Cell-free clostridial neurotoxin assay

Abstract

The present invention relates to cell-free methods, such as methods for determining clostridial neurotoxin activity of a composition, determining whether a composition comprises a clostridial neurotoxin polypeptide, and / or determining whether a clostridial neurotoxin polypeptide or a portion thereof comprised in a composition has an activity altering characteristic. The invention also relates to an isolated capture substrate for clostridial neurotoxins, uses thereof, therapeutic or cosmetic clostridial neurotoxin compositions, and methods of making them.

Description

Technical Field

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

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

[0003] In nature, clostridial neurotoxins (such as botulinum neurotoxins [BoNTs]) are initially synthesized as single - chain polypeptides and are modified by post - translational proteolytic cleavage to form two polypeptide chains linked by a disulfide bond. The cleavage occurs at specific cleavage sites, usually referred to as activation sites, which are located between the cysteine residues that form the inter - chain disulfide bond. This double - chain form is the active form of the toxin. The two chains are respectively called 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 contains an N - terminal translocation component (HN domain) and a C - terminal targeting component (HC domain). The cleavage site is located between the L chain and the translocation domain component. After the HC domain binds to the target neuron, the bound toxin enters the cell through endosomes, the HN domain transports the L chain across the endosomal membrane into the cytoplasm, and the L chain exerts protease function (also known as non - cytotoxic protease).

[0004] The non - cytotoxic protease acts by proteolytically cleaving intracellular transport proteins - SNARE proteins (such as SNAP - 25, VAMP or Syntaxin). SNARE is an abbreviation for Soluble NSF Attachment Receptor, where NSF refers to N - ethylmaleimide - sensitive factor. SNARE proteins are essential for intracellular vesicle fusion and are thus crucial for cells to secrete molecules through vesicular transport. The function of the protease is a zinc - dependent endopeptidase activity with high substrate specificity for SNARE proteins.

[0005] In the production and formulation of Clostridial neurotoxins for therapeutic and / or cosmetic purposes, it is necessary to accurately assess the activity of a given composition.

[0006] The mouse median lethal dose (LD50) assay has been the mainstay for assessing the activity of Clostridial neurotoxins. This assay simultaneously tests the action of all three domains of the toxin (i.e., binding, translocation, and protease). Specifically, it defines the median intraperitoneal injection toxin dose that kills 50% of the experimental animals at a specific time point after administration (usually 2 - 4 days) (activity is expressed in mouse LD50 units). However, unfortunately, the LD50 assay requires the use of a large number of animals. Additionally, LD50 units are not absolute measurements as they are not biological constants and are highly dependent on the assay conditions. In particular, the error associated with this assay can be as high as 60% between different testing institutions (Sesardic et al., 2003; Biologicals 31(4):265 - 276).

[0007] The mouse flaccid paralysis assay, also known as the "mouse ptosis assay", correlates the activity of Clostridial neurotoxins with the degree of abdominal bulge observed after subcutaneous injection of the toxin into the left inguinal region of the mouse, and the degree of paralysis is dose - related. This method has been proposed as an improvement over the mouse LD50 assay as it employs a humane endpoint. This assay is approximately 10 - fold more sensitive than the LD50 assay, uses sub - lethal doses of the toxin, and is faster than the LD50 assay as it can provide results within 24 to 48 hours, while a typical LD50 assay takes 72 to 96 hours. The results of this assay are in good agreement with the LD50 values (Sesardic et al., 1996; Pharmacology & Toxicology 78(5):283 - 8). Although this assay uses 20% of the number of animals used in the LD50 assay, it still requires the use of animals.

[0008] Assays such as the mouse / rat phrenic nerve - hemidiaphragm assay (based on the use of isolated nerve / muscle specimens) correlate the activity of Clostridial neurotoxins with the reduction in the amplitude of the twitch response after application of the specimen to a maintenance medium. The usual endpoint of this assay is the time required to observe a 50% reduction in amplitude. However, unfortunately, the hemidiaphragm assay (like the LD50 assay) results in the use of a large number of animals. Additionally, this assay requires highly trained professionals who are proficient in using complex and expensive equipment.

[0009] All of the above detection methods have certain drawbacks, especially animal welfare issues. In addition, none of the above detection methods are suitable for high-throughput detection. Therefore, there is a need in the art for alternative and / or improved clostridial neurotoxin detection methods. In addition, when performing cell-based detection, clostridial neurotoxins need to be formulated in cell growth medium. Therefore, this detection method is not suitable for characterizing clostridial neurotoxin preparations for therapeutic and / or cosmetic purposes, for example, for determining optimal excipients and their concentrations.

[0010] Cell-free assays known in the art typically involve incubating the tested clostridial neurotoxin with SNARE proteins and determining the amount of SNARE proteins cleaved by the tested clostridial neurotoxin using conventional techniques such as SDS-PAGE and protein immunoblotting. Alternatively, assays are known for detecting binding of clostridial neurotoxins to cell receptors, such as enzyme-linked immunosorbent assays (ELISAs), which use receptor substrates expressed in prokaryotic host cells (usually Escherichia coli), which lack the post-translational processing machinery of mammalian cells. Traditional cell-free assays may lack the sensitivity to detect small differences in activity. This may be particularly important when characterizing clostridial neurotoxin preparations for therapeutic and / or cosmetic purposes, as differences in activity between preparations with different excipients may be small but significant in therapeutic or cosmetic terms.

[0011] The use of clostridial neurotoxins in therapeutic and cosmetic treatments of humans and other mammals is expected to expand to a wider range of diseases and conditions that could benefit from the properties of these toxins. In view of this, there is an increasing need for large-scale production of clostridial neurotoxins and suitable formulations thereof.

[0012] The large-scale production of biotherapeutic agents, particularly Clostridial neurotoxins, is challenging, and unwanted polypeptide modifications and / or degradation can occur at multiple stages of the production process. If such unwanted polypeptide modifications and / or degradation are present, the activity per picogram of Clostridial neurotoxin can be significantly reduced compared to a composition that does not contain such modifications and / or degradation. One such unwanted modification is oxidation, which can occur during the cell expression, purification, bioprocessing, formulation, and / or storage of Clostridial neurotoxins. In fact, oxidation is one of the major degradation pathways of biotherapeutic agents. Oxidizing agents 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, Drug Research 31, 541-553). During bioprocessing and formulation, metal catalysts can originate from metal-contaminated buffers and / or metal contact surfaces, and it has been demonstrated that the oxidation of histidine and methionine residues catalyzed by metals leads to loss of activity, for example due to aggregation and / or precipitation of the oxidized polypeptide. Additionally, in large-scale production, oxidizing agents are commonly used for disinfection. Clostridial neurotoxins are large polypeptides with many surface-exposed amino acid residues, all of which can be oxidized.

[0013] For therapeutic and / or cosmetic purposes, the amount of unwanted polypeptide modifications and / or degradation present in a Clostridial neurotoxin composition must meet strictly defined criteria. Therefore, there is a need to sensitively and / or specifically determine whether such unwanted polypeptide modifications and / or degradation are present in the composition. As previously mentioned, traditional cell-free assays typically only test one aspect of neurotoxin activity, such as the L-chain proteolytic activity or the HC domain binding affinity. Therefore, the value of traditional assays in this regard may be limited because they do not provide a comprehensive and in-depth understanding of the nature of the Clostridial neurotoxin polypeptides present in the composition. For example, such assays cannot determine: the amount of free L-chain or H-chain contaminants present in the composition; and / or whether the Clostridial neurotoxin present in the composition is degraded and / or has undergone unwanted modifications (such as oxidation). In other words, traditional assays do not provide the technician with sufficient information to determine whether any activity observed in a Clostridial neurotoxin composition is due to unwanted polypeptide modifications and / or degradation.

[0014] The present invention overcomes one or more of the above problems.

[0015] Summary of the Invention

[0016] The present inventors have developed a novel cell-free method for determining the activity of Clostridium neurotoxins in a composition. Advantageously, the cell-free method may be particularly sensitive and / or specific, enabling the determination of small differences in activity between different compositions. This may be particularly advantageous when characterizing Clostridium neurotoxin preparations for therapeutic and / or cosmetic use, as the differences in activity between preparations with different excipients may be small but significant therapeutically or cosmetically.

[0017] Additionally or alternatively, the activity results obtained using the cell-free method of the present invention are surprisingly similar to those determined using cell-based methods, while avoiding the disadvantages of cell-based methods. In particular, unlike cell-based methods, the method of the present invention is suitable for characterizing Clostridium neurotoxin preparations for therapeutic and / or cosmetic use, such as for determining the optimal excipients and their concentrations, as the Clostridium neurotoxin being tested does not need to be formulated in a growth medium.

[0018] Advantageously, the cell-free method of the present invention can also be used to determine whether a composition has properties that alter activity (such as unwanted polypeptide modifications and / or degradation (such as oxidation)). For example, the cell-free method has been shown to accurately predict the loss of potency associated with the oxidation of Clostridium neurotoxins.

[0019] Furthermore, in embodiments where the step of determining L-chain activity is performed in an assay sample comprising dissociated L-chain polypeptide and a complex comprising a capture substrate and the Clostridium neurotoxin receptor-binding domain (HC domain), advantageously, the number of plates required for a given assay can be reduced. Thus, the cleavage assay can reduce waste and cost and is more suitable for high-throughput screening.

[0020] Detailed description of the present invention

[0021] In one aspect, the present invention provides a cell-free method for determining the activity of Clostridium neurotoxin in a composition comprising a Clostridium neurotoxin polypeptide, the method comprising:

[0022] (a) providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0023] (b) contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate;

[0024] (c) removing unbound Clostridium neurotoxin polypeptide;

[0025] (d) adding a reducing agent to dissociate the light chain (L-chain) polypeptide of the bound Clostridium neurotoxin polypeptide; and

[0026] (e) determining the amount of a cleavable substrate cleaved by the L-chain polypeptide in the assay sample, thereby determining the activity of the Clostridium neurotoxin in the composition.

[0027] On the one hand, the present invention provides a cell-free method for determining whether a composition contains a Clostridium neurotoxin polypeptide, the method comprising:

[0028] (a) providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0029] (b) contacting the capture substrate with the composition so that the Clostridium neurotoxin polypeptide binds to the capture substrate;

[0030] (c) removing any unbound Clostridium neurotoxin polypeptide;

[0031] (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of any bound Clostridium neurotoxin polypeptide; and

[0032] (e) determining that a cleavable substrate has been cleaved, thereby determining that the composition contains a Clostridium neurotoxin polypeptide, or determining that the cleavable substrate has not been cleaved, thereby determining that the composition does not contain a Clostridium neurotoxin polypeptide.

[0033] In the above aspect, when the composition does not contain a Clostridium neurotoxin polypeptide, there is no unbound Clostridium neurotoxin polypeptide in step (c), and there is no dissociated L chain polypeptide in step (d).

[0034] On the one hand, the present invention provides a cell-free method for measuring the Clostridium neurotoxin activity in a composition containing a Clostridium neurotoxin polypeptide, the method comprising:

[0035] (a) providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0036] (b) contacting the capture substrate with the composition so that the Clostridium neurotoxin polypeptide binds to the capture substrate;

[0037] (c) removing the unbound Clostridium neurotoxin polypeptide;

[0038] (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound Clostridium neurotoxin polypeptide, thereby providing an assay sample containing the dissociated L chain polypeptide and a complex containing the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain); and

[0039] (e) measuring the amount of a cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby measuring the Clostridium neurotoxin activity in the composition.

[0040] On the one hand, the present invention provides a cell-free method for determining whether a composition contains a Clostridium neurotoxin polypeptide, the method comprising:

[0041] (a) providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0042] (b) Contact the capture substrate with the composition so that the Clostridium neurotoxin polypeptide binds to the capture substrate;

[0043] (c) Remove any unbound Clostridium neurotoxin polypeptide;

[0044] (d) Add a reducing agent to dissociate the light chain (L-chain) polypeptide of any bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising any dissociated L-chain polypeptide and any complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain); and

[0045] (e) Determine that the cleavable substrate in the assay sample has been cleaved, thereby determining that the composition comprises a Clostridium neurotoxin polypeptide, or determine that the cleavable substrate in the assay sample has not been cleaved, thereby determining that the composition does not comprise a Clostridium neurotoxin polypeptide.

[0046] In the above aspect, when the composition does not comprise a Clostridium neurotoxin polypeptide, there is no unbound Clostridium neurotoxin polypeptide in step (c), no dissociated L-chain polypeptide in step (d), and no complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain) in step (d). In other words, when the composition does not comprise a Clostridium neurotoxin polypeptide, the assay sample may not comprise a dissociated L-chain polypeptide and a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain).

[0047] In one embodiment, the present invention provides a cell-free method for determining that a composition comprises a Clostridium neurotoxin polypeptide, the method comprising:

[0048] (a) Provide a capture substrate for the Clostridium neurotoxin polypeptide;

[0049] (b) Contact the capture substrate with the composition so that the Clostridium neurotoxin polypeptide binds to the capture substrate;

[0050] (c) Remove the unbound Clostridium neurotoxin polypeptide;

[0051] (d) Add a reducing agent to dissociate the light chain (L-chain) polypeptide of the bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L-chain polypeptide and a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain); and

[0052] (e) Determine that the cleavable substrate in the assay sample has been cleaved, thereby determining that the composition comprises a Clostridium neurotoxin polypeptide.

[0053] The cleavable substrate and the capture substrate are separate (e.g., there is no covalent linkage between them, such as being different polypeptides), and can be added before, during, or after the step of adding a reducing agent (preferably during the addition of the reducing agent). Thus, the cleavable substrate is preferably added simultaneously with the reducing agent.

[0054] For example, a cell-free method for determining the activity of a Clostridium neurotoxin polypeptide in a composition comprising the Clostridium neurotoxin polypeptide may include:

[0055] (a) providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0056] (b) contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate;

[0057] (c) removing unbound Clostridium neurotoxin polypeptide;

[0058] (d) adding a cleavable substrate and a reducing agent to dissociate the light chain (L chain) polypeptide of the bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L chain polypeptide and a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain); and

[0059] (e) determining the amount of the cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby determining the activity of the Clostridium neurotoxin in the composition.

[0060] 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 carried out in cells or cell lysates. Thus, the components of the present invention (e.g., the capture substrate and the cleavable substrate) are preferably recombinantly prepared and isolated from cells. Advantageously, this constitutes a purer system and may be easier to control.

[0061] In some embodiments, the components of the present invention (e.g., the capture substrate and the cleavable substrate) have been purified. The term "purified" can be used to refer to a "substantially pure" substance such as a polypeptide. Thus, in a composition, these components can account for at least 90%, 95%, 99%, or 99.9% of the total biological material present (e.g., fatty acids, nucleic acids, and / or polypeptides).

[0062] In practicing the methods of the invention, the capture substrate can be contacted with the composition under conditions suitable for binding of the Clostridium neurotoxin polypeptide, if present in the composition, to the capture substrate. For example, a capture substrate at 20 - 500 nM can be contacted with a Clostridium neurotoxin polypeptide at 0.1 - 150 pM. The capture substrate and the composition can be contacted for at least 5 minutes, 10 minutes, 30 minutes, or 45 minutes, preferably at least 50 minutes. The capture substrate and the composition can be contacted for ≤5 hours, ≤4 hours, ≤3 hours, ≤2 hours, or ≤1.5 hours, preferably ≤75 minutes. The capture substrate and the composition can be contacted for 5 minutes to 5 hours, 10 minutes to 4 hours, 10 minutes to 3 hours, 30 minutes to 2 hours, or 45 minutes to 75 minutes, preferably 50 minutes to 70 minutes (e.g., 60 minutes). During the contact, the capture substrate and the composition can be incubated at 20 - 45°C or 30 - 40°C, preferably at 35 - 40°C (e.g., 37°C). Thus, the capture substrate can be contacted for 50 - 70 minutes and incubated at 35 - 40°C during the contact. Preferably, the capture substrate and the composition are agitated during the contact, e.g., by using a plate shaker. The agitation speed can be 100 - 1000 rpm, 400 - 800 rpm, or 500 - 700 rpm, preferably 550 - 650 rpm (e.g., 600 rpm). In one embodiment, when the capture substrate is immobilized on a solid support, any supernatant can be removed before contacting the capture substrate with the composition.

[0063] The methods described herein can tolerate non-Clostridium neurotoxin components (e.g., buffers and / or excipients) that may be present in the composition. However, suitable components can 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 detergents (e.g., Tween-20, such as 0.025% - 0.1% (preferably 0.05%) Tween-20). Preferably, the composition contains at least BSA (e.g., 0.5 - 2% (preferably 1%) BSA). Advantageously, the use of BSA can increase the sensitivity of the method ( Figure 2 ), e.g., when compared to casein.

[0064] The method may include removing Clostridium neurotoxin polypeptides that have not bound to a capture substrate, such as Clostridium neurotoxin polypeptides that have not bound to the capture substrate through their HCC domain (such as the HC domain). Those skilled in the art will understand that any step of "removing unbound Clostridium neurotoxin polypeptides" may not necessarily remove all unbound Clostridium neurotoxin polypeptides. Thus, the method may include removing substantially all unbound Clostridium neurotoxin polypeptides. The term "substantially all unbound Clostridium neurotoxin polypeptides" may refer to at least 90%, 95%, 98%, or 99% of the unbound Clostridium neurotoxin polypeptides. Preferably, the method includes removing 100% of the unbound Clostridium neurotoxin polypeptides. The removal can be achieved by any suitable technique known in the art. In one embodiment, after the capture substrate immobilized on a solid support has been contacted with the composition according to the invention, the supernatant can be removed to thereby remove unbound Clostridium neurotoxin polypeptides. The removal can also additionally or alternatively include a washing step with a suitable buffer, optionally incubated at a suitable temperature and / or under suitable stirring conditions. For example, removing unbound Clostridium neurotoxin polypeptides can include a washing step with a composition (such as a washing buffer) containing the same non-Clostridium neurotoxin components as in the composition contacted with the capture substrate. Exemplary compositions for the washing step can include: a buffer (such as Dulbecco's phosphate buffered saline (DPBS)), a polypeptide (such as bovine serum albumin (BSA), such as 0.5 - 2% (preferably 1%) BSA), and a detergent (such as Tween-20, such as 0.025% - 0.1% (preferably 0.05%) Tween-20). The washing can be repeated if necessary. In one embodiment, after the capture substrate immobilized on a solid support has been contacted with the composition according to the invention, the supernatant can be removed, a washing buffer can be added, and then the washing buffer can be removed to thereby remove unbound Clostridium neurotoxin polypeptides.

[0065] The method can include adding a reducing agent to dissociate the light chain (L-chain) polypeptide of any Clostridium neurotoxin polypeptide bound to a capture substrate. Any suitable reducing agent can be used as long as it can reduce the disulfide bond between the L-chain and H-chain of the Clostridium neurotoxin without significantly reducing the activity of the L-chain of the Clostridium neurotoxin. Suitable reducing agents can be dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). The reducing agent can be present at any suitable concentration. However, exemplary concentrations of DTT include 2.5 - 7.5 mM, preferably 5 mM. The reducing agent can be contacted with any Clostridium 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 can be contacted with any Clostridium neurotoxin polypeptide bound to the capture substrate for ≤ 6 hours, ≤ 5 hours, or ≤ 4 hours, preferably ≤ 3 hours. The reducing agent can be contacted with any Clostridium neurotoxin polypeptide bound to the 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). During the contact, the reducing agent and any Clostridium neurotoxin polypeptide bound to the capture substrate can be incubated at 20 - 45 °C or 30 - 40 °C, preferably at 35 - 40 °C (e.g., 37 °C). Thus, the reducing agent can be contacted with any Clostridium neurotoxin polypeptide bound to the capture substrate for 2.5 - 3.5 hours and incubated at 35 - 40 °C during the contact. Preferably, the reducing agent and any Clostridium neurotoxin polypeptide bound to the capture substrate are stirred during the contact, for example, by using a plate shaker. The stirring speed can be 100 - 1000 rpm, 400 - 800 rpm, or 500 - 700 rpm, preferably 550 - 650 rpm (e.g., 600 rpm).

[0066] The method can include the step of separating the L-chain polypeptide from the complex of the capture substrate and the Clostridium neurotoxin HCC domain (such as the HC domain) (e.g., a complex comprising the capture substrate and the Clostridium neurotoxin HCC domain (such as the HC domain)) after contacting with the reducing agent. 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 (such as a vial or a well of a microplate). Then, the L-chain polypeptide can be contacted with a cleavable substrate.

[0067] However, most preferably, after contact with the reducing agent, the dissociated L-chain polypeptide does not separate from the complex of the capture substrate and the Clostridial neurotoxin HCC domain (such as the HC domain) (e.g., a complex comprising the capture substrate and the Clostridial neurotoxin HCC domain (such as the HC domain)) (e.g., does not transfer to a separate container such as a vial or a well of a microtiter plate). In other words, although the L-chain polypeptide is not part of the complex, it can remain in the solution containing the complex (e.g., the complex can be immobilized). Advantageously, the inventors have found that this step can significantly improve the sensitivity of the cell-free assay. This is especially the case when the cleavable substrate comprises the first luciferase domain described herein, a linker comprising the Clostridial neurotoxin cleavage site, and the second luciferase domain. Prior to this discovery, it was expected that non-specific background binding would be significant and the specificity of the method would be negatively affected. Unexpectedly, this was not the case, and it was found that using this method not only significantly improved the sensitivity of the assay, but also achieved this without significantly increasing the non-specific background, as detailed in this example. In view of this discovery, advantageously, the number of plates used in a given assay can also be reduced. Thus, such methods can reduce waste and cost and are more suitable for high-throughput screening. In these (most preferred) embodiments, the activity of the L-chain is evaluated in the presence of a complex of the capture substrate and the Clostridial neurotoxin HCC domain (such as the HC domain) (e.g., a complex comprising the capture substrate and the Clostridial neurotoxin HCC domain (such as the HC domain)). The combination comprising the dissociated L-chain polypeptide, the capture substrate, and the complex of the Clostridial neurotoxin HCC domain (such as the HC domain) (e.g., as described herein, e.g., a complex comprising the capture substrate and the Clostridial neurotoxin HCC domain (such as the HC domain)) may be referred to herein as an "assay sample". After the step of adding the reducing agent for dissociating the light chain (L-chain) polypeