Cell-free clostridial neurotoxin assay

Abstract

The present invention is directed to cell-free methods, such as methods for determining the clostridial neurotoxin activity of a composition, determining whether a composition contains a clostridial neurotoxin polypeptide, and / or determining whether a clostridial neurotoxin polypeptide or portion thereof contained in a composition contains an activity-altering characteristic. The present invention is also directed to isolated capture substrates for clostridial neurotoxins, uses thereof, therapeutic or cosmetic clostridial neurotoxin compositions, and methods for making same.

Description

[Technical Field]

[0001] The present invention particularly relates to clostridial neurotoxins and methods for determining their activity. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can intoxicate neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes A to G and X (see WO 2018 / 009903 A2), as well as the neurotoxins produced by C. baratii and C. butyricum. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, particularly the release of neurotransmitters. Botulinum neurotoxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0003] In nature, clostridial neurotoxins (e.g., botulinum neurotoxin [BoNT]) are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H chain), with a molecular weight of approximately 100 kDa, and the light chain (L chain), with a molecular weight of approximately 50 kDa. The H chain is connected to an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain component. C The toxin binds to the target neuron, and the bound toxin is internalized into the cell via endosomes. NThe domain translocates the L chain across the endosomal membrane into the cytoplasm, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0004] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin). The acronym SNARE is derived from Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and, therefore, for the secretion of molecules from cells by vesicular trafficking. The protease function is a zinc-dependent endopeptidase activity, and it exhibits high substrate specificity for SNARE proteins.

[0005] When manufacturing and formulating clostridial neurotoxins for therapeutic and / or cosmetic purposes, there is a need to accurately assess the activity of a given composition.

[0006] Mouse LD 50 The LD50 assay has historically been the primary assay for assessing clostridial neurotoxin activity. The assay simultaneously tests the activity of all three domains (e.g., binding, translocation, or protease). More specifically, it defines the median intraperitoneal lethal dose of the toxin at a specific time point, usually 2-4 days after administration (activity is measured as the mouse LD50). 50 However, unfortunately, LD 50 The assay uses a large number of animals. 50 The units are not biological constants and are therefore not absolute measurements, but are highly dependent on assay conditions. In particular, the error associated with this assay can be as high as 60% between different testing facilities (Sesardic et al. 2003; Biologicals 31(4):265-276).

[0007] The mouse flaccid paralysis assay, also known as the "mouse abdominal ptosis assay," correlates the activity of clostridial neurotoxins with the degree of abdominal distention observed after subcutaneous injection of the toxin into the left groin of mice. The degree of paralysis is dose-dependent. This approach relies on a humane endpoint and is therefore not suitable for mouse LD. 50 This assay is proposed as an improvement to the LD 50 It is approximately 10 times more sensitive than the conventional LD ​​assay, uses sublethal doses of toxins, and 50 LD because results are available in 24-48 hours compared to 72-96 hours for testing 50 The results of this assay are 50 The LD values ​​are in excellent agreement with those of the 1996 study (Sesardic et al., 1996; Pharmacol Toxicol, 78(5): 283-8). 50 It uses 20% of the animals used in the assay, but still requires the use of animals.

[0008] Assays such as the mouse / rat phrenic nerve hemidiaphragm assay (based on the use of ex vivo nerve / muscle preparations) correlate the activity of clostridial neurotoxins with a decrease in the amplitude of the twitch response of the preparation after application to a maintenance medium. The usual endpoint of the assay is the time until a 50% decrease in amplitude is observed. Unfortunately, however, the hemidiaphragm assay (LD) 50 Such assays (like the ELISA assay) involve the use of large numbers of animals. Furthermore, such assays require highly skilled personnel trained in the use of sophisticated and expensive equipment.

[0009] All of the above assays have certain drawbacks, particularly animal welfare issues. Furthermore, none of the above assays are suitable for high-throughput testing. Therefore, there is a need in the art for alternative and / or improved clostridial neurotoxin assays. Furthermore, when performing cell-based assays, clostridial neurotoxins are formulated in cell growth media. Therefore, such assays are not amenable to characterization of therapeutic and / or cosmetic clostridial neurotoxin formulations, for example, to identify optimal excipients and their concentrations.

[0010] Cell-free assays known in the art typically involve incubating a test clostridial neurotoxin with a SNARE protein and determining the amount of SNARE protein cleaved by the test clostridial neurotoxin using conventional techniques such as SDS-PAGE or Western blotting. Alternatively, assays for testing the binding of clostridial neurotoxins to cellular receptors are also known, such as ELISAs that use receptor substrates expressed in prokaryotic host cells (typically Escherichia coli), which lack the post-translational machinery of mammalian cells. Traditional cell-free assays may lack the sensitivity to detect small differences in activity. This may be particularly relevant in the context of characterizing therapeutic and / or cosmetic clostridial neurotoxin formulations, where small but potentially therapeutically / cosmetically significant differences in activity for formulations using different excipients.

[0011] The use of clostridial neurotoxins in the therapeutic and cosmetic treatment of humans and other mammals is expected to continually expand the range of diseases and ailments that can benefit from the properties of these toxins, creating an increasing demand for large-scale production of clostridial neurotoxins and their appropriate formulations.

[0012] Large-scale production of biotherapeutics, particularly clostridial neurotoxins, is challenging due to the potential for undesired polypeptide modification and / or degradation at multiple stages of the process. In the presence of such undesired polypeptide modification and / or degradation, the activity per pg of clostridial neurotoxin can be significantly reduced compared to compositions lacking such modification and / or degradation. One such undesired modification is oxidation, which can occur during cellular expression, purification, bioprocessing, formulation, and / or storage of clostridial neurotoxins. Indeed, oxidation is one of the major degradation pathways for biotherapeutics. Oxidants such as peroxides, dissolved oxygen, metal ions, light, and free radicals can catalyze the oxidation of amino acids such as methionine, cysteine, histidine, tryptophan, tyrosine, and phenylalanine (Torosantucci et al. (2014), Pharm Res, 31, 541-553). In bioprocessing and formulation, metal catalysts may originate from metal-contaminated buffers and / or metal-contacting surfaces, and metal-catalyzed oxidation of histidine and methionine residues has been demonstrated to cause loss of activity, for example, due to aggregation and / or precipitation of the oxidized polypeptide. Furthermore, oxidizing agents are commonly utilized for decontamination in large-scale manufacturing. Clostridial neurotoxins are large polypeptides with many surface-exposed amino acid residues that are candidates for oxidation.

[0013] It is important for therapeutic and / or cosmetic purposes that the amount of undesired polypeptide modification and / or degradation present in a clostridial neurotoxin composition meets strictly defined criteria. Therefore, there is a need to determine with high sensitivity and / or specificity whether a composition contains such undesired polypeptide modification and / or degradation. As mentioned above, conventional cell-free assays usually measure L-chain proteolytic activity or H-chain proteolytic activity. COnly one aspect of neurotoxin activity, such as domain binding affinity, is examined. Thus, conventional assays may be somewhat limited in value in this respect because they do not provide comprehensive insight into the nature of the clostridial neurotoxin polypeptides present in the composition. For example, such assays cannot determine: the amount of uncontaminated light or heavy chains present in the composition; and / or whether the clostridial neurotoxin present in the composition has been degraded and / or has undergone undesirable modification (such as oxidation). In other words, conventional assays do not provide sufficient information for a skilled artisan to determine whether any activity observed in a clostridial neurotoxin composition results from undesirable polypeptide modification and / or degradation.

[0014] The present invention solves one or more of the problems set forth above. Summary of the Invention

[0015] The present inventors have developed a novel cell-free method for determining the clostridial neurotoxin activity of a composition. Advantageously, the cell-free method is particularly sensitive and / or specific, allowing for the determination of small differences in activity between compositions. This may be particularly advantageous in the context of characterizing therapeutic and / or cosmetic clostridial neurotoxin formulations, where activity differences for formulations with different excipients may be small but may be therapeutically / cosmetically important.

[0016] Additionally or alternatively, activity results obtained using the cell-free methods of the present invention are surprisingly similar to those determined using cell-based methods, while avoiding the drawbacks associated with cell-based methods. In particular, unlike cell-based methods, the methods of the present invention do not require the Clostridial neurotoxin to be formulated in a growth medium for testing, thereby enabling the characterization of therapeutic and / or cosmetic Clostridial neurotoxin formulations, for example, to identify optimal excipients and their concentrations.

[0017] Advantageously, the cell-free methods of the invention may be used to determine whether a composition contains an activity-altering characteristic, such as undesirable modification and / or degradation (e.g., oxidation) of a polypeptide. For example, the cell-free methods have been shown to accurately predict the loss of potency associated with oxidation of clostridial neurotoxins.

[0018] Furthermore, the dissociated L chain polypeptide and the capture substrate and clostridial neurotoxin receptor binding domain (H C In embodiments where assays for light chain activity are performed on complexes comprising the cleavage domain and assay samples comprising the cleavage domain, the number of plates used for a given assay can be advantageously reduced. Thus, cleavage assays are associated with reduced waste and costs and may be more amenable to high-throughput testing. DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, the present invention provides a cell-free method for determining clostridial neurotoxin activity of a composition comprising a clostridial neurotoxin polypeptide, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide; and (e) determining the amount of cleavable substrate cleaved by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition.

[0020] In one aspect, the present invention provides a cell-free method for determining whether a composition contains a Clostridial neurotoxin polypeptide, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing any unbound clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate any light chain (L chain) polypeptides of any bound Clostridial neurotoxin polypeptides; and (e) determining that the cleavable substrate has been cleaved, thereby determining that the composition contains a clostridial neurotoxin polypeptide, or determining that the cleavable substrate has not been cleaved, thereby determining that the composition does not contain a clostridial neurotoxin polypeptide.

[0021] In the above embodiment, when the composition does not contain a clostridial neurotoxin polypeptide, there are no unbound clostridial neurotoxin polypeptides in step (c) and no dissociated L chain polypeptides in step (d).

[0022] In one aspect, the present invention provides a cell-free method for determining clostridial neurotoxin activity of a composition comprising a clostridial neurotoxin polypeptide, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L chain polypeptide from the capture substrate and the clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining the amount of cleavable substrate in the assay sample that is cleaved by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition.

[0023] In one aspect, the present invention provides a cell-free method for determining whether a composition contains a Clostridial neurotoxin polypeptide, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing any unbound clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate any light chain (L chain) polypeptides of any bound Clostridial neurotoxin polypeptides, thereby separating any dissociated L chain polypeptides from the capture substrate and the Clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H C providing an assay sample comprising any complex comprising the nucleotide sequence (domain); and (e) determining that the cleavable substrate is cleaved in the assay sample, thereby determining that the composition contains a clostridial neurotoxin polypeptide, or determining that the cleavable substrate is not cleaved in the assay sample, thereby determining that the composition does not contain a clostridial neurotoxin polypeptide.

[0024] In the above-described embodiment, when the composition does not contain a clostridial neurotoxin polypeptide, there is no unbound clostridial neurotoxin polypeptide in step (c), there is no dissociated L chain polypeptide in step (d), and there is no capture substrate and a clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H CIn other words, when the composition does not contain a clostridial neurotoxin polypeptide, the assay sample contains a complex containing the dissociated L chain polypeptide and the capture substrate and the clostridial neurotoxin receptor-binding domain (H domain). CC Domain, e.g. H C domain), and may not include.

[0025] In one embodiment, the present invention provides a cell-free method for determining that a composition contains a Clostridial neurotoxin polypeptide, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L chain polypeptide from the capture substrate and the clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining that the cleavable substrate was cleaved in the assay sample, thereby determining that the composition contains a clostridial neurotoxin polypeptide.

[0026] The cleavable substrate is separate from the capture substrate (e.g., not covalently attached to the capture substrate, e.g., a different polypeptide) and may be added before, during, or after (preferably during) the step of adding the reducing agent. Thus, preferably, the cleavable substrate is added simultaneously with the reducing agent.

[0027] For example, a cell-free method for determining the clostridial neurotoxin activity of a composition comprising a clostridial neurotoxin polypeptide may include: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a cleavable substrate and a reducing agent to dissociate the light (L) chain polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L chain polypeptide and the captured substrate and clostridial neurotoxin receptor-binding domain (H) from the cleavable substrate and a reducing agent to dissociate the light (L) chain polypeptide of the bound clostridial neurotoxin polypeptide. CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining the amount of cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby determining the clostridial neurotoxin activity of the composition.

[0028] The cell-free method of the present invention is an in vitro method. As used herein, the term "cell-free" means that the method is not performed in cells or cell lysates. Therefore, the components of the present invention (e.g., capture substrates and cleavable substrates) are preferably prepared recombinantly and separated from cells. Advantageously, this constitutes a much more flawless system that can be better controlled.

[0029] In some embodiments, the components of the invention (e.g., capture substrates and cleavable substrates) are purified. The term "purified" may be used to indicate that a substance, such as a polypeptide, is "substantially pure." Thus, in a composition, the component may represent at least 90%, 95%, 99%, or 99.9% of the total biological material (e.g., fatty acids, nucleic acids, and / or polypeptides) present.

[0030] When practicing the methods of the present invention, the capture substrate and the composition may be contacted under conditions suitable for binding of the clostridial neurotoxin polypeptide (if present in the composition) to the capture substrate. For example, 20 to 500 nM of the capture substrate may be contacted with 0.1 to 150 pM of the clostridial neurotoxin polypeptide. The capture substrate and the composition may be contacted for at least 5, 10, 30, or 45 minutes, preferably at least 50 minutes. The capture substrate and the composition may be contacted for ≦5 hours, ≦4 hours, ≦3 hours, ≦2 hours, or ≦1.5 hours, preferably ≦75 minutes. The capture substrate and the composition may be contacted for 5 to 5 hours, 10 to 4 hours, 10 to 3 hours, 30 to 2 hours, or 45 to 75 minutes, preferably 50 to 70 minutes (e.g., 60 minutes). The capture substrate and the composition may be contacted at 20 to 45°C or 30 to 40°C, preferably 35 to 40°C (e.g., 37°C). Therefore, the capture substrate may be contacted for 50 to 70 minutes, and the contact may be incubated at 35 to 40°C. The capture substrate and the composition are preferably agitated during contact, for example, using a plate shaker. Agitation at 100 to 1000 rpm, 400 to 800 rpm, or 500 to 700 rpm, preferably 550 to 650 rpm (e.g., 600 rpm), is preferred. In one embodiment, when the capture substrate is immobilized on a solid support, any supernatant may be removed before contacting the capture substrate with the composition.

[0031] The methods herein may tolerate non-clostridial neurotoxin components (e.g., buffers and / or excipients) that may be present in the composition. Nevertheless, suitable components may include buffers (e.g., Dulbecco's phosphate-buffered saline (DPBS)), polypeptides (e.g., bovine serum albumin (BSA), such as 0.5-2% (preferably 1%) BSA), and surfactants (e.g., Tween-20, such as 0.025%-0.1% (preferably 0.05%) Tween-20). Preferably, the composition includes at least BSA (e.g., 0.5-2% (preferably 1%) BSA). Advantageously, the use of BSA may improve the sensitivity of the method, for example, when compared to casein (Figure 2).

[0032] The method comprises: CC Domain (e.g. H CThe capture substrate may include removing clostridial neurotoxin polypeptides that are not bound to the capture substrate, such as clostridial neurotoxin polypeptides that are not bound via a clostridial domain. Those skilled in the art will understand that any step of "removing unbound clostridial neurotoxin polypeptides" does not necessarily remove all unbound clostridial neurotoxin polypeptides. Thus, the method may include removing substantially all unbound clostridial neurotoxin polypeptides. The term "substantially all unbound clostridial neurotoxin polypeptides" may mean at least 90%, 95%, 98%, or 99% of the unbound clostridial neurotoxin polypeptides. Preferably, the method includes removing 100% of the unbound clostridial neurotoxin polypeptides. Removal may be achieved by any suitable technique known in the art. In one embodiment, once the capture substrate immobilized on a solid support is contacted with the composition of the present invention, the supernatant may be removed, thereby removing unbound clostridial neurotoxin polypeptides. Removal may additionally or alternatively include a washing step with a suitable buffer, optionally incubated at a suitable temperature and / or under suitable agitation conditions. For example, removal of unbound clostridial neurotoxin polypeptides may include a washing step using a composition (e.g., a wash buffer) containing the same non-clostridial neurotoxin components present in the composition contacted with the capture substrate. An exemplary composition for the washing step may include a buffer (e.g., Dulbecco's phosphate-buffered saline (DPBS)), a polypeptide (e.g., bovine serum albumin (BSA), such as 0.5% to 2% (preferably 1%) BSA), and a detergent (e.g., Tween-20, such as 0.025% to 0.1% (preferably 0.05%) Tween-20). The wash may be repeated as necessary. In one embodiment, when a capture substrate immobilized on a solid support is contacted with a composition according to the present invention, the supernatant may be removed and a wash buffer may be added, followed by removal of the wash buffer, thereby removing unbound clostridial neurotoxin polypeptides.

[0033] The method may include adding a reducing agent to dissociate the light chain (L chain) polypeptide of any clostridial neurotoxin polypeptide bound to the capture substrate. Any suitable reducing agent may be used as long as it is capable of reducing the disulfide bond between the L chain and H chain of the clostridial neurotoxin without significantly reducing the L chain activity of the clostridial neurotoxin. Suitable reducing agents may be dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). The reducing agent may be present at any suitable concentration, but exemplary DTT concentrations include 2.5 to 7.5 mM, preferably 5 mM. The reducing agent may contact any clostridial neurotoxin polypeptide bound to the capture substrate for at least 30 minutes, 60 minutes, or 120 minutes, preferably at least 160 minutes. The reducing agent may contact any clostridial neurotoxin polypeptide bound to the capture substrate for ≦6 hours, ≦5 hours, or ≦4 hours, preferably ≦3 hours. The reducing agent may be contacted with any clostridial neurotoxin polypeptide bound to a capture substrate for 30 minutes to 6 hours, 1 hour to 5 hours, or 2 hours to 5 hours, preferably 2.5 hours to 3.5 hours (e.g., 3 hours). The reducing agent and any clostridial neurotoxin polypeptide bound to a capture substrate may be incubated at 20 to 45°C or 30 to 40°C, preferably 35 to 40°C (e.g., 37°C) during contact. Therefore, the reducing agent may be contacted with any clostridial neurotoxin polypeptide bound to a capture substrate for 2.5 to 3.5 hours, and the contact may be incubated at 35 to 40°C. Preferably, the reducing agent and any clostridial neurotoxin polypeptide bound to a capture substrate are stirred during contact, for example, using a plate shaker. The stirring may be at 100 to 1000 rpm, 400 to 800 rpm, or 500 to 700 rpm, preferably 550 to 650 rpm (e.g., 600 rpm).

[0034] The method includes contacting the reducing agent with a capture substrate and Clostridial neurotoxin H. CC Domain (e.g. H C domain) complex (e.g., the capture substrate and Clostridial neurotoxin H CC Domain (e.g. HC The method may further include separating the L chain polypeptide from the complex containing the capture substrate (the L chain polypeptide). In one embodiment, when the capture substrate is immobilized on a solid support, the supernatant containing the L chain polypeptide can be placed in a separate container (e.g., a vial or well). The L chain polypeptide may then be contacted with a cleavable substrate.

[0035] However, most preferably, after contact with a reducing agent, the dissociated L chain polypeptide binds to the capture substrate and Clostridial neurotoxin H. CC Domain (e.g. H C domain) and complexes (e.g., capture substrates and Clostridial neurotoxin H CC Domain (e.g. H C The L chain polypeptide is not separated from the complex (e.g., transferred to another container, such as a vial or well) but remains in solution with the complex (e.g., the complex may be immobilized, although it is not part of the complex). Advantageously, the inventors have discovered that this process significantly improves the sensitivity of cell-free assays. This may be particularly true when the cleavable substrate comprises a first luciferase domain, a linker comprising a Clostridial neurotoxin cleavage site, and a second luciferase domain, as described herein. Prior to this discovery, it was expected that nonspecific background binding would be significant, adversely affecting the specificity of the method. Unexpectedly, this was not the case, and it was found that employing this method not only significantly improved the sensitivity of the assay, but also, as detailed in this Example, did so without significantly increasing nonspecific background. In light of this discovery, the number of plates used in a given assay can also be advantageously reduced. Thus, such methods are associated with reduced waste and costs, and may be more amenable to high-throughput testing. In such an embodiment (which is most preferred), the activity of the L chain is determined by the binding of the capture substrate and the Clostridial neurotoxin H CC Domain (e.g. H C domain) complex (e.g., a capture substrate and a Clostridial neurotoxin H CC Domain (e.g. H CThe activity of the dissociated L chain polypeptide and the clostridial neurotoxin H is assessed in the presence of a complex comprising the dissociated L chain polypeptide and the clostridial neurotoxin H domain. CC Domain (e.g. H C domain) complex (e.g., as described herein, e.g., a capture substrate and a Clostridial neurotoxin H CC Domain (e.g. H C The combination comprising the complex comprising the light chain (L chain) polypeptide, the capture substrate, and the complex comprising the light chain (L chain) polypeptide may be referred to herein as an "assay sample." The cleavable substrate may be added to the assay sample after the step of adding a reducing agent to dissociate the light chain (L chain) polypeptide. Preferably, however, the cleavable substrate is added at the same time as the reducing agent (e.g., simultaneously with the reducing agent). Thus, preferably, the assay sample comprises the dissociated L chain polypeptide, the capture substrate, and the Clostridial neurotoxin H. CC Domain (e.g. H C The combination comprises a cleavable substrate (domain) complex and a cleavable substrate. The cleavable substrate may be present in the assay sample for at least 30 minutes, 60 minutes, or 120 minutes, preferably at least 160 minutes. The cleavable substrate may be present in the assay sample for ≦6 hours, ≦5 hours, or ≦4 hours, preferably ≦3 hours. The cleavable substrate may be present in the assay sample for 30 minutes to 6 hours, 1 hour to 5 hours, or 2 hours to 5 hours, preferably 2.5 hours to 3.5 hours (e.g., 3 hours). The assay sample containing the cleavable substrate may be incubated at 20 to 45°C or 30 to 40°C, preferably 35 to 40°C (e.g., 37°C) during contact. Thus, the cleavable substrate may be present in the assay sample for 2.5 to 3.5 hours, and the assay sample containing the cleavable substrate may be incubated at 35 to 40°C during the contact. It is preferred that the assay sample containing the cleavable substrate is agitated during the contact, for example, using a plate shaker. The stirring may be at 100 to 1000 rpm, 400 to 800 rpm, or 500 to 700 rpm, preferably 550 to 650 rpm (for example, 600 rpm).

[0036] After contact with a reducing agent, the light chain polypeptide captures the substrate and clostridial neurotoxin H.CC Domain (e.g. H C Methods that do not separate the target domain from the target protein complex may be more practical and easier to perform and / or may be associated with reduced variability (assay noise). 50 The improvement in the clostridial neurotoxin H activity (measured by the clostridial neurotoxin H activity) may allow the testing of compositions containing very low amounts of clostridial neurotoxin polypeptides. Such compositions may be formulation compositions produced by dilution of the drug substance (obtainable after purification of the clostridial neurotoxin polypeptide). Any such advantage may be achieved by separating the L-chain polypeptide from the capture substrate and clostridial neurotoxin H activity after contact with a reducing agent. CC Domain (e.g. H C This can be clarified by comparison with an equivalent method in which the protein is separated from the (domain) complex (e.g., transferred to a separate vial / well).

[0037] The capture substrates described herein (e.g., in the context of the methods, uses of capture substrates, or isolated capture substrates) comprise a combination of the capture substrate and a Clostridial neurotoxin receptor-binding domain (H CC Domain or H C The complex may be part of a complex comprising a capture substrate and a full-length clostridial neurotoxin polypeptide, including or consisting of its light and heavy chains. The complex preferably comprises a capture substrate and a heavy chain (e.g., a translocation domain [H N domain] and H C When a reducing agent is added to a complex containing a capture substrate and a clostridial neurotoxin polypeptide, the complex preferably lacks the light chain of the clostridial neurotoxin. In the complex, the heavy chain H C Domain or H CC The domain preferably binds to the capture substrate (eg, by non-covalent interactions).

[0038] Thus, the assay sample described herein preferably comprises a dissociated L chain polypeptide (e.g., if present) and a complex comprising a capture substrate and a clostridial neurotoxin heavy chain. The heavy chain preferably comprises the translocation domain (H) of the clostridial neurotoxin. N domain) and H C It comprises or consists of domains.

[0039] The cleavable substrate may be used in the method of the present invention (eg, may be present in the assay sample) at a concentration of 1 to 1000 nM, for example 10 to 500 nM, preferably 50 to 150 nM (eg 100 nM).

[0040] The cleavable substrate is preferably not directly or indirectly immobilized on a solid support (e.g., on a plate, e.g., in a well thereof). Avoiding such immobilization (e.g., by releasing the cleavable substrate into solution) can increase sensitivity. In other words, directly or indirectly immobilizing the cleavable substrate on a solid support (e.g., on a plate, e.g., in a well thereof) can decrease sensitivity. The decrease in sensitivity can occur by reducing the total number of available binding sites on the solid support (e.g., on a plate, e.g., in a well thereof).

