (about a 1:978 molar ratio of clostridial neurotoxin to trypsin) or with Lys-C at ≧200 units / mg (about a 1:1174 molar ratio of clostridial neurotoxin to Lys-C) in a 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 No. 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 trypsin (Trypzean) is defined as the amount of enzyme that produces a change in absorbance at 253 nm of 0.003 per minute in a 3.2 mL reaction volume at pH 7.6 and 25°C using 0.23 mM Na-benzoyl-L-arginine ethyl ester solution (BAEE) as substrate.

[0025] The Lys-C used is preferably commercially available Lys-C (Sigma No. 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 set forth as SEQ ID NO: 48. One unit of the 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 one or more cleavage products are observed by SDS-PAGE (preferably when stained with Coomassie or a dye of equivalent sensitivity) in addition to the cleavage products of the H and L chains of the clostridial neurotoxin, it is confirmed that trypsin or Lys-C hydrolyzes a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin. Preferably, if at least 3, 4, 5, 6, 7, 8, 9, or 10 cleavage products are observed by SDS-PAGE in addition to the cleavage products of the H and L chains of the clostridial neurotoxin after performing the above assay, it is confirmed that trypsin or Lys-C hydrolyzes a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin.

[0027] Additionally or alternatively, an intrinsic activation loop is confirmed to be inefficiently proteolytically cleaved by trypsin or Lys-C if less than 70% of the intrinsic activation loop is proteolytically processed by trypsin or Lys-C to yield a dichain clostridial neurotoxin (as assessed by SDS-PAGE after performing the above assay, preferably stained with Coomassie or a dye of comparable sensitivity). Preferably, a clostridial neurotoxin can be characterized as having an inefficiently proteolytically cleaved intrinsic activation loop by trypsin or Lys-C if less than 60%, 50%, 40%, 30%, 10%, or 5% of the intrinsic activation loop is proteolytically processed by trypsin or Lys-C (as assessed by SDS-PAGE after performing the above assay). More preferably, if less than 30% of the intrinsic activation loop is proteolytically processed by trypsin or Lys-C (as assessed by SDS-PAGE after performing the above assays), the clostridial neurotoxin can be characterized as having an inefficient proteolytic cleavage of the intrinsic activation loop by trypsin or Lys-C.

[0028] The clostridial neurotoxin (before modification) preferably has peptide bonds (either within or outside the activation loop) that are not 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 clostridial neurotoxin present in the reaction contains peptide bonds that are hydrolyzed by enterokinase or factor Xa in the methods of the invention.

[0029] In one embodiment, the method of the invention further comprises contacting the modified clostridial neurotoxin with enterokinase or factor Xa (more preferably, enterokinase), thereby producing the corresponding two-chain modified clostridial neurotoxin.

[0030] In one aspect, the present invention provides a modified Clostridial neurotoxin (e.g., obtainable by a method of the present invention), wherein the intrinsic activation loop of the Clostridial neurotoxin is replaced by an exogenous activation loop, thereby producing the modified Clostridial neurotoxin, wherein the exogenous activation loop has the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - Contains Cys (SEQ ID NO: 1).

[0031] In one embodiment, the clostridial neurotoxin (before modification) is characterized in that a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin is hydrolyzed by trypsin or Lys-C. In one embodiment, the clostridial neurotoxin (before modification) is characterized in that the intrinsic activation loop is inefficiently proteolytically processed by trypsin or Lys-C. In another embodiment, the clostridial neurotoxin (before modification) is characterized in that a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin is hydrolyzed by trypsin or Lys-C and the intrinsic activation loop is inefficiently proteolytically processed by trypsin or Lys-C. These characteristics of the clostridial neurotoxin (before modification) are preferably confirmed by the assays described above.

[0032] The present invention can involve replacing the intrinsic activation loop of any clostridial neurotoxin with the exogenous activation loop described herein. Preferably, the clostridial neurotoxin is not BoNT / C1. The clostridial neurotoxin can be a botulinum neurotoxin or a tetanus neurotoxin. Preferably, the clostridial neurotoxin is a botulinum neurotoxin (BoNT), such as BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X.

[0033] In one embodiment, the clostridial neurotoxin used in the present invention is BoNT / X, BoNT / E, or BoNT / A1C1 hybrid. Preferably, the clostridial neurotoxin is BoNT / X or BoNT / E, both of which are characterized in that trypsin and / or Lys-C hydrolyze the peptide bond outside their intrinsic activation loop, and / or both clostridial neurotoxins contain intrinsic activation loops that are inefficiently proteolytically processed by trypsin and / or Lys-C. Most preferably, the clostridial neurotoxin used in the present invention is BoNT / X.

[0034] The term "intrinsic activation loop," as used herein, refers to the activation loop present in a subject Clostridial neurotoxin, e.g., a subject Clostridial neurotoxin of the indicated serotype. For example, BoNT / A1 comprises a BoNT / A1 heavy chain and a light chain, and therefore, the intrinsic activation loop of BoNT / A1 is the A1 activation loop. For a Clostridial neurotoxin chimera or hybrid, one skilled in the art would recognize, for example, the L chain and the H chain. N The "endogenous activation loop" can be identified by determining the serotype(s) from which the domain is derived. In some embodiments, a chimeric or hybrid clostridial neurotoxin may have an endogenous activation loop that is a fusion of activation loops from two different serotypes. As an example, a chimeric clostridial neurotoxin, such as BoNT / A1C1, has a BoNT / A1 light chain and a translocation 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, an "endogenous activation loop" is any activation loop that is not SEQ ID NO: 1. In one embodiment, an "endogenous activation loop" is any activation loop that is not SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0036] In contrast, "exogenous activation loop," as used herein, refers to an activation loop that is different from the endogenous activation loop present in a subject Clostridial neurotoxin, e.g., a subject Clostridial neurotoxin of a designated 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. For a Clostridial neurotoxin chimera or hybrid, one skilled in the art would recognize, for example, the L chain and the H chain. N By determining the serotype(s) from which the domain is derived, it can be determined whether the activation loop is an "exogenous activation loop." NIf the domain is derived from BoNT / D, the intrinsic activation loop may have a portion of the BoNT / B sequence and a portion of the BoNT / D sequence, and if the activation loop (e.g., the C1 activation loop) is different, 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 a subject Clostridial neurotoxin with the activation loop and determining whether the activation loop is present in the subject Clostridial neurotoxin sequence. If not, the activation loop can be identified as an exogenous activation loop.

[0038] Preferably, the entire endogenous activation loop is replaced with an exogenous activation loop described herein, however, in some embodiments, only a portion of the endogenous activation loop is replaced, e.g., 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) can be deleted, and an exogenous activation loop can be inserted, preferably at the position originally occupied by the endogenous activation loop. Alternatively, the endogenous activation loop can be retained in the modified clostridial neurotoxin of the present invention, and preferably inactivated (e.g., by mutation). It is preferred that the endogenous activation loop (or a portion thereof, more preferably the entire endogenous activation loop) is not present in the modified clostridial neurotoxin of the present invention. It is preferred that the exogenous activation loop occupies the position in the clostridial neurotoxin originally occupied by the endogenous activation loop.

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

[0041] In one embodiment, the intrinsic activation loop comprises a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to 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. In one embodiment, the intrinsic activation loop comprises a polypeptide sequence having at least 95% sequence identity to 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 intrinsic activation loop comprises the polypeptide sequence set forth 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 intrinsic activation loop comprises a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to 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 intrinsic activation loop comprises a polypeptide sequence having at least 95% sequence identity to 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 intrinsic activation loop comprises the polypeptide sequence set forth 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 intrinsic activation loop comprises a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:24. In one embodiment, the intrinsic activation loop comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:24. Preferably, the intrinsic activation loop comprises the polypeptide sequence set forth as SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:24.

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

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

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

[0047] The present invention relates to a method for treating a cysteine-containing nucleotide sequence comprising administering to a subject an intrinsic activation loop, such as a cysteine-containing nucleotide sequence, a cysteine-containing nucleotide sequence, or a cysteine-containing nucleotide sequence, such as a cysteine-containing nucleotide sequence, ... a -Ile-Asp / Glu-Gly-Arg-(Yaa) b The present invention encompasses methods and clostridial neurotoxins in which an exogenous activation loop comprising a polypeptide designated as -Cys (SEQ ID NO: 1) has been replaced by an exogenous activation loop comprising a polypeptide designated 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 designated by the letters "a" and "b," respectively. In one embodiment, "a" and "b" can be any integer that allows proteolytic cleavage of the activation loop to result in an active di-chain clostridial 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] It is not intended that Xaa or Yaa be limited to only one type of amino acid. Thus, one or more residues present at position Xaa can be independently selected from the standard amino acids, i.e., 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, i.e., 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, immediately C-terminal to the Arg residue in SEQ ID NO: 1) is not proline.

[0050] Alternatively / additionally, one or more residues present at positions Xaa or Yaa can be independently selected from non-canonical amino acids (amino acids that are not part of the canonical set of 20 amino acids listed above). By way of example, non-standard amino acids may include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, L-ornithine, L-2-amino-3-guanidinopropionic acid or D-isomers of lysine, arginine and / or ornithine and 4-fluorophenylalanine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.

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

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

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

[0054] In one embodiment, the amino acid residue Xaa immediately N-terminal to Ile of 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. Thus, in embodiments where "a" is at least 2, Xaa may include at least Lys-Ala, where Ala is immediately N-terminal to Ile of SEQ ID NO:1.

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

[0056] In one embodiment, the amino acid residue Yaa immediately C-terminal to Arg of 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 to it. The uncharged hydrophobic amino acid is preferably leucine. Thus, in embodiments where "b" is at least 2, Yaa may include at least Ser-Leu, where Ser is immediately C-terminal to Arg of 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 to SEQ ID NO: 2. In one embodiment, the exogenous activation loop has at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 2. Preferably, the exogenous activation loop has at least 95% sequence identity to SEQ ID NO: 2. More preferably, the exogenous activation loop has at least 99% sequence identity to SEQ ID NO: 2.

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

[0060] The extrinsic loop can also be a variant of SEQ ID NO:2, such as SEQ ID NO:3, or a sequence having at least 70% sequence identity thereto. 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 extrinsic activation loop has at least 80%, 85%, or 90% sequence identity to SEQ ID NO:3. Preferably, the extrinsic activation loop has at least 95% sequence identity to SEQ ID NO:3. More preferably, the extrinsic activation loop has at least 99% sequence identity to SEQ ID NO:3.

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

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

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

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

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

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

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

[0068] The term "enterokinase" or "EK" encompasses the enterokinase described herein as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) that is capable of hydrolyzing the peptide bond of SEQ ID NO: 1. A suitable enterokinase is the enterokinase light chain, commercially available from NEB (#P8070). One unit may be defined as the amount of enzyme required to cleave 25 μg of MBP-EK-paramyosin-ΔSal substrate to 95% completion in 16 hours at 25°C in a total reaction volume of 25 μl (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 (pH 8.0 at 25°C)).

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

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

[0071] The term "factor Xa" encompasses factor Xa as described herein as well as any protease with structural and / or functional similarity (preferably structural and functional similarity) that is capable of hydrolyzing the peptide bond of SEQ ID NO: 1. A suitable factor Xa is commercially available from NEB (No. P8010). One unit can be defined as the amount of factor Xa required to cleave 50 μg of the MBP fusion protein test substrate, MBP-ΔSal (substrate MBP-ΔSal is a maltose-binding protein fused to a truncated form of paramyosin, with the amino acids Ile-Glu-Gly-Arg at the fusion junction) to 95% completion in a 50 μl reaction volume (20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl (pH 8.0)) at 23°C for no longer than 6 hours.

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

[0073] The contacting can occur under any suitable conditions that result in the production of more than 30%, 40%, 50%, or 60% (preferably, more than 70%) of the proteolytically processed single-chain clostridial neurotoxin into the corresponding di-chain clostridial neurotoxin, with or without substantial hydrolysis of peptide bonds outside the activation loop of the clostridial neurotoxin. "Without substantial hydrolysis" can mean that less than 5%, 4%, 3%, 2%, or 1% of the contacted clostridial neurotoxin is hydrolyzed by the protease in the methods of the invention at peptide bonds 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 clostridial neurotoxin to achieve the above. Optimization of such conditions can be empirically determined using routine techniques, such as visual analysis of SDS-PAGE (e.g., stained with Coomassie or similarly sensitive dyes) or spectroscopic techniques (e.g., mass spectrometry) of the reaction products after the contacting.

[0075] The methods of the invention preferably result in the production of only clostridial neurotoxin light and heavy chains, as assessed by SDS-PAGE (eg, stained with Coomassie or similarly sensitive dyes).

[0076] In one embodiment, proteolytic processing by a protease in the methods of the invention results in the production of fewer than five degradation products of a clostridial neurotoxin light or heavy chain, more preferably fewer than four, three, two or one degradation products, and preferably the light and heavy chains produced by the methods of the invention are full-length light and heavy chains.

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

[0078] In one embodiment, the contacting occurs for at least 1 hour, eg, at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 hours.

[0079] In one embodiment, the contacting 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, the contacting occurs at a temperature of 1 to 10° C. (preferably, about 4° C.). Preferably, the contacting 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, the contacting occurs at a temperature of 15 to 25° C. (preferably, about 20° C.). Preferably, the contacting occurs at a temperature of 15 to 25° C. (more preferably, about 20° C.) for 10 to 25 hours (preferably, 15 to 20 hours).

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

[0083] The contacting step of the method of the present invention can include the use of at least 1 μg of protease per mg of clostridial neurotoxin. In one embodiment, the contacting 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 mg of clostridial neurotoxin. Preferably, the contacting 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 mg of clostridial neurotoxin.

[0084] In one embodiment, the contacting step of the method of the present invention comprises using ≦20 μg of protease per mg of clostridial neurotoxin. In one embodiment, the contacting step of the method of the present invention comprises using ≦15 μg of protease per mg of clostridial neurotoxin. Preferably, the contacting step of the method of the present invention comprises using ≦10 μg of protease per mg of clostridial neurotoxin. More preferably, the contacting step of the method of the present invention comprises using ≦7 μg of protease per mg of clostridial neurotoxin.

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

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

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

[0088] The contacting step of the methods of the present invention may involve the use of at least 0.02, 0.04, 0.06, or 0.08 units of Lys-C per mg of clostridial neurotoxin. Preferably, the contacting step of the methods of the present invention may involve the use of at least 0.04 units of Lys-C per mg of clostridial neurotoxin. In some embodiments, the contacting step of the methods of the present invention may involve the use of ≦0.5, ≦0.4, or ≦0.2 units of Lys-C per mg of clostridial neurotoxin. Preferably, the contacting step of the methods of the present invention may involve the use of ≦0.2 units of Lys-C per mg of clostridial neurotoxin. The contacting step of the methods of the present invention may involve the use of 0.02 to 0.5 units of Lys-C per mg of clostridial neurotoxin. Preferably, the methods of the present invention may involve the use of 0.04 to 0.2 units of Lys-C per mg of clostridial neurotoxin.

[0089] The contacting step of the methods of the present invention may involve the use of at least 0.1, 0.2, 0.3, or 0.4 units of trypsin per mg of clostridial neurotoxin. Preferably, the contacting step of the methods of the present invention may involve the use of at least 0.4 units of trypsin per mg of clostridial neurotoxin. In some embodiments, the contacting step of the methods of the present invention may involve the use of ≦2.5, ≦2.3, ≦2.1, or ≦1.9 units of trypsin per mg of clostridial neurotoxin. Preferably, the contacting step of the methods of the present invention may involve the use of ≦1.8 units of trypsin per mg of clostridial neurotoxin. The contacting step of the methods of the present invention may involve the use of 0.1 to 2.5 units of trypsin per mg of clostridial neurotoxin. Preferably, the methods of the present invention may involve the use of 0.3 to 2 units (more preferably, 0.4 to 1.8 units) of trypsin per mg of clostridial neurotoxin.

[0090] In one embodiment, the clostridial neurotoxin (e.g., before modification) can be BoNT / X. A reference BoNT / X sequence is provided as SEQ ID NO: 33. A histidine-tagged version of BoNT / X is provided as SEQ ID NO: 34. A reference nucleotide sequence encoding BoNT / X is provided as SEQ ID NO: 32.

[0091] In one embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / A. A reference BoNT / A sequence is shown as SEQ ID NO:35.

[0092] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / B. A reference BoNT / B sequence is shown as SEQ ID NO:36.

[0093] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / C. The reference BoNT / C1 sequence is shown as SEQ ID NO:37.

[0094] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / D. A reference BoNT / D sequence is shown as SEQ ID NO:38.

[0095] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / E. A reference BoNT / E sequence is shown as SEQ ID NO:39.

[0096] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / F. A reference BoNT / F sequence is shown as SEQ ID NO:40.

[0097] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be BoNT / G. A reference BoNT / G sequence is shown as SEQ ID NO:41.

[0098] In another embodiment, the Clostridial neurotoxin (e.g., before modification) can be TeNT. A reference TeNT sequence is shown as SEQ ID NO:42.

[0099] As discussed above, clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) having a molecular weight of approximately 100 kDa and a light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H chain). C domain) and N-terminal cartwheel component (H N domain).

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

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

[0102] The reference sequences identified above should be considered as a guide, as slight variations may occur according to serovar. For example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different Clostridial sequences. Botulinum type A neurotoxin: amino acid residues M1 to K448 Botulinum type B neurotoxin: amino acid residues M1 to K441 Botulinum type C1 neurotoxin: amino acid residues M1 to K449 Botulinum type D neurotoxin: amino acid residues M1 to R445 Botulinum type E neurotoxin: amino acid residues M1 to R422 Botulinum type F neurotoxin: amino acid residues M1 to K439 Botulinum type G neurotoxin: amino acid residues M1 to K446 Tetanus neurotoxin: amino acid residues M1 to A457.

[0103] A translocation domain is a molecule that allows the translocation of a protease to a target cell, resulting in functional expression of protease activity in the cytosol of the target cell. Whether any molecule (e.g., a protein or peptide) has the requisite 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 translocation function can be easily monitored. +and / or confirmed by the release of labeled NAD from the liposomes [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

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

[0106] Further methodology that allows for the evaluation of membrane fusogenicity and therefore the identification of translocation domains suitable for use in the present invention is 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 variant translocation domains, so long as the variant domain still demonstrates the required translocation activity. By way of example, variants may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with the reference translocation domain. The term fragment, when used in connection with a translocation domain, refers to 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 translocation domain. In the case of a clostridial translocation domain, the fragment preferably comprises a fragment of the reference translocation domain (e.g., H NA translocation "fragment" of the present invention includes fragments of variant translocation domains based on a reference sequence.

[0108] The translocation domain is preferably capable of forming an ion-permeable pore in a lipid membrane under conditions of low pH. It has been found to be advantageous to use only those parts of the protein molecule that are capable of forming a pore in the endosomal membrane.

[0109] The translocation domain can be obtained from a microbial protein source, in particular a bacterial or viral protein source. Thus, in one embodiment, the translocation domain is the translocation domain of an enzyme, for example, a bacterial toxin or a viral protein.

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

[0111] The translocation domain may be of clostridial origin, e.g., H N domain (or functional component thereof). N " refers to a portion or fragment of the H chain of a clostridial neurotoxin equivalent to approximately the amino-terminal half of the H chain, or a domain corresponding to that fragment in an intact H chain. In one embodiment, the H C The function is H C It can be removed by deletion of the amino acid sequence (either at the level of DNA synthesis by nuclease or protease treatment, or at the post-synthetic level). C The function can be inactivated by chemical or biochemical treatment. Thus, in some embodiments, the heavy chain can be incapable of binding to the binding site on the target cell to which the native Clostridial neurotoxin (i.e., the holotoxin) binds.

[0112] Examples of suitable (reference) translocation domains include: Botulinum 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 type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879).

[0113] The reference sequences identified above should be considered as a guide, as slight variations may occur according to serovar. For example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different Clostridial sequences. Botulinum type A neurotoxin - amino acid residues (A449 to K871) Botulinum type B neurotoxin - amino acid residues (A442 to S858) Botulinum type C neurotoxin - amino acid residues (T450 to N866) Botulinum type D neurotoxin - amino acid residues (D446 to N862) Botulinum type E neurotoxin - amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440 to K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458 to V879).

[0114] In the context of the present invention, various Clostridial neurotoxins H containing translocation domains in embodiments of the present invention are NThese domains may be useful insofar as these active fragments facilitate the release of non-cytotoxic proteases (e.g., clostridial light chains) from intracellular vesicles into the cytoplasm of target cells, thereby allowing the clostridial neurotoxin to participate in the overall cellular mechanism by which it proteolytically cleaves its substrate. N The region is approximately 410-430 amino acids long and contains the translocation domain. Studies have shown that the H N It has been shown that the entire length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment include, for example, Clostridial neurotoxin H containing a translocation 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 Other aspects of this embodiment include, for example, Clostridial neurotoxin H that includes a translocation 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, we 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 moieties, as well as modified H N Modified H having non-naturally occurring amino acid sequences and / or synthetic amino acid residues, so long as the moiety still demonstrates the translocation function described above, may be used. N Includes parts.

[0117] Alternatively, the translocation domain may be of non-clostridial origin. Examples of non-clostridial (reference) translocation domains include the diphtheria toxin translocation domain [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 Pseudomonas exotoxin type A translocation domain [Prior et al. Biochemistry (1992) 31, 3555-3559], and the anthrax toxin translocation domain [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 1000-1101]. Examples of suitable translocation domains include, but are not limited to, various membrane-fusogenic or hydrophobic peptides with translocation function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al. (1992) PNAS, 89, pp. 7934-7938] and amphipathic peptides [Murata et al. (1992) Biochem., 31, pp. 1986-1992]. Translocation domains can closely resemble translocation domains present in naturally occurring proteins or can contain amino acid mutations, so long as the mutations do not destroy the translocation ability of the translocation domain.

[0118] Specific examples of viral (reference) translocation domains suitable for use in the present invention include specific translocation domains of membrane fusion proteins expressed by viruses. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e., membrane fusion and vesiculation) functions of several membrane fusion and amphipathic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other virally expressed membrane fusion proteins known to have desirable translocation activity include the translocation domain of the membrane fusion peptide of Semliki Forest virus (SFV), the translocation domain of vesicular stomatitis virus (VSV) glycoprotein G, the translocation domain of SER virus F protein, and the translocation domain of foamy virus envelope glycoprotein. Virus-encoded aspike proteins have particular application in the present invention, such as the E1 protein of SFV and the G protein of VSV.

[0119] Use of the translocation domains listed in the table (below) (see) includes use of sequence variants thereof. Variants may contain one or more conservative nucleic acid substitutions and / or nucleic acid deletions or insertions, so long as the variant retains the required translocation function. Variants may also contain one or more amino acid substitutions and / or amino acid deletions or insertions, so long as the variant retains the required translocation function. [Table B]

[0120] Clostridial neurotoxin H C Examples of domain reference sequences include:

[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] For the recently identified BoNT / X, C The domain corresponds to amino acids 893-1306, and the domain boundary has been reported to vary by approximately 25 amino acids (e.g., 868-1306 or 918-1306).

[0123] The clostridial neurotoxins described herein may further comprise a translocation-facilitating domain, which facilitates delivery of a non-cytotoxic protease to the cytosol of a target cell, as described, for example, in WO08 / 008803 and WO08 / 008805, each of which is incorporated herein by reference.

[0124] For example, a suitable translocation-facilitating domain includes an enveloped virus membrane fusion peptide domain, and examples of suitable membrane fusion peptide domains include influenza virus membrane fusion peptide domains (e.g., influenza A virus membrane fusion peptide domain of 23 amino acids), alphavirus membrane fusion peptide domains (e.g., Semliki Forest virus membrane fusion peptide domain of 26 amino acids), vesiculovirus membrane fusion peptide domains (e.g., vesicular stomatitis virus membrane fusion peptide domain of 21 amino acids), respirovirus membrane fusion peptide domains (e.g., Sendai virus membrane fusion peptide domain of 25 amino acids), and monovirus membrane fusion peptide domains (e.g., 25 amino acids). Examples of such a domain include a rubirivirus membrane fusion peptide domain (e.g., a 25-amino acid canine distemper virus membrane fusion peptide domain), an avulavirus membrane fusion peptide domain (e.g., a 25-amino acid Newcastle disease virus membrane fusion peptide domain), a henipavirus membrane fusion peptide domain (e.g., a 25-amino acid Hendra virus membrane fusion peptide domain), a metapneumovirus membrane fusion peptide domain (e.g., a 25-amino acid human metapneumovirus membrane fusion peptide domain), or a spumavirus membrane fusion peptide domain, such as a simian foamy virus membrane fusion peptide domain or a fragment or variant thereof.

[0125] As a further example, the translocation facilitating domain may be a translocation facilitating domain of Clostridial neurotoxin H CN The neurotoxin may comprise a domain or a fragment or variant thereof. More particularly, the neurotoxin may comprise a clostridial neurotoxin H CN The translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. CN The translocation facilitating 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. Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127).

[0126] The above sequence positions may vary slightly according to serotype / subtype and are appropriate (see reference) Clostridial neurotoxin H CN Further examples of domains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum 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 type E neurotoxin - amino acid residues (848-1085) Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127).

[0127] Any of the above facilitating domains can be combined with any of the previously described translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide, or with a clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The translocation facilitating domain can be combined with a non-clostridial translocation domain peptide.CN The facilitating domain can be combined with a clostridial translocation domain peptide, examples of which include: Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum 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 type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127).

[0128] In some embodiments, the clostridial neurotoxin of the present invention comprises a functional H C Thus, the clostridial neurotoxin binds to rat synaptosomal membranes in the binding assay described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82 (Clostridial H C In one embodiment, the clostridial neurotoxin preferably lacks the last 50 C-terminal amino acids of the clostridial neurotoxin holotoxin. In another embodiment, the clostridial 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 clostridial neurotoxin holotoxin. Alternatively, C Binding activity can be abolished / reduced by mutagenesis - for example, referring to BoNT / A for convenience, modification of one or two amino acid residue mutations in the ganglioside binding pocket (W1266 to L and Y1267 to F) can abolish / reduce binding activity of H CSimilar mutations can be made to the peptide components of non-serotype A clostridia, for example, constructs based on botulinum B (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F) with mutations. Other mutations to the active site (e.g., Y1267S in botulinum type A toxin and corresponding highly conserved residues in other clostridial neurotoxins) can be made to the peptide components of non-serotype A clostridia, for example, constructs based on botulinum B (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F). Other mutations to the active site (e.g., Y1267S in botulinum type A toxin and corresponding highly conserved residues in other clostridial neurotoxins) can be made to the peptide components of non-serotype A clostridia. C This mutation achieves the same loss of receptor binding activity. Details of this and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which is incorporated herein by reference.

[0129] Natural Clostridial Neurotoxin H C The peptide contains approximately 400-440 amino acid residues and is divided into two functionally distinct domains, each of approximately 25 kDa: the N-terminal domain (H CN peptide or domain) and the C-terminal region (H CCIt consists of a set of peptides (commonly called domains). This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: 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 CC It is well documented that the clostridial heavy chain H is involved in the binding of clostridial neurotoxins to their natural cellular receptors, i.e., nerve endings at the neuromuscular junction, a fact also confirmed by the publications mentioned above. Therefore, throughout this specification, it is referred to as a clostridial heavy chain being a functional heavy chain H. C The absence of a peptide (or domain) that prevents the heavy chain from binding to the cell surface receptor to which native clostridial neurotoxins bind means that the clostridial heavy chain is simply a functional H CC This means that the peptide is missing. CC The peptide region may be partially or completely deleted or otherwise modified (e.g., by conventional chemical or proteolytic treatment) to inactivate its natural binding ability to nerve endings at the neuromuscular junction.

[0130] Thus, in one embodiment, the clostridial neurotoxin H of the present invention NThe peptide is the C-terminal peptide portion of the clostridial neurotoxin (H CC ) and therefore lacks the H of natural clostridial neurotoxins. C For example, in one embodiment, the C-terminally extended Clostridium H N The peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of the Clostridial neurotoxin heavy chain. N The peptide is the C-terminal peptide portion of the clostridial neurotoxin (H CC ) and therefore lacks the H of natural clostridial neurotoxins. C By way of example, in one embodiment, Clostridium H N The peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of the clostridial neurotoxin heavy chain. N The peptide is a Clostridium H selected from the group consisting of: CC Lacking a reference sequence. Botulinum type A neurotoxin - amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin - amino acid residues (Y1098 to E1291) Botulinum type C neurotoxin - amino acid residues (Y1112 to E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086 to K1252) Botulinum type F neurotoxin - amino acid residues (Y1106 to E1274) Botulinum type G neurotoxin - amino acid residues (Y1106 to E1297) Tetanus neurotoxin - amino acid residues (Y1128 to D1315).

[0131] The reference sequences identified above should be considered as a guide, as slight variations may occur according to serosubtype.

[0132] The present invention is suitable for application to many different varieties of clostridial neurotoxins. Thus, in the context of the present invention, the term "clostridial neurotoxin" includes 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 clostridial neurotoxins or derivatives derived from any of the above. The term "clostridial neurotoxin" also includes botulinum neurotoxin serotype H. Preferably, the clostridial neurotoxin is not BoNT / C1.

[0133] Botulinum neurotoxins (BoNTs) are produced by C. botulinum in the form of large protein complexes, consisting of BoNT itself and several accessory proteins complexed together. There are currently nine distinct classes of botulinum neurotoxins, namely, botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage of amino acid sequence identity.

[0134] BoNTs are absorbed in the gastrointestinal tract and enter the systemic circulation, where they bind to the presynaptic membranes of cholinergic nerve terminals and prevent 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 synaptosomal-associated protein 25 kDa (SNAP-25). BoNT / C1 cleaves syntaxin. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

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

[0136] The term "clostridial neurotoxin" also encompasses modified clostridial neurotoxins and derivatives thereof, including, but not limited to, those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that are modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. For example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains compared to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the toxin, such as biological activity or persistence. Thus, in one embodiment, the modified clostridial neurotoxin of the present invention is a modified clostridial neurotoxin or a modified clostridial neurotoxin derivative or a modified clostridial neurotoxin derivative.

[0137] Modified clostridial neurotoxins may be modified by modifying the amino acid sequence of the heavy chain (e.g., modified H CThe modified heavy chain may have one or more modifications in the H domain, and the modified heavy chain binds to target neurons with greater or less affinity than the native (unmodified) Clostridial neurotoxin. C Such modifications in the domain are C This may involve modifying residues in the ganglioside-binding site of the domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to ganglioside receptors and / or protein receptors on target neurons. Examples of such modified Clostridial neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

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

[0139] The modified clostridial neurotoxin may contain one or more modifications that increase / decrease the biological activity and / or biological persistence of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin may contain a leucine- or tyrosine-based motif, which increases or decreases the biological activity and / or biological persistence of the modified clostridial 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), where x is any amino acid. Suitable tyrosine-based motifs include Yxx-Hy (SEQ ID NO: 78), where Hy is a hydrophobic amino acid. Examples of modified clostridial neurotoxins containing leucine- and tyrosine-based motifs are described in WO2002 / 08268, the entire contents of which are incorporated herein by reference.

[0140] The term "clostridial neurotoxin" is intended to encompass hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of a light chain derived from one clostridial neurotoxin or subtype and at least a portion of a heavy chain derived from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may contain an entire light chain derived from one clostridial neurotoxin subtype and a heavy chain derived from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may contain a portion of the heavy chain (e.g., a binding domain) derived from one clostridial neurotoxin subtype, with another portion of the heavy chain derived from another clostridial neurotoxin subtype. Similarly, or alternatively, a therapeutic element may comprise a light chain portion derived from a different clostridial neurotoxin. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to patients who are immunologically resistant to a given clostridial neurotoxin subtype, to patients who may have a lower than average concentration of receptors for a given clostridial neurotoxin heavy chain binding domain, 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 clostridial neurotoxins are described in US8,071,110, and this publication is incorporated herein by reference in its entirety.Therefore, in one embodiment, the modified clostridial neurotoxin of the present invention is a modified hybrid clostridial neurotoxin or a modified chimeric clostridial neurotoxin.

[0141] In particularly preferred embodiments, the clostridial neurotoxin is a BoNT / X that includes at least one domain from a non-BoNT / X clostridial neurotoxin (eg, a BoNT / X hybrid or chimera).

[0142] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. BoNT / XL chain and non-BoNT / XH N and H C domain, ii.BoNT / XH N Domains and non-BoNT / XL chains and H C domain, iiiBoNT / XH C Domains and non-BoNT / XL chains and H N domain, iv. BoNT / XL chain 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 domains and non-BoNT / XL chains.

[0143] In one embodiment, the modified Clostridial neurotoxin of the present invention comprises a BoNT / XL chain and a H N Domains and BoNT / AH C In one embodiment, the BoNT / XL chain and H domain N Domains and BoNT / AH C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence that contains at least 70% sequence identity to SEQ ID NO: 6. In one embodiment, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxins of the present invention containing domains are encoded by nucleotide sequences that contain at least 80% or 90% sequence identity to SEQ ID NO: 6. Preferably, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 6. In one embodiment, the BoNT / XL chain and HN Domains and BoNT / AH C The modified Clostridial neurotoxin of the present invention containing a domain comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 7. In one embodiment, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxins of the present invention containing domains comprise polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 7. Preferably, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxin containing domain of the present invention comprises (more preferably consists of) the polypeptide sequence shown as SEQ ID NO:7.

[0144] In one embodiment, the modified Clostridial neurotoxin of the present invention comprises a BoNT / XL chain and a H N Domains and BoNT / BH C In one embodiment, the BoNT / XL chain and H domain N Domains and BoNT / AH C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence that contains at least 70% sequence identity to SEQ ID NO: 8. In one embodiment, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxins of the present invention containing domains are encoded by nucleotide sequences that contain at least 80% or 90% sequence identity to SEQ ID NO: 8. Preferably, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 8. In one embodiment, the BoNT / XL chain and H N Domains and BoNT / AH CThe modified Clostridial neurotoxin of the present invention containing a domain comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 9. In one embodiment, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxins of the present invention containing domains comprise polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 9. Preferably, the BoNT / XL chain and H N Domains and BoNT / AH C The modified Clostridial neurotoxin containing domain of the present invention comprises (more preferably consists of) the polypeptide sequence shown as SEQ ID NO:9.

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

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

[0147] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i.BoNT / AL chain and non-BoNT / AH 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 chains.

[0148] In one embodiment, the modified Clostridial neurotoxin of the present invention comprises a BoNT / AL chain and a H N domain and BoNT / C1 H C In one embodiment, the BoNT / AL chain and H domains N domain and BoNT / C1 H C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence that contains at least 70% sequence identity to SEQ ID NO: 12. In one embodiment, the BoNT / AL chain and H N domain and BoNT / C1 H CThe modified Clostridial neurotoxins of the present invention containing domains are encoded by nucleotide sequences that contain at least 80% or 90% sequence identity to SEQ ID NO: 12. Preferably, the BoNT / AL chain and H N domain and BoNT / C1 H C The modified Clostridial neurotoxin of the present invention containing a domain is encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 12. In one embodiment, the BoNT / AL chain and H N domain and BoNT / C1 H C The modified Clostridial neurotoxin of the present invention containing a domain comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 13. In one embodiment, the BoNT / AL chain and H N domain and BoNT / C1 H C The modified Clostridial neurotoxins of the present invention containing domains comprise polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 13. Preferably, the BoNT / AL chain and H N domain and BoNT / C1 H C The modified Clostridial neurotoxin containing domain of the present invention comprises (more preferably consists of) the polypeptide sequence shown as SEQ ID NO:13.

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

[0150] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. BoNT / BL chains and non-BoNT / BH N and H C domain, ii. BoNT / BH N Domains and non-BoNT / BL chains and H C domain, iii. BoNT / BH C Domains and non-BoNT / BL chains and H N domain, iv. BoNT / BL chain and H N Domains and non-BoNT / BH C domain, v.BoNT / BL chain and H C Domains and non-BoNT / BH N domain, or vi.BoNT / BH N Domain and H C domains and non-BoNT / BL chains.

[0151] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. BoNT / DL chain and non-BoNT / XD H N and H C domain, ii. BoNT / DH N Domains and non-BoNT / DL chains and H C domain, iii. BoNT / DH C Domains and non-BoNT / DL chains 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 domain and non-BoNT / DL chain.

[0152] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i.BoNT / EL chain and non-BoNT / EH N and H C domain, ii.BoNT / EH N Domains and non-BoNT / EL chains and H C domain, iii. BoNT / EH C Domains and non-BoNT / EL chains 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 chains.

[0153] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. BoNT / FL chain and non-BoNT / FH N and H C domain, ii. BoNT / FH N domains and non-BoNT / FL chains and H C domain, iii. BoNT / FH C domains and non-BoNT / FL chains and H N domain, iv. BoNT / FL chain and H N Domain and non-BoNT / FH C domain, v.BoNT / FL chain and H C Domain and non-BoNT / FH N domain, or vi.BoNT / FH N Domain and H C domains and non-BoNT / FL chains.

[0154] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. BoNT / GH chain and non-BoNT / GH N and H C domain, ii. BoNT / GH N Domains and non-BoNT / GL chains and H C domain, iii. BoNT / GH C Domains and non-BoNT / GL chains and H N domain, iv. BoNT / GL chain and H N Domains and non-BoNT / GH C domain, v.BoNT / GL chain and H C Domains and non-BoNT / GH N domain, or vi. BoNT / GH N Domain and H C domains and non-BoNT / GL chains.

[0155] For example, in one embodiment, a clostridial neurotoxin of the present invention (containing an extrinsic activation loop) may comprise: i. TeNT L chain and non-TeNT H chain N and H C domain, ii.TeNT H N Domains and non-TeNT L and H chains C domain, iii.TeNT H C Domains and non-TeNT L and H chains 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 domains as well as non-TeNT light chains.

[0156] The term "clostridial neurotoxin" also encompasses newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the toxin encoded by Enterococcus, which has closest sequence identity to BoNT / X, the toxin encoded by Weissella oryzae, which cleaves VAMP2 at W89-W90, designated 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 "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In a retargeted clostridial neurotoxin, the clostridial neurotoxin has been modified to include an exogenous ligand known as a targeting moiety (TM). The TM is selected to provide binding specificity for the desired target cell, and as part of the retargeting process, the clostridial neurotoxin's native binding moiety (e.g., H C Domain or H CCRetargeting techniques are described, for example, in EP-B-0689459, WO1994 / 021300, EP-B-0939818, US 6,461,617, US 7,192,596, WO1998 / 007864, EP-B-0826051, US 5,989,545, US 6,395,513, US 6,962, 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. Thus, in one embodiment, the modified clostridial neurotoxin of the present invention is a modified retargeted clostridial neurotoxin. The modified clostridial neurotoxin of the present invention is a modified retargeted clostridial neurotoxin that is capable of targeting the functional H of a clostridial neurotoxin. C The polypeptide lacks the native binding function of a clostridial neurotoxin and may also lack any functionally equivalent TM. The polypeptide thus lacks the native binding function of a clostridial neurotoxin and binds to rat synaptosomal membranes (Clostridial H) in the binding assay described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82. C In one embodiment, the TM is preferably not a wheat germ agglutinin (WGA) peptide.

[0158] In one embodiment, the modified Clostridial neurotoxin of the present invention is a modified LH neurotoxin as described herein. N It may comprise a polypeptide.

[0159] In one embodiment, the modified Clostridial neurotoxin is a modified LH neurotoxin described herein. N It may comprise a polypeptide and a targeting moiety (TM).

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

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

[0162] Thus, in one embodiment, the modified clostridial neurotoxin of the present invention comprises a clostridial neurotoxin non-cytotoxic protease domain, a clostridial neurotoxin translocation domain (e.g., the LH domain of a clostridial neurotoxin), N ) and a TM comprising PA or a fragment thereof. The modified Clostridial neurotoxin may comprise a polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - an extrinsic activation loop containing Cys (SEQ ID NO: 1).

[0163] Thus, in one embodiment, the modified Clostridial neurotoxin comprises 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 / TeNT, the endogenous clostridial neurotoxin activation loop is the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - an exogenous activation loop containing Cys (SEQ ID NO: 1).

[0164] Lys-C hydrolyzes one or more peptide bonds outside the intrinsic activation loop of the clostridial neurotoxin. For example, Lys-C has been shown to hydrolyze one or more peptide bonds in PA TM. Therefore, Lys-C may be involved in the activation of LH N and is not suitable for use with conventional clostridial neurotoxins, including PA TM.

[0165] Thus, in some embodiments, the modified Clostridial neurotoxin comprises PA (or a fragment thereof) and: i. amino acid residues 1 to 871 of SEQ ID NO: 35; ii. amino acid residues 1 to 858 of SEQ ID NO: 36; iii. amino acid residues 1 to 862 of SEQ ID NO: 38; iv. amino acid residues 1 to 845 of SEQ ID NO: 39; v. amino acid residues 1 to 864 of SEQ ID NO: 40; vi. amino acid residues 1 to 863 of SEQ ID NO: 41; vii. amino acid residues 1 to 879 of SEQ ID NO: 42; or viii. Amino acid residues 1 to 924 of SEQ ID NO: 33 Including, The activation loop of endogenous clostridial neurotoxins consists of the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1) has been replaced with an exogenous activation loop containing

[0166] In one embodiment, the modified Clostridial 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 Clostridial neurotoxin has a polypeptide sequence that has 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 Clostridial 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 Clostridial 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 Clostridial neurotoxin has a polypeptide sequence that has 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 Clostridial neurotoxin has a polypeptide sequence 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.

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

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

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

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

[0176] The present invention also encompasses clostridial neurotoxins having non-natural protease cleavage sites. In such clostridial 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 to the clostridial neurotoxin (i.e., an exogenous cleavage site). Such sites require an exogenous protease for cleavage, allowing for improved control over the timing and location of the cleavage event. Non-natural protease cleavage sites that can be used in clostridial neurotoxins include the following: TEV (Tobacco Etch Virus) (ENLYFQ↓G) (SEQ ID NO: 79) Thrombin (LVPR↓GS) (SEQ ID NO: 80) PreScission (LEVLFQ↓GP) (SEQ ID NO: 81).

[0177] Other protease cleavage sites include the recognition sequence that is cleaved by non-cytotoxic proteases, for example, by the light chain of clostridial neurotoxins.These include the SNARE (for example, SNAP-25, syntaxin, VAMP) protein recognition sequence that is cleaved by non-cytotoxic proteases, for example, by the light chain of clostridial neurotoxins.Clostridial neurotoxins that contain 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.In addition, the self-cleaving sequence, intein, is also encompassed by the term protease cleavage site.Self-splicing reaction can be controlled, for example, by changing the concentration of reducing agent present.

[0178] The present invention also encompasses clostridial neurotoxins containing a "destructive cleavage site." In the clostridial neurotoxin, a non-natural protease cleavage site is incorporated into the clostridial neurotoxin at a position such that cleavage at the site reduces or inactivates the activity of the clostridial neurotoxin. The destructive protease cleavage site may be susceptible to cleavage by a local protease in the event that the clostridial neurotoxin migrates to a non-target location after administration. Suitable non-natural protease cleavage sites include those described above. Clostridial 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 clostridial neurotoxins of the present invention, particularly their light chain components, can be PEGylated, which can help increase the stability of the light chain component, e.g., its duration of action. PEGylation is particularly preferred when the light chain comprises BoNT / A, B, or C1 protease. PEGylation preferably involves adding PEG to the N-terminus of the light chain component. For example, the N-terminus of the light chain can be extended with one or more amino acid (e.g., cysteine) residues, which can be the same or different. One or more of the amino acid residues can have their own PEG molecule attached thereto (e.g., covalently attached). An example of this technology is described in WO2007 / 104567, the entire text of which is incorporated herein by reference.

[0180] The modified clostridial neurotoxins of the present invention may be free of complexing proteins present in a naturally occurring clostridial neurotoxin complex.

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

[0182] In another aspect, the present invention provides a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a modified Clostridial neurotoxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and 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: 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 produced using any suitable process known in the art. Thus, the nucleic acid molecules can be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be produced using molecular biology techniques.

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

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

[0188] In one aspect, the present invention provides a nucleotide sequence encoding a modified Clostridial neurotoxin of the invention. The nucleotide sequence comprises a sequence having at least 70% sequence identity to 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 comprises a sequence having at least 80% or 90% sequence identity to 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 comprises (more preferably consists of) 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 comprising SEQ ID NO:1.

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

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

[0192] The present invention therefore provides a di-chain clostridial neurotoxin obtainable by the method of the present invention.

[0193] The term "obtainable" as used herein also encompasses the term "obtained." In one embodiment, the term "obtainable" means obtained.

[0194] The clostridial neurotoxins of the present invention suitably find utility in medicine or cosmetics. In use, the clostridial neurotoxin is preferably in a di-chain form.

[0195] The (modified) Clostridial neurotoxins of the present invention can be used to prevent or treat certain medical or cosmetic diseases and conditions. Thus, in a further aspect, the present invention provides a (modified) Clostridial neurotoxin as described above for use in medicine.

[0196] In a related aspect, the present invention provides a (modified) Clostridial neurotoxin as described above for use in the prevention or treatment of a disease or condition selected from the following: conditions associated with unwanted immune secretion, strabismus, blepharospasm, esotropia, dystonia (e.g., spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapeutic (cosmetic) applications benefiting from cell / muscle incapacitation (by SNARE downregulation or inactivation), oculomotor neuromuscular disorders or conditions (e.g., concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, thyroid myopathy), writer's cramp, blepharospasm, teeth grinding, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis. Symptoms of bladder aches and pains include: atrophy of the bladder, spasms resulting in abnormal bladder control, animus, back spasms, cramps, tension headaches, levator pelvic 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, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological diseases, chronic neurogenic inflammation and smooth muscle disorders.

[0197] When the (modified) clostridial neurotoxin of the present invention comprises a BoNT / X sequence (or a portion thereof), the clostridial neurotoxin may be able to target other types of secretory cells besides neurons due to its ability to cleave VAMP4, VAMP5, and / or Ykt6. In some embodiments, the targeted secretory cells are secretory immune cells. As used herein, "secretory immune cells" refers to immune cells that secrete cytokines, chemokines, or antibodies. Such secretory immune cells may be innate immune cells, including, but not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Antibody-secreting secretory immune cells (e.g., leukocytes) may also be targeted by the clostridial neurotoxin of the present disclosure. Non-limiting examples of antibody-secreting cells include, but are not limited to, plasma B cells, plasma cells, plasma cells, and effector B cells. In some embodiments, the clostridial neurotoxin can modulate an immune response. Thus, further contemplated herein is the therapeutic use of the Clostridial neurotoxins of the present invention to treat conditions associated with unwanted secretions, preferably unwanted immune secretions, including, but not limited to, inflammation, psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.

[0198] In one aspect, the present invention provides a pharmaceutical composition comprising a (modified) clostridial neurotoxin or a di-chain clostridial neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt.

[0199] The (modified) Clostridial neurotoxins of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection can be in the form of a solution, suspension, or emulsion, or a dry powder that is dissolved or suspended in a suitable vehicle before use.

[0200] In the case of a (modified) Clostridial neurotoxin to be delivered locally, the (modified) Clostridial neurotoxin may be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0201] Local delivery means can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of (modified) Clostridial neurotoxins allow for delivery to the lungs and / or other nasal passages and / or bronchi or airways.

[0202] The (modified) Clostridial neurotoxins of the present invention may be administered to a patient by intrathecal or epidural injection into the spinal column at the level of the spinal segment involved in innervating the affected organ.

[0203] The preferred routes of administration are laparoscopic and / or by localized, especially intramuscular, injection.

[0204] The dosage ranges for administration of the (modified) Clostridial neurotoxins of the present invention are those that produce the desired therapeutic effect. It will be appreciated that the dosage range required will vary depending on the precise nature of the (modified) Clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent, or severity of the patient's condition, if any, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0205] Suitable daily dosages (per kg of patient body weight) are in the range 0.0001-1 ng / kg, preferably 0.0001-0.5 ng / kg, more preferably 0.002-0.5 ng / kg, and especially preferably 0.004-0.5 ng / kg. Unit dosages can vary from less than 1 picogram to 30 ng, but will typically be 0.01-1 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 the (modified) Clostridial neurotoxin as a 1× dose, in which context preferred dosages are in the range 1× to 100× (i.e., 0.05 to 5 ng).

[0207] Fluid dosage forms are usually prepared using a (modified) clostridial neurotoxin and a pyrogen-free sterile vehicle. The (modified) clostridial neurotoxin can be dissolved or suspended in the vehicle depending on the vehicle and concentration used. In preparing a solution, the (modified) clostridial neurotoxin is dissolved in the vehicle, and the solution is made isotonic by adding sodium chloride, if necessary, and sterilized by filtration through a sterile filter using aseptic techniques, and then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is appropriate, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, lubricants, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics can be dissolved in the vehicle.

[0208] Dry powders that are dissolved or suspended in a suitable vehicle before use can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic techniques in a sterile area. Alternatively, the ingredients can be dissolved in suitable containers using aseptic techniques in a sterile area. The product is then lyophilized and the containers are 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 accomplished by filtration. The components may be isolated in a sterile state or may be sterilized after isolation, for example, by gamma irradiation.

[0210] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in the composition(s) to promote uniform distribution of the components.

[0211] Administration according to the present invention can utilize a variety of delivery techniques, including microparticle encapsulation, viral delivery systems, or high-pressure aerosol impaction.

[0212] Embodiments relating to various methods of the present invention are intended to apply equally to other methods, clostridial neurotoxins, e.g., modified clostridial neurotoxins (whether in single-chain or double-chain form), uses or pharmaceutical compositions, and vice versa.

[0213] sequence homology

[0214] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global, local, and hybrid methods, such as segmental approaches. Protocols for determining percent identity are routine within the skill of those in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., 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, e.g., 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)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box, see e.g., 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 e.g., C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0215] Thus, percent sequence identity is determined by conventional methods. See, e.g., 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 opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are designated by standard single-letter codes):

[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, 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 can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences.The alignment comparison and identity percentage determination of two or more sequences can be carried out using a specific mathematical algorithm, for example, BLAST, which will be familiar to those skilled in the art.

number

[0217] The percent identity is then calculated as:

number

[0218] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, such as conservative amino acid substitutions, other substitutions that do not significantly affect the folding or activity of the polypeptide, small deletions, usually from 1 to about 30 amino acids, small amino- or carboxyl-terminal extensions, e.g., an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag (see below). Conservative amino acid substitutions Basicity: Arginine lysine histidine Acidic: glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Balin 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) can be substituted for amino acid residues in the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

[0220] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. The transcription and translation of the plasmid containing nonsense mutation is carried out in a cell-free system containing Escherichia coli (E. coli) S30 extract and commercially available enzymes and other reagents. Protein is 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 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 natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to unnatural species by in vitro chemical modification. To further expand the scope of substitution, chemical modification can be combined with site-directed mutagenesis (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0221] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0222] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). In addition to mutations of putative contact site amino acids, sites of biological interaction can also be determined by physical analysis of the structure, such as those determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling. See, e.g., 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 essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0223] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as 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 methods in which two or more positions in a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the range of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. 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 meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. 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 a general dictionary of many of the terms used in this disclosure.

[0225] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in practicing or testing embodiments of the present disclosure. Numeric ranges include the numbers defining the range. Unless otherwise specified, any nucleic acid sequence is written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation.

[0226] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0227] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or single-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "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 interchangeably herein. Conventional single-letter and three-letter codes for amino acid residues may be used in the present disclosure and claims. The three-letter codes for amino acids are defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0228] Other definitions of terms may appear throughout this specification. Before exemplary embodiments are described in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0229] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between a stated value or intervening value in a stated range and any other stated or intervening value within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range where either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0230] It should be noted that, herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, reference to "a clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0231] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0232] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief explanation of the drawings]

[0233] [Figure 1]Figure 1 shows a protein sequence comparison of the activation loop of all BoNT serotypes and tetanus toxin, showing the two adjacent cysteines that form the disulfide bridge connecting the light and heavy chains of the toxin molecule. The factor Xa cleavage site (IDGR) in BoNT / C1 and BoNT / CD is underlined. [Figure 2] Figure 2 shows that all four proteases tested, trypsin (TrypZean), Lys-C, factor Xa (FXa), and enterokinase (EK), are capable of cleaving the BoNT / C1 activation loop and generating a double-stranded molecule compared to a C-protease-untreated control. (A, B) BoNT / C1 (0) (SEQ ID NO: 15) treated with factor Xa (FXa), enterokinase (EK), and trypsin (1-16 hour time course) as indicated, as examined by SDS-PAGE under non-reducing (A) and reducing (B) conditions. Similarly, proteolytic digestion with Lys-C generates a double-stranded molecule 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]3(A) shows BoNT / X cleavage by Lys-C. Samples were tested under non-reducing and reducing (+DTT) conditions. 1 - Benchmark (5 μl), 2 - No protease 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 - No protease 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 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 - No protease control, 3 - TrypZean 0.125 μg / ml, 4 - TrypZean 0.25 μg / ml, 5 - No protease 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 - No protease control + DTT, 12 - TrypZean 0.125 μg / ml + DTT, 13 - TrypZean 0.25 μg / ml + DTT, 14 - No protease 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 proteases and a successful BoNT duplex formation event, comparing non-reducing (-DTT) and reducing (+DTT) conditions. Lanes 4-7 show in-process samples. Lanes 8-11 show 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 BoNT / E tested with 10 μg / ml of Lysobacter enzymogenes ("Lys-C") and Pseudomonas aeruginosa ("rLys-C") endoprotease Lys-C for 2 hours at 37°C. Samples were tested under non-reducing and reducing (+TCEP) conditions. Figure 5(B) shows BoNT / E treated with the indicated amounts of trypsin for 7 hours at 20°C. (C) Samples stored at -20°C. (T) No protease control at 20°C. Samples were tested under reducing (+DTT) conditions. [Figure 6] 6 shows modified BoNT / E (SEQ ID NO: 11) treated with the indicated proteases and successful BoNT duplex formation events, with a comparison between 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 duplex 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 the positive control (double-chain BoNT / A1). [Figure 8] FIG. 8 shows BoNT / A1C1 and BoNT / C1 dose-dependent inhibition of glutamate release from primary rat neurons. [Figure 9] FIG. 9 depicts the intact mass analysis of enterokinase-activated, non-reduced modified BoNT / E (SEQ ID NO: 11) with an indicated mass of 143853 Da. [Figure 10] FIG. 10 shows the intact mass analysis of enterokinase-activated, reduced, modified BoNT / E (SEQ ID NO: 11) with the indicated masses of 47,518 Da and 96,338 Da. [Figure 11] FIG. 11 shows the intact mass analysis of non-reduced modified BoNT / E (SEQ ID NO: 11) activated by factor Xa, with an indicated mass of 143,850 Da. [Figure 12] FIG. 12 shows the intact mass analysis of reduced, modified BoNT / E (SEQ ID NO: 11) activated by factor Xa, with the indicated masses of 47,518 Da and 96,335 Da. [Figure 13]13 shows LHN / A1 treated with EK containing an EK cleavage site inserted in the activation loop (SEQ ID NO:44) compared to the native A1 loop treated with Lys-C (SEQ ID NO:46): 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] FIG. 14 shows the activation of modified BoNT / XA (SEQ ID NO: 7) with FXa. [Figure 15] FIG. 15 shows the activation of modified BoNT / XB (SEQ ID NO: 9) with FXa.

[0234] Sequence Listing

[0235] Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, the presence of said residue / codon is optional.

[0236] SEQ ID NO: 1 (C1 activation loop consensus sequence) Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys

[0237] SEQ ID NO: 2 (C1 activation loop) CHKAIDGRSLYNKTLDC

[0238] SEQ ID NO: 3 (C1 activation loop mutant) CHKAIEGRSLYNKTLDC

[0239] SEQ ID NO: 4 (nucleic acid sequence, BoNT / X, with C1 activation loop)

[0240] SEQ ID NO: 5 (polypeptide sequence, BoNT / X, with C1 activation loop)

[0241] SEQ ID NO: 6 (nucleic acid sequence, BoNT / XA [LH N XH C A], with C1 activation loop)

[0242] SEQ ID NO: 7 (polypeptide sequence, BoNT / XA [LH N XH C A], with C1 activation loop)

[0243] SEQ ID NO: 8 (nucleic acid sequence, BoNT / XB [LH N XH C B], with C1 activation loop)

[0244] SEQ ID NO: 9 (polypeptide sequence, BoNT / XB [LH N XH C B], with C1 activation loop)

[0245] SEQ ID NO: 10 (nucleic acid sequence, BoNT / E, with C1 activation loop)

[0246] SEQ ID NO: 11 (polypeptide sequence, BoNT / E, with C1 activation loop) [ka]

[0247] SEQ ID NO: 12 (nucleic acid sequence, BoNT / A1C1, with C1 activation loop)

[0248] SEQ ID NO: 13 (polypeptide sequence, BoNT / A1C1, with C1 activation loop) [ka]

[0249] SEQ ID NO: 14 (nucleic acid sequence, BoNT / C1(0) (Endonegative))

[0250] SEQ ID NO: 15 (Polypeptide sequence, BoNT / C1(0) (Endonegative))

[0251] SEQ ID NO: 16 (nucleic acid sequence, BoNT / C1)

[0252] SEQ ID NO: 17 (polypeptide sequence, BoNT / C1)

[0253] SEQ ID NO: 18 (enterokinase and factor Xa cleavage site) IDGR

[0254] SEQ ID NO: 19 (Enterokinase and Factor Xa cleavage site mutant) IEGR

[0255] SEQ ID NO: 20 (BoNT / X activation loop) CPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGC

[0256] SEQ ID NO: 21 (BoNT / A1 & A6 activation loop) CVRGIITSKTKSLDKGYNKALNDLC

[0257] SEQ ID NO: 22 (BoNT / B2, B3 & B6 activation loop) CKSVRAPGIC

[0258] SEQ ID NO: 23 (BoNT / D activation loop) CLRLTKNSRDDSTC

[0259] SEQ ID NO: 24 (BoNT / E1 to E5, E9 & E12 activation loop) CKNIVSVKGIRKSIC

[0260] SEQ ID NO: 25 (BoNT / F1 and F6 activation loop) CKSVIPRKGTKAPPRLC

[0261] SEQ ID NO: 26 (BoNT / F2 and F3 activation loop) CKSIIPRKGTKQSPSLC

[0262] SEQ ID NO: 27 (BoNT / F4 activation loop) CKSIIPRKGTKAPPRLC

[0263] SEQ ID NO: 28 (BoNT / F5 activation loop) CLNSSFKKNTKKPLC

[0264] SEQ ID NO: 29 (BoNT / F7 activation loop) CKSIVSKKGTKNSLC

[0265] SEQ ID NO: 30 (TeNT activation loop) CKKIIPPTNIRENLYNRTASLTDLGGELC

[0266] SEQ ID NO: 31 (BoNT / G activation loop) CKPVMYKNTGKSEQC

[0267] SEQ ID NO: 32 (nucleic acid sequence, wild-type BoNT / X-10HT)

[0268] SEQ ID NO: 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] SEQ ID NO: 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] SEQ ID NO: 37 (BoNT / C - UniProt P18640)

[0273] SEQ ID NO: 38 (BoNT / D - UniProt P19321)

[0274] SEQ ID NO: 39 (BoNT / E - UniProt Q00496)

[0275] SEQ ID NO: 40 (BoNT / F - UniProt A7GBG3)

[0276] SEQ ID NO: 41 (BoNT / G - UniProt Q60393)

[0277] SEQ ID NO: 42 (TeNT - UniProt P04958)

[0278] SEQ ID NO: 43 (nucleic acid sequence, LH N / A1, with EK cleavage site)

[0279] SEQ ID NO: 44 (polypeptide sequence, LH N / A1, with EK cleavage site) MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKT KSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKY TMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIF SGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEAHHHHHHHHHH

[0280] SEQ ID NO: 45 (nucleic acid sequence, BoNT, with native A1 loop)

[0281] SEQ ID NO: 46 (polypeptide sequence, BoNT, with native 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] SEQ ID NO: 49 (polypeptide sequence, enterokinase light chain) IVGGSDSREGAWPWVVALYFDDQQVCGASLVSRDWLVSAAHCVYGRNMEPSKWKAVLGLHMASNLTSPQIETRLIDQIVINRHYNKRRKNNDIAMMHLEMKVNYTDYIQPICLPEEN QVFPPGRICSIAGWGALIYQGSTADVLQEADVPLLSNEKCQQQMPEYNITENMVCAGYDAGGVDSCQGDSGGPLMCQENNRWLLAGVTSFGYQCALPNRPGVYARVPRFTEWIQSFLH

[0285] SEQ ID NO: 50 (polypeptide sequence, factor Xa heavy chain) IVGGRDCAEGECPWQALLVNEENEGFCGGTILNEFYVLTAAHCLHQAKRFTVRVGDRNTEQEEGNEMAHEVEMTVKHSRFVKETYDFDIAVLRLKTPIRFRRNVAPACLPEKDWAEATLMTQKTGIVSG FGRTHEKGRLSSTLKMLEVPYVDRSTCKLSSSFTITPNNMFCAGYDTQPEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKFGVYTKVSNFLKWIDKIMKARAGAAGSRGHSEAPATWTVPPPLPL

[0286] SEQ ID NO: 51 (polypeptide sequence, factor Xa light chain) ANSFLEEVKQGNLERECLEEACSLEEAREVFEDAEQTDEFWSKYKDGDQCEGHPCLNQGHCKDGIGDYTCTCAEGFEGKNCEFSTREICSLDNGGCDQFCREERSEVRCSCAHGYVLGDDSKSCVSTERFPCGKFTQGRS

[0287] SEQ ID NO: 52 (Polypeptide 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] SEQ ID NO: 53 (LH N / A, with C1 activation loop) [ka]

[0289] SEQ ID NO: 54 (LH N / B, with C1 activation loop) [ka]

[0290] SEQ ID NO: 55 (LH N / D, with C1 activation loop) [ka]

[0291] SEQ ID NO: 56 (LH N / E, with C1 activation loop) [ka]

[0292] SEQ ID NO: 57 (LH N / F, with C1 activation loop) [ka]

[0293] SEQ ID NO: 58 (LH N / G, with C1 activation loop) [ka]

[0294] SEQ ID NO: 59 (LH N / TeNT, with C1 activation loop) [ka]

[0295] SEQ ID NO: 60 (LH N / X, with C1 activation loop) [ka] [Example]

[0296] material and method

[0297] material

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

[0299] Protein purification

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

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

[0302] The bacterial pellet was disrupted 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 with 6 μg / 1 mg BoNT factor Xa (NEB) at 4 °C, followed by polishing using Phenyl HP resin (GE).

[0303] BoNT / E with C1 loop (SEQ ID NO: 11)

[0304] The bacterial pellet was disrupted 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), followed by polishing using CHT type II resin (Biorad).

[0305] BoNT / X (SEQ ID NO: 5)

[0306] The bacterial pellet was disrupted by sonication in lysis buffer (50 mM Tris-HCl pH = 8) and clarified by centrifugation. The target protein was captured using a HisTrap HP column (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), followed by polishing using a 1 ml HisTrap column (GE).

[0307] LC / MS

[0308] Prior to analysis, samples were buffer exchanged into 50 mM ammonium bicarbonate. Samples were either intact protein or reduced by incubation with 10 mM DTT for 30 minutes at 37°C. Samples were run using a Waters Acquity H-Class UPLC system coupled to a Waters Xevo G2-XS QToF mass spectrometer. Mobile phase A: 0.1% formic acid in water 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 demonstrated cleavage within the activation loop. BoNT / C1(0) (SEQ ID NO: 15) was digested overnight with enterokinase (EK) or factor Xa (FXa) at 4°C and 25°C. BoNT / C1(0) was further digested with trypsin over a 16-hour time course at 20°C (Figures 2A and 2B). All three proteases were able to cleave the BoNT / C1 activation loop and generate a double-stranded molecule, compared to the protease-untreated control. However, additional cleavage products were visible 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 increasing amounts 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 failed to cleave the protein into its two-chain form (Figure 3B, lanes 18 and 19, respectively).

[0316] In an attempt to improve the activity of BoNT / X, the BoNT / X activation loop was replaced with the BoNT / C1 activation loop (SEQ ID NO: 2), creating the modified BoNT protein SEQ ID NO: 5. The modified BoNT was purified using several chromatography steps and treated with enterokinase (EK) or factor Xa (FXa) to confirm that the presence of the BoNT / C loop allows for the generation of a double-chain molecule. Surprisingly, Figure 4 shows that EK and FXa specifically cleave the modified BoNT / X into a double-chain 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 cleaves / degrades BoNT / E imprecisely. Treatment with trypsin resulted in truncation of BoNT / E, meaning that an additional purification step was required to separate the full-length protein from the truncated product (Figure 5B).

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

[0321] [Example 4]

[0322] Proteolytic activation of BoNT / A1C1 chimera

[0323] To facilitate proteolytic protein activation, the BoNT / C loop is N / A1 and C1 H C The BoNT / A1C1 activation loop was introduced into a BoNT / A1C1 chimera (with the BoNT / A1C1 domain). The BoNT / A1 activation loop of BoNT / A1C1 was replaced with the BoNT / C1 loop to create the modified BoNT protein SEQ ID NO: 13. The modified BoNT was purified using several chromatography steps and treated with factor Xa (FXa) to confirm that the presence of the BoNT / C loop allows for the generation of a double-chain molecule. Figure 7A shows that FXa specifically cleaves the modified BoNT / A1C1 into a double-chain form. For comparison, wild-type MetBoNT / A1 (commercially available from Metabiologics A1080116), which contains 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] BoNTs containing the C1 loop retain SNARE cleavage activity

[0326] Rat primary cortical neurons were treated with BoNT / A1C1 (SEQ ID NO: 13) of Example 4, which contains the BoNT / C1 activation loop, for 24 hours, and recombinant BoNT / C1 (SEQ ID NO: 17) was purified. After incubation, SNARE-dependent glutamate release from cells stimulated with potassium chloride was measured (FIG. 8). These data confirm the activity of clostridial neurotoxins modified to contain the 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 disulfide bridges 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 generate 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 sites into endogenous loops

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

[0333] The native activation loops from each serotype have evolved to allow protease accessibility and processing of the toxin into a dichain form by Clostridium. Without wishing to be bound by theory, it is believed that mutating these loops to create protease recognition sites may lead to conformational changes that may negatively affect cleavage efficiency.

[0334] To test this hypothesis, BoNT / A1 light chain and polypeptide with translocation domain (LH) were N / A1) was modified to contain the EK protease recognition sequence DDDDK (SEQ ID NO: 44). N The efficiency of proteolytic cleavage of / A1 with EK was assessed and compared to cleavage of the wild-type A1 activation loop with Lys-C (note that a direct comparison using EK is not possible due to the lack of an EK recognition site in the wild-type loop). Figure 13 shows that cleavage of the modified loop is significantly less efficient than the wild-type loop.

[0335] [Example 8]

[0336] Proteolytic activation of BoNT / XA chimeras

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

[0338] [Example 9]

[0339] Proteolytic activation of BoNT / XB chimeras

[0340] A BoNT / XB chimera was generated containing the light chain and translocation 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 two-chain form of the modified BoNT / XB chimera was produced after activation with FXa.

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

[0342] item [Item 1] 1. A method for proteolytically processing a single-chain clostridial neurotoxin into the corresponding two-chain clostridial neurotoxin, comprising: a. providing a single-chain clostridial neurotoxin; b. contacting a single-chain clostridial neurotoxin with enterokinase or factor Xa; Including, Single-chain clostridial neurotoxins have the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b - an activation loop containing Cys (SEQ ID NO: 1), wherein a=1 to 10 and b=4 to 15; The method wherein enterokinase or factor Xa hydrolyzes the peptide bond of the activation loop, thereby generating a di-chain clostridial neurotoxin. [Item 2] Item 1. The method according to item 1, wherein a=2 to 4. [Item 3] 3. The method according to item 1 or 2, wherein a=3. [Item 4] The method according to any one of the preceding items, wherein b=6 to 10. [Item 5] Item 10. The method of any one of the preceding items, wherein b=8. [Item 6] The method of any one of the preceding items, wherein the activation loop comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 7] The method of any one of the preceding items, wherein the activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 8] The method of any one of the preceding items, wherein the activation loop comprises a polypeptide sequence having SEQ ID NO:2 or SEQ ID NO:3. [Item 9] The method of any one of the preceding items, wherein the activation loop comprises SEQ ID NO:2. [Item 10] The method of any one of the preceding items, wherein the activation loop consists of SEQ ID NO:2. [Item 11] The method of any one of the preceding items, wherein the single-chain clostridial neurotoxin is contacted with enterokinase. [Item 12] Clostridial neurotoxins are functional H C The method of any one of the preceding items, wherein the polypeptide lacks a domain. [Item 13] The method of any one of the preceding items, wherein the clostridial neurotoxin is a retargeted clostridial neurotoxin comprising a non-clostridial targeting moiety (TM). [Item 14] The method of any one of the preceding items, wherein the Clostridial neurotoxin is a retargeted Clostridial neurotoxin comprising a TM, and the TM comprises anthrax toxin protective antigen (PA) or a fragment thereof. [Item 15] Clostridial neurotoxins a. SEQ ID NO: 52 or a fragment thereof, 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 15. The method of claim 14, wherein the polypeptide sequence has at least 70% sequence identity to a polypeptide comprising: [Item 16] The method of any one of the preceding items, wherein the Clostridial neurotoxin is not BoNT / C1. [Item 17] The method of any one of the preceding items, wherein the Clostridial neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X and TeNT. [Item 18] The method of any one of the preceding items, wherein the clostridial neurotoxin is BoNT / X, BoNT / E, a chimeric BoNT, or a hybrid BoNT. [Item 19] Single-chain clostridial neurotoxins a. encoded by a nucleotide sequence having at least 70% sequence identity to 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 of any one of the preceding items, comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. [Item 20] 1. A method of making a modified Clostridial neurotoxin, comprising the steps of: a. identifying the intrinsic activation loop of a clostridial neurotoxin, wherein the clostridial neurotoxin i. The peptide bond outside the intrinsic activation loop of clostridial neurotoxins is hydrolyzed by trypsin or Lys-C. ii. Inefficient proteolytic processing of the intrinsic activation loop by trypsin or Lys-C characterized by; b. Replacing the endogenous activation loop with an exogenous activation loop, thereby providing a modified Clostridial neurotoxin, wherein the exogenous activation loop has the polypeptide sequence Cys-(Xaa) a-Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1), a=1 to 10, and b=4 to 15. [Item 21] 21. The method of item 20, wherein the intrinsic activation loop is one or more selected from 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. [Item 22] 22. The method of claim 20 or 21, wherein the clostridial neurotoxin is identified as suitable for use in the method by contacting the clostridial neurotoxin with trypsin or Lys-C and confirming hydrolysis of a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin. [Item 23] 23. The method of any one of items 20 to 22, further comprising contacting the modified clostridial neurotoxin with enterokinase or factor Xa, thereby producing the corresponding two-chain modified clostridial neurotoxin. [Item 24] 24. The method according to any one of items 20 to 23, wherein a=2 to 4. [Item 25] 25. The method according to any one of items 20 to 24, wherein a=3. [Item 26] 26. The method according to any one of items 20 to 25, wherein b=6 to 10. [Item 27] 27. The method according to any one of items 20 to 26, wherein b=8. [Item 28] 28. The method of any one of items 20 to 27, wherein the extrinsic activation loop comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 29] 29. The method of any one of items 20 to 28, wherein the extrinsic activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 30] 30. The method of any one of items 20 to 29, wherein the extrinsic activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3. [Item 31] 31. The method of any one of items 20 to 30, wherein the extrinsic activation loop comprises SEQ ID NO:2. [Item 32] 32. The method of any one of items 20 to 31, wherein the extrinsic activation loop consists of SEQ ID NO:2. [Item 33] 33. The method of any one of paragraphs 23 to 32, wherein the modified Clostridial neurotoxin is contacted with enterokinase. [Item 34] Clostridial neurotoxins are functional H C 34. The method of any one of items 20 to 33, wherein the polypeptide lacks a domain. [Item 35] 35. The method of any one of paragraphs 20 to 34, wherein the clostridial neurotoxin is a retargeted clostridial neurotoxin comprising a non-clostridial targeting moiety (TM). [Item 36] 36. The method of any one of items 20 to 35, wherein the Clostridial neurotoxin is a retargeted Clostridial neurotoxin comprising a TM, and the TM comprises anthrax toxin protective antigen (PA) or a fragment thereof. [Item 37] Clostridial neurotoxins a. SEQ ID NO: 52 or a fragment thereof, 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 37. The method of claim 36, wherein the polypeptide sequence has at least 70% sequence identity to a polypeptide comprising: [Item 38] 38. The method of any one of items 20 to 37, wherein the clostridial neurotoxin is not BoNT / C1. [Item 39] 39. The method of any one of items 20 to 38, wherein the clostridial neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X and TeNT. [Item 40] 40. The method of any one of items 20 to 39, wherein the clostridial neurotoxin is BoNT / X, BoNT / E, a chimeric BoNT or a hybrid BoNT. [Item 41] The modified clostridial neurotoxin a. encoded by a nucleotide sequence having at least 70% sequence identity to 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 of any one of items 20 to 40, comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. [Item 42] an endogenous activation loop of a clostridial neurotoxin is replaced with an exogenous activation loop, thereby providing a modified clostridial neurotoxin; The extrinsic activation loop is the polypeptide sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 1), a=1 to 10, and b=4 to 15, The clostridial neurotoxin is a modified clostridial neurotoxin (e.g., obtainable by the method of any one of items 20 to 41), characterized in that a peptide bond outside the intrinsic activation loop of the clostridial neurotoxin is hydrolyzed by trypsin or Lys-C and / or the intrinsic activation loop is inefficiently proteolytically processed by trypsin or Lys-C. [Item 43] 43. The modified Clostridial neurotoxin of item 42, wherein the intrinsic activation loop is selected from one or more of 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. [Item 44] 44. The modified Clostridial neurotoxin according to item 42 or 43, wherein a=2 to 4. [Item 45] 45. The modified Clostridial neurotoxin according to any one of items 42 to 44, wherein a=3. [Item 46] 46. ​​The modified Clostridial neurotoxin according to any one of items 42 to 45, wherein b=6 to 10. [Item 47] 47. The modified Clostridial neurotoxin according to any one of items 42 to 46, wherein b=8. [Item 48] 48. The modified Clostridial neurotoxin of any one of items 42 to 47, wherein the extrinsic activation loop comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 49] 49. The modified Clostridial neurotoxin of any one of items 42 to 48, wherein the extrinsic activation loop comprises a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3. [Item 50] 50. The modified Clostridial neurotoxin of any one of items 42 to 49, wherein the extrinsic activation loop comprises a polypeptide sequence having SEQ ID NO: 2 or SEQ ID NO: 3. [Item 51] 51. The modified Clostridial neurotoxin of any one of items 42 to 50, wherein the extrinsic activation loop comprises SEQ ID NO:2. [Item 52] 52. The modified Clostridial neurotoxin of any one of items 42 to 51, wherein the extrinsic activation loop consists of SEQ ID NO:2. [Item 53] Clostridial neurotoxins are functional H C 53. The modified Clostridial neurotoxin according to any one of items 42 to 52, which lacks a domain. [Item 54] 54. The modified clostridial neurotoxin of any one of paragraphs 42 to 53, wherein the clostridial neurotoxin is a retargeted clostridial neurotoxin comprising a non-clostridial targeting moiety (TM). [Item 55] 55. The modified clostridial neurotoxin of any one of items 42 to 54, wherein the clostridial neurotoxin is not BoNT / C1. [Item 56] 56. The modified Clostridial neurotoxin of any one of items 42 to 55, wherein the Clostridial neurotoxin is selected from BoNT / A, BoNT / B, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X and TeNT. [Item 57] 57. The modified Clostridial neurotoxin of any one of items 42 to 56, wherein the Clostridial neurotoxin is BoNT / X, BoNT / E, a chimeric BoNT or a hybrid BoNT. [Item 58] Single-chain clostridial neurotoxins a. encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12; or b. The modified Clostridial neurotoxin of any one of items 42 to 57, comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. [Item 59] 59. The modified Clostridial neurotoxin of any one of items 42 to 58, wherein the modified Clostridial neurotoxin is a retargeted Clostridial neurotoxin comprising a targeting moiety (TM), wherein the TM comprises anthrax toxin protective antigen (PA) or a fragment thereof. [Item 60] The modified clostridial neurotoxin a. SEQ ID NO: 52 or a fragment thereof, 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 60. The modified Clostridial neurotoxin according to item 59, having a polypeptide sequence having at least 70% sequence identity to a polypeptide comprising: [Item 61] 61. A method for proteolytically processing the modified clostridial neurotoxin of any one of items 42 to 60 into the corresponding two-chain clostridial neurotoxin, comprising contacting the modified clostridial neurotoxin with enterokinase or factor Xa, thereby producing the two-chain clostridial neurotoxin. [Item 62] 62. A double-chain Clostridial neurotoxin obtainable by the method of any one of items 1 to 19, 23 to 41 or 61. [Item 63] 62. A pharmaceutical composition comprising the modified clostridial neurotoxin of any one of items 42 to 60 or the two-chain clostridial neurotoxin of item 62 and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt. [Item 64] Conditions associated with unwanted immune secretion, strabismus, blepharospasm, esotropia, dystonia (e.g., spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), aesthetic therapeutic (cosmetic) applications that benefit from cell / muscle incapacitation (via SNARE downregulation or inactivation), oculomotor neuromuscular disorders or conditions (e.g., concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, thyroid abnormalities), writer's cramp, blepharospasm, teeth grinding, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasms, cramps, tension headaches, levator pelvis 64. The modified Clostridial neurotoxin of any one of items 42 to 60, the double-chain Clostridial neurotoxin of item 62 or the pharmaceutical composition of item 63 for use in treating one or more of: urinary tract 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 headache), wrinkled forehead, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological diseases, chronic neurogenic inflammation and smooth muscle disorders. [Item 65] 19. Use of enterokinase to hydrolyze peptide bonds of a polypeptide comprising the polypeptide sequence shown as SEQ ID NO:18 or SEQ ID NO:19. [Item 66] A nucleotide sequence encoding a modified Clostridial neurotoxin comprising a sequence having at least 70% sequence identity to 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 end SEQ ID NO: Sequence number [Figure 2] 20℃ Time course (hr): 20℃ time course (hr) intact: intact [Figure 4] intact: intact [Figure 5] Benchmark: Benchmark -ve control: negative control +ve control: positive control Trypsin: Trypsin intact: intact [Figure 6] intact: intact [Figure 7] intact: intact [Figure 8] Glutamate: glutamic acid Stimulated: Stimulated Basal: Basal [Figures 9-12] Intensity [Counts]: Intensity [Counts] Mass [Da]: Mass [Da] [Figure 14] Bench Mark ladder: Benchmark ladder HisTrap load: HisTrap load Control: Control 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. 1. A method for proteolytically processing a single-chain modified botulinum neurotoxin serotype X (BoNT / X) or BoNT / X chimera into a corresponding two-chain modified BoNT / X or BoNT / X chimera, comprising: contacting a single-chain modified BoNT / X or BoNT / X chimera with enterokinase or factor Xa; The single-chain modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 1), where a is at least 1 and b is at least 1, and The method wherein enterokinase or factor Xa hydrolyzes the peptide bond of the activation loop, thereby generating a two-chain modified BoNT / X or BoNT / X chimera.

2. 1. A method for producing a modified botulinum neurotoxin serotype X (BoNT / X) or a BoNT / X chimera, comprising the steps of: replacing the endogenous activation loop of a BoNT / X or BoNT / X chimera with an exogenous activation loop, thereby providing a modified BoNT / X or BoNT / X chimera; wherein the modified BoNT / X or BoNT / X chimera is i. A peptide bond outside the endogenous activation loop of BoNT / X or a BoNT / X chimera is hydrolyzed by trypsin or Lys-C; and / or ii. Inefficient proteolytic processing of the intrinsic activation loop by trypsin or Lys-C and The extrinsic activation loop has the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa) It comprises b-Cys (SEQ ID NO: 1), where a is at least 1 and b is at least 1.

3. The method of claim 2, wherein the BoNT / X or BoNT / X chimera is identified as suitable for use in the method by contacting the BoNT / X or BoNT / X chimera with trypsin or Lys-C and confirming hydrolysis of a peptide bond outside the intrinsic activation loop of the BoNT / X or BoNT / X chimera.

4. The method of claim 2 or 3, further comprising contacting the modified BoNT / X or BoNT / X chimera with enterokinase or factor Xa, thereby producing the corresponding two-chain modified BoNT / X or BoNT / X chimera.

5. an endogenous activation loop of a botulinum neurotoxin serotype X (BoNT / X) or BoNT / X chimera is replaced with an exogenous activation loop, thereby providing a modified BoNT / X or BoNT / X chimera; the extrinsic activation loop comprises the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 1), wherein a is at least 1 and b is at least 1; Modified BoNT / X or BoNT / X chimeras.

6. 5. The method of any one of claims 2 to 4, wherein the intrinsic activation loop is SEQ ID NO:

20.

7. 6. The modified BoNT / X or BoNT / X chimera of claim 5, wherein the intrinsic activation loop is SEQ ID NO:

20.

8. a. the activation loop comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3; b. the activation loop comprises a polypeptide sequence having SEQ ID NO:2 or SEQ ID NO:3; c. the activation loop comprises SEQ ID NO:2, and / or d. the activation loop consists of SEQ ID NO:2; The method of claim 1.

9. a. the extrinsic activation loop comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3; b. the extrinsic activation loop comprises a polypeptide sequence having SEQ ID NO:2 or SEQ ID NO:3; c. the extrinsic activation loop comprises SEQ ID NO:2, and / or d. the extrinsic activation loop consists of SEQ ID NO:2; 7. The method according to any one of claims 2 to 4 or 6.

10. a. the extrinsic activation loop comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3; b. the extrinsic activation loop comprises a polypeptide sequence having SEQ ID NO:2 or SEQ ID NO:3; c. the extrinsic activation loop comprises SEQ ID NO:2; d. the extrinsic activation loop consists of SEQ ID NO:2, and / or e. The BoNT / X or BoNT / X chimera is characterized in that a peptide bond outside the intrinsic activation loop of the BoNT / X or BoNT / X chimera is hydrolyzed by trypsin or Lys-C and / or the intrinsic activation loop is inefficiently proteolytically processed by trypsin or Lys-C. The modified BoNT / X or BoNT / X chimera of claim 5 or 7.

11. a. The modified BoNT / X or BoNT / X chimera is a functional H C lacking a domain, and / or b. The modified BoNT / X or BoNT / X chimera is a retargeted clostridial neurotoxin containing a non-clostridial targeting moiety (TM); 10. The method of any one of claims 1 to 4, 6, 8, or 9.

12. a. The modified BoNT / X or BoNT / X chimera is a functional H of a clostridial neurotoxin. C lacking a domain, and / or b. The modified BoNT / X or BoNT / X chimera is a retargeted clostridial neurotoxin containing a non-clostridial targeting moiety (TM); 11. The modified BoNT / X or BoNT / X chimera of claim 5, 7, or 10.

13. The modified BoNT / X or BoNT / X chimera is i. encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; or ii. comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9; and When in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 1), where a is at least 1 and b is at least 1.

12. The method of any one of claims 1 to 4, 6, 8, 9, or 11.

14. The modified BoNT / X or BoNT / X chimera is i. encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; or ii. comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9; and When in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 1), where a is at least 1 and b is at least 1.

13. The modified BoNT / X or BoNT / X chimera of any one of claims 5, 7, 10, or 12.

15. A modified botulinum neurotoxin serotype X (BoNT / X) or BoNT / X chimera comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, wherein when in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO:1), where a is at least 1 and b is at least 1.

16. 16. A method for proteolytically processing a modified BoNT / X or BoNT / X chimera encoded by a nucleic acid encoding a modified Clostridial neurotoxin comprising the sequence of any one of claims 5, 7, 10, 12, 14, or 15, or a modified BoNT / X or BoNT / X chimera comprising at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, wherein when in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO:1), where a is at least 1 and b is at least 1, into a corresponding two-chain modified BoNT / X or BoNT / X chimera, the method comprising contacting the modified BoNT / X or BoNT / X chimera with a protease, thereby generating the two-chain modified BoNT / X or BoNT / X chimera.

17. 17. A double-stranded modified BoNT / X or BoNT / X chimera obtainable by the method of any one of claims 1 to 4, 6, 8, 9, 11, 13, or 16.

18. a. a modified BoNT / X or BoNT / X chimera according to any one of claims 5, 7, 10, 12, 14, or 15; or b. A modified BoNT / X or BoNT / X chimera encoded by a nucleic acid encoding a modified clostridial neurotoxin comprising a sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, wherein when in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO:1), where a is at least 1 and b is at least 1; A double-chain modified BoNT / X or BoNT / X chimera obtained by contacting a protease with the

19. A pharmaceutical composition comprising a modified BoNT / X or BoNT / X chimera described in any one of claims 5, 7, 10, 12, 14, or 15, or a two-stranded modified BoNT / X or BoNT / X chimera described in claim 17 or 18, and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

20. Conditions associated with unwanted immune secretions, strabismus, blepharospasm, esotropia, dystonia, torticollis, cosmetic therapeutic applications that benefit from cellular / muscle incapacitation, oculomotor neuromuscular disorders or conditions, writer's cramp, teeth grinding, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, back spasms, cramps, tension headaches, pelvic levator 20. A pharmaceutical comprising the modified BoNT / X or BoNT / X chimera of any one of claims 5, 7, 10, 12, 14, or 15, the two-stranded modified BoNT / X or BoNT / X chimera of claim 17 or 18, or the pharmaceutical composition of claim 19, for use in treating one or more of the following: idiopathic urinary tract syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, anal fissures, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain, headache pain, wrinkled forehead, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary-neurological diseases, chronic neurogenic inflammation, and smooth muscle disorders.

21. a. the dystonia is selected from spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, and cervical dystonia; b. The torticollis is spasmodic torticollis. c. the ocular motility condition is selected from concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, and thyroid dysplasia; d. The muscle pain is pain from muscle spasms; e. The headache pain is a tension headache, f. Cell / muscle incapacitation is due to SNARE downregulation or inactivation, or g. Beauty therapy is beauty; The pharmaceutical composition of claim 20.

22. A nucleic acid encoding a modified botulinum neurotoxin serotype X (BoNT / X) or BoNT / X chimera comprising a sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, wherein when in single-chain form, the modified BoNT / X or BoNT / X chimera has an activation loop comprising the polypeptide sequence Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO:1), wherein a is at least 1 and b is at least 1.

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