[0041] The capture substrate may be any substrate capable of binding to a clostridial neurotoxin. Suitably, the capture substrate for use in the methods of the present invention is capable of binding to the clostridial neurotoxin and / or clostridial neurotoxin H being assayed. CC Domain (e.g. H CCThe capture substrate may be selected against a clostridial neurotoxin receptor polypeptide or a ganglioside to which a clostridial neurotoxin binds. The method of the present invention may employ the use of a combination of a capture substrate comprising a clostridial neurotoxin receptor polypeptide and a capture substrate comprising a ganglioside. The capture substrate comprising a clostridial neurotoxin receptor polypeptide and the capture substrate comprising a ganglioside may form a complex (e.g., via a non-covalent interaction). Such complex formation may occur when the clostridial neurotoxin polypeptide or a portion thereof (e.g., H C or H CCThe capture substrate may be immobilized directly or indirectly on a solid support, while the ganglioside-containing capture substrate is added thereto. The ganglioside-containing capture substrate may then form a complex with the immobilized clostridial neurotoxin receptor polypeptide-containing capture substrate. However, it is preferred that the capture substrate comprises a clostridial neurotoxin receptor polypeptide. Thus, in some embodiments, it is preferred that the method does not involve the use of a capture substrate that comprises (or consists of) a ganglioside (e.g., GT1b). Indeed, the combined use of a clostridial neurotoxin receptor polypeptide-containing capture substrate (e.g., comprising the extracellular portion of a neuronal clostridial neurotoxin receptor polypeptide) and a ganglioside-containing capture substrate has been shown to reduce the sensitivity of the method, for example, when the clostridial neurotoxin receptor polypeptide (e.g., the extracellular portion of a neuronal clostridial neurotoxin receptor polypeptide) used is SV2c and the ganglioside is GT1b. In contrast, the combined use of a capture substrate comprising a clostridial neurotoxin receptor polypeptide (e.g., comprising the extracellular portion of a neuronal clostridial neurotoxin receptor polypeptide) and a capture substrate comprising a ganglioside has been shown to improve the sensitivity of the method when the clostridial neurotoxin receptor polypeptide (e.g., comprising the extracellular portion of a neuronal clostridial neurotoxin receptor polypeptide) is SYT-I. Thus, preferably, when the capture substrate comprises SYT-I (e.g., its extracellular portion), a capture substrate comprising a ganglioside (preferably GT1b) may be used. The capture substrate is preferably a substrate comprising a clostridial neurotoxin receptor polypeptide (e.g., a modified BoNT / BH receptor polypeptide) as described herein. CC This can be particularly advantageous when it involves domains.

[0042] In some examples herein, a "capture substrate" is referred to. However, this indicates that one or more capture substrate molecules are present when the method is performed. It does not necessarily indicate that two or more types of capture substrates are present, but this is encompassed. Thus, the capture substrates may be of one type (e.g., all of the capture substrates include a receptor polypeptide or ganglioside for a first clostridial neurotoxin) or of multiple types (e.g., some of the capture substrates include a receptor polypeptide for a first clostridial neurotoxin and some of the capture substrates include a receptor polypeptide for a second, different clostridial neurotoxin). Preferably, the capture substrate is of one type.

[0043] Gangliosides are oligoglycosylceramides derived from lactosylceramide and containing sialic acid residues, such as N-acetylneuraminic acid ("NANA" or "SA" or "Neu5Ac" or "NeuAc"). In some embodiments, the sialic acid component is N-glycolylneuraminic acid (Neu5Gc) or a Neu5Ac analog in which the amine group is replaced with an OH (3-deoxy-D-glycero-D-galacto-nonulosonic acid, abbreviated "KDN"). Gangliosides are defined according to the naming system proposed by Svennerholm, in which M, D, T, and Q refer to mono-, di-, tri-, and tetrasialogangliosides, respectively, and the numbers 1, 2, 3, etc. refer to the migration order of gangliosides in thin-layer chromatography. For example, the migration order of monosialogangliosides is GM3 > GM2 > GM1. Additional terms, such as GM1a and GD1b, are added to indicate variations within the basic structure. Glycosphingolipids with zero, one, two, and three sialic acid residues attached to the internal galactose unit are called asialo- (or O-), a-, b-, and c-series gangliosides, respectively. Gangliosides with sialic acid residues attached to the internal N-galactosamine residue are classified as α-series gangliosides. The biosynthetic pathways for the O-, a-, b-, and c-series gangliosides involve the sequential activity of sialyltransferases and glycosyltransferases, as elucidated, for example, by Ledeen et al., 2015 (Ledeen, Robert W., and Gusheng Wu. "The multitasked life of GM1 ganglioside, a true factotum of nature." Trends in biochemical sciences 40.7 (2015): 407-418). Further sialylation of each series and at different positions on the carbohydrate chain can lead to an increasingly complex and heterogeneous range of products, including the α-series gangliosides, which have sialic acid residues attached to internal N-acetylgalactosamine residues.

[0044] In the cellular context, gangliosides are delivered to the outer layer of the cell membrane via a transport system involving vesicle formation. Gangliosides are present and concentrated on the cell surface, with the two hydrocarbon chains of the ceramide moiety embedded in the cell membrane and the oligosaccharides located on the extracellular surface, providing a point of recognition for extracellular molecules or the surface of neighboring cells. The sialoglycan component of gangliosides extends from the cell surface, where it can participate in intermolecular interactions. They function by recognizing specific molecules on the cell surface and regulating the activity of proteins in the cell membrane. Gangliosides also specifically bind to viral and bacterial toxins, such as those derived from botulinum, tetanus, and cholera. For example, the cell surface receptor specific for cholera toxin is ganglioside GM1 (or GM1a):Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer.

[0045] BoNT is H CC In the H domain, BoNT / A, / B, / E, / F, and / G have two independent binding domains for gangliosides and neuronal protein receptors. CCWhile most BoNTs possess a conserved ganglioside-binding site consisting of an "E(Q)...H(K)...SXWY...G" motif in the ATP domain, BoNT / C, / D, and / DC possess two independent ganglioside-binding sites (Lam, Kwok-Ho, et al. "Diverse binding modes, same goal: The receptor recognition mechanism of botulinum neurotoxin." Progress in biophysics and molecular biology 117.2 (2015): 225-231). Most BoNTs bind only to gangliosides with a 2,3-linked N-acetylneuraminic acid residue (Sia5) attached to Gal4 in the oligosaccharide core, whereas the corresponding ganglioside-binding pocket on TeNT can also bind to GM1a, a ganglioside lacking the Sia5 sugar residue. It has been shown that introducing the H1241K mutation into recombinant BoNT / F confers GM1-binding ability (Benson, Marc A., et al. "Unique ganglioside recognition strategies for clostridium neurotoxins." Journal of Biological Chemistry 286.39 (2011): 34015-34022). It has also been confirmed that BoNT / D binds to GM1a and GD1a (Kroken, Abby R., et al. "Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons." Journal of Biological Chemistry 286.30 (2011): 26828-26837).

[0046] The ganglioside may be GM1 (e.g., GM1a or GM1b), GM2, GM3 (e.g., NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1b, GQ1b, GD2, or GD3.

[0047] Combining data from ganglioside-deficient mice and biochemical assays reveals that BoNT / A, E, F, and G prefer the terminal NAcGal-Gal-NAcNeu moiety present in GD1a and GT1b, whereas BoNT / B, C, D, and TeNT require the disialyl motif found in GD1b, GT1b, and GQ1b.

[0048] Thus, gangliosides can contain terminal NAcGal-Gal-NAcNeu moieties or disialyl motifs. Gangliosides can contain GD1a, GT1b, GD1b, GQ1b, or GM1 (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer). For example, gangliosides can contain GD1a, GT1b, GD1b, or GQ1b.

[0049] Suitable gangliosides may be selected from those described in WO2018 / 060351.

[0050] The ganglioside-containing capture substrate may be used at a concentration of 0.1 to 500 μM, for example, 1 to 250 μM, 1 to 100 μM, 10 to 50 μM, or 20 to 30 μM, for example, 24.1 μM.

[0051] The capture substrate preferably comprises a clostridial neurotoxin receptor polypeptide. The term "clostridial neurotoxin receptor polypeptide" can encompass a full-length clostridial neurotoxin receptor polypeptide or a portion thereof. The clostridial neurotoxin receptor polypeptide may be a neuronal clostridial neurotoxin receptor polypeptide, such as a full-length neuronal clostridial neurotoxin receptor polypeptide or a portion thereof. Preferably, the clostridial neurotoxin receptor polypeptide comprises (or consists of) the extracellular portion of a neuronal clostridial neurotoxin receptor, more preferably comprises (or consists of) the extracellular portion of a neuronal botulinum neurotoxin receptor.

[0052] Advantageously, the use of a capture substrate comprising a clostridial neurotoxin receptor polypeptide (and optionally a capture substrate comprising a ganglioside) can improve the methods of the present invention. In particular, the method can improve the determination of whether a composition contains an activity-changing characteristic (e.g., undesired polypeptide modification and / or degradation (e.g., oxidation)). For example, a capture substrate comprising a clostridial neurotoxin receptor polypeptide (and optionally a capture substrate comprising a ganglioside) can be more sensitive to an activity-changing characteristic of a clostridial neurotoxin (e.g., undesired polypeptide modification and / or degradation (e.g., oxidation)) compared to an antibody-capture substrate. An antibody may be unable to detect or distinguish between a clostridial neurotoxin that contains an activity-changing characteristic (e.g., undesired polypeptide modification and / or degradation (e.g., oxidation)) and a clostridial neurotoxin that does not.

[0053] The capture substrate may include the non-extracellular portion of the neuronal Clostridial neurotoxin receptor, but typically only includes the non-extracellular portion that is soluble in aqueous solution. Indeed, the absence of the non-extracellular portion is preferred, e.g., to improve solubility and facilitate recombinant production and / or handling.

[0054] The neuronal Clostridial neurotoxin receptor polypeptide may be a human neuronal Clostridial neurotoxin receptor polypeptide (e.g., its extracellular portion). The neuronal Clostridial neurotoxin receptor polypeptide may be synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), or synaptotagmin II (SYT-II). Preferably, the neuronal Clostridial neurotoxin receptor polypeptide comprises (or consists of) the extracellular portion of SV2a, SV2b, SV2c, SYT-I, or SYT-II. Thus, the capture substrate of the present invention may comprise the extracellular portion of SV2a, SV2b, SV2c, SYT-I, or SYT-II. The polypeptide sequence of the neuronal Clostridial neurotoxin receptor polypeptide (e.g., its extracellular portion) may be the same as the wild-type (e.g., human) polypeptide sequence. In another embodiment, the polypeptide of the neuronal Clostridial neurotoxin receptor polypeptide (e.g., its extracellular portion) may contain one or more modifications compared to a wild-type (e.g., human) polypeptide sequence. Preferably, when the Clostridial neurotoxin receptor polypeptide (e.g., its extracellular portion) is human SYT-II, the polypeptide sequence contains a leucine 51 to phenylalanine substitution (L51F), as set forth in SEQ ID NO:21.

[0055] The clostridial neurotoxin composition is CC (Preferably H C In the case of a Clostridial neurotoxin containing a SV2 domain, the capture substrate may contain SV2c, SV2a, or SV2b, preferably the extracellular portion of SV2c. There are three SV2 isoforms in humans: SV2a, SV2b, and SV2c, but BoNT / A has the highest affinity for SV2c. BoNT / A binds to the β chain of SV2-LD4 and the BoNT H C It may specifically bind to the luminal domain 4 of SV2 (SV2-LD4) through direct backbone-to-backbone interactions between the β-strand of and through interactions with the N559-linked glycan.

[0056] The clostridial neurotoxin composition is CC (Preferably H C In the case of a clostridial neurotoxin containing a SYT-II domain, the capture substrate may comprise the extracellular portion of SYT-II or SYT-I, preferably SYT-II. BoNT / B binds to SYT-I and SYT-II, but SYT-II is thought to be abundant on human neuronal cells. Thus, SYT-II may be a preferred neuroclostridial neurotoxin receptor polypeptide for use in the present invention.

[0057] The clostridial neurotoxin composition is CC (Preferably H C In the case of a Clostridial neurotoxin containing a SV2c, SV2a, or SV2b domain, the capture substrate may comprise the extracellular portion of SV2c, SV2a, or SV2b.

[0058] The clostridial neurotoxin composition is CC (Preferably H C In the case of a clostridial neurotoxin containing a SV2b or SV2c domain, the capture substrate may comprise the extracellular portion of SV2b or SV2c.

[0059] The clostridial neurotoxin composition is CC (Preferably H C In the case of a Clostridial neurotoxin containing a SV2c, SV2a, or SV2b domain, the capture substrate may comprise the extracellular portion of SV2c, SV2a, or SV2b.

[0060] The clostridial neurotoxin composition is CC (Preferably H C In the case of a Clostridial neurotoxin containing a SYT-II domain, the capture substrate may comprise the extracellular portion of SYT-II or SYT-I, preferably SYT-II.

[0061] The extracellular portion of SYT-II may comprise the extracellular portion of amino acid residues 1-75, 1-70, 1-65, 1-64, or 1-61 (preferably 1-61) of full-length SYT-II.

[0062] The extracellular portion of human SYT-II may refer to amino acid residues 1-61 of full-length human SYT-II. The residue positions may be determined by alignment of the amino acid sequence against SEQ ID NO:23.

[0063] Full length human SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:23. In one embodiment, full length human SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:23. Preferably, full length human SYT-II may comprise SEQ ID NO:23. Full length human SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:23. In one embodiment, full length human SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:23. Preferably, full length human SYT-II may consist of SEQ ID NO:23.

[0064] The extracellular portion of mouse SYT-II may refer to amino acid residues 1-64 of full-length mouse SYT-II. The residue positions may be determined by alignment of the amino acid sequence to SEQ ID NO:24.

[0065] Full length mouse SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:24. In one embodiment, full length mouse SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:24. Preferably, full length mouse SYT-II may comprise SEQ ID NO:24. Full length mouse SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:24. In one embodiment, full length mouse SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:24. Preferably, full length mouse SYT-II may consist of SEQ ID NO:24.

[0066] Thus, a capture substrate comprising the extracellular portion of mouse SYT-II may comprise a polypeptide sequence having at least 70% or more sequence identity to SEQ ID NO: 22. In one embodiment, a capture substrate comprising the extracellular portion of mouse SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22. Preferably, a capture substrate comprising the extracellular portion of mouse SYT-II may comprise SEQ ID NO: 22. Thus, a capture substrate comprising the extracellular portion of mouse SYT-II may consist of a polypeptide sequence having at least 70% or more sequence identity to SEQ ID NO: 22. In one embodiment, a capture substrate comprising the extracellular portion of mouse SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22. Preferably, a capture substrate comprising the extracellular portion of mouse SYT-II may consist of SEQ ID NO: 22.

[0067] The SYT-II is preferably human SYT-II.

[0068] The extracellular portion of human SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 19. In one embodiment, the extracellular portion of human SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 19. Preferably, the extracellular portion of human SYT-II may comprise SEQ ID NO: 19. The extracellular portion of human SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 19. In one embodiment, the extracellular portion of human SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 19. Preferably, the extracellular portion of human SYT-II may consist of SEQ ID NO: 19.

[0069] Thus, a capture substrate comprising the extracellular portion of human SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 18. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 18. Preferably, a capture substrate comprising the extracellular portion of human SYT-II may comprise SEQ ID NO: 18. Thus, a capture substrate comprising the extracellular portion of human SYT-II may consist of a polypeptide sequence having at least 70% or more sequence identity to SEQ ID NO: 18. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 18. Preferably, a capture substrate comprising the extracellular portion of human SYT-II may consist of SEQ ID NO: 18.

[0070] As indicated above, when the SYT-II is human SYT-II, the polypeptide sequence preferably comprises a leucine 51 to phenylalanine substitution (L51F). In other words, the human SYT-II is preferably modified human SYT-II. The position of residue 51 may be determined by alignment of the amino acid sequence to SEQ ID NO: 19. The extracellular portion of modified human SYT-II may comprise an L51F substitution and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 21. In one embodiment, the extracellular portion of modified human SYT-II may comprise an L51F substitution and a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 21. Preferably, the extracellular portion of modified human SYT-II may comprise SEQ ID NO: 21. The extracellular portion of modified human SYT-II may comprise an L51F substitution and consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 21. In one embodiment, the extracellular portion of modified human SYT-II may comprise an L51F substitution and may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 21. Preferably, the extracellular portion of modified human SYT-II may consist of SEQ ID NO: 21. The extracellular portion of modified human SYT-II may comprise an L51F substitution and may be encoded by a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 50. In one embodiment, the extracellular portion of modified human SYT-II may comprise an L51F substitution and may be encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 50. Preferably, the extracellular portion of modified human SYT-II may comprise an L51F substitution and may be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 50.

[0071] Thus, a capture substrate comprising the extracellular portion of modified human SYT-II may comprise an L51F substitution and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 20. In one embodiment, a capture substrate comprising the extracellular portion of modified human SYT-II may comprise an L51F substitution and a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 20. Preferably, a capture substrate comprising the extracellular portion of modified human SYT-II may comprise SEQ ID NO: 20. Thus, a capture substrate comprising the extracellular portion of modified human SYT-II may comprise an L51F substitution and may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 20. In one embodiment, a capture substrate comprising the extracellular portion of modified human SYT-II may comprise an L51F substitution and may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 20. Preferably, a capture substrate comprising the extracellular portion of modified human SYT-II may consist of SEQ ID NO: 20.

[0072] The extracellular portion of SYT-I may include the extracellular portion of amino acid residues 1-57 (eg, amino acid residues 33-54) of full-length SYT-I.

[0073] The extracellular portion of mouse SYT-I may include the extracellular portion of amino acid residues 1-59 of full-length mouse SYT-I. The extracellular portion of mouse SYT-I may refer to amino acid residues 1-59 of full-length mouse SYT-I. The residue positions may be determined by alignment of the amino acid sequence to SEQ ID NO:82.

[0074] The extracellular portion of mouse SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-59 of SEQ ID NO: 82. In one embodiment, the extracellular portion of mouse SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-59 of SEQ ID NO: 82. Preferably, the extracellular portion of mouse SYT-I may comprise amino acid residues 1-59 of SEQ ID NO: 82. The extracellular portion of mouse SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-59 of SEQ ID NO: 82. In one embodiment, the extracellular portion of mouse SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-59 of SEQ ID NO: 82. Preferably, the extracellular portion of mouse SYT-I may consist of amino acid residues 1-59 of SEQ ID NO:82.

[0075] Full length mouse SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 82. In one embodiment, full length mouse SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 82. Preferably, full length mouse SYT-I may comprise SEQ ID NO: 82. Full length mouse SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 82. In one embodiment, full length mouse SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 82. Preferably, full length mouse SYT-I may consist of SEQ ID NO: 82.

[0076] The extracellular portion of mouse SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 83. In one embodiment, the extracellular portion of mouse SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 83. Preferably, the extracellular portion of mouse SYT-I may comprise SEQ ID NO: 83. The extracellular portion of mouse SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 83. In one embodiment, the extracellular portion of mouse SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 83. Preferably, the extracellular portion of mouse SYT-I may consist of SEQ ID NO: 83.

[0077] Thus, a capture substrate comprising the extracellular portion of mouse SYT-I can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 84. In one embodiment, a capture substrate comprising the extracellular portion of mouse SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 84. Preferably, a capture substrate comprising the extracellular portion of mouse SYT-I can comprise SEQ ID NO: 84. Thus, a capture substrate comprising the extracellular portion of mouse SYT-I can consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 84. In one embodiment, a capture substrate comprising the extracellular portion of mouse SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 84. Preferably, a capture substrate comprising the extracellular portion of mouse SYT-I can consist of SEQ ID NO: 84.

[0078] The SYT-I is preferably human SYT-I. The extracellular portion of human SYT-I may refer to amino acid residues 1-57 (e.g., amino acid residues 33-54) of full-length human SYT-I. Thus, the extracellular portion of human SYT-I may include amino acid residues 1-57 (e.g., amino acid residues 33-54) of full-length human SYT-I. Residue positions may be determined by amino acid sequence alignment to SEQ ID NO:28.

[0079] The extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. In one embodiment, the extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. Preferably, the extracellular portion of human SYT-I may comprise amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. The extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. In one embodiment, the extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. Preferably, the extracellular portion of human SYT-I can consist of amino acid residues 1-57 (e.g., amino acid residues 33-54) of SEQ ID NO: 28.

[0080] Preferably, the extracellular portion of SYT-I may comprise the extracellular portion of amino acid residues 1-60 (e.g., amino acid residues 33-54) of full-length SYT-I. The extracellular portion of human SYT-I may refer to amino acid residues 1-60 (e.g., amino acid residues 33-54) of full-length human SYT-I. The residue positions may be determined by amino acid sequence alignment to SEQ ID NO:28.

[0081] The extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. In one embodiment, the extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. Preferably, the extracellular portion of human SYT-I may comprise amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. The extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. In one embodiment, the extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28. Preferably, the extracellular portion of human SYT-I can consist of amino acid residues 1-60 (e.g., amino acid residues 33-54) of SEQ ID NO: 28.

[0082] Full length human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 28. In one embodiment, full length human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 28. Preferably, full length human SYT-I may comprise SEQ ID NO: 28. Full length human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 28. In one embodiment, full length human SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 28. Preferably, full length human SYT-I may consist of SEQ ID NO: 28.

[0083] The extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 80. In one embodiment, the extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 80. Preferably, the extracellular portion of human SYT-I may comprise SEQ ID NO: 80. The extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 80. In one embodiment, the extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 80. Preferably, the extracellular portion of human SYT-I may consist of SEQ ID NO: 80.

[0084] Thus, a capture substrate comprising the extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 81. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 81. Preferably, a capture substrate comprising the extracellular portion of human SYT-I may comprise SEQ ID NO: 81. Thus, a capture substrate comprising the extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 81. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 81. Preferably, a capture substrate comprising the extracellular portion of human SYT-I may consist of SEQ ID NO: 81.

[0085] The extracellular portion of SV2a may include the extracellular portion of amino acid residues 469-599 of full-length SV2a (e.g., amino acid residues 469-598 or 469-595). The extracellular portion of human SV2a may refer to amino acid residues 469-599 of full-length human SV2a (e.g., amino acid residues 469-598 or 469-595). The residue positions may be determined by amino acid sequence alignment to SEQ ID NO:25.

[0086] Full-length human SV2a may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 25. In one embodiment, full-length human SV2a may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 25. Preferably, full-length human SV2a may comprise SEQ ID NO: 25. Full-length human SV2a may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 25. In one embodiment, full-length human SV2a may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 25. Preferably, full-length human SV2a may consist of SEQ ID NO: 25.

[0087] The extracellular portion of SV2b may include the extracellular portion of amino acid residues 410-539 of full-length SV2b. The extracellular portion of human SV2b may refer to amino acid residues 410-539 of full-length human SV2b. The residue positions may be determined by alignment of the amino acid sequence to SEQ ID NO:26.

[0088] Full-length human SV2b can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 26. In one embodiment, full-length human SV2b can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 26. Preferably, full-length human SV2b can comprise SEQ ID NO: 26. Full-length human SV2b can consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 26. In one embodiment, full-length human SV2b can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 26. Preferably, full-length human SV2b can consist of SEQ ID NO: 26.

[0089] The extracellular portion of SV2c can include the extracellular portion of amino acid residues 400-600, 450-590, 460-580, 470-570, or 500-570 of full-length SV2c. The extracellular portion of human SV2c can include at least luminal domain 4 (e.g., amino acid residues 519-563 of SV2c). Thus, the extracellular portion of human SV2c can refer to amino acid residues 519-563 of full-length human SV2c. The extracellular portion of human SV2c including at least luminal domain 4 can include amino acid residues 473-567 of full-length human SV2c. Residue positions can be determined by amino acid sequence alignment to SEQ ID NO:27.

[0090] Full-length human SV2c can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 27. In one embodiment, full-length human SV2c can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 27. Preferably, full-length human SV2c can comprise SEQ ID NO: 27. Full-length human SV2c can consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 27. In one embodiment, full-length human SV2c can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 27. Preferably, full-length human SV2c can consist of SEQ ID NO: 27.

[0091] The extracellular portion of human SV2c may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 30. In one embodiment, the extracellular portion of human SV2c may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 30. Preferably, the extracellular portion of human SV2c may comprise SEQ ID NO: 30. The extracellular portion of human SV2c may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 30. In one embodiment, the extracellular portion of human SV2c may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 30. Preferably, the extracellular portion of human SV2c may consist of SEQ ID NO: 30.

[0092] Thus, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 29. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 29. Preferably, a capture substrate comprising the extracellular portion of human SV2c can comprise SEQ ID NO: 29. Thus, a capture substrate comprising the extracellular portion of human SV2c can consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 29. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 29. Preferably, a capture substrate comprising the extracellular portion of human SV2c can consist of SEQ ID NO: 29.

[0093] A capture substrate comprising the extracellular portion of human SV2c can be encoded by a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 51. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can be encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 51. Preferably, a capture substrate comprising the extracellular portion of human SV2c can be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 51.

[0094] Thus, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 40. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 40. Preferably, a capture substrate comprising the extracellular portion of human SV2c can comprise SEQ ID NO: 40. Thus, a capture substrate comprising the extracellular portion of human SV2c can consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 40. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 40. Preferably, a capture substrate comprising the extracellular portion of human SV2c can consist of SEQ ID NO: 40.

[0095] Of the capture substrates comprising the extracellular portion of human SV2c, SEQ ID NO: 40 is most preferred.

[0096] Prior to carrying out the methods of the present invention, the capture substrate may be immobilized on a solid support. Such immobilization may be achieved by any means known in the art, but preferably by a tag (e.g., a GST tag) present on the capture substrate and a suitable binding partner (e.g., glutathione or a derivative thereof) present on the solid support. Other suitable tags, such as His tags, are known in the art. Suitable solid supports are known in the art. Advantageously, this allows for convenient washing of any capture substrate-clostridial neurotoxin complexes and easy removal of the supernatant with minimal loss of such complexes. The solid support may be a plastic support, such as the inner surface (in typical use) of a plate (e.g., a multiwell plate such as a microtiter plate), a column, or a tube (e.g., a microcentrifuge tube). Preferably, the solid support is the inner surface (in typical use) of a multiwell plate. Multiwell plates are convenient for handling large numbers of samples and are suitable for use in high-throughput techniques.

[0097] The capture substrate of the present invention may include post-translational modifications such as glycosylation. Glycosylation is preferably N-linked glycosylation. N-linked glycosylation preferably occurs at an asparagine residue present in the clostridial neurotoxin receptor polypeptide included in the capture substrate. Preferably, the clostridial neurotoxin receptor polypeptide includes the extracellular portion of SV2c glycosylated at N559. The residue position can be determined by amino acid sequence alignment with SEQ ID NO: 27. Glycosylation may include Man-5 glycan, G0f glycan, G1f glycan, or G2f glycan. The glycosylation preferably further includes N-acetylglucosamine (GlcNAc), e.g., G0f-GlcNAc.

[0098] A capture substrate containing post-translational modifications may be produced by expressing a nucleic acid encoding the capture substrate in a suitable host cell. The capture substrate may then be isolated from the host cell using standard techniques. For example, the host cell may be derived from a mammalian cell (e.g., from a mammalian cell line), preferably a human host cell, such as a human cell line host cell. Such host cells may be particularly advantageous when assaying clostridial neurotoxin compositions for human therapeutic and / or cosmetic use, as the capture substrate preferably exhibits post-translational modifications more similar to those found in humans compared to capture substrates produced using alternative methods, such as capture substrates produced by prokaryotic (e.g., E. coli) expression.

[0099] Advantageously, when the capture substrate comprises SV2c (e.g., its extracellular portion) that contains glycosylation, the capture substrate may enable improved (e.g., greater sensitivity) differentiation between oxidized and non-oxidized clostridial neurotoxin polypeptides (compared to an equivalent capture substrate that does not contain glycosylation).

[0100] Suitable host cells for producing capture substrates containing post-translational modifications may be HEK293 cells, Chinese hamster ovary (CHO) cells, or Drosophila cells, preferably HEK293 cells. In some embodiments, nucleic acids encoding the capture substrates may be expressed using Drosophila expression systems known in the art.

[0101] The nucleic acid may have at least 70% sequence identity to SEQ ID NO: 50. In one embodiment, the nucleic acid may have at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 50. Preferably, the nucleic acid may comprise (and more preferably consist of) SEQ ID NO: 50.

[0102] The nucleic acid may have at least 70% sequence identity to SEQ ID NO: 51. In one embodiment, the nucleic acid may have at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 51. Preferably, the nucleic acid may comprise (and more preferably consist of) SEQ ID NO: 51.

[0103] The cleavable substrate for use in the present invention is any substrate that can be cleaved by a clostridial neurotoxin. The cleavable substrate is appropriately selected to suit the clostridial neurotoxin and / or clostridial neurotoxin L-chain being assayed. The cleavable substrate includes a clostridial neurotoxin cleavage site, such as a SNARE scissile bond. Suitable cleavable substrates and their components may be those described in WO2018 / 075783A2, which is incorporated herein by reference.

[0104] A clostridial neurotoxin cleavage site may further include multiple amino acids located N-terminal and / or C-terminal, preferably N-terminal and C-terminal, of a scissile bond in a SNARE. Thus, a clostridial neurotoxin cleavage site may include at least SNARE amino acid residues P3P2P1P1'P2'P3', where P1 and P1' are residues located N-terminal and C-terminal to the scissile bond (scissile peptide bond) cleaved by a clostridial neurotoxin, such as Gln197 and Arg198 of SNAP-25, which are cleaved by the light chain of BoNT / A.

[0105] Thus, a clostridial neurotoxin cleavage site may further comprise at least 2 (e.g., the cleavage site includes at least P3P2P1P1'), 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues N-terminal to the amino acid residues that form the scissile bond in a SNARE. A clostridial neurotoxin cleavage site may further comprise at least 2 (e.g., the cleavage site includes at least P1P1'P2'P3'), 5, 10, 15, 20, 25, 30, 40, 50, or 100 amino acid residues C-terminal to the amino acid residues that form the scissile bond in a SNARE. For example, a clostridial neurotoxin cleavage site may further comprise: up to 5 amino acid residues N-terminal and up to 5 amino acid residues C-terminal to the amino acid residues that form the scissile bond in the SNARE; up to 10 amino acid residues N-terminal and up to 10 amino acid residues C-terminal to the amino acid residues that form the scissile bond in the SNARE; up to 25 amino acid residues N-terminal and up to 25 amino acid residues C-terminal to the amino acid residues that form the scissile bond in the SNARE; up to 50 amino acid residues N-terminal and up to 50 amino acid residues C-terminal to the amino acid residues that form the scissile bond in the SNARE; or up to 100 amino acid residues N-terminal and up to 100 amino acid residues C-terminal to the amino acid residues that form the scissile bond in the SNARE. For the avoidance of doubt, the term "up to" as used in this context is inclusive of the indicated number, for example, "up to 5 amino acid residues" includes "5 amino acid residues."

[0106] Preferably, a clostridial neurotoxin cleavage site includes 8 amino acid residues C-terminal to the amino acid residues that form the scissile bond in a SNARE and up to 55 amino acid residues N-terminal to the amino acid residues that form the scissile bond in a SNARE (e.g., a clostridial neurotoxin cleavage site may include up to a 65 amino acid portion of a SNARE).

[0107] A clostridial neurotoxin cleavage site comprising a SNARE scissile bond may comprise (or consist of) at least 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues of a SNARE described herein, preferably at least 55 amino acid residues of a SNARE described herein. A clostridial neurotoxin cleavage site comprising a SNARE scissile bond may comprise (or consist of) <200, <150, <100, <90, <80, <70, <60, <50, <40, <30, <20, <10, <9, <8, or <7 amino acid residues of a SNARE described herein. For example, a clostridial neurotoxin cleavage site comprising a SNARE scissile bond may comprise (or consist of) 6-200, 10-150, 20-100, 50-85, 55-80, or 60-75 amino acid residues of a SNARE as described herein. Preferably, a clostridial neurotoxin cleavage site comprising a SNARE scissile bond may comprise (or consist of) 60-70 amino acid residues of a SNARE as described herein.

[0108] Thus, in some embodiments, the clostridial neurotoxin cleavage site is derived from a SNARE (e.g., a fragment of a SNARE). The SNARE may be SNAP-25, synaptobrevin (VAMP), or syntaxin. Thus, the clostridial neurotoxin cleavage site may comprise the scissile bond of SNAP-25, synaptobrevin (VAMP), or syntaxin.

[0109] The SNARE for use in the present invention is preferably SNAP-25. Examples of SNAP-25 polypeptides include NCBI gene number 6616, NCBI reference sequence NP570824.1 (human SNAP25b), and NCBI reference sequence NP_112253.1 (rat SNAP25b). Preferably, the SNAP-25 is human SNAP-25. SNAP-25 may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:4. In one embodiment, SNAP-25 may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:4. Preferably, SNAP-25 may comprise SEQ ID NO:4. SNAP-25 may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:4. In one embodiment, SNAP-25 can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 4. Preferably, SNAP-25 can consist of SEQ ID NO: 4.

[0110] The scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by the L chain of BoNT / A may be Gln197-Arg198. The scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by the L chain of BoNT / C1 may be Arg198-Ala199. The scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by the L chain of BoNT / E may be Arg180-Ile181.

[0111] A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:4. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:4. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise SEQ ID NO:4. A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:4. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:4. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of SEQ ID NO: 4. Preferably, when a clostridial neurotoxin cleavage site is included in a cleavable substrate of the invention, the N-terminal methionine residue (residue 1) of SEQ ID NO: 4 is absent.

[0112] A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 5. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise SEQ ID NO: 5. A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 5. Preferably, the clostridial neurotoxin cleavage site comprising the scissile bond of SNAP-25 may consist of SEQ ID NO:5.

[0113] The SNARE may be VAMP. The VAMP may be VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, or YKT6. The VAMP may be human VAMP. Exemplary VAMP polypeptide sequences are shown in the table below. Thus, a VAMP may comprise a polypeptide sequence having at least 70% sequence identity to any of SEQ ID NOs: 31-36. In one embodiment, a VAMP may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any of SEQ ID NOs: 31-36. Preferably, a VAMP may comprise any of SEQ ID NOs: 31-36. Thus, a VAMP may consist of a polypeptide sequence having at least 70% sequence identity to any of SEQ ID NOs: 31-36. In one embodiment, a VAMP may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any of SEQ ID NOs: 31-36. Preferably, VAMP can consist of any of SEQ ID NOs: 31 to 36.

[0114] The scissile bonds in VAMP cleaved by the L chains of the indicated clostridial neurotoxins are shown in the table below: [Table A]

[0115] Human VAMP1, VAMP2, and VAMP3 can be cleaved by the light chains of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT. VAMP4, VAMP5, and YKT6 can be cleaved by the light chain of BoNT / X.

[0116] The SNARE may be syntaxin. The syntaxin may be syntaxin 1A or syntaxin 1B.

[0117] Syntaxin 1A may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 37. In one embodiment, syntaxin 1A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 37. Preferably, syntaxin 1A may comprise SEQ ID NO: 37. Syntaxin 1A may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 37. In one embodiment, syntaxin 1A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 37. Preferably, syntaxin 1A may consist of SEQ ID NO: 37.

[0118] Syntaxin 1B may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 38. In one embodiment, syntaxin 1B may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 38. Preferably, syntaxin 1B may comprise SEQ ID NO: 38. Syntaxin 1B may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 38. In one embodiment, syntaxin 1B may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 38. Preferably, syntaxin 1B may consist of SEQ ID NO: 38.

[0119] Syntaxin 1A and syntaxin 1B can be cleaved by BoNT / C1. The scissile bond in syntaxin 1A (SEQ ID NO: 37) cleaved by the light chain of BoNT / C1 can be Lys253-Ala254. The scissile bond in syntaxin 1B (SEQ ID NO: 38) cleaved by the light chain of BoNT / C1 can be Lys252-Ala253.

[0120] In embodiments where the polypeptide sequence of a clostridial neurotoxin cleavage site differs from a described SNARE polypeptide sequence (SEQ ID NO:) due to sequence identity, the clostridial neurotoxin cleavage site still includes the relevant scissile bond of the SNARE polypeptide sequence.

[0121] The cleavable substrate may further comprise an element indicating the presence or absence of cleavage of the cleavable substrate at the clostridial neurotoxin cleavage site. The element may comprise two distinct states: one indicating the presence of cleavage and the other indicating the absence of cleavage. The element may comprise a single module or multiple modules (preferably two modules). The multiple modules may be different from each other. Preferably, the element comprises two modules, the first module being N-terminal to the linker comprising (or consisting of) the clostridial neurotoxin cleavage site, and the second module being C-terminal to the linker comprising (or consisting of) the clostridial neurotoxin cleavage site. When in a non-cleavable form, the cleavable substrate is preferably a single-chain polypeptide (e.g., a fusion polypeptide).

[0122] Advantageously, cleavable substrates that include a moiety that indicates the presence or absence of cleavage exhibit improved properties, for example, when used in the methods of the invention. In contrast, methods that use cleavable substrates and antibodies that bind to the cleaved (and uncleaved) forms of the substrate may be associated with high levels of background signal, which may result from nonspecific antibody binding.

[0123] The element may include a detectable label. The detectable label may be a label that is visually detectable due to the label's optical properties. The detectable label may be a fluorescent label. Such labels may be detected using fluorescence techniques, such as fluorescence microscopy. Thus, in particularly preferred embodiments, the detectable label is a fluorophore (e.g., a fluorescent dye) or a fluorescent polypeptide. The fluorescent dye may be HiLyte fluorescent dyes (available from AnaSpec), AlexaFluor (available from Thermo Fisher), Atto (available from Sigma-Aldrich), Quantum Dot (available from Sigma-Aldrich), or Janelia Fluor dyes (available from Janelia, US). The fluorescent polypeptide may be cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP), or red fluorescent protein (RFP).

[0124] In one embodiment, the first module of the element is a fluorescent label (e.g., a fluorescent polypeptide), and the second module of the element is a different fluorescent label (e.g., a different fluorescent polypeptide). For example, the first module of the element can be YFP, and the second module of the element can be CFP. The fluorescent labels can be carefully selected to function as a fluorescence resonance energy transfer (FRET) donor and acceptor pair. Thus, in one embodiment, when the cleavable substrate is intact and not cleaved by a clostridial neurotoxin, the fluorescent labels are in close proximity and FRET occurs; when the cleavable substrate is cleaved by a clostridial neurotoxin, the fluorescent labels are no longer in close proximity and FRET does not occur. Therefore, measuring a change in the FRET signal (e.g., the fluorescent properties of the cleavable substrate) can indicate whether the cleavable substrate has been cleaved. Suitable cleavable substrates and related FRET methodologies are taught in EP 2332959 A2, incorporated herein by reference.

[0125] In a preferred embodiment, the element comprises a luciferase. As used herein, the term "luciferase" refers to an enzyme that catalyzes a bioluminescent reaction, for example, by catalyzing the oxidation of luciferin to release oxyluciferin, emitting light. Luciferases can be naturally occurring or artificially produced. As used herein, a "functional" luciferase is one that is capable of catalyzing a reaction in the presence of an appropriate substrate.

[0126] Exemplary luciferases include Nanoluc (SEQ ID NO: 1), firefly luciferase (e.g., Photinus pyralis luciferase), bacterial luciferase (e.g., Vibrio fischieri or Vibrio harveyi luciferase), sea pansy luciferase (e.g., Renilla reniformis luciferase), dinoflagellate luciferase, Gaussia luciferase, and copepod luciferase. Other suitable luciferases are described in U.S. Patent No. 8,557,970, which is incorporated herein by reference.

[0127] Preferably, the luciferase comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. In one embodiment, the luciferase may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 1. Preferably, the luciferase may comprise SEQ ID NO: 1. Preferably, the luciferase consists of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. In one embodiment, the luciferase may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 1. Preferably, the luciferase may consist of SEQ ID NO: 1.

[0128] The luciferase may be present as two separate modules of an element to indicate the presence or absence of cleavage of a cleavable substrate at a clostridial neurotoxin cleavage site, wherein the first module of the element may be a first luciferase domain and the second module of the element may be a second luciferase domain.

[0129] Thus, in a particularly preferred embodiment, the cleavable substrate is a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain. In one embodiment, (i) when the linker is intact (i.e., when the clostridial neurotoxin cleavage site is not cleaved by the clostridial neurotoxin), the linker operably links the first and second luciferase domains, thereby providing a functional luciferase (i.e., the cleavable substrate has luciferase activity); (ii) when the linker is cleaved (i.e., when the clostridial neurotoxin cleavage site is cleaved by the clostridial neurotoxin), the linker no longer operably links the first and second luciferase domains, resulting in a loss of luciferase activity (i.e., the cleavable substrate does not have luciferase activity). The term "single-chain" as used in the context of a cleavable substrate may refer to a single polypeptide molecule having a series of amino acid residues joined together by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In other words, each described element of a single-chain polypeptide may be joined to other elements by peptide bonds.

[0130] The term "operably linked," as used in the context of first and second luciferase domains, means joining the first and second luciferase domains in such a way that any intervening sequences do not prevent the two fragments from forming an active and functional tertiary structure. The operably joined first and second luciferase domains exhibit luciferase activity, e.g., as in the absence of a linker.

[0131] The first luciferase domain is preferably N-terminal to the linker containing the clostridial neurotoxin cleavage site. Preferably, the first luciferase domain is the most N-terminal element of the cleavable substrate. The first luciferase domain may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2. In one embodiment, the first luciferase domain may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:2. Preferably, the first luciferase domain comprises SEQ ID NO:2. The first luciferase domain may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2. In one embodiment, the first luciferase domain may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:2. Preferably, the first luciferase domain may consist of SEQ ID NO:2.

[0132] The second luciferase domain is preferably C-terminal to the linker containing the clostridial neurotoxin cleavage site. Preferably, the second luciferase domain is the most C-terminal element of the cleavable substrate. The second luciferase domain may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:3. In one embodiment, the second luciferase domain may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:3. Preferably, the second luciferase domain comprises SEQ ID NO:3. The second luciferase domain may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:3. In one embodiment, the second luciferase domain may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:3. Preferably, the second luciferase domain may consist of SEQ ID NO:3.

[0133] In some embodiments, the linker may consist of a clostridial neurotoxin cleavage site. However, it is preferred that the linker further comprises one or more spacers. Most preferably, the linker comprises (and more preferably consists of) a first spacer N-terminal to the clostridial neurotoxin cleavage site and a second spacer C-terminal to the clostridial neurotoxin cleavage site. When one or more spacers are present, the spacers may have the same or different polypeptide sequences (preferably the same polypeptide sequence).

[0134] Selection of an appropriate spacer sequence and size is within the ability of one skilled in the art. The spacer may be of any suitable length, such as 3-20, 2-15, 5-15, or 4-8 amino acids in length. The spacer may comprise (or consist of) glycine and serine residues. In particular, the spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 39. In one embodiment, the spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 39. Preferably, the spacer may comprise SEQ ID NO: 39. In particular, the spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 39. In one embodiment, the spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 39. Preferably, the spacer may consist of SEQ ID NO: 39.

[0135] Preferably, a cleavable substrate of the invention comprises (from N-terminus to C-terminus): (i) a first luciferase domain; (ii) a first spacer; (iii) a clostridial neurotoxin cleavage site; (iv) a second spacer; and (v) a second luciferase domain. More preferably, a cleavable substrate of the invention may consist of (from N-terminus to C-terminus): (i) a first luciferase domain; (ii) a first spacer; (iii) a clostridial neurotoxin cleavage site; (iv) a second spacer; and (v) a second luciferase domain. In such an embodiment, a linker is formed from elements (ii), (iii), and (iv). The first and second spacers preferably have the same polypeptide sequence.

[0136] The cleavable substrate may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. In one embodiment, the cleavable substrate may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 6. Preferably, the cleavable substrate may comprise SEQ ID NO: 6.

[0137] The cleavable substrate may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. In one embodiment, the cleavable substrate may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 6. Preferably, the cleavable substrate may consist of SEQ ID NO: 6.

[0138] In some examples herein, a "cleavable substrate" is referred to. However, this indicates that multiple "cleavable substrates" may be present when carrying out the method. It does not necessarily indicate that two or more different types of cleavable substrates exist but are encompassed. Thus, the cleavable substrate may be of one type (e.g., all cleavable substrates comprising: (i) a first luciferase domain; (ii) a linker comprising a SNAP-25 clostridial neurotoxin cleavage site; and (iii) a second luciferase domain), or of multiple types (e.g., a portion of a cleavable substrate comprising: (i) a first luciferase domain; (ii) a linker comprising a SNAP-25 clostridial neurotoxin cleavage site; and (iii) a portion of a cleavable substrate comprising a second luciferase domain and: (i) YFP; (ii) a linker comprising a SNAP-25 clostridial neurotoxin cleavage site; and (iii) GFP). Preferably, the cleavable substrate is of one type.

[0139] The method may include determining whether the cleavable substrate has been cleaved. In particular, the method may include determining the amount of cleavable substrate (preferably in an assay sample) cleaved by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition. The manner in which such a determination is made will depend on the particular properties of the cleavable substrate used in the method. For example, if the cleavable substrate contains a detectable label, a difference in detection characteristics may indicate whether the cleavable substrate has been cleaved and / or the amount of cleavable substrate that has been cleaved. The difference may be determined by comparison with an appropriate control. For example, determining whether a cleavable substrate containing a fluorescent label has been cleaved may involve comparing the fluorescence of the cleavable substrate to a control and determining whether there is a difference in fluorescence (e.g., a loss of FRET signal).

[0140] A suitable control may be a negative control. A negative control may be provided by performing the method in a similar manner, but the composition being tested does not contain a clostridial neurotoxin polypeptide. A negative control may also be a negative standard sample. A negative standard sample may correspond to a theoretically or experimentally determined value that represents a negative result in the method of the invention. The value may be determined before performing the method of the invention, or may be determined simultaneously with or after performing the method of the invention.

[0141] In one embodiment, when the control is a negative control, a difference in the properties of the cleavable substrate compared to the negative control indicates that the composition being tested comprises a light chain polypeptide (preferably a clostridial neurotoxin polypeptide). A difference in the properties of the cleavable substrate compared to the negative control indicates that the composition comprises a heavy chain polypeptide (or H C or H CC In such cases, this may indicate that the composition contains a Clostridial neurotoxin polypeptide.

[0142] In one embodiment, when the control is a negative control, a difference in the properties of the cleavable substrate compared to the negative control indicates that the composition tested does not contain a light chain polypeptide (preferably does not contain a clostridial neurotoxin polypeptide). In one embodiment, when the control is a negative control, no difference in the properties of the cleavable substrate compared to the negative control indicates that the composition tested does not contain a light chain polypeptide and / or a heavy chain polypeptide (or H C or H CC In such a case, it can be determined that the composition does not contain a Clostridial neurotoxin polypeptide.

[0143] In one embodiment, when the control is a negative control, a difference in the properties of the cleavable substrate compared to the negative control indicates that the composition being tested contains an active L chain polypeptide (preferably, an active Clostridial neurotoxin polypeptide). The extent of the difference can be quantified and indicates the number of active L chain polypeptides (e.g., the number of active Clostridial neurotoxin polypeptides). A difference in the properties of the cleavable substrate compared to the negative control indicates that the composition contains an H chain polypeptide (or H chain polypeptides) that does not contain an activity-reducing property. C or H CC The degree of difference is quantified and indicates that the H chain polypeptide (or H domain polypeptide) that does not contain the activity-reducing property is also included. C or H CC The degree of difference can be quantified and indicate the activity level of a composition comprising a clostridial neurotoxin polypeptide. Preferably, the greater the difference compared to the negative control, the greater the number of active L chain polypeptides (e.g., the number of active clostridial neurotoxin polypeptides). Preferably, the greater the difference compared to the negative control, the greater the number of H chain polypeptides (or H domain polypeptides) that do not contain the activity-reducing property. C or H CC Preferably, the greater the difference compared to the negative control, the greater the activity level of the composition comprising the clostridial neurotoxin polypeptide.

[0144] In one embodiment, when the control is a negative control, the absence of a difference in the properties of the cleavable substrate compared to the negative control indicates that the composition being tested comprises an inactive light chain polypeptide (preferably an inactive clostridial neurotoxin polypeptide). In one embodiment, when the control is a negative control, the absence of a difference in the properties of the cleavable substrate compared to the negative control indicates that the composition being tested comprises an inactive light chain polypeptide and / or a heavy chain polypeptide (or H) comprising an activity-reducing property. C or H CC Such compositions may be considered inactive compositions.

[0145] In one embodiment, when the control is a negative control, the difference in the properties of the cleavable substrate compared to the negative control may be used to indicate the activity level of the composition. The difference may be used to indicate the number of active L chain polypeptides (e.g., the number of active Clostridial neurotoxin polypeptides) present in the composition. The difference may be used to indicate the number of H chain polypeptides (or H chain polypeptides) without the activity-reducing properties present in the composition. C or H CC The number of domain polypeptides may also be indicated.

[0146] In some embodiments, the difference in the properties of the cleavable substrate compared to a control may be quantified to indicate the activity level of the composition (e.g., the number of active clostridial neurotoxin polypeptides present in the composition).

[0147] A suitable control may be a positive control. A positive control may be provided by performing the method in a similar manner, where the composition comprises a clostridial neurotoxin polypeptide, preferably associated with a known activity level. A positive control may also be a positive standard sample. A positive standard sample may correspond to a value determined theoretically or experimentally and representing a positive result in the method of the present invention. A positive result may represent an ideal activity level of a composition comprising a clostridial neurotoxin polypeptide. Such a value may be used for quality control purposes. The value may be determined before performing the method of the present invention, or may be determined simultaneously with or after performing the method of the present invention. Thus, when a "property of a cleavable substrate" or "luminescence" (e.g., luminescence level) is used herein with respect to a control, this may refer to the "property of a cleavable substrate" or "luminescence" (e.g., luminescence level) represented by the control.

[0148] In one embodiment, when the control is a positive control, the absence of a difference in the properties of the cleavable substrate compared to the positive control indicates that the composition tested contains a light chain polypeptide (preferably comprising a clostridial neurotoxin polypeptide). The absence of a difference in the properties of the cleavable substrate compared to the positive control indicates that the composition does not contain a heavy chain polypeptide (or H C or H CC In such cases, this may allow for a determination that the composition contains a Clostridial neurotoxin polypeptide.

[0149] In one embodiment, when the control is a positive control, the absence of a difference in the properties of the cleavable substrate when compared to the positive control indicates that the composition being tested contains an active L chain polypeptide (preferably an active Clostridial neurotoxin polypeptide). The absence of a difference in the properties of the cleavable substrate when compared to the positive control indicates that the composition does not contain an H chain polypeptide (or H chain polypeptide) that does not contain an activity-reducing property. C or H CC Such compositions may be considered active compositions.

[0150] In one embodiment, when the control is a positive control, the difference in the properties of the cleavable substrate compared to the positive control can be used to indicate the activity level of the composition. The difference can be used to indicate the number of active L chain polypeptides (e.g., the number of active Clostridial neurotoxin polypeptides) present in the composition. The difference can be used to indicate the number of H chain polypeptides (or H chain polypeptides) without the activity-reducing properties present in the composition. C or H CC The number of domain polypeptides may also be indicated.

[0151] In one embodiment, when the control is a positive control, the absence of a difference in the properties of the cleavable substrate when compared to the positive control may be used to indicate that the composition has the same level of activity as the positive control. This may be used to indicate the number of active L chain polypeptides (e.g., the number of active Clostridial neurotoxin polypeptides) present in the composition. This may be used to indicate the number of H chain polypeptides (or H chain polypeptides) without the activity-reducing properties present in the composition. C or H CC The number of domain polypeptides may also be indicated.

[0152] When the cleavable substrate comprises (i) a first luciferase domain; (ii) a linker containing a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain, determining whether the cleavable substrate has been cleaved can be assessed by measuring luciferase activity. This can be accomplished by adding an appropriate luciferase substrate (preferably, the luciferase substrate is added to the assay sample). Preferably, the luciferase substrate is furimazine (2-furanylmethyl-deoxy-coelenterazine). In such cases, determining whether the cleavable substrate has been cleaved can be assessed by measuring luminescence. For example, the cleavable substrate can be contacted with furimazine (preferably in the assay sample) at a concentration of 1 to 100 μM, preferably 10 to 50 μM (e.g., 37.5 μM). Luminescence can be measured using any suitable technique, such as an automated plate reader (e.g., a BMG Labtech CLARIOstar plate reader).

[0153] Thus, in a preferred embodiment, the property of the cleavable substrate may be luciferase activity. The difference in the property of the cleavable substrate may be a difference in the level of luciferase activity. As mentioned above, this may be determined by measuring luminescence.

[0154] Thus, in one embodiment, when the control is a negative control, a lower luminescence level compared to the negative control indicates that the composition tested contains a light chain polypeptide (preferably a clostridial neurotoxin polypeptide). A lower luminescence level compared to the negative control indicates that the composition tested contains a heavy chain polypeptide (or a heavy C or H CC In such cases, this may indicate that the composition contains a Clostridial neurotoxin polypeptide.

[0155] In one embodiment, when the control is a negative control, an identical luminescence compared to the negative control indicates that the composition tested does not contain a light chain polypeptide (preferably does not contain a clostridial neurotoxin polypeptide). In one embodiment, when the control is a negative control, an identical luminescence compared to the negative control indicates that the composition tested does not contain a light chain polypeptide and / or a heavy chain polypeptide (or H chain polypeptide). C or H CC In such a case, it can be determined that the composition does not contain a clostridial neurotoxin polypeptide.

[0156] In one embodiment, when the control is a negative control, a lower level of luminescence compared to the negative control indicates that the composition tested contains an active L chain polypeptide (preferably, an active Clostridial neurotoxin polypeptide). The degree of the lower level can be quantified to indicate the number of active L chain polypeptides (e.g., the number of active Clostridial neurotoxin polypeptides). A lower level of luminescence compared to the negative control indicates that the composition contains an H chain polypeptide (or H chain polypeptides) that does not contain the activity-reducing property. C or H CCThe extent of the low level can be quantified to indicate the activity level of the composition comprising the clostridial neurotoxin polypeptide. Preferably, the lower the luminescence level compared to the negative control, the higher the number of active L chain polypeptides (e.g., the number of active clostridial neurotoxin polypeptides). Preferably, the lower the luminescence level compared to the negative control, the higher the number of active H chain polypeptides (or H chain polypeptides) that do not contain the activity-reducing property. C or H CC Preferably, the lower the level of luminescence compared to the negative control, the higher the activity level of the composition comprising the clostridial neurotoxin polypeptide.

[0157] In one embodiment, when the control is a negative control, an identical luminescence compared to the negative control indicates that the composition tested comprises an inactive light chain polypeptide (preferably, an inactive clostridial neurotoxin polypeptide). In one embodiment, when the control is a negative control, an identical luminescence compared to the negative control indicates that the composition tested comprises an inactive light chain polypeptide and / or a heavy chain polypeptide (or H) comprising an activity-reducing property. C or H CC domain polypeptide).

[0158] Thus, in one embodiment, when the control is a positive control, an identical (or lower) level of luminescence compared to the positive control indicates that the composition being tested contains a light chain polypeptide (preferably a clostridial neurotoxin polypeptide). An identical (or lower) level of luminescence compared to the positive control indicates that the composition contains a heavy chain polypeptide (or a heavy C or H CC In such cases, this may indicate that the composition contains a Clostridial neurotoxin polypeptide.

[0159] In one embodiment, when the control is a positive control, an identical (or lower) level of luminescence compared to the positive control indicates that the composition being tested contains an active L chain polypeptide (preferably, an active Clostridial neurotoxin polypeptide). An identical (or lower) level of luminescence compared to the positive control indicates that the composition contains an H chain polypeptide (or H chain polypeptide) that does not contain the activity-reducing property. C or H CC It may also be shown that the polypeptide comprises a polypeptide domain.

[0160] An identical luminescence level compared to the positive control may indicate that the composition tested contains the same number of active L chain polypeptides (e.g., active Clostridial neurotoxin polypeptides) as the positive control. An identical luminescence level compared to the positive control may indicate that the composition tested contains the same number of H chain polypeptides (or H chain polypeptides) that do not contain the activity-reducing property as the positive control. C or H CC An identical level of luminescence compared to the positive control can indicate that the composition has the same level of activity as the positive control.

[0161] A lower level of luminescence compared to the positive control may indicate that the composition tested contains more active L chain polypeptides (e.g., active Clostridial neurotoxin polypeptides) than the positive control. A lower level of luminescence compared to the positive control may indicate that the composition tested contains more H chain polypeptides (or H chain polypeptides) that do not contain the activity-reducing property. C or H CC domain polypeptide) than a positive control and / or a heavy chain polypeptide (or H domain polypeptide) having increased activity properties. C or H CCA lower luminescence level compared to the positive control may indicate that the composition has a higher activity level than the positive control. The extent of the lower level may be quantified to indicate the activity level of the composition containing the clostridial neurotoxin polypeptide. Preferably, the lower the luminescence level compared to the positive control, the greater the number of active L chain polypeptides (e.g., the number of active clostridial neurotoxin polypeptides). Preferably, the lower the luminescence level compared to the positive control, the greater the number of active H chain polypeptides (or H chain polypeptides) that do not contain the activity-reducing property. C or H CC Preferably, the lower the luminescence level compared to the positive control, the higher the number of H chain polypeptides (or H domain polypeptides). C or H CC Preferably, the lower the level of luminescence compared to the positive control, the higher the activity level of the composition comprising the clostridial neurotoxin polypeptide.

[0162] In one embodiment, when the control is a positive control, a higher level of luminescence compared to the positive control indicates that the composition tested does not contain a light chain polypeptide (preferably contains a clostridial neurotoxin polypeptide). In one embodiment, when the control is a positive control, a higher level of luminescence compared to the positive control indicates that the composition tested does not contain a light chain polypeptide (preferably contains a clostridial neurotoxin polypeptide) and / or contains a heavy chain polypeptide (or H chain polypeptide). C or H CC In such cases, this may be used to determine that the composition does not contain a Clostridial neurotoxin polypeptide. To make a more definitive determination, a comparison with a negative control may also be made, as described herein.

[0163] In one embodiment, when the control is a positive control, a higher level of luminescence compared to the positive control indicates that the composition tested comprises an inactive L chain polypeptide (preferably, an active Clostridial neurotoxin polypeptide). In one embodiment, when the control is a positive control, a higher level of luminescence compared to the positive control indicates that the composition tested comprises an inactive L chain polypeptide (preferably, an active Clostridial neurotoxin polypeptide) and / or an H chain polypeptide (or H chain polypeptide) comprising an activity-reducing property. C or H CC domain polypeptide).

[0164] A higher luminescence level compared to the positive control may indicate that the composition tested contains less active L chain polypeptide (e.g., active Clostridial neurotoxin polypeptide) than the positive control. A higher luminescence level compared to the positive control may indicate that the composition tested contains less active L chain polypeptide (e.g., active Clostridial neurotoxin polypeptide) than the positive control. C or H CC A higher luminescence level compared to the positive control may indicate that the composition has a lower activity level than the positive control. The degree of high level may be quantified to indicate the activity level of the composition comprising the clostridial neurotoxin polypeptide. Preferably, the higher the luminescence level compared to the positive control, the fewer the number of active L chain polypeptides (e.g., the number of active clostridial neurotoxin polypeptides). Preferably, the higher the luminescence level compared to the positive control, the fewer the number of H chain polypeptides (or H chain polypeptides) that do not contain the activity-reducing property. C or H CC Preferably, the higher the level of luminescence compared to the positive control, the lower the activity level of the composition comprising the clostridial neurotoxin polypeptide.

[0165] As used herein, the term "different" (and related terms such as "change," "altered," "difference," and their synonyms) can refer to a difference that is substantially different from a comparison standard (e.g., a control described herein). A difference (and related terms such as "change," "altered," "difference," and their synonyms) can refer to a statistically significant difference when compared to a comparison standard (e.g., a control described herein). A "substantial difference" can be at least a 5%, 10%, 15%, 20%, 25%, or 30% difference when compared to a comparison standard (e.g., a control described herein). The term "no difference" (and related terms such as "unchanged" and "same," and their synonyms) can refer to the absence of a substantial difference when compared to a comparison standard (e.g., a control described herein). No difference (and related terms such as "unchanged" and "same," and their synonyms) can refer to the absence of a statistically significant difference when compared to a comparison standard (e.g., a control described herein). As used herein, the term "low" (and related terms such as "less than") can mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% lower when compared to a comparison standard (e.g., a control described herein). The term "low" (and related terms such as "less than") can mean statistically significantly lower when compared to a comparison standard (e.g., a control described herein). As used herein, the term "high" (and related terms such as "higher than") can mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% higher when compared to a comparison standard (e.g., a control described herein). The term "high" (and related terms such as "higher than") can mean statistically significantly higher when compared to a comparison standard (e.g., a control described herein).

[0166] The final step of the methods of the invention can include assigning a measured activity value. The measured activity value can be a luciferase activity value (e.g., measured by luminescence). Preferably, the measured activity value is a measured luminescence value. In some embodiments, the measured activity value (e.g., luminescence value) is compared to a control value. The methods of the invention can be used to determine potency values ​​(EC 50 To obtain the potency value, the method may be performed using at least a second, third, and / or fourth composition comprising different concentrations of a clostridial neurotoxin polypeptide, determining activity values ​​for at least the second, third, and / or fourth composition, and comparing the measured activity value (of the first composition) with the measured luciferase activity value of at least the second, third, and / or fourth composition to obtain a composition potency value (EC 50 ) may be determined.

[0167] "EC 50 " may refer to a Clostridial neurotoxin (e.g., its amount or concentration in a composition) that elicits a response halfway between the baseline and maximum after a specific exposure time. It is often used as a measure of efficacy. The EC of a graded dose-response curve 50 represents the concentration at which 50% of the maximal effect of the clostridial neurotoxin is observed.

[0168] In some examples herein, "clostridial neurotoxin polypeptide" and "light chain polypeptide" are referred to (respectively). However, this indicates that one or more clostridial neurotoxin polypeptides or light chain polypeptides (respectively) may be present when carrying out the method (e.g., in a composition). It is not intended to necessarily indicate that two or more different types of clostridial neurotoxin polypeptides or light chain polypeptides (respectively) are present, although this is encompassed. Thus, the clostridial neurotoxin polypeptide or light chain polypeptide may be one type (e.g., all clostridial neurotoxin polypeptides (respectively) are BoNT / A polypeptides, or all light chain polypeptides are BoNT / A light chain polypeptides), or multiple types (e.g., some clostridial neurotoxin polypeptides are BoNT / B polypeptides and some BoNT / A polypeptides, or some light chain polypeptides are BoNT / A light chain polypeptides and some BoNT / D light chain polypeptides, respectively). Preferably, there is one type of clostridial neurotoxin polypeptide. Preferably, there is one type of light chain polypeptide. The above applies equally to some examples herein that refer to "botulinum neurotoxin polypeptides."

[0169] The clostridial neurotoxin according to the present invention is a botulinum neurotoxin or a tetanus neurotoxin (TeNT)H. CC The clostridial neurotoxins of the present invention may comprise a BoNT / AH domain. CC Domain, BoNT / BH CC Domain, BoNT / C1H CC Domain, BoNT / DH CC Domain, BoNT / EH CC Domain, BoNT / FH CC Domain, BoNT / GH CC Domain, BoNT / XH CC Domain or TeNT / H CC Preferably, the clostridial neurotoxin of the present invention comprises a BoNT / BH domain. CC Domain or BoNT / AH CCdomain, more preferably BoNT / BH CC Includes the domain.

[0170] The clostridial neurotoxin according to the present invention is a botulinum neurotoxin or a tetanus neurotoxin (TeNT)H. C The clostridial neurotoxins of the present invention may comprise a BoNT / AH domain. C Domain, BoNT / BH C Domain, BoNT / C1H C Domain, BoNT / DH C Domain, BoNT / EH C Domain, BoNT / FH C Domain, BoNT / GH C Domain, BoNT / XH C Domain or TeNT / H C Preferably, the clostridial neurotoxin of the present invention comprises a BoNT / BH domain. C Domain or BoNT / AH C domain, more preferably BoNT / BH C Includes the domain.

[0171] The term "clostridial neurotoxin" encompasses toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F). The reference BoNT / A sequence is set forth as SEQ ID NO: 12. The reference BoNT / B sequence is set forth as SEQ ID NO: 13. The reference BoNT / C1 (also referred to herein as BoNT / C) sequence is set forth as SEQ ID NO: 41. The reference BoNT / D sequence is set forth as SEQ ID NO: 42. The reference BoNT / E sequence is set forth as SEQ ID NO: 43. The reference BoNT / F sequence is set forth as SEQ ID NO: 44. The reference BoNT / G sequence is set forth as SEQ ID NO: 45. The reference TeNT sequence is set forth as SEQ ID NO: 46. The reference BoNT / X sequence is set forth as SEQ ID NO: 47. The term "clostridial neurotoxin" may also include newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the enterococcus-encoded toxin with closest sequence identity to BoNT / X, the Weissella oryzae-encoded toxin called BoNT / Wo (NCBI Ref. Seq: WP_027699549.1) that cleaves VAMP2 at W89-90, the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1) that cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero-encoded toxin (NCBI Ref. Seq: WP_034687872.1).

[0172] Thus, the clostridial neurotoxin may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Accordingly, the compositions of the present invention may comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT. Thus, the clostridial neurotoxin polypeptide may be a BoNT / A polypeptide, a BoNT / B polypeptide, a BoNT / C polypeptide, a BoNT / D polypeptide, a BoNT / E polypeptide, a BoNT / F polypeptide, a BoNT / G polypeptide, a BoNT / X polypeptide, or a TeNT polypeptide. Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. Accordingly, the compositions of the present invention may comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X. Thus, the clostridial neurotoxin polypeptide may be a BoNT / A polypeptide, a BoNT / B polypeptide, a BoNT / C polypeptide, a BoNT / D polypeptide, a BoNT / E polypeptide, a BoNT / F polypeptide, a BoNT / G polypeptide, or a BoNT / X polypeptide.

[0173] Clostridial neurotoxins are formed from two polypeptide chains: a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal translocation component (H Ndomains). Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum in the form of large protein complexes consisting of BoNT itself complexed with numerous accessory proteins. There are currently eight different classes of botulinum neurotoxins, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, 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 this serotype classification correlates with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further classified into different subtypes based on amino acid percentage sequence identity.

[0174] BoNTs are absorbed in the gastrointestinal tract and enter the systemic circulation, where they bind to the presynaptic membrane of cholinergic nerve terminals and inhibit 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 25 kDa synaptosomal-associated protein (SNAP-25); and BoNT / C1 cleaves syntaxin. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1. Tetanus toxin is produced by a single serotype by C. tetani. C. butyricum produces BoNT / E, and C. baratii produces BoNT / F.

[0175] 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 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

[0176] For the recently identified BoNT / X, the light chain is reported to correspond to amino acids 1-439 of the molecule, and the boundary of the light chain can vary by approximately 25 amino acids (eg, 1-414 or 1-464).

[0177] The BoNT / AL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-448 of SEQ ID NO: 12. The BoNT / BL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-440 of SEQ ID NO: 13. The BoNT / C1L chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-441 of SEQ ID NO: 41. The BoNT / DL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-445 of SEQ ID NO: 42. The BoNT / EL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-422 of SEQ ID NO: 43. The BoNT / FL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-439 of SEQ ID NO: 44. The BoNT / GL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-441 of SEQ ID NO: 45. The BoNT / XL chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-439 of SEQ ID NO: 47. The BoNT / L chain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1-457 of SEQ ID NO: 46.

[0178] The above reference sequences should be considered as a guideline as slight variations may occur depending on the serotype. As an example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different Clostridial sequences: Botulinum type A neurotoxin: amino acid residues M1-K448 Botulinum type B neurotoxin: amino acid residues M1-K441 Botulinum type C1 neurotoxin: amino acid residues M1-K449 Botulinum type D neurotoxin: amino acid residues M1-R445 Botulinum type E neurotoxin: amino acid residues M1-R422 Botulinum type F neurotoxin: amino acid residues M1-K439 Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457

[0179] The translocation domain is a fragment of the H chain of a clostridial neurotoxin approximately equal to the amino-terminal half of the H chain, or the domain corresponding to that fragment within an intact H chain. C The function is H C It can be removed by deletion of the amino acid sequence (either at the level of DNA synthesis or post-synthesis by treatment with nucleases or proteases). C The function may be inactivated by chemical or biological treatment, and thus, in some embodiments, the heavy chain may not be able to bind to the binding site on the target cell to which the native clostridial neurotoxin (i.e., the holotoxin) binds.

[0180] 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)

[0181] BoNT / AH N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 449-871 of SEQ ID NO: 12. N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 441-858 of SEQ ID NO: 13. BoNT / C1H N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 442-866 of SEQ ID NO: 41. BoNT / DH N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 446-862 of SEQ ID NO: 42. BoNT / EH N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 423-845 of SEQ ID NO: 43. BoNT / FH N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 440-864 of SEQ ID NO: 44. BoNT / GH NThe domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 442-863 of SEQ ID NO: 45. BoNT / XH N The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 440-892 of SEQ ID NO: 47. N The domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 458-879 of SEQ ID NO:46.

[0182] The above reference sequences should be considered as a guideline as slight variations may occur depending on the serotype, for example US2007 / 0166332 (hereby incorporated by reference) cites slightly different Clostridial sequences. Botulinum type A neurotoxin - amino acid residues (A449-K871) Botulinum type B neurotoxin - amino acid residues (A442-S858) Botulinum type C neurotoxin - amino acid residues (T450-N866) Botulinum type D neurotoxin - amino acid residues (D446-N862) Botulinum type E neurotoxin - amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440-K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458-V879)

[0183] In the context of the present invention, various clostridial neurotoxins H containing a translocation domain N Regions that may be useful in embodiments of the present invention include H from the heavy chain of a clostridial neurotoxin. NThe region is approximately 410-430 amino acids long and contains the translocation domain. 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 Clostridial neurotoxin H that contain a translocation domain having, for example, 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 Clostridial neurotoxin H that include translocation domains having, for example, lengths of up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, and up to 425 amino acids. N It can contain regions.

[0184] For further details on the genetic basis of toxin production in C. botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0185] Term H N is a naturally occurring neurotoxin H N modified H having a moiety and a non-naturally occurring amino acid sequence and / or synthetic amino acid residues. N In one embodiment, the modified H N The moiety still exhibits the translocation function described above.

[0186] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include: 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

[0187] For the recently identified BoNT / X, H C The domain is reported to correspond to amino acids 893-1306, with the domain boundary potentially varying by approximately 25 amino acids (eg, 868-1306 or 918-306).

[0188] BoNT / AH C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 872-1296 of SEQ ID NO: 12. C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 859-1291 of SEQ ID NO: 13. BoNT / C1H C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 867-1291 of SEQ ID NO: 41. BoNT / DH C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 863-1276 of SEQ ID NO: 42. BoNT / EH C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 846-1252 of SEQ ID NO: 43. BoNT / FH C The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 865-1274 of SEQ ID NO: 44. BoNT / GH CThe domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 864-1297 of SEQ ID NO: 45. BoNT / XH C The domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 893-1306 of SEQ ID NO: 47. C The domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 880-1315 of SEQ ID NO:48.

[0189] Clostridial neurotoxin heavy chain (e.g., H C The light chain portion (domain portion) may further comprise a translocation facilitating domain (or a fragment thereof may be a translocation facilitating domain fragment), which facilitates delivery of the light chain to the cytoplasm of a target cell, and is described, for example, in WO08 / 008803 and WO08 / 008805, each of which is incorporated herein by reference.

[0190] As an example, the translocation facilitating domain is 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. Specific (non-limiting) examples include: 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)

[0191] The above sequence positions may vary slightly depending on the serotype / subtype, and the appropriate (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)

[0192] Any of the above-described facilitating domains may be conjugated to any of the aforementioned translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be conjugated to a non-clostridial translocation domain peptide or a clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The translocation facilitating domain may be conjugated to a non-clostridial translocation domain peptide. CN The facilitating domain may be linked to 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)

[0193] Naturally occurring clostridial neurotoxin H C The peptide contains approximately 400-440 amino acid residues and is divided into two functionally distinct domains of approximately 25 kDa each: the N-terminal domain (generally H CN peptide or domain) and the C-terminal region (generally H CCIt consists of a peptide or domain. 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 established, and this fact is supported by the above publications, that the clostridial heavy chain is responsible for the binding of the clostridial neurotoxins to their natural cellular receptors, i.e., the nerve endings of the neuromuscular junction. Therefore, it is clear that the clostridial heavy chain is a functional heavy chain H. C References throughout this specification to a heavy chain lacking a peptide (or domain) such that the heavy chain is unable to bind to the cell surface receptor to which native clostridial neurotoxins bind are intended to be interpreted as meaning that the clostridial heavy chain is merely a functional H CC This means that the peptide is missing. CC The peptide region may be partially or entirely deleted or otherwise modified (eg, by conventional chemical or proteolytic treatment) to reduce its natural binding ability to nerve endings at the neuromuscular junction.

[0194] H CC The reference sequence is shown below: Botulinum type A neurotoxin - amino acid residues (Y1111-L1296) Botulinum type B neurotoxin - amino acid residues (Y1098-E1291) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315).

[0195] The above reference sequences should be considered as a guideline as slight variations may occur depending on the subserotype.

[0196] BoNT / AH CC The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1111-1296 of SEQ ID NO: 12. CC The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1098-1291 of SEQ ID NO: 13. BoNT / C1H CC The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1112-1291 of SEQ ID NO: 41. BoNT / DH CC The domain can comprise a polypeptide sequence having 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1099-1276 of SEQ ID NO: 42. BoNT / EH CCThe domain can comprise a polypeptide sequence having 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1086-1252 of SEQ ID NO: 43. CC The domain can comprise a polypeptide sequence having 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1106-1274 of SEQ ID NO: 44. BoNT / GH CC The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1106-1297 of SEQ ID NO: 45. CC The domain can comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% sequence identity to amino acid residues 1128-1315 of SEQ ID NO:46.

[0197] The term "clostridial neurotoxin" is intended to encompass modified clostridial neurotoxins and derivatives thereof, including, but not limited to, those described below. A modified clostridial neurotoxin or derivative thereof may contain one or more modified amino acids compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids not present in the native (unmodified) form of the clostridial neurotoxin. By way of example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may alter the functional aspects of the toxin, such as biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the present invention is a modified clostridial neurotoxin or a modified clostridial neurotoxin derivative or a clostridial neurotoxin derivative.

[0198] Modified clostridial neurotoxins have one or more modifications in the amino acid sequence of the heavy chain (modified H Cdomain, etc.), 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 H domain alter binding to ganglioside receptors and / or protein receptors on target neurons. C These may include modifications of residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridial neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated by reference in their entirety.

[0199] Therefore, BoNT / AH CC The domain is preferably a modified BoNT / AH CC domain, more preferably a modified BoNT / AH C domain. Thus, preferably, the clostridial neurotoxin according to the present invention is a modified BoNT / A. Preferably, the modified clostridial neurotoxin comprises one or more modifications that increase the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications. Suitable modified clostridial neurotoxins are described below and in WO2015 / 004461A1 and WO2016 / 110662A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 14-17 (preferably SEQ ID NO: 14 - mrBoNT / A) as described herein.

[0200] The modified BoNT / A may include a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. Such modified BoNT / As may exhibit reduced or no side effects compared to the use of known BoNT / As, and may exhibit improved tissue retention properties, thereby improving potency and / or duration of action, providing further advantages by allowing lower doses to be used (or higher doses without additional adverse effects) compared to known clostridial toxin therapeutics.

[0201] The modification may be a modification relative to the BoNT / A set forth as SEQ ID NO: 12, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO: 12. Because the presence of a methionine residue at position 1 in SEQ ID NO: 12 (as well as SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, one of skill in the art will consider the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO: 12 contains a methionine, the numbering of that position is as defined above (e.g., ASN886 is ASN886 in SEQ ID NO: 12). Alternatively, if a methionine is not present in SEQ ID NO: 12, the numbering of the amino acid residue should be adjusted by -1 (e.g., ASN886 becomes ASN885 in SEQ ID NO: 12). Similar considerations apply to the presence / absence of a methionine at position 1 in the other polypeptide sequences described herein, and one of skill in the art will readily determine the correct numbering of the amino acid residues using techniques routinely employed in the art.

[0202] Alignments for determining amino acid residue numbering as described herein may be performed using any of the methods described herein for determining sequence homology and / or percent sequence identity.

[0203] The amino acid residues indicated as the target for modification above are surface-exposed amino acid residues.

[0204] The modified BoNT / A may include a modification at one or more amino acid residues selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0205] The term "one or more amino acid residues" as used in the context of a modified BoNT / A preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residues. Thus, a modified BoNT / A may contain modifications at at least 2, 3, 4, 5, 6, or 7 (preferably 7) of the indicated amino acid residues. A modified BoNT / A may contain 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residues" as used in the context of a modified BoNT / A means all of the indicated amino acid residues.

[0206] Preferably, other than one or more amino acid modifications at the indicated amino acid residues, the modified BoNT / A contains no further amino acid modifications when compared to SEQ ID NO:12.

[0207] The modifications are selected from the following: i. Substitution of acidic surface-exposed amino acid residues with basic amino acid residues; ii. Substitution of acidic surface-exposed amino acid residues with uncharged amino acid residues; iii. Substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. Deletion of acidic surface-exposed amino acid residues.

[0208] The above modifications result in modified BoNT / As with increased surface positive charge and elevated isoelectric points compared to the corresponding unmodified BoNT / As (e.g., SEQ ID NO: 12). Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding of the polypeptide to the cell surface and increasing retention at the site of administration and / or duration of action.

[0209] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are composed of specific sequences of amino acids (also called amino acid residues within the protein). Each amino acid in the standard set of 20 has a different side chain (or R group), meaning that each amino acid residue within a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be affected by the surrounding chemical environment, such as temperature and pH. The overall chemical properties of a protein are determined by the sum of these various factors.

[0210] Certain amino acid residues (more on this below) have ionizable side chains that can acquire a charge depending on the pH of their surroundings. Whether such a side chain is charged at a given pH depends on the pKa of the associated ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) of a particular proton from the conjugate base.

[0211] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value can depend on temperature, ionic strength, and the microenvironment of the ionizable group). Thus, these side chains exhibit a negative charge at pH 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains become protonated and lose their charge.

[0212] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chains with pKa values ​​of approximately 10–12. Therefore, these side chains exhibit a positive charge at pH 7.4. At higher pH values, these side chains become deprotonated and lose their charge.

[0213] Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their surface exposure) and the surrounding pH. A change in the surrounding pH will change the overall charge of the protein. Therefore, for every protein, there is a certain pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry that is well known to those skilled in the art.

[0214] Therefore, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a zero net charge. An increase in pI means that a higher pH value is required for the protein to exhibit a zero net charge. Therefore, an increase in pI represents an increase in the net charge of a protein at a given pH. Conversely, a decrease in pI means that a lower pH value is required for the protein to exhibit a zero net charge. Therefore, a decrease in pI represents a decrease in the net charge of a protein at a given pH.

[0215] Methods for determining the pI of a protein are known in the art and are familiar to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations can be performed using computer programs known in the art, such as the Compute pI / MW Tool in ExPASy (https: / / web.expasy.org / compute_pi / ), which is a preferred method for calculating pI according to the present invention. Comparisons of pI values ​​between different molecules should be performed using the same calculation method / program.

[0216] Where appropriate, the calculated pI of a protein can be confirmed experimentally using the technique of isoelectric focusing ("observed pI"). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which the net charge is zero, which is the protein's pI. Because the results obtained by isoelectric focusing are typically relatively low resolution in nature, the inventors believe that results obtained by calculated pI (as described above) are more appropriate to use.

[0217] Throughout this specification, unless otherwise specified, "pI" means "calculated pI."

[0218] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups exposed on its surface. This can be achieved by altering one or more amino acids of the protein. For example, decreasing the number of acidic residues or increasing the number of basic residues can increase the pI.

[0219] A modified BoNT / A of the present invention may have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than BoNT / A (e.g., SEQ ID NO: 12). Preferably, the modified BoNT / A may have a pI of at least 6.6, such as at least 6.8.

[0220] The properties of the 20 common amino acids are shown in the table below: [Table B]

[0221] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0222] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0223] The following amino acids are considered uncharged polar (meaning they can participate in hydrogen bonds) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0224] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0225] In an amino acid insertion, an additional amino acid residue (one not normally present) is incorporated into a BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from a Clostridial toxin amino acid sequence, thereby decreasing the total number of amino acid residues in said sequence.

[0226] Preferably, the modification is a substitution, advantageously retaining the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The substituted amino acid residue may be one of the 20 standard amino acids, as described above. Alternatively, the substituted amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the 20 standard set described above). By way of example, the substituted amino acid may be a basic non-standard amino acid such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an E. coli auxotrophic expression host.

[0227] In one embodiment, the substitution is selected from the following: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment where the substitution is substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is substituted with the corresponding uncharged amide amino acid residue (i.e., aspartic acid is substituted with asparagine, glutamic acid is substituted with glutamine).

[0228] Preferably, the basic amino acid residue is a lysine or arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0229] After modification according to the present invention, the modified BoNT / A is preferably capable of binding to the target cell receptor to which unmodified BoNT / A (eg, SEQ ID NO: 12) binds.

[0230] Preferably, the modified BoNT / A for use in the present invention is a clostridial toxin H CN The modified BoNT / A preferably contains 4 to 40 amino acid modifications located in the nucleotide sequence of ...

[0231] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. As an example, amino acid modifications may be introduced by modifying a DNA sequence encoding a polypeptide (e.g., encoding unmodified BoNT / A or a fragment thereof). This can be achieved by standard molecular cloning techniques, such as site-directed mutagenesis, in which short DNA strands (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence with a polymerase enzyme, or by inserting / deleting portions of a gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized.

[0232] The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOS: 14-17. In one embodiment, the modified BoNT / A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOS: 14-17. Preferably, the modified BoNT / A may comprise any one of SEQ ID NOS: 14-17. The modified BoNT / A may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOS: 14-17. In one embodiment, the modified BoNT / A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOS: 14-17. Preferably, the modified BoNT / A may consist of any one of SEQ ID NOS: 14-17. Of the listed SEQ ID NOS, SEQ ID NOS: 14 is most preferred. One skilled in the art will understand that when the polypeptide sequence of a modified BoNT / A changes in percent sequence identity compared to a given sequence number, at least one modification (e.g., one that increases pI) will still be present (e.g., unmodified) in the variant of the modified BoNT / A.

[0233] Thus, the compositions of the present invention preferably comprise a modified BoNT / A, such as those described above. In some embodiments, the clostridial neurotoxin polypeptide is a modified BoNT / A polypeptide, the L chain is a BoNT / AL chain, and the H chain is a modified BoNT / A polypeptide. N The domain is BoNT / AH N domain, H CC Domain (e.g., H C domain) is a modified BoNT / AH CC domain (e.g., modified BoNT / AH CC domain).

[0234] The modified BoNT / A can be encoded by a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 49. In one embodiment, the modified BoNT / A can be encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 49. Preferably, the modified BoNT / A can be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 49. One of skill in the art will understand that when the nucleotide sequence encoding a modified BoNT / A varies by percent sequence identity compared to a given SEQ ID NO, the encoded modified BoNT / A still comprises at least one modification (e.g., one that increases pI), and therefore the relevant region of the nucleotide sequence encoding the at least one modification will still be present (e.g., unmodified) in the variant nucleotide sequence.

[0235] The modified BoNT / A can include substitutions at one or more (preferably, two or more, three or more, four or more, five or more, or six or more, more preferably all) of positions 930, 955, 991, 1026, 1052, 1229, and 886. Preferably, the modified BoNT / A includes a lysine or arginine (more preferably, a lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the modified BoNT / A includes a lysine or arginine (more preferably, a lysine) at at least two, three, four, five, six, or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the modified BoNT / A contains lysine or arginine (more preferably lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0236] A clostridial neurotoxin may comprise (or consist of) a hybrid or chimeric clostridial neurotoxin. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may comprise the entire light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may comprise a portion of the heavy chain (e.g., the binding domain) of one clostridial neurotoxin subtype, with another portion of the heavy chain from another clostridial neurotoxin subtype. Similarly or alternatively, a therapeutic element may comprise light chain subgroups from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic effects of such clostridial neurotoxins to subjects who are immunologically tolerant to a given clostridial neurotoxin subtype, who may have a lower-than-average receptor concentration for a given clostridial neurotoxin heavy chain binding domain, or who may have protease-resistant mutants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Pat. No. 8,071,110, which is incorporated herein by reference in its entirety.

[0237] The clostridial neurotoxins of the present invention are BoNT / BH CC domains (e.g., BoNT / BH CC The neurotoxin may be a chimeric clostridial neurotoxin containing a BoNT / BH domain, preferably a BoNT / BH C Thus, in a particularly preferred embodiment, the clostridial neurotoxin of the present invention is a chimeric clostridial neurotoxin comprising a BoNT / A light chain and a translocation domain (LH N domain), and BoNT / B receptor binding domain (H CThe BoNT / BH may be a chimeric Clostridial neurotoxin comprising (preferably consisting of) the BoNT / BH domain. C The domain contains the following substitutions: E1191M and S1199Y. Suitable chimeric Clostridial neurotoxins may be those taught in WO2017 / 191315A1, incorporated herein by reference. Preferred such sequences include SEQ ID NOs: 7-11, with SEQ ID NO: 7 being most preferred.

[0238] BoNT / A LH N The domain is BoNT / BH C The chimeric BoNT / A may be covalently linked to the domain. The chimeric BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0239] LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N Domain and H C Separating domains and 10 It may correspond to the first amino acid residue of the helix, H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N Domain and H C Separating domains and 10 It may correspond to the second amino acid residue of the helix.

[0240] As used herein, "BoNT / A LH N Domain and H C Separating domains and 10 The reference to the "first amino acid residue of the helix" is N Domain and H C Separating domains and 10 It refers to the N-terminal residue of the helix.

[0241] As used herein, "BoNT / B LH" N Domain and H C Separating domains and 10 The reference to the "second amino acid residue of the helix" isN Domain and H C Separating domains and 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0242] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with alpha helices, beta sheets, and reverse turns. 3 10 The amino acids in the helix are arranged in a right-handed helix, with each full turn completed by three residues and 10 atoms separating the intramolecular hydrogen bonds between them. A 120° rotation in the helix (i.e., the helix has three residues per turn) corresponds to a translation of 2.0 Å (= 0.2 nm) along the helix axis, resulting in 10 atoms in the ring formed by the hydrogen bonds. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it; this repeated i+3→i hydrogen bond results in 3 10 Define a helix. 3 10 A helix is ​​a standard concept in structural biology that is familiar to those skilled in the art.

[0243] These three 10 A helix corresponds to four residues that form the actual helix, plus two cap (or transition) residues, one at each end of these four residues. As used herein, "LH" refers to a helix that is formed by a helix. N Domain and H C Separating Domains 3 10 The term "helix" consists of those six residues.

[0244] Through structural analysis and sequence alignment, LH N Domain and H C Separating Domains 3 10 The helix was identified. 10 The helix is ​​located at its N-terminus (i.e., the LH N The C-terminal part of the domain is surrounded by an α-helix, and the C-terminal (i.e., the H C The N-terminal part of the domain is surrounded by β-strands. 10The first (N-terminal) residue of the helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0245] LH N Domain and H C Separating Domains 3 10 The helices can be determined, for example, from the published crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0246] The LH sequences of other neurotoxins were identified using publicly available in silico modeling and alignment tools, such as the homology modeling servers LOOPP (Learn, Observe, and Output Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / Similarity Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program ClustalOmega (http: / / www.clustal.org / omega / ), and many other tools / services listed in the Internet Resources for Molecular and Cellular Biologists (http: / / molbiol-tools.ca / ). N Domain and H C Separating Domains 3 10 It is also possible to determine the position of helices. N / H CN The highly conserved structure of the perijunctional region makes it an ideal region for overlapping of different serotypes.

[0247] For example, the following method can be used to identify this 3 10 The sequence of the helix may be determined: 1. Obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb) using the structural homology modeling tool LOOP (http: / / loopp.org); 2. Copy the structural (pdb) file obtained in this way to H CN The N-terminus of the domain and the preceding (H N By editing the protein to contain only about 80 residues (part of the H domain), the highly structurally conserved H N / H CN ” Maintaining territory; 3. Superimpose each serotype onto the corresponding 3BTA.pdb structure using the protein superposition server SuperPose ( http: / / wishart.biology.ualberta.ca / superpose / ); 4. Examine the overlapping pdb files to find the H of the BoNT / A1. N The domain begins with the 3 10 positioning the helices and identifying corresponding residues in other serotypes; 5. Other BoNT serum sequences are aligned in ClustalOmega to ensure corresponding residues are correct.

[0248] LH determined by this method N , H C and 3 10 An example of a helical domain is shown below: [Table C]

[0249] Using structural analysis and sequence alignment, LH N Domain and H C Separating Domains 3 10 The beta strand following the helix is ​​a conserved structure in all botulinum neurotoxins and tetanus neurotoxins, and LHN Domain and H C Separating Domains 3 10 It was found to begin at the eighth residue when starting from the first residue of the helix (e.g., BoNT / A1 residue 879).

[0250] BoNT / AB chimera is a BoNT / B-derived H C LH from BoNT / A covalently bound to the domain N It may contain a domain, Here, the LH N The C-terminal amino acid residue of the domain is the H C It corresponds to the eighth amino acid residue from the N-terminus of the β chain located at the beginning (N-terminus) of the domain, and C The N-terminal amino acid residue of the domain is the H C It corresponds to the seventh amino acid residue from the N-terminus of the β-chain located at the beginning (N-terminus) of the domain.

[0251] BoNT / AB chimera is a BoNT / B-derived H C LH from BoNT / A covalently bound to the domain N It may contain a domain, Here, the LH N The C-terminal amino acid residue of the domain is the same as that of the LH of BoNT / A. N The C-terminal amino acid residue of the α-helix is ​​located at the end (C-terminus) of the domain. C The N-terminal amino acid residue of the domain is the same as that of the LH of BoNT / B. N It corresponds to the amino acid residue that is adjacent to the C-terminal amino acid residue of the α-helix at the end (C-terminus) of the domain.

[0252] The rationale for the design process of the BoNT / AB chimera is to ensure that the secondary structure is intact, thereby attempting to minimize any changes to the tertiary structure. 10It is hypothesized that not disrupting the four central amino acid residues of the helix ensures an optimal conformation for the chimeric neurotoxin.

[0253] BoNT / A-derived LH N The domain may be a polypeptide sequence corresponding to amino acid residues 1 to 872 of SEQ ID NO: 12, or having at least 70% sequence identity thereto. N The domain may be a polypeptide sequence corresponding to amino acid residues 1 to 872 of SEQ ID NO: 12, or having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the LH domain is derived from BoNT / A. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:12.

[0254] BoNT / B-derived H C The domain may be a polypeptide sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 13 or having at least 70% sequence identity thereto. C The domain may be a polypeptide sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 13, or having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / B C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:13.

[0255] Preferably, LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 12), and C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 13).

[0256] Preferably, BoNT / BH C The H domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CCIt further includes substitution, addition, or deletion of at least one amino acid residue in a domain (e.g., a subdomain). CC Suitable amino acid residue substitutions, additions or deletions in the domains are disclosed in WO2013 / 180799 and WO2016 / 154534 (both of which are incorporated herein by reference).

[0257] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in a domain include substitution mutations selected from the group consisting of: V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0258] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in a domain further include combinations of two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0259] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the domain also include a combination of the three substitution mutations E1191M, S1199W, and W1178Q.

[0260] Preferably, BoNT / BHCC Suitable amino acid residue substitutions, additions, or deletions in the domain include a combination of two substitution mutations: E1191M and S1199Y.

[0261] The modification may be a modification relative to the unmodified BoNT / B set forth as SEQ ID NO: 13, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO: 13. Because the presence of a methionine residue at position 1 of SEQ ID NO: 13 is optional, one of skill in the art would take the presence / absence of the methionine residue into consideration when determining the numbering of the amino acid residues. For example, if SEQ ID NO: 13 contains a methionine, the numbering of that position would be as defined above (e.g., E1191 would become E1191 in SEQ ID NO: 13). Alternatively, if a methionine is not present in SEQ ID NO: 13, the numbering of that amino acid residue should be corrected by −1 (e.g., E1191 would become E1190 in SEQ ID NO: 13). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art would readily determine the correct numbering of the amino acid residues using routine techniques in the art.

[0262] A chimeric Clostridial neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOS: 7-11. In one embodiment, a chimeric Clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOS: 7-11. Preferably, a chimeric Clostridial neurotoxin may comprise any one of SEQ ID NOS: 7-11. A chimeric Clostridial neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOS: 7-11. In one embodiment, a chimeric Clostridial neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOS: 7-11. Preferably, a chimeric Clostridial neurotoxin may consist of any one of SEQ ID NOS: 7-11. Of the listed SEQ ID NOS, SEQ ID NOS: 7 is most preferred. Those skilled in the art will recognize that at least one BoNT / BH CC If the polypeptide sequence of the chimeric Clostridial neurotoxin containing the domain mutation changes compared to a given SEQ ID NO in terms of percent sequence identity, then at least one BoNT / BH CC It will be understood that the domain mutations (preferably E1191M and S1199Y) are present (eg, unmodified) in the chimeric Clostridial neurotoxin variant.

[0263] Thus, the compositions of the present invention most preferably comprise a chimeric clostridial neurotoxin, such as those described above. In some embodiments, the clostridial neurotoxin polypeptide is a chimeric clostridial neurotoxin polypeptide, the L chain is a BoNT / AL chain, and the H N The domain is BoNT / AH N domain, H CC Domain (e.g. H C Domain) is BoNT / BH CC domain (e.g. BoNT / BH CC domain).

[0264] In another embodiment, the clostridial neurotoxin of the present invention comprises a BoNT / X light chain and a translocation domain (LH N domain), and receptor binding domain (H C domain), or chimeric clostridial neurotoxins comprising portions thereof from different (i.e., non-BoNT / X) clostridial neurotoxins. Suitable chimeric and / or hybrid clostridial neurotoxins may be those taught in WO2020 / 065336A1, which is incorporated herein by reference.

[0265] In embodiments in which the clostridial neurotoxins described herein have a tag (eg, a His tag) and / or a linker for purification, the tag and / or linker are optional.

[0266] The clostridial neurotoxins of the present invention may not include complex proteins present in a naturally occurring clostridial neurotoxin complex.

[0267] The clostridial neurotoxins of the present invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, the clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin.

[0268] In one embodiment, a nucleic acid (e.g., DNA) is provided that comprises a nucleic acid sequence encoding a Clostridial neurotoxin. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0269] In a preferred embodiment, the vector has a promoter selected from the following: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002-0.4%) T7-lac operator / IPTG / 0.2 mM (0.05-2.0 mM)

[0270] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002-0.4%) T7-lac operator / IPTG / 0.2 mM (0.05-2.0 mM) T5-lac operator / IPTG / 0.2 mM (0.05-2.0 mM)

[0271] The nucleic acid molecules may be produced using any suitable procedure known in the art. Thus, the nucleic acid molecules may be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention may be produced using molecular biology techniques.

[0272] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0273] The above-mentioned nucleic acid sequence information is optionally modified to account for codon bias in the expression system of the final host cell (eg, E. coli) used.

[0274] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably herein. Preferably, the nucleotide sequence is a DNA sequence.

[0275] The clostridial neurotoxins of the present invention preferably have an L chain connected to an H chain (or a component thereof, e.g., H) via a disulfide bond. NThe clostridial neurotoxin of the present invention exists as a di-chain clostridial neurotoxin linked to a protease (domain). Thus, the clostridial neurotoxin of the present invention may be a clostridial neurotoxin or variant thereof (expressed by percent sequence identity to a given SEQ ID NO:) described herein that has been cleaved by a protease at its activation loop (or sites).

[0276] The clostridial neurotoxin preferably comprises an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and can be obtained by (e.g., obtained by) cleaving a polypeptide having at least 70% sequence identity to SEQ ID NO: 14 with a protease at one or more sites within its activation loop. In one embodiment, the clostridial neurotoxin comprises an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and can be obtained by (e.g., obtained by) cleaving a polypeptide having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 14 with a protease at one or more sites within its activation loop. Preferably, the clostridial neurotoxin comprises an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and can be obtained by (e.g., obtained by) cleaving a polypeptide comprising SEQ ID NO: 14 with a protease at one or more sites within its activation loop.

[0277] The clostridial neurotoxin is most preferably obtainable (e.g., obtained by) cleaving a polypeptide comprising an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and having at least 70% sequence identity to SEQ ID NO: 7, at one or more sites within its activation loop with a protease. In one embodiment, the clostridial neurotoxin is obtainable (e.g., obtained by) cleaving a polypeptide comprising an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 7, at one or more sites within its activation loop with a protease. Preferably, the clostridial neurotoxin is obtainable (e.g., obtained by) cleaving a polypeptide comprising an L chain and an H chain, the L chain and the H chain being linked by a disulfide bond, and comprising SEQ ID NO: 7, at one or more sites within its activation loop with a protease.

[0278] In particularly preferred embodiments, the di-chain clostridial neurotoxin comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 77 or 78 (preferably SEQ ID NO: 77) and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 79, wherein the light and heavy chains are linked by a disulfide bond. More preferably, the di-chain clostridial neurotoxin comprises (or consists of) a light chain comprising SEQ ID NO: 77 or 78 (preferably SEQ ID NO: 77) and a heavy chain comprising SEQ ID NO: 79, wherein the light and heavy chains are linked by a disulfide bond. Even more preferably, the di-chain clostridial neurotoxin comprises (or consists of) a light chain having SEQ ID NO: 77 and a heavy chain having SEQ ID NO: 79, wherein the light and heavy chains are linked by a disulfide bond. The disulfide bond is preferably formed by and / or between the cysteine ​​residue at position 429 of SEQ ID NO: 77 or 78 and the cysteine ​​residue at position 6 of SEQ ID NO: 79. The di-chain clostridial neurotoxin may correspond to the di-chain form of SEQ ID NO: 7.

[0279] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to generate dichain clostridial neurotoxins are taught in WO2014 / 080206, WO2014 / 079495, and EP2677029A2, which are incorporated herein by reference.

[0280] Suitable activation loop sequences are shown in the following table: [Table D]

[0281] Lys-C may cleave the C-terminus of the activation loop at one or more lysine residues contained therein. Those skilled in the art will understand that if Lys-C cleaves the activation loop multiple times, small peptides in the activation loop of the dichain clostridial neurotoxin may not be present when compared to the SEQ ID NOs set forth herein. For example, SEQ ID NOs: 75 and 76 may not be present.

[0282] The present invention provides a method for producing a single-chain clostridial neurotoxin having a light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cells to provide a host cell homogenate comprising a single-chain clostridial neurotoxin, and isolating the single-chain clostridial neurotoxin. In one aspect, the present invention provides a method for proteolytically processing a clostridial neurotoxin described herein, the method comprising contacting the clostridial neurotoxin with a protease that hydrolyzes a peptide bond within the activation loop of the clostridial neurotoxin, thereby converting the (single-chain) clostridial neurotoxin into the corresponding two-chain clostridial neurotoxin (e.g., the light chain and heavy chain are linked by a disulfide bond).

[0283] Thus, the present invention provides a di-chain clostridial neurotoxin obtainable by the method of the present invention.

[0284] Where an initial methionine amino acid residue or corresponding initial codon is shown in any of the SEQ ID NOs disclosed herein, said residue / codon is optional. Preferably, said initial methionine amino acid residue or corresponding initial codon is not present.

[0285] The composition used in the methods of the invention may be a first clostridial neurotoxin formulation that includes one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

[0286] The method may further include determining the clostridial neurotoxin activity of at least a second clostridial neurotoxin formulation, wherein the at least second clostridial neurotoxin formulation comprises the same clostridial neurotoxin present in the same amount as the first clostridial neurotoxin formulation and one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts, wherein the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts are different from the one or more pharmaceutically acceptable carriers, excipients, adjuvants, adjuvants, and / or salts present in the first clostridial neurotoxin formulation.

[0287] The method can include comparing the clostridial neurotoxin activity of a first clostridial neurotoxin formulation with the clostridial neurotoxin activity of at least a second clostridial neurotoxin formulation, and selecting one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts if the clostridial neurotoxin formulation containing them exhibits the highest activity.

[0288] The above methods may enable one to select a preferred pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt for formulating a Clostridial neurotoxin.

[0289] The method may further include determining the clostridial neurotoxin activity of at least a second clostridial neurotoxin formulation, wherein the at least a second clostridial neurotoxin formulation comprises the same clostridial neurotoxin present in the same amount as the first clostridial neurotoxin formulation, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts, wherein the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts are the same as but present in different amounts (e.g., different concentrations) than the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts present in the first clostridial neurotoxin formulation.

[0290] The method may include comparing the clostridial neurotoxin activity of a first clostridial neurotoxin formulation with the clostridial neurotoxin activity of at least a second clostridial neurotoxin formulation, and selecting amounts of one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts when the clostridial neurotoxin formulation containing them exhibits the highest activity.

[0291] The above methods may enable one to select preferred amounts of pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts for formulating a Clostridial neurotoxin.

[0292] In one aspect, the present invention provides a method for producing a clostridial neurotoxin composition for therapeutic or cosmetic use, the method comprising: (a) obtaining the results of the methods described in the present invention and, if the clostridial neurotoxin activity (e.g., activity level) is the same as or higher than a positive control, formulating and / or packaging the composition for therapeutic or cosmetic use; or (b) if the activity of the clostridial neurotoxin is less than that of a positive control, further purifying the composition and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

[0293] In one aspect, a therapeutic or cosmetic Clostridial neurotoxin composition obtainable by the method of the present invention is provided, optionally wherein the therapeutic or cosmetic Clostridial neurotoxin composition is packaged.

[0294] As used herein, the term "obtainable" also encompasses the term "obtained."

[0295] In one aspect, the present invention provides an isolated capture substrate for a clostridial neurotoxin, wherein the capture substrate comprises an extracellular portion of a clostridial neurotoxin receptor polypeptide that includes an amino acid modification and / or a post-translational modification. The isolated capture substrate may form a complex with a clostridial neurotoxin, wherein the H of the clostridial neurotoxin isCC Domain (e.g. H C The isolated capture substrate contains a Clostridial neurotoxin receptor-binding domain (H domain) bound to the extracellular portion of the Clostridial neurotoxin receptor polypeptide. CC Domain or H C The isolated capture substrate may form a complex with the SYT-II domain. Preferably, the isolated capture substrate comprises the extracellular portion of SYT-II comprising the L51F substitution described herein. Alternatively, the isolated capture substrate may comprise the extracellular portion of SV2c comprising a post-translational modification (preferably glycosylation) described herein. An isolated capture substrate may refer to a capture substrate isolated from a cell. Such a capture substrate may be recombinantly produced and isolated using standard techniques. Thus, in some embodiments, the term "isolated capture substrate" is intended to encompass a capture substrate in an in vitro environment. Preferably, the isolated capture substrate is immobilized on a solid support, as described herein for capture substrates in general.

[0296] In one aspect, the present invention provides the use of an isolated capture substrate for a clostridial neurotoxin to determine the presence or absence of an activity altering characteristic of a clostridial neurotoxin polypeptide contained in a composition, wherein the isolated capture substrate comprises an extracellular portion of a clostridial neurotoxin receptor polypeptide that includes an amino acid modification and / or a post-translational modification.

[0297] The activity-altering trait may be an activity-increasing trait or an activity-decreasing trait, preferably an activity-decreasing trait. In one embodiment, the activity-altering trait may be any trait that alters the activity of a clostridial neurotoxin polypeptide when compared to an otherwise identical clostridial neurotoxin (preferably an otherwise identical active bichain clostridial neurotoxin) that lacks the trait. In one embodiment, the activity-altering trait is an activity-altering trait that alters the activity of a portion of a clostridial neurotoxin (e.g., a light chain or heavy chain, or H chain) that lacks the trait when compared to an otherwise identical portion of a clostridial neurotoxin that lacks the trait. C or HCC The activity-altering property may be any property that alters the activity of a Clostridial neurotoxin polypeptide (e.g., a portion thereof, such as a domain). Thus, the activity-altering property may be a topological change and / or a structural change. The activity-altering property may be a modification (e.g., a post-translational modification), such as a covalent modification and / or a non-covalent modification. In one embodiment, the activity-altering property is deamidation, fragmentation (e.g., truncation of the Clostridial neurotoxin polypeptide, e.g., resulting in loss of a domain), aggregation, oxidation (e.g., oxidation of a methionine residue or a tryptophan), glycation (e.g., glycation of a lysine residue), lactosylation, incorrect disulfide bond formation (e.g., resulting in loss of a light chain or loss of a heavy chain), incorrect charge (e.g., incorrect charge profile), and / or an intact activation loop (e.g., the composition may be contaminated with a single-chain Clostridial neurotoxin polypeptide). Such an activity-altering property is preferably an activity-reducing property. Preferably, the activity-reducing property is oxidation.

[0298] An example of an activity-altering property of an L chain may include an intact activation loop resulting in an inactive L chain polypeptide, which may occur if proteolytic cleavage of a single-chain clostridial neurotoxin into an active two-chain form is unsuccessful and / or inefficient. Other activity-altering properties of an L chain include topological changes, conformational changes, deamidation, fragmentation, aggregation, oxidation (e.g., oxidation of methionine residues or tryptophan), glycation (e.g., glycation of lysine residues), lactosylation, incorrect disulfide bond formation, and / or incorrect charge (e.g., incorrect charge profile).

[0299] H chain (e.g., H C Examples of activity-altering properties of a polypeptide (e.g., a polypeptide domain) may include a topological change, a conformational change, deamidation, fragmentation, aggregation, oxidation (e.g., oxidation of methionine residues or tryptophan), glycation (e.g., glycation of lysine residues), lactosylation, and / or an incorrect charge (e.g., an incorrect charge profile).

[0300] Amino acids that can be oxidized include methionine, cysteine, histidine, tryptophan, tyrosine, and / or phenylalanine (Torosantucci et al (2014), Pharm Res, 31, 541-553).

[0301] The activity-altering property may be a clostridial neurotoxin (or a portion thereof, e.g., a light chain or a heavy chain, or H C or H CC The activity of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property can be compared with that of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property. C or H CC The activity-increasing property may be a property that changes the activity of a Clostridial neurotoxin polypeptide (or a portion thereof, e.g., a light chain or heavy chain, or H chain) by at least 1%, 2%, 5%, 10%, 25%, 50%, or 100% when compared to the activity of a Clostridial neurotoxin polypeptide (or a portion thereof, e.g., a light chain or heavy chain, or H chain). C or H CC The activity of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property can be compared with that of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property. C or H CC The activity-reducing property may be at least 1%, 2%, 5%, 10%, 25%, 50%, or 100% increased when compared to a Clostridial neurotoxin polypeptide (or a portion thereof, such as a light chain or heavy chain, or H chain). C or H CC The activity of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property can be compared with that of an otherwise identical Clostridial neurotoxin (or a portion thereof, e.g., an L chain or H chain, or H domain) lacking said property. C or H CC The property may be a property that reduces the number of times ...

[0302] The activity-altering property is preferably an activity-altering property of the H chain (e.g., an activity-altering modification of the H chain), and more preferably an H C H, such as activity-reducing modifications present in the domain CThe activity-reducing properties of the domain.

[0303] The inventors have discovered that by combining the methods of the present invention with one or more additional methods, valuable insight into the properties of clostridial neurotoxin compositions can be gained (see, e.g., Example 3). This allows the quality and / or efficacy of such compositions to be examined in a domain-specific manner. Advantageously, this is a significant improvement over cell-based assays, which cannot provide mechanistic insight into the specific causes of potency variation.

[0304] Thus, in one embodiment, the method of the present invention may further comprise obtaining results of a cell-free substrate cleavage assay. In one embodiment, the method of the present invention may further comprise obtaining results of a heavy chain binding assay. Preferably, the method of the present invention may further comprise obtaining results of a cell-free substrate cleavage assay and results of a heavy chain binding assay.

[0305] In one embodiment, the results may be obtained prior to carrying out the methods of the invention. In some embodiments, a negative result, i.e., a result lower than the control as determined by the cell-free substrate cleavage assay and / or heavy chain binding assay, may require carrying out the methods of the invention.

[0306] The results of the cell-free substrate cleavage assay (e.g., endopeptidase assay) and / or the results of the heavy chain binding assay may be compared with the results of the present invention (e.g., the determined clostridial neurotoxin activity of the composition). The comparison may determine whether the clostridial neurotoxin polypeptide (or a portion thereof, e.g., the light chain or heavy chain, or the heavy chain) contained in the composition is a clostridial neurotoxin polypeptide. C or H CC The comparison may allow a determination as to whether a Clostridial neurotoxin polypeptide (or a portion thereof, e.g., a light chain or heavy chain, or H chain) contained in the composition contains an activity-changing characteristic. C or H CCPreferably, the results of the cell-free substrate cleavage assay and the results of the heavy chain binding assay can be compared with the results of the present invention (e.g., the determined clostridial neurotoxin activity of the composition).

[0307] The heavy chain binding control and / or (preferably and) substrate cleavage assay control may be a negative control. The heavy chain binding control and / or (preferably and) substrate cleavage assay control is preferably a positive control. Suitable positive controls are described herein. The heavy chain binding positive control is a control that measures the activity-altering properties of the clostridial neurotoxin H chain, preferably H. C Preferably, the positive control for substrate cleavage represents a composition comprising a Clostridial neurotoxin polypeptide that does not contain the activity modifier characteristic of the L chain.

[0308] If the results of the heavy chain binding assay show that the heavy chain binding of the composition is weaker compared to the heavy chain binding positive control, the results of the cell-free substrate cleavage assay show that the L chain activity of the composition is the same compared to the substrate cleavage positive control, and the results of the method of the present invention show that the clostridial neurotoxin activity of the composition is lower compared to a positive control (e.g., a positive standard sample), then the clostridial neurotoxin polypeptide contained in the composition is a heavy chain, preferably an H chain. C It may be determined that the domain contains an activity-reducing property.

[0309] If the results of the heavy chain binding assay indicate that the composition has stronger heavy chain binding compared to the heavy chain binding positive control, the results of the cell-free substrate cleavage assay indicate that the composition has the same L chain activity compared to the substrate cleavage positive control, and the results of the method of the present invention indicate that the composition has higher clostridial neurotoxin activity compared to a positive control (e.g., a positive standard sample), then the clostridial neurotoxin polypeptide contained in the composition is a heavy chain, preferably an H chain. C It may be determined that the domain contains an activity-increasing property.

[0310] If the results of the heavy chain binding assay show that the heavy chain binding of the composition is the same as that of the heavy chain binding positive control, the results of the cell-free substrate cleavage assay show that the L chain activity of the composition is lower as compared to the substrate cleavage positive control, and the results of the method of the present invention show that the clostridial neurotoxin activity of the composition is lower as compared to a positive control (e.g., a positive standard sample), then the clostridial neurotoxin polypeptide contained in the composition can be determined to contain the L chain activity-reducing property.

[0311] If the results of the heavy chain binding assay indicate that the heavy chain binding of the composition is the same as that of the heavy chain binding positive control, the results of the cell-free substrate cleavage assay indicate that the L chain activity of the composition is higher as compared to the substrate cleavage positive control, and the results of the method of the present invention indicate that the clostridial neurotoxin activity of the composition is higher as compared to a positive control (e.g., a positive standard sample), it can be determined that the clostridial neurotoxin polypeptide contained in the composition contains an L chain activity-increasing property.

[0312] One skilled in the art will understand that any difference can be quantified to determine the amount of polypeptide in the composition that contains a given activity altering profile.

[0313] Heavy chain binding assays include: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) contacting the capture substrate with a composition comprising the clostridial neurotoxin polypeptide to bind the clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; and (d) determining the amount of Clostridial neurotoxin polypeptide bound to the capture substrate.

[0314] Thus, in one aspect, the present invention provides a (cell-free) method for assaying heavy chain binding of a Clostridial neurotoxin polypeptide, said method comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) combining the capture substrate with the clostridial neurotoxin polypeptide or at least the H of the clostridial neurotoxin polypeptide. CC or H C contacting the Clostridial neurotoxin polypeptide or portion thereof with a composition comprising a domain, thereby binding the Clostridial neurotoxin polypeptide or portion thereof to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides or portions thereof; and (d) determining the amount of Clostridial neurotoxin polypeptide or portion thereof bound to the capture substrate.

[0315] The amount of clostridial neurotoxin polypeptide (or a portion thereof) bound to the capture substrate may be determined using any suitable method. For example, an antibody (suitably a polyclonal antibody) that specifically binds to the clostridial neurotoxin polypeptide (or a portion thereof) may be used. The antibody may be detected using a secondary antibody that specifically binds to the antibody (suitably a polyclonal antibody) that specifically binds to the clostridial neurotoxin polypeptide (or a portion thereof). The secondary antibody may include a suitable detection means, such as a conjugated peroxidase enzyme (e.g., horseradish peroxidase) that can be detected by incubation with a chromogenic (e.g., DAB, TMB, OPD), fluorogenic (e.g., ADHP), or chemiluminescent (e.g., ECL) substrate. A chromogenic, fluorescent, and / or chemiluminescent change (indicative of catalysis by peroxidase) may be used to quantify the amount of clostridial neurotoxin polypeptide (or a portion thereof) bound to the capture substrate. Preferably, the substrate is TMB (3,3',5,5'-tetramethylbenzidine).

[0316] The heavy chain binding assay may be an ELISA as known in the art. A suitable methodology is provided in Example 3 herein. Another heavy chain binding assay technique may be or include bilayer interferometry. Such a technique may use the Octet Red 96e system.

[0317] The term "specific," as used in the context of an antibody that specifically binds to an antigen (e.g., a Clostridial neurotoxin polypeptide), can mean that the antibody binds to the antigen with greater specificity and affinity than it binds to a non-antigen. In some embodiments, the binding can be at least 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold greater than binding to a non-antigen.

[0318] The cell-free substrate cleavage assay preferably involves the use of a cleavable substrate as described herein. In one embodiment, the cell-free substrate cleavage assay may be carried out in the same manner as the methods of the invention, but omitting the capture substrate and contacting them with a composition to remove unbound Clostridial neurotoxin polypeptide. The cell-free substrate cleavage assay may include: (a) contacting a composition comprising a clostridial neurotoxin polypeptide and a reducing agent with a cleavable substrate; and (b) determining the amount of cleavable substrate cleaved by the light chain polypeptide of the clostridial neurotoxin polypeptide, thereby determining the light chain activity of the composition.

[0319] Thus, in one aspect, the present invention provides a cell-free method for assaying substrate cleavage comprising: (a) contacting a composition comprising a clostridial neurotoxin polypeptide and a reducing agent with a cleavable substrate; or (b) contacting a composition comprising at least the light chain of a clostridial neurotoxin polypeptide with a cleavable substrate; and (c) determining the amount of cleavable substrate cleaved by the L chain polypeptide (e.g., the L chain of a clostridial neurotoxin polypeptide), thereby determining the L chain activity of the composition.

[0320] Due to the presence of a reducing agent, the composition may comprise an L chain polypeptide dissociated from the corresponding H chain polypeptide of the clostridial neurotoxin polypeptide. Determining the amount of the cleavable substrate cleaved by the L chain polypeptide of the clostridial neurotoxin polypeptide may be suitably carried out using any of the methods described herein. Preferably, the same cleavable substrate is used, and the methodology used is the same as in the method of the present invention.

[0321] A suitable cell-free substrate cleavage assay methodology is described in Example 3 herein.

[0322] In one aspect, the present invention provides a method for treating a clostridial neurotoxin comprising administering to a subject ... C or H CC The present invention provides the use of an isolated capture substrate for a clostridial neurotoxin to determine the presence or absence of oxidation of a clostridial neurotoxin receptor polypeptide (domain), wherein the isolated capture substrate comprises an extracellular portion of a clostridial neurotoxin receptor polypeptide that includes an amino acid modification and / or a post-translational modification.

[0323] In one aspect, the present invention provides a clostridial neurotoxin polypeptide or portion thereof (e.g., H thereof) that is included in a composition. C or H CC The present invention provides a cell-free method for determining whether a heavy chain (e.g., a heavy chain domain) contains an activity-altering property, the method comprising: (a) Clostridial neurotoxin polypeptides or portions thereof (e.g., H C or H CC providing a capture substrate for the heavy chain (e.g., heavy chain domain); (b) contacting the capture substrate with the composition to capture a Clostridial neurotoxin polypeptide or portion thereof (e.g., its H C or H CCbinding a heavy chain (e.g., a heavy chain domain) to the capture substrate; (c) Unbound Clostridial neurotoxin polypeptide or portion thereof (e.g., H C or H CC domains, etc.) of the heavy chain; (d) a Clostridial neurotoxin polypeptide or portion thereof (e.g., H) bound to the capture substrate; C or H CC determining the amount of heavy chain (e.g., domain); (e) Conjugated Clostridial neurotoxin polypeptides or portions thereof (e.g., H C or H CC comparing the amount of the heavy chain (e.g., heavy chain domain) with that of a control; and (f) Based on the comparison, the clostridial neurotoxin polypeptide or portion thereof (e.g., H) contained in the composition is C or H CC determining whether a heavy chain (e.g., heavy chain, domain, etc.) contains an activity-altering feature (e.g., an activity-altering feature of the heavy chain).

[0324] Preferably, the present invention provides a cell-free method for determining whether a Clostridial neurotoxin polypeptide contained in a composition contains an activity-altering property, the method comprising: (a) providing a capture substrate for said clostridial neurotoxin polypeptide; (b) contacting the capture substrate with the composition to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) determining the amount of Clostridial neurotoxin polypeptide bound to the capture substrate; (e) comparing the amount of bound Clostridial neurotoxin polypeptide with a control; and (f) determining, based on the comparison, whether a Clostridial neurotoxin polypeptide contained in the composition contains an activity-altering characteristic (e.g., an activity-altering characteristic of an H chain).

[0325] A suitable control may be any control described herein. A negative control may be a Clostridial neurotoxin polypeptide or portion thereof (e.g., H) containing an activity-reducing property. C or H CC In such embodiments, the composition may represent a composition comprising a clostridial neurotoxin polypeptide or portion thereof (e.g., an H chain, such as an H domain) bound to a capture substrate. C or H CC If the amount of a clostridial neurotoxin polypeptide or a portion thereof (e.g., H chain, such as H domain) contained in the composition is higher than that of the negative control, C or H CC In such embodiments, a clostridial neurotoxin polypeptide or portion thereof (e.g., an H chain, such as an H domain) bound to a capture substrate can be determined to be free of the activity-reducing properties of the H chain. C or H CC If the amount of the clostridial neurotoxin polypeptide or the portion thereof (e.g., H chain, such as H domain) contained in the composition is the same as or less than the negative control, C or H CC It can be determined that a domain (such as a heavy chain) contains the activity-reducing characteristic of the heavy chain.

[0326] The control is preferably a positive control, more preferably a heavy chain binding positive control as described herein.

[0327] In one embodiment, a clostridial neurotoxin polypeptide or portion thereof (e.g., H) is bound to a capture substrate. C or H CC If the amount of a clostridial neurotoxin polypeptide or portion thereof (e.g., H chain, H domain, etc.) contained in the composition is higher than that of the heavy chain binding positive control, C or H CC It can be determined that a domain (such as a heavy chain) contains an activity-enhancing characteristic of the heavy chain.

[0328] In one embodiment, a clostridial neurotoxin polypeptide or portion thereof (e.g., H) is bound to a capture substrate. C or H CCIf the amount of a clostridial neurotoxin polypeptide or portion thereof (e.g., H chain, such as a H domain) contained in the composition is less than that of the heavy chain-binding positive control, C or H CC It can be determined that a domain (such as an H chain domain) contains the activity-reducing property of the H chain.

[0329] In one embodiment, a clostridial neurotoxin polypeptide or portion thereof (e.g., H) is bound to a capture substrate. C or H CC If the amount of a clostridial neurotoxin polypeptide or a portion thereof (e.g., H chain, such as a H domain) contained in the composition is the same as that of a heavy chain-binding positive control, C or H CC It can be determined that the H chain domains (e.g., H chain domains) do not contain activity-altering properties of the H chain.

[0330] A clostridial neurotoxin polypeptide or portion thereof (e.g., H) bound to a capture substrate. C or H CC The amount of a heavy chain (e.g., a domain) may be determined using any suitable method, such as those described above.

[0331] Any of the methods described herein may further comprise comprising a clostridial neurotoxin polypeptide or portion thereof (e.g., H) in the composition. C or H CC It can be determined whether a specific domain (e.g., H chain) contains an activity-altering property.

[0332] The method may further include: (i) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide; and (ii) determining the amount of cleavable substrate cleaved by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition.

[0333] The method may further include: (i) adding a reducing agent to dissociate a light (L) chain polypeptide of the bound clostridial neurotoxin polypeptide, thereby combining the dissociated L chain polypeptide with the capture substrate and the clostridial neurotoxin receptor-binding domain (H) CC Domain, e.g. H C and (ii) determining the amount of cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby determining the clostridial neurotoxin activity of the composition.

[0334] In one embodiment, where the method involves the use of a capture substrate (e.g., comprising the extracellular portion of modified human SYT-II), the method may include comparing the amount of cleavable substrate cleaved by the L chain polypeptide with a control.

[0335] A suitable control may be any control described herein. A negative control may represent a composition comprising a Clostridial neurotoxin polypeptide that comprises an activity-reducing property.

[0336] The control is preferably a positive control, more preferably a substrate cleavage positive control as described herein.

[0337] In one embodiment, if the clostridial neurotoxin activity of the composition is lower than the substrate cleavage positive control, it can be determined that the clostridial neurotoxin polypeptide contained in the composition contains an activity-reducing characteristic of the L chain.

[0338] In one embodiment, if the clostridial neurotoxin activity of the composition is greater than the substrate cleavage positive control, it can be determined that the clostridial neurotoxin polypeptide contained in the composition contains an activity-increasing L chain characteristic.

[0339] In one embodiment, if the clostridial neurotoxin activity of the composition is the same as that of the substrate cleavage positive control, it can be determined that the clostridial neurotoxin polypeptide contained in the composition does not contain an activity-altering characteristic of the L chain.

[0340] In one aspect, the present invention provides a method for producing a therapeutic or cosmetic Clostridial neurotoxin composition, the method comprising: (a) obtaining the results of the method according to the present invention; and (b) formulating and / or packaging a composition for therapeutic or cosmetic use when the clostridial neurotoxin polypeptide contained in the composition does not contain an activity-altering property; or (c) further purifying the composition if the Clostridial neurotoxin polypeptide contained in the composition has an activity-altering characteristic; and (d) formulating and / or packaging such further refined composition for therapeutic or cosmetic use;

[0341] In one embodiment, when the term "obtaining a result" or "obtaining a set of results" of a method or assay is used herein, the method performed to obtain said result may be performed as part of a method of the invention.

[0342] In one aspect, the present invention provides a kit comprising: (a) an isolated capture substrate described herein; and (b) optionally, a means for detecting binding of the botulinum neurotoxin to the capture substrate; and / or (c) Optionally, instructions for its use.

[0343] Various method-related embodiments of the present invention are intended to apply equally to alternative methods, products, and / or applications, and vice versa.

[0344] sequence homology

[0345] Percent identity can be determined using any of a variety of sequence alignment methods, including, but not limited to, global methods, local methods, and hybrid methods such as segmental approaches. Protocols for determining percent identity are routine procedures 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 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 input sequences.Non-limiting methods include, for example, Matchbox (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)).

[0346] 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 and the alignment score is optimized 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 follows (amino acids are indicated by standard single-letter code); preferably, as described herein, the method is used to align sequences with the SEQ ID NOs set forth herein and define amino acid position numbers.

[0347] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, percent identity may be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. The calculation of percent sequence identity may further take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Comparison of sequences and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which will be familiar to those skilled in the art.

[0348] Alignment scores for determining sequence identity

number

[0349] The percent identity is then calculated as follows: [total number of identical matches × 100] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0350] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically from 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0351] 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

[0352] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, etc.) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues in the polypeptides. The polypeptides of the invention can also include unnatural amino acid residues.

[0353] Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Individual methods for incorporating unnatural amino acid residues into proteins are known in the art. For example, an in vitro system can be utilized in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing Escherichia coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, e.g., 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 a second method, translation is carried out in Xenopus oocytes by microinjecting mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of a desired unnatural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine), which is incorporated into the polypeptide in place of its natural counterpart.See Koide et al., Biochem. 33:7470-6, 1994. Natural amino acid residues can be converted to non-natural species by in vitro chemical modification. Combining chemical modification with site-directed mutagenesis can further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0354] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, unnatural amino acids, and non-naturally occurring amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0355] 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). Biological interaction sites can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutation of putative contact site amino acids. 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 identities of essential amino acids can also be inferred from homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0356] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, 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 within a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the range of substitutions allowed 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 WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0357] 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 of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0358] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, any nucleic acid sequence is written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0359] The headings provided herein are not intended to limit the various aspects or embodiments of the present disclosure.

[0360] As used herein, amino acids are represented using amino acid names, three-letter abbreviations, or one-letter abbreviations. As used herein, the term "protein" 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 cases, "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." As used herein, the terms "protein" and "polypeptide" are used interchangeably. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by one or more nucleotide sequences.

[0361] Other definitions of terms may appear throughout this specification. Before proceeding in more detail with respect to exemplary embodiments, it is to be understood that this disclosure is not limited to particular embodiments described, 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.

[0362] When a range of values ​​is given, each intervening value between the upper and lower limit of that range is also understood to be expressly disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is included within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each of the smaller ranges including either, neither, or both of those limits is also included within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0363] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a modified botulinum neurotoxin" includes a plurality of such candidate agents, reference to "the modified botulinum neurotoxin" includes a reference to one or more modified botulinum neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0364] 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.

[0365] 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]

[0366] [Figure 1] Figure 1 shows a schematic diagram of an example assay. BoNT (3 / 4 circle) binds to a capture substrate (triangle), and after appropriate washing steps, the light chain is released by adding dithiothreitol (DTT) to reduce the disulfide bond between the BoNT heavy and light chains. The light chain polypeptide is transferred to a separate vial (e.g., well) and incubated with a cleavable substrate containing SNAP-25 flanked by cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Activity is determined by measuring the CFP / YFP ratio. [Figure 2] FIG. 2 shows toxin binding as an increase in optical density (OD) value when casein or 1% bovine serum albumin (BSA) was used as the carrier protein blocking buffer. [Figure 3] FIG. 3 shows the binding of mrBoNT / A to the SV2c capture substrate alone or to SV2c and GT1b. [Figure 4] Figure 4 shows the amount of cleaved SNAP-25 (represented by the decrease in the CFP / YFP ratio) in the assay described in Figure 1 when the tested composition contained full-length mrBoNT / A (circles) or only the L chain (squares). [Figure 5] FIG. 5 shows assay qualification for both mrBoNT / A (A) and mrBoNT / AB (B), displaying linearity, accuracy, and precision between 150% and 50% levels. [Figure 6] Figure 6 shows the percent activity of mrBoNT / A compositions after forced oxidation for the times indicated on the x-axis when tested in a cell-based assay (circles), an endopeptidase assay (triangles), an ELISA (inverted triangles), or a binding and cleavage assay (diamonds) as in Figure 1. Also shown over time is the amount of oxidized mrBoNT / A peptides (specifically, % unmodified / unoxidized) encompassing at least a portion of the HC domain (squares). [Figure 7] Figure 7 shows a bilayer interferometry (BLI) binding kinetics summary of unoxidized (control) or oxidized (0.01% HO for 48 hours) mrBoNT / A to capture substrates containing the extracellular portion of nonglycosylated GST-SV2c expressed in E. coli (top panel) or the extracellular portion of glycosylated HEK293-expressed GST-SV2c (bottom panel). [Figure 8] 8 shows the percent activity of mrBoNT / AB compositions when tested in a cell-based assay (triangles) or in an ELISA (circles) using a capture substrate containing the extracellular portion of L51F mutant human SYT-II. Also shown is the amount of oxidized mrBoNT / AB peptide (specifically, % unmodified / unoxidized) encompassing at least a portion of the HC domain (squares). [Figure 9] FIG. 9 shows a bilayer interferometry (BLI) binding kinetics summary of unoxidized (control) or oxidized (0.001% H 2 O 2 for 72 hours) mrBoNT / AB to a capture substrate containing the extracellular portion of the L51F mutant human SYT-II capture substrate. [Figure 10] FIG. 10 shows the correlation of BoNT activity results comparing the cell-free assay described in FIG. 1 with the cell-based assay. [Figure 11] 11 shows: (A) a cleavable substrate having: a first luciferase domain; a linker containing a SNAP-25 cleavage site flanked by a spacer; and a second luciferase domain; and (B) a schematic diagram of an exemplary assay. BoNT (¾ circle) binds to the capture substrate (triangle), and after appropriate washing steps, the light chain is released by reducing the disulfide bond between the BoNT heavy and light chains with the addition of dithiothreitol (DTT) and the cleavable substrate. The light chain is not removed. The luciferase substrate is then added, and luminescence is measured to calculate the BoNT activity of the composition. [Figure 12]Figure 12 shows: (A) the BoNT activity of the composition when assessed using the cleavable substrate (comprising CFP and YFP) shown in Figure 1; (B) the BoNT activity of the same composition when assessed using the cleavable substrate (comprising a first and second luciferase domain) shown in Figure 11A, except that after DTT reduction, the L chain polypeptide is transferred to a separate vial (e.g., a well) and incubated with the cleavable substrate; and (C) the BoNT activity of the same composition when assessed using the method shown in Figure 11B (i.e., if the L chain is not removed, the cleavable substrate described in Figure 11A is added to the vial (e.g., a well)). [Figure 13] FIG. 13 shows the assay qualification of the mrBoNT / AB method shown in FIG. 11B, displaying linearity, accuracy, and precision between the 150% and 50% levels. [Figure 14] Figure 14 shows: (A) a reference curve generated when evaluated according to Figure 1 without using the cleavable substrate (comprising the first and second luciferase domains) shown in Figure 11A; and (B) mrBoNT / AB targeted recovery 5 ng / mL in formulation buffer, which reproduces the increased rebinding in saline. [Figure 15] Figure 15 shows the linearity and parallelism of the formulation analytical assay changes between the 130% and 75% levels. [Figure 16] FIG. 16 shows the combined results of various assay formats for forced degradation samples of mrBoNT / A, (A) and mrBoNT / AB (0.001% H 2 O 2 for 48 hours), compared with (B) cell-based assay data (control). [Figure 17] 17 shows the percent activity of oxidized mrBoNT / AB in a modified binding and cleavage assay (see Example 2) utilizing either human SYT-I (squares) or human SYT-II (L51F modified - circles) capture substrates. The activity is expressed relative to the percentage of clostridial neurotoxin polypeptide in the composition that is oxidized at the reference amino acid residue.

[0367] Sequence Listing

[0368] In any of the SEQ ID NOs below, where a first Met amino acid residue or corresponding first codon is shown, said residue / codon is optional. Preferably, said first Met amino acid residue or corresponding first codon is not present.

[0369] SEQ ID NO:1 - polypeptide sequence, luciferase MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL

[0370] SEQ ID NO:2 - polypeptide sequence, luciferase domain 1 MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS

[0371] SEQ ID NO:3 - Polypeptide sequence, luciferase domain 2 VTGYRLFEEIL

[0372] SEQ ID NO: 4 Polypeptide sequence, full length SNAP-25 MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

[0373] SEQ ID NO:5 - Polypeptide sequence, 65 amino acids SNAP-25 RENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRAT KMLGSG

[0374] SEQ ID NO:6 - Polypeptide sequence, luciferase-cleavable substrate MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFD GKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSSGGGGSRENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGSGGGGSVTGYRLFEEIL

[0375] SEQ ID NO: 7 - Polypeptide sequence, mrBoNT / AB

[0376] SEQ ID NO:8 - Polypeptide sequence, BoNT / AB mutant 2 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0377] SEQ ID NO: 9 - Polypeptide sequence, BoNT / AB mutant 3 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0378] SEQ ID NO: 10 - Polypeptide sequence, BoNT / AB mutant 4 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0379] SEQ ID NO: 11 - Polypeptide sequence, BoNT / AB mutant 5 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPANLIGNMLYKDDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNNLNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0380] sequence number 12 - polypeptide sequence, natural BoNT / A (BoNT / A)

[0381] SEQ ID NO: 13 - Polypeptide sequence, BoNT / B MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNNLNNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0382] SEQ ID NO: 14 - Polypeptide sequence, mrBoNT / A

[0383] SEQ ID NO: 15 - Polypeptide sequence, cationic BoNT / A mutant 2

[0384] SEQ ID NO: 16 - Polypeptide sequence, cationic BoNT / A mutant 3

[0385] SEQ ID NO: 17 - Polypeptide sequence, cationic BoNT / A mutant 4

[0386] SEQ ID NO: 18 - Polypeptide sequence, capture substrate containing the extracellular portion (amino acids 1-61) of human wild-type SYTII MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTY LNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLP

[0387] SEQ ID NO: 19 - Polypeptide sequence, extracellular portion of human wild-type SYTII (amino acids 1-61) MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLP

[0388] SEQ ID NO: 20 - Polypeptide sequence, capture substrate containing the extracellular portion (amino acids 1-61) of human modified SYTII (L51F) MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTY LNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKFFNEINKIPLP

[0389] SEQ ID NO: 21 - Polypeptide sequence, extracellular portion (amino acids 1-61) of human modified SYTII (L51F) MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKFFNEINKIPLP

[0390] SEQ ID NO: 22 - Polypeptide sequence, capture substrate containing the extracellular portion of mouse SYTII (amino acids 1-64) MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLN GDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNGEPNVAPATTTATMPLAPVAPADNSTESTGPGESQEDMFAKLKEKFFNEINKIPLP

[0391] SEQ ID NO: 23 - Polypeptide sequence, full length human wild-type SYTII MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPL PPWALIAIAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDDDDA ETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVK VFLLPDKKKKYETKVHRKTLNPAFNETFTFKVPYQELGGKTLVMAIYDFDRFSKHDIIGE VKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKM DVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVVTVL DYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGKNK

[0392] SEQ ID NO: 24 - Polypeptide sequence, full length mouse wild-type SYTII MRNIFKRNQEPNVAPATTTATMPLAPVAPADNSTESTGPGESQEDMFAKLKEKFFNEINK IPLPPWALIAMAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDD DDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDP YVKVFLLPDKKKKYETKVHRKTLNPAFNETFTFKVPYQELAGKTLVMAIYDFDRFSKHDI IGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNL KKMDVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVV TVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGK NK

[0393] SEQ ID NO: 25 - Polypeptide sequence, full length human SV2a MEEGFRDRAAFIRGAKDIAKEVKKHAAKKVVKGLDRVQDEYSRRSYSRFEEEDDDDDFPA PSDGYYRGEGTQDEEEGGASSDATEGHDEDDEIYEGEYQGIPRAESGGKGERMADGAPLA GVRGGLSDGEGPPGGRGEAQRRKEREELAQQYEAILRECGHGRFQWTLYFVLGLALMADG VEVFVVGFVLPSAEKDMCLSDSNKGMLGLIVYLGMMVGAFLWGGLADRLGRRQCLLISLS VNSVFAFFSSFVQGYGTFLFCRLLSGVGIGGSIPIVFSYFSEFLAQEKRGEHLSWLCMFW MIGGVYAAAMAWAIIPHYGWSFQMGSAYQFHSWRVFVLVCAFPSVFAIGALTTQPESPRF FLENGKHDEAWMVLKQVHDTNMRAKGHPERVFSVTHIKTIHQEDELIEIQSDTGTWYQRW GVRALSLGGQVWGNFLSCFGPEYRRITLMMMGVWFTMSFSYYGLTVWFPDMIRHLQAVDY ASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVT SSNTFFRNCTFINTVFYNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAYMVYFV SFLGTLAVLPGNIVSALLMDKIGRLRMLAGSSVMSCVSCFFLSFGNSESAMIALLCLFGG VSIASWNALDVLTVELYPSDKRTTAFGFLNALCKLAAVLGISIFTSFVGITKAAPILFAS AALALGSSLALKLPETRGQVLQ

[0394] SEQ ID NO: 26 - Polypeptide sequence, full length human SV2b MDDYKYQDNYGGYAPSDGYYRGNESNPEEDAQSDVTEGHDEEDEIYEGEYQGIPHPDDVK AKQAKMAPSRMDSLRGQTDLMAERLEDEEQLAHQYETIMDECGHGRFQWILFFVLGLALM ADGVEVFVVSFALPSAEKDMCLSSSSKKGMLGMIVYLGMMAGAFILGGLADKLGRKRVLSM SLAVNASFASLSSFVQGYGAFLFCRLISGIIGGALPIVFAYFSEFLSREKRGEHLSWLG IFWMTGGLYASAMAWSIIPHYGWGFSMGTNYHFHSWRVFVIVCALPCTVSMVALKFMPES PRFLLEMGKHDEAWMILKQVHDTNMRAKGTPEKVFTVSNIKTPKQMDEFIEIQSSTGTWY QRWLVRFKTIFKQVWDNALYCVMGPYRMNTLILAVVWFAMAFSYYGLTVWFPDMIRYFQD EEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFDECYFE DVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDNDFLIYL VSFLGSLSVLPGNIISALLMDRIGRLKMIGGSMLISAVCCFFLFFGNSESAMIGWQCLFC GTSIAAWNALDVITVELYPTNQRATAFGILNGLCKFGAILGNTIFASFVGITKVVPILLA AASLVGGGLIALRLPETREQVLM

[0395] SEQ ID NO: 27 - Polypeptide sequence, full length human SV2c MEDSYKDRTSLMKGAKDIAREVKKQTVKKVNQAVDRAQDEYTQRSYSRFQDEEDDDDYYP AGETYNGEANDDEGSSEATEGHDEDDEIYEGEYQGIPSMNQAKDSIVSVGQPKGDEYKDR RELESERRADEEELAQQYELIIQECGHGRFQWALFFVLGMALMADGVEVFVVGFVLPSAE TDLCIPNSGSGWLGSIVYLGMMVGAFFWGGLADKVGRKQSLLICMSVNGFFAFLSSFVQG YGFFLFCRLLSGFGIGGAIPTVFSYFAEVLAREKRGEHLSWLCMFWMIGGIYASAMAWAI IPHYGWSFSMGSAYQFHSWRVFVIVCALPCVSSVVALTFMPESPRFLLEVGKHDEAWMIL KLIHDTNMRARGQPEKVFTVNKIKTPKQIDELIEIESDTGTWYRRCFVRIRTELYGIWLT FMRCFNYPVRDNTIKLTIVWFTLSFGYYGLSVWFPDVIKPLQSDEYALLTRNVERDKYAN FTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDT VFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAYWIYFVNFLGTLAVLPGNIVS ALLMDRIGRLTMLGGSMVLSGISCFFLWFGTSESMMIGMLCLYNGLTISAWNSLDVVTVE LYPTDRRATGFGFLNALCKAAAVLGNLIFGSLVSITKSIPILLASTVLVCGGLVGLCLPD TRTQVLM

[0396] SEQ ID NO: 28 - Polypeptide sequence, full length human SYT-I MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPPWA LIAIAIVAVLLVLTCCFCICKKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALK DDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDP YVKVFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDI IGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNL KKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVV TVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEVDAMLAV KK

[0397] SEQ ID NO: 29 - Polypeptide sequence, capture substrate containing the extracellular portion of SV2c (amino acids 473-567) MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLSSGLEVLFQGPVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0398] SEQ ID NO: 30 - Polypeptide sequence, extracellular portion of SV2c (amino acids 473-567) VERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0399] SEQ ID NO: 31 - Polypeptide sequence, VAMP1_human (P23763) MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT

[0400] SEQ ID NO: 32 - Polypeptide sequence, VAMP2_human (P63027) MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

[0401] SEQ ID NO: 33 - Polypeptide sequence, VAMP3_human (Q15836) MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

[0402] SEQ ID NO: 34 - Polypeptide sequence, VAMP4_human (O75379) MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

[0403] SEQ ID NO: 35 - Polypeptide sequence, VAMP5_human (O95183) MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN

[0404] SEQ ID NO: 36 - Polypeptide sequence, YKT6_human (O15498) MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

[0405] SEQ ID NO: 37 - Polypeptide sequence, syntaxin 1A MKDRTQELRTAKDSDDDDDVAVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEEVKRKHSA ILASPNPDEKTKEELEELMSDIKKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIRKTQH STLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGRTTTSEELEDMLESGNPAIFASG IIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFDMDMAMLVESQGEMIDRIEYNVEHA VDYVERAVSDTKKAVKYQSKARRKKIMIIICCVILGIVIASTVGGIFA

[0406] SEQ ID NO: 38 - Polypeptide sequence, syntaxin 1B MKDRTQELRSAKDSDDEEEVVHVDRDHFMDEFFEQVEEIRGCIEKLSEDVEQVKKQHSAI LAAPNPDEKTKQELEDLTADIKKTANKVRSKLKAIEQSIEQEEGLNRSSADLRIRKTQHS TLSRKFVEVMTEYNATQSKYRDRCKDRIQRQLEITGRTTTNEELEDMLESGKLAIFTDDI KMDSQMTKQALNEIETRHNEIIKLETSIRELHDMFVDMAMLVESQGEMIDRIEYNVEHSV DYVERAVSDTKKAVKYQSKARRKKIMIICCVVLGVVLASSIGGTLGL

[0407] SEQ ID NO: 39 - polypeptide sequence, spacer SGGGGS

[0408] SEQ ID NO: 40 - Polypeptide sequence, capture substrate containing the extracellular portion of SV2c (amino acids 473-567) MGWSCIILFLVATATGVHSGGGGSSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPD FMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLSSGLEVLFQGPVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0409] SEQ ID NO: 41 - Polypeptide sequence, BoNT / C - UniProt P18640 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGGFALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYIRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRK DINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN VDYLNSYYYLESQKLSDNVEDFFTTRSIEEALDNSAKVYTYFPTLAKVNAGVQGGLFLM WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNIN KFIRECSVTYLFKNMLPKVIDENLEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSF QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYFAKEL DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNN DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYA IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFGNENISGICSIGTYFRLGGDWYR HNYLVPTVKQGNYASLLESTSTHWGFVPVSE

[0410] sequence number 42 - polypeptide sequence, BoNT / D - UniProt P19321 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALKHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE VVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFP EFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIR ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTTDTIYATQGGECSQNCVYALKLQSNLGNY GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS TSSFWKFISRDPGWVE

[0411] SEQ ID NO: 43 - Polypeptide sequence, BoNT / E - UniProt Q00496 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

[0412] SEQ ID NO: 44 - Polypeptide sequence, BoNT / F - UniProt A7GBG3 MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN EHTPINEFHPPVTRTTSVNIKSSTVNKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY DPSNDGFGSINIVTFSPEYETFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANF KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNDINTKEIDDTTNLN NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL IPTILVFTIKSFIGSENKNIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERF ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC IRNNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS SNGCFWSFISKEHGWQEN

[0413] SEQ ID NO: 45 - Polypeptide sequence, BoNT / G - UniProt Q60393 MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGH SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNL AGEYGIKTRYSYFSYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAY EEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAAYN TQNNTIENNFSIDQLILDNDLSSGIDLPNETPFTNFDDIDIPVYIKQSALKKIFVDGD SLFEYLHAQTFPSNIENLQLTNSLNDALRNNKVYTFFSTNLVEKANTVVGASLFVNWVK GVIDDFTSESTQKSTIDKVSDVSIIIPYIGPANLVGNETAKENFKNAFEIGGAAILMEFI PELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDS IPFDLSLYTKDTILIQVFNNYISNISSNAILSSYRGGRLIDSGYGATMNVGSDVIFND IGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNDIQTYLQNEYTII SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVS SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAI NYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYD NYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

[0414] SEQ ID NO: 46 - Polypeptide sequence, TeNT - UniProt P04958 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFN PPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGN SYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDN KNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLH GLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYK AIANKLSQVTSCNDPNIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTE IELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNT NAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAE KNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPPVTKGIPYAP EYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVI SKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPANLIVKQGYEGN FIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYK LVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKN KLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIG ITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDIS GFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPK VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP DKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNN NQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDV QLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYV SYNNNEHIVGYPKDGNAFNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDD KNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

[0415] SEQ ID NO: 47 - 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

[0416] SEQ ID NO: 48 - His-TEV sequence MHHHHHHDDDDK

[0417] SEQ ID NO: 49 - nucleic acid sequence, encoding mrBoNT / A

[0418] SEQ ID NO: 50 - nucleic acid sequence, encoding the extracellular portion (amino acids 1-61) of human modified SYTII (L51F) ATGCGTAACATCTTCAAACGTAACCAAGAGCCGATTGTTGCGCCGGCGACCACCACCGCGACCATGCCGATTGGTCCGGTTGACAACAGCACCGAAAGCGGTGGCGCGGTGAAAGCCAAGAAGATATGTTTGCGAAGCTGAAAGAGAAGTTCTTTAACGAAATCAACAAGATTCCGCTGCCG

[0419] SEQ ID NO: 51 - nucleic acid sequence, encoding the SV2c capture substrate atgggctggagctgcattattctgtttctggtggcgaccgcgaccggcgtgcatagcggc ggcggcggcagcagcccgattctgggctattggaaaattaaaggcctggtgcagccgacc cgcctgctgctggaatatctggaagaaaaatatgaagaacatctgtatgaacgcgatgaa ggcgataaatggcgcaacaaaaaatttgaactgggcctggaatttccgaacctgccgtat tatattgatggcgatgtgaaactgaccgagcatggcgattattcgctatattgcggat aaacataacatgctgggcggctgcccgaaagaacgcgcggaaattagcatgctggaaggc gcggtgctggatattcgctatggcgtgagccgcattgcgtatagcaaagattttgaaacc ctgaaagtggattttctgagcaaactgccggaaatgctgaaaatgtttgaagatcgcctg tgccataaaacctatctgaacggcgatcatgtgacccatccggattttatgctgtatgat gcgctggatgtggtgctgtatatggatccgatgtgcctggatgcgtttccgaaactggtg tgctttaaaaaacgcattgaagcgattccgcagattgataaatatctgaaaagcagcaaa tatattgcgtggccgctgcagggctggcaggcgacctttggcggcggcgatcatccgccg aaaagcgatctgagcagcggcctggaagtgctgtttcagggcccggtggaacgcgataaa tatgcgaactttaccattaactttaccatggaaaaccagattcataccggcatggaatat gataacggccgctttattggcgtgaaatttaaaagcgtgacctttaaagatagcgtgttt aaaagctgcacctttgaagatgtgaccagcgtgaacacctattttaaaaactgcaccttt attgataccgtgtttgataacaccgattttgaaccgtataaatttattgatagcgaattt aaaaactgcagcttttttcataacaaaacc

[0420] SEQ ID NO: 75 - C-terminal side L-chain fragment TKSLDKGYNK

[0421] SEQ ID NO: 76 - C-terminal side L-chain fragment 2 SLDKGYNK

[0422] SEQ ID NO: 77 - Double-chain L-chain 1 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0423] Sequence number 78 - Double-stranded L chain 2 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0424] Sequence number 79 - Double-stranded H chain ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLR AQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAV ILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNR IIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKI QSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYK YFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0425] SEQ ID NO: 80 - Polypeptide sequence, extracellular portion of human SYT-I (amino acids 1-60) MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPPWA

[0426] SEQ ID NO: 81 - Polypeptide sequence, capture substrate containing the extracellular portion of human SYT-I (amino acids 1-60) MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTY LNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPPWA

[0427] SEQ ID NO:82 - Polypeptide sequence, full length mouse SYT-I MVSASRPEALAAPVTTVATLVPHNATEPASPGEGKEDAFSKLKQKFMNELHKIPLPWALIAIAIVAVLLVVTCCFCVCKKCLFKKKNKKKGKEKGGKNAINMKD VKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQF TFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVGGLSDPYVKIH LMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEVDAMLAVKK

[0428] SEQ ID NO: 83 - Polypeptide sequence, extracellular portion of mouse SYT-I (amino acids 1-59) MVSASRPEALAAPVTTVATLVPHNATEPASPGEGKEDAFSKLKQKFMNELHKIPLPPPWA

[0429] SEQ ID NO: 84 - Polypeptide sequence, capture substrate containing the extracellular portion of mouse SYT-I (amino acids 1-59) MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKT YLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMVSASRPEALAAPVTTVATLVPHNATEPASPGEGKEDAFSKLKQKFMNELHKIPLPWA [Example]

[0430] Example 1

[0431] Materials and Methods

[0432] The relevant capture substrate for a neurotoxin serotype was immobilized to a clear Maxisorp 96-well microtiter plate at 5 μg / mL in Dulbecco's PBS with 600 rpm shaking for 1 hour at 37°C before blocking with 150 μL 1% BSA Dulbecco's PBS, 0.05% Tween 20. Serial dilutions of the relevant neurotoxin test sample were then applied to the plate in 1% BSA Dulbecco's PBS, 0.05% Tween 20. After 1 hour of shaking at 600 rpm at 37°C, unbound neurotoxin was removed from the plate by washing once with 1% BSA Dulbecco's PBS, 0.05% Tween 20. Bound toxin was then released from the capture substrate by reduction with 50 mM Hepes-NaOH, pH 7.1, 5 mM NaCl, 0.1% Tween-20, 10 μM ZnCl2, 5 mM DTT with 15 minutes of shaking at 600 rpm at 37°C. The supernatants were then transferred to black opaque microtiter plates containing SNAP25 substrate sandwiched between 2 μM CFP / YFP (see Figure 1 ) before being incubated at 37°C and 600 rpm for 3 h, and then fluorescence was measured at excitation / emission 434 / 526 (FRET signal) and 434 / 470 (CFP signal).

[0433] result

[0434] A novel plate-based assay platform was developed to assess the receptor affinity and endopeptidase activity of clostridial neurotoxins (Figure 1). The assay utilizes the commercially available BoTest endopeptidase reporter system (BioSentinel) in combination with custom-made, recombinantly expressed receptor substrates (capture substrates) to measure the quality of the toxin's light and heavy chains, respectively. The objectives of this method are to: (i) provide accurate sample potency predictions that limit the demands on cell-based assays; and (ii) characterize and understand the mechanism of action during the degradation of toxin samples.

[0435] substrate

[0436] For binding of mrBoNT / A (SEQ ID NO: 14, converted to a dichain toxin by incubation with Lys-C; see WO 2014 / 080206, incorporated herein by reference), the toxin-binding region containing SV2c (519-563) was used. Recombinant GST-SV2c (amino acids 473-567), shown as SEQ ID NO: 29, was expressed in E. coli.

[0437] To generate analogs with eukaryotic peptide glycosylation, the GST-SV2c(473-567) substrate (SEQ ID NO: 40) was also produced in HEK293 mammalian cells. Mass spectrometry analysis was performed and showed a mixture of glycans G0F, G1F, and G2F, suggesting consistent but heterogeneous glycosylation of the GST-SV2c(473-567) substrate when expressed in mammalian HEK293 cells (see Table below). [Table E]

[0438] In particular, glycosylation was observed at the physiologically relevant amino acid residue N559.

[0439] For binding of mrBoNT / AB (SEQ ID NO: 7, converted to a dichain toxin by incubation with Lys-C), a synaptotagmin II (SYTII) substrate was produced in E. coli in which SYTII(1-61) was GST-tagged. The SYTII was human, carrying the L51F amino acid mutation (SEQ ID NO: 20).

[0440] Characterization of capture substrate binding

[0441] Following serial dilutions of the toxin and application to the plate, the captured toxin was probed with an anti-BoNT antibody before secondary detection with a horseradish peroxidase (HRP)-conjugated anti-species antibody. The level of binding to the capture receptor was proportional to the affinity of the neurotoxin for the capture substrate. The effect of the carrier protein blocking buffer on assay sensitivity was evaluated. Surprisingly, the use of BSA (1%) as a blocker improved binding when compared with casein (Figure 2).

[0442] To enhance toxin binding, various attempts were made to co-immobilize ganglioside GT1b onto plates with a capture substrate containing the extracellular portion of SV2c. Neither pre-coating the plate with the SV2c portion capture substrate nor co-immobilization of GT1b with the SV2c portion capture substrate improved toxin capture beyond that of the SV2c portion capture substrate alone. In fact, surprisingly, co-immobilization with GT1b decreased the sensitivity of the assay (Figure 3). Therefore, to improve sensitivity, it seemed advantageous to eliminate the use of gangliosides (e.g., GT1b) in the binding and cleavage assays.

[0443] Binding and cleavage assays

[0444] After assessing binding characteristics, we characterized the complete capture substrate binding and cleavage assays. The assays were performed as described above. Briefly, capture substrates were immobilized in the wells of a multiwell plate before incubation with mrBoNT / A or mrBoNT / AB, respectively. The BoNT-capture substrate complexes were washed, and the light chains were released from the bound toxin by adding a buffer containing 50 mM HEPES-NaOH, pH 7.1, 5 mM NaCl, 0.1% Tween-20, 10 μM ZnCl2, and 5 mM DTT. The supernatant was then transferred to an additional assay plate containing a CFP / YFP FRET-based cleavable substrate (Figure 1). This allowed us to measure the light chain endopeptidase activity of the toxin specifically bound to each capture substrate, eliminating the contribution of free light chain polypeptides that may be generated by toxin degradation or impurities.

[0445] To assess the contribution of nonspecific endopeptidase activity carried over from the toxin binding step, a recombinant light chain-only control was used. Figure 4 shows that no endopeptidase activity was detected in the sample containing only the recombinant light chain, indicating no carryover of neurotoxin that was not bound to the specific receptor. This demonstrates that the assay has little or no nonspecific background.

[0446] Assay Qualification

[0447] To assess accuracy, precision, and reproducibility, binding and cleavage assays were performed in triplicate for mrBoNT / A and mrBoNT / AB, respectively, across five levels ranging from 50% to 150%. As shown in Figure 5A, the assays demonstrate high accuracy and precision across the five levels. The standard deviation across the five levels for triplicates averaged less than 6%, and the average recovery for the target drifted less than 5%. This indicates that the assays have good linearity, precision, and precession between 50% and 150%. As shown in Figure 5B, assay compositions also demonstrate comparable accuracy, linearity, and precision when performed using compositions containing mrBoNT / AB. The following table summarizes the assays performed using mrBoNT / A or mrBoNT / AB compositions. [Table F]

[0448] Determining the Stability of Clostridial Neurotoxin Compositions

[0449] To test the assay's stability-indicating, comparability- and potency-predicting properties, mrBoNT / A and degraded samples of mrBoNT / A were tested and compared with mass spectrometry and cell-based methods, as well as orthogonal binding methods.

[0450] Oxidized mrBoNT / A

[0451] Figure 6 shows that the binding and cleavage assays show high comparability with existing cell-based methods, with determined activity levels correlating with the amount of oxidized mrBoNT / A present in the composition. While forced oxidation of mrBoNT / A did not appear to affect only light chain activity when tested in the standard BoTest, potency estimates when tested in the binding and cleavage assays closely matched the overall physiological potency when tested in the cell-based assay.

[0452] mrBoNT / A samples from the same oxidation study were also evaluated by bilayer interferometry (BLI) using the Octet Red 96e system with both glycosylated and nonglycosylated capture substrates containing the GST-tagged extracellular portion of SV2c.

[0453] As shown in Figure 7, the unglycosylated capture substrate bound to mrBoNT / A faster than the glycosylated form. However, the glycosylated capture substrate dissociated from the toxin at a significantly slower rate. Oxidized mrBoNT / A, both in its glycosylated and unglycosylated forms, exhibited decreased capture substrate binding. The toxin dissociation rate was slowed by oxidized mrBoNT / A and both types of capture substrate (glycosylated and unglycosylated).

[0454] Thus, taking the results of Figures 6 and 7 together, the binding and cleavage assays allow for the detection of differences in the activity of oxidized BoNT, likely the result of reduced capture substrate binding.

[0455] Oxidized mrBoNT / AB

[0456] The forced oxidation samples of mrBoNT / AB were then analyzed by ELISA as described above using a capture substrate containing the extracellular portion of L51F mutant human SYTII. C We show that domain oxidation is associated with reduced activity as assessed in cell-based assays and reduced receptor binding by ELISA.

[0457] Similarly, forced oxidation samples were tested in parallel with BLI for mrBoNT / A (FIG. 9). The binding affinity of oxidized mrBoNT / AB to capture substrates containing the L51F mutant SYTII was reduced.

[0458] Thus, these results demonstrate that oxidized mrBoNT / AB has a different receptor binding profile than the equivalent non-oxidized mrBoNT / AB. Therefore, the binding and cleavage assays could be used to distinguish between the activity levels of oxidized and non-oxidized mrBoNT / AB. Furthermore, the binding and cleavage assays could be used to attribute loss of activity to specific parts of the toxin's mechanism of action (e.g., binding and / or proteolytic activity), providing mechanistic insight into the underlying cause of a composition's loss of activity.

[0459] Correlation with cell-based activity results

[0460] Because the binding and cleavage assay measures both the heavy chain affinity of a toxin composition and the endopeptidase activity of the toxin bound to its respective receptor, samples that generate a signal in this assay are likely physiologically active. To confirm this, a given composition was tested using the binding and cleavage assay and simultaneously tested using a cell-based assay. Figure 10 shows a highly significant correlation between the activity data obtained using the binding and cleavage assay and the activity data obtained using the cell-based assay (P=0.0001). This dataset included 27 samples, including mrBoNT / AB or mrBoNT / A samples subjected to forced degradation (including oxidation, glycation, and heat stress) or samples obtained at various points in the mrBoNT / AB manufacturing process, including samples with various purity levels or expressed or isolated under different conditions.

[0461] conclusion

[0462] In conclusion, the binding and cleavage assay reliably predicts the physiological activity of clostridial neurotoxin compositions. Because the data correlated strongly with cell-based assay results, the binding and cleavage assay may also be used as a high-throughput screening tool to triage large numbers of compositions prior to cell-based testing. Throughout the testing of samples, the assay provided stability indicators for various covalent post-translational modifications of the toxin, such as oxidation and glycation, that could be attributed to specific domains. The assay also provided stability indicators for changes in the toxin manufacturing process that involve unknown structural / biological differences. The results of this example suggest that loss of neurotoxin potency is predictable and can be specifically attributed to domains of toxin samples using a combined approach (see Example 3).

[0463] Example 2

[0464] Materials and Methods

[0465] The relevant capture substrate for the neurotoxin serotype was immobilized to a white Maxisorp 96-well microtiter plate at 5 μg / mL in Dulbecco's PBS with 600 rpm shaking for 1 hour at 37°C before blocking with 150 μL 1% BSA Dulbecco's PBS, 0.05% Tween 20. Serial dilutions of the relevant neurotoxin test sample were then applied to the plate in 1% BSA Dulbecco's PBS, 0.05% Tween 20. After 1 hour of shaking at 600 rpm at 37°C, unbound neurotoxin was then removed from the plate by washing once with 1% BSA Dulbecco's PBS, 0.05% Tween 20. The bound toxin was then released from the capture substrate by reduction with 50 mM Hepes-NaOH, pH 7.1, 0.1 mg / mL BSA, 0.1% Tween-20, 50 μM ZnCl, and 5 mM DTT with 0.1 μM cleavage substrate containing the first luciferase domain, the second luciferase domain, and a SNAP-25 cleavage site (SEQ ID NO: 6 with an N-terminal His-TEV tag [SEQ ID NO: 48]). The assay plate was then incubated at 37°C and 600 rpm for 3 hours. Next, 50 μL of 37.5 μM furimazine diluted in 50 mM HEPES-NaOH, 0.1% Tween-20, 50 μM ZnCl, and 5 mM DTT was added to all wells. Luminescence was then captured using a plate reader-based method.

[0466] result

[0467] Cleavable substrates

[0468] A new cleavable substrate (containing SEQ ID NO: 6) was generated containing an N-terminal first luciferase domain (SEQ ID NO: 2) flanking a linker containing a SNAP-25 cleavage site flanked by two spacers and a C-terminal second luciferase domain (SEQ ID NO: 3) (Figure 11A).

[0469] Modification binding and cleavage assays

[0470] The binding and cleavage assay was performed according to Example 1, but substituting the CFP / YFP cleavage substrate shown in Figure 11A (and luminescence measured in a white Maxisorp 96-well microtiter plate), although the assay was found to be significantly less sensitive (Figure 12B) when compared to the same assay using the CFP / YFP cleavage substrate with the same mrBoNT / AB composition (Figure 12A).

[0471] As part of the assay characterization procedure, the assay was performed as shown in Figure 11B and described in the Materials and Methods section of Example 2. Specifically, a luciferase-based cleavage substrate was added to the wells (containing the clostridial neurotoxin-captured substrate complex) and incubated with DTT. Luciferase substrate (furimazine [Carbosynth ZEC04024]) was then added and luminescence was assessed.

[0472] Prior to conducting this experiment, it was anticipated that nonspecific background binding would be significant, adversely affecting the specificity of the assay. Surprisingly, this was not the case, and it was confirmed that employing this method not only significantly improved the sensitivity of the assay (Figure 12C), but also did not result in an increase in nonspecific background, which was minimal as demonstrated by a control assay in which no capture substrate was immobilized on the assay plate (Figure 12C, squares). Indeed, sensitivity was greatly improved when compared to the assay of Example 1, in which the CFP / YFP cleavage substrate was used (Figure 12A).

[0473] In light of this discovery, the number of plates used for a given assay can be advantageously reduced, meaning that the improved binding and cleavage assays are more suitable for high-throughput testing, leading to reduced losses and costs.

[0474] The improved binding and cleavage assay was performed in triplicate at five levels between 50% and 150% for mrBoNT / AB to further examine the assay's accuracy, precision, and reproducibility. As shown in Figure 13, the assay exhibited high accuracy and precision across five levels. The standard deviation across the five levels for the triplicates averaged 8.6%, and the average recovery for the target drifted by less than 0.4%. Therefore, this demonstrated good linearity, accuracy, and precision between 50% and 150%.

[0475] Characterization of clostridial neurotoxin preparations.

[0476] Figure 14A shows a reference curve for an mrBoNT / AB composition generated using the assay according to Example 1, but utilizing the cleavable substrate shown in Figure 11A (containing first and second luciferase domains and measuring luminescence in a white Maxisorp 96-well microtiter plate). When evaluating various cosmetic or therapeutic clostridial neurotoxin formulations, assay interference from various excipients and saline-based formulation buffers can occur. The assay according to Example 1 lacked the sensitivity necessary to achieve a parallel linear potency assay.

[0477] In contrast, with the increased sensitivity, the improved binding and cleavage assay exhibits linearity capable of detecting very small differences in activity (Figure 15), as shown in Figure 11B. As shown in Figure 14B, the improved binding and cleavage assay was not significantly affected by increasing saline adjustments that altered excipient and buffer concentrations. Thus, the improved binding and cleavage assay is particularly suitable for characterizing / screening manufacturing-appropriate excipients and / or excipient concentrations for formulating therapeutic and / or cosmetic clostridial neurotoxin compositions.

[0478] Example 3

[0479] Materials and Methods

[0480] Heavy chain receptor affinity ELISA

[0481] The relevant capture substrate for the neurotoxin serotype was immobilized onto a clear Maxisorp 96-well microtiter plate at 5 μg / mL in Dulbecco's PBS with shaking at 600 rpm for 1 hour at 37°C before blocking with 150 μL 1% BSA Dulbecco's PBS, 0.05% Tween 20. Serial dilutions of the relevant neurotoxin test sample were then applied to the plate in 1% BSA Dulbecco's PBS, 0.05% Tween 20. After shaking at 600 rpm for 1 hour at 37°C, unbound neurotoxin was removed from the plate with one wash with 1% BSA Dulbecco's PBS, 0.05% Tween 20. The captured toxin was fixed with 2% paraformaldehyde for 10 minutes at room temperature before an additional wash with 1% BSA Dulbecco's PBS, 0.05% Tween 20. Anti-BoNT antibodies were then applied at 2 μg / mL in 1% BSA Dulbecco's PBS, 0.05% Tween 20, followed by incubation at 37°C for 1 hour with 600 rpm shaking. Plates were then washed once with 1% BSA Dulbecco's PBS, 0.05% Tween 20 before the addition of 0.4 μg / mL of the relevant anti-species HRP-conjugated antibody. Following a final incubation at 37°C for 1 hour with 600 rpm shaking, plates were washed twice with Dulbecco's PBS before being treated with pre-warmed TMB (3,3',5,5'-tetramethylbenzidine) substrate (Thermo Fisher N301) for 5 minutes and then quenched with ELISA stop solution (0.16 M sulfuric acid, TMB stop solution, Thermo Fisher N600).

[0482] Endopeptidase activity only

[0483] The relevant BoNT neurotoxin preparations were serially diluted in 50 mM HEPES NaOH, 50 μM ZnCl, 0.1% Tween 20, and 5 mM DTT. The serial dilutions were then added to a white 96-well microtiter plate pre-loaded with 10 μL of 1 μM cleavage substrate (containing SEQ ID NO: 6) containing the first luciferase domain, the second luciferase domain, and a SNAP-25 cleavage site, diluted in 50 mM HEPES NaOH, 50 μM ZnCl, 0.1% Tween 20, and 5 mM DTT. The plate was then incubated at 37°C for 3 hours with shaking at 600 rpm. Next, 50 μL of 37.5 μM furimazine diluted in 50 mM HEPES NaOH, 50 μM ZnCl, 0.1% Tween 20, and 5 mM DTT was added to all wells, and luminescence was subsequently captured using a plate reader-based method.

[0484] result

[0485] Samples of mrBoNT / AB and mrBoNT / A (respectively) were subjected to forced digestion and tested by heavy chain receptor affinity ELISA, in vitro endopeptidase assay, or binding and cleavage assays as described herein.

[0486] Figure 16 combines: (i) heavy chain receptor affinity ELISA; (ii) in vitro endopeptidase assay; and (ii) the binding and cleavage assay described in Example 2, allowing for domain-specific assessment of the quality and potency of neurotoxin formulations. A decrease in receptor affinity for either mrBoNT / A (Figure 16A) or mrBoNT / AB (Figure 16B) was readily detected using a receptor capture sandwich ELISA format. In cases where there was evidence of endopeptidase activity comparable to that of a control (e.g., a standard sample), the loss of potency observed in the binding and cleavage assay (confirmed by a conventional cell-based assay) was attributed to a topological change or post-translational covalent modification of the neurotoxin heavy chain binding to the relevant receptor (Figure 16B). Conversely, in cases where there was comparable heavy chain receptor affinity and reduced endopeptidase activity (Figure 16A), the loss of potency detected in the binding and cleavage assay and confirmed by a conventional cell-based assay could be attributed to a loss of endopeptidase kinetic activity.

[0487] conclusion

[0488] Mouse LD 50 Traditional neurotoxin analysis techniques, such as assays and more recent cell-based assays, that focus on formulation potency, cannot easily identify the specific reasons for changes in potency. Well-publicized and well-characterized neurotoxin domains, each with specific functions in the mode of action, are all essential to producing the exceptional potency and clinical efficacy of clostridial neurotoxin products. Reduced affinity for the relevant neurotoxin receptor results in reduced cell binding, thereby reducing cell penetration and hindering therapeutic response. Conversely, reduced catalytic activity of a neurotoxin formulation results in slower or no cleavage of SNARE proteins in the cytoplasm. In either case, mouse LD 50 The cause of the decreased or increased potency cannot be diagnosed using the assay or cell-based assay.

[0489] Cell-free binding and cleavage assays can predict physiological potency by examining key functions of the neurotoxin domain, making them comparable to cell-based assays in determining potency. Furthermore, by combining them with heavy chain receptor affinity ELISA and in vitro endopeptidase assays, decreased or increased activity can be easily detected. Therefore, unlike cell-based assays, changes in potency can be attributed to specific functions of the neurotoxin subunits.

[0490] Example 4

[0491] Materials and Methods

[0492] To test binding to SYT-I receptor substrates, mrBoNT / AB (SEQ ID NO: 14, converted to a dichain toxin by incubation with Lys-C) or wild-type BoNT / B (purchased from Metabiologics, Madison, USA) were assessed by bilayer interferometry (BLI) using the Octet Red 96e system. SYT-I receptor substrates, both mouse (SEQ ID NO: 84) and human (SEQ ID NO: 81) sequences, were immobilized on His- or GST-tagged capture biosensor probes and exposed to serial dilutions of mrBoNT / AB (starting at a concentration of 300 μg / ml, with half-dilution serial dilutions) or wild-type BoNT / B (starting at a concentration of 500 μg / ml). All data presented represent the average of 3n, where the data were fitted to a 1:1 ...

Claims

1. A cell-free method for determining the clostridial neurotoxin activity of a composition comprising a clostridial neurotoxin polypeptide, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L chain polypeptide from the capture substrate and the clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining the amount of cleavable substrate cleaved in the assay sample by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of said composition.

2. The method of claim 1, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide, wherein the capture substrate comprises a clostridial neurotoxin receptor polypeptide or a ganglioside to which a clostridial neurotoxin binds; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L chain polypeptide from the capture substrate and the clostridial neurotoxin receptor-binding domain (H CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining the amount of cleavable substrate cleaved in the assay sample by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition, wherein the cleavable substrates each comprise a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker operably links the first and second luciferase domains, thereby providing a functional luciferase.

3. A cell-free method for determining the clostridial neuroactivity of a composition comprising a clostridial neurotoxin polypeptide, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound clostridial neurotoxin polypeptide; and (e) determining the amount of cleavable substrate cleaved by said L chain polypeptide, thereby determining the clostridial neurotoxin activity of said composition.

4. The method of claim 3, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide, wherein the capture substrate comprises a clostridial neurotoxin receptor polypeptide or a ganglioside to which a clostridial neurotoxin binds; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the light chain polypeptide of the bound clostridial neurotoxin polypeptide; and (e) determining the amount of cleavable substrate cleaved by the L chain polypeptide, thereby determining the clostridial neurotoxin activity of the composition, wherein the cleavable substrates each comprise a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker operably links the first and second luciferase domains, thereby providing a functional luciferase.

5. A cell-free method for determining whether a composition contains a Clostridial neurotoxin polypeptide, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the clostridial neurotoxin polypeptide to the capture substrate; (c) removing any unbound clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate any light chain (L chain) polypeptides of any bound clostridial neurotoxin polypeptides; and (e) determining that the cleavable substrate is cleaved, thereby determining that the composition contains a clostridial neurotoxin polypeptide, or determining that the cleavable substrate is not cleaved, thereby determining that the composition does not contain a clostridial neurotoxin polypeptide.

6. The method of claim 5, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide, wherein the capture substrate comprises a clostridial neurotoxin receptor polypeptide or a ganglioside to which a clostridial neurotoxin binds; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing any unbound clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate any light chain polypeptides of any bound clostridial neurotoxin polypeptides; and (e) determining that the cleavable substrate is cleaved, thereby determining that the composition contains a clostridial neurotoxin polypeptide, or determining that the cleavable substrate is not cleaved, thereby determining that the composition does not contain a clostridial neurotoxin polypeptide, wherein the cleavable substrates each comprise a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker operably links the first and second luciferase domains, thereby providing a functional luciferase.

7. The method of claim 5, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing any unbound clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate any L chain polypeptides of any bound Clostridial neurotoxin polypeptides, thereby separating any dissociated L chain polypeptides from the capture substrate and the Clostridial neurotoxin receptor domain (H CC Domain, e.g. H C providing an assay sample comprising any complex comprising the nucleotide sequence (domain); and (e) determining that the cleavable substrate is cleaved in the assay sample, thereby determining that the composition contains a clostridial neurotoxin polypeptide, or determining that the cleavable substrate is not cleaved in the assay sample, thereby determining that the composition does not contain a clostridial neurotoxin polypeptide.

8. The method of claim 5 or 7, comprising: (a) providing a capture substrate for a clostridial neurotoxin polypeptide, wherein the capture substrate comprises a clostridial neurotoxin receptor polypeptide or a ganglioside to which a clostridial neurotoxin binds; (b) contacting the composition with the capture substrate to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound clostridial neurotoxin polypeptides; (d) adding a reducing agent to dissociate the L-chain polypeptide of the bound clostridial neurotoxin polypeptide, thereby separating the dissociated L-chain polypeptide from the capture substrate and the clostridial neurotoxin receptor domain (H CC Domain, e.g. H C providing an assay sample comprising a complex comprising the nucleotide sequence (domain); and (e) determining that the cleavable substrate is cleaved in the assay sample, thereby determining that the composition comprises a Clostridial neurotoxin polypeptide, wherein the cleavable substrates are each single-chain polypeptides comprising: (i) a first luciferase domain; (ii) a linker comprising a Clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker operably links the first and second luciferase domains, thereby providing a functional luciferase.

9. 10. The method of any one of the preceding claims, wherein the cleavable substrate is added simultaneously with the reducing agent.

10. 10. The method of any one of the preceding claims, wherein the cleavable substrate comprises (and is preferably such a single chain polypeptide) comprising: (i) a first luciferase domain; (ii) a linker comprising a Clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; said linker operably links the first and second luciferase domains, thereby providing a functional luciferase, preferably (i) when the linker is cleaved, the construct is unable to exhibit luciferase activity; and (ii) When the linker is intact, the construct is capable of exhibiting luciferase activity.

11. A clostridial neurotoxin polypeptide or portion thereof (e.g., H thereof) in a composition comprising: C or H CC Cell-free methods for determining whether a heavy chain (e.g., a domain) contains an activity-altering property: (a) Clostridial neurotoxin polypeptides or portions thereof (e.g., H C or H CC providing a capture substrate for the heavy chain (e.g., heavy chain domain); (b) a clostridial neurotoxin polypeptide or a portion thereof (e.g., its H C or H CC contacting the capture substrate with the composition to bind a heavy chain (e.g., a heavy chain, such as a domain) to the capture substrate; (c) Unbound Clostridial neurotoxin polypeptide or portion thereof (e.g., its H C or H CC domains, etc.) of the heavy chain; (d) a Clostridial neurotoxin polypeptide or portion thereof (e.g., its H) bound to the capture substrate. C or H CC determining the amount of heavy chain (e.g., domain); (e) a conjugated clostridial neurotoxin polypeptide or portion thereof (e.g., its H C or H CC comparing the amount of a heavy chain (e.g., a heavy chain domain) with a control; and (f) determining whether the clostridial neurotoxin polypeptide or a portion thereof (e.g., its H) is present in the composition based on the comparison; C or H CC domain) of the heavy chain has the activity-changing property (e.g., H C or H CC determining whether the heavy chain comprises a domain or domains that alter the activity of the heavy chain.

12. 10. The method according to any one of the preceding claims, wherein the capture substrate is directly or indirectly immobilized on a solid support, preferably a plastic support.

13. 10. The method of claim 1, wherein a capture substrate comprising a Clostridial neurotoxin receptor polypeptide is used in combination with a capture substrate comprising a ganglioside.

14. 10. The method of any one of the preceding claims, wherein the Clostridial neurotoxin polypeptide is a botulinum neurotoxin (BoNT) polypeptide.

15. 10. The method of claim 1, wherein the capture substrate comprises the extracellular portion of a BoNT receptor polypeptide, such as the extracellular portion of a neuronal BoNT receptor polypeptide.

16. The method of claim 15, wherein the extracellular portion of the BoNT receptor polypeptide of the neuronal cell comprises an amino acid modification and / or a post-translational modification.

17. 17. The method of claim 15 or 16, wherein the extracellular portion of the BoNT receptor polypeptide is the extracellular portion of synaptotagmin II (SYTII).

18. 18. The method of claim 17, wherein the extracellular portion of SYTII comprises amino acid residues 1 to 61 of SYTII.

19. 19. The method of any one of claims 15 to 18, wherein the extracellular portion of the BoNT receptor polypeptide is the extracellular portion of human SYTII containing an L51F substitution.

20. 17. The method of claim 15 or 16, wherein the extracellular portion of the BoNT receptor polypeptide is the extracellular portion of synaptotagmin I (SYT-I).

21. The BoNT polypeptides are each BoNT / BH CC The method of any one of claims 14 to 20, comprising a domain.

22. 17. The method of claim 16, wherein the post-translational modification is glycosylation, preferably N-linked glycosylation.

23. 23. The method of any one of claims 15-16 or 22, wherein the extracellular portion of the BoNT receptor polypeptide is the extracellular portion of SV2c, and preferably the extracellular portion comprises SV2c luminal domain 4 (e.g., amino acid residues 519-563 of SV2c).

24. 24. The method of claim 23, wherein the extracellular portion comprises amino acid residues 473 to 567 of SV2c.

25. 25. The method of claims 1-16, 23 or 24, wherein the extracellular portion of the neuronal BoNT receptor polypeptide is the extracellular portion of SV2c comprising a glycosylation at N559.

26. 26. The method of claims 22 to 25, wherein the glycosylation comprises Man-5 glycan, G0f glycan, G1f glycan, or G2f glycan, preferably wherein the glycan further comprises N-acetylglucosamine (GlcNAc), such as G0f-GlcNAc.

27. 10. The method according to any one of the preceding claims, wherein the capture substrate is recombinantly produced in a mammalian cell, preferably a human cell.

28. The BoNT polypeptides are each BoNT / AH CC 28. The method of any one of claims 14 to 16 or claims 22 or 27, comprising a domain.

29. 10. The method according to any one of the preceding claims, which does not involve the use of gangliosides, preferably does not involve the use of GT1b.

30. 10. The method of any one of the preceding claims, wherein the composition is a first Clostridial neurotoxin formulation comprising one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

31. 31. The method of claim 30, wherein the clostridial neurotoxin activity of at least a second clostridial neurotoxin formulation is determined using the method of any one of the preceding claims, wherein the at least a second clostridial neurotoxin formulation comprises the same clostridial neurotoxin present in the same amount as the first clostridial neurotoxin formulation and one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts, wherein the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts are different from or the same as but present in a different amount (e.g., a different concentration) as the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts present in the first clostridial neurotoxin formulation.

32. The method of claim 31, wherein the clostridial neurotoxin activity of the first clostridial neurotoxin preparation is compared with the clostridial neurotoxin activity of at least a second clostridial neurotoxin preparation, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts, or amounts thereof, are selected when the clostridial neurotoxin preparation containing them exhibits the highest activity.

33. 33. The method of any one of claims 1-4, 9-10, or 12-32, further comprising obtaining results from a heavy chain binding assay and / or a cell-free substrate cleavage assay.

34. 34. The method of any one of claims 1-4, 9-10, or 12-33, further comprising comparing the results of the method of any one of claims 1-4, 9-10, or 12-33 (e.g., the determined Clostridial neurotoxin activity of the composition) with the results of a heavy chain binding assay and / or a cell-free substrate cleavage assay.

35. The clostridial neurotoxin polypeptide (or a portion thereof, e.g., a light chain or a heavy chain, or H) contained in the composition C or H CC whether a Clostridial neurotoxin polypeptide (or a portion thereof, e.g., the L chain or H chain, or the H domain) comprises an activity-modifying feature, or whether a Clostridial neurotoxin polypeptide (or a portion thereof, e.g., the L chain or H chain, or the H domain) comprised in a composition that comprises an activity-modifying feature. C or H CC 35. The method of claim 34, wherein the comparison allows a determination to be made about the amount of the signal (part thereof, such as a domain) in the signal.

36. The clostridial neurotoxin polypeptide or portion thereof (e.g., its H) contained in the composition C or H CC Use of an isolated capture substrate for a clostridial neurotoxin to determine the presence or absence of an activity-altering characteristic of a heavy chain (e.g., a domain), wherein the isolated capture substrate comprises an extracellular portion of a clostridial neurotoxin receptor polypeptide that includes an amino acid modification and / or a post-translational modification.

37. The use according to claim 36, wherein the activity-altering characteristic is an activity-reducing heavy chain modification, more preferably activity-reducing heavy chain oxidation.

38. 1. A method for producing a therapeutic or cosmetic Clostridial neurotoxin composition, comprising: (a) obtaining the results of the method of any one of claims 1 to 4, 9 to 10, or 12 to 35, and if the clostridial neurotoxin activity is the same as or higher than a positive control (e.g., a positive standard sample), formulating and / or packaging the composition for therapeutic or cosmetic use; or (b) if the clostridial neurotoxin activity is lower than a positive control (e.g., a positive standard sample), further purifying the composition and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

39. 1. A method for producing a therapeutic or cosmetic Clostridial neurotoxin composition, comprising: (a) obtaining the results of the method of any one of claims 11 to 35; and (b) formulating and / or packaging the composition for therapeutic or cosmetic use when the clostridial neurotoxin polypeptide contained in the composition does not contain the activity-altering property; or (c) further purifying the composition if the Clostridial neurotoxin polypeptide contained in the composition has the activity-changing characteristic; and (d) formulating and / or packaging the further purified composition for therapeutic or cosmetic use;

40. 40. A therapeutic or cosmetic Clostridial neurotoxin composition obtainable by the method of claim 38 or 39, optionally wherein the therapeutic or cosmetic Clostridial neurotoxin composition is packaged.

41. Isolated capture substrate for a clostridial neurotoxin: Wherein the capture substrate comprises an extracellular portion of a clostridial neurotoxin receptor polypeptide, the extracellular portion comprising an amino acid modification and / or a post-translational modification.

42. Kit includes: (a) an isolated capture substrate according to claim 41; and (b) optionally, a means for detecting binding of a Clostridial neurotoxin (e.g., a botulinum neurotoxin) to the capture substrate; and / or (c) Optionally, instructions for its use.

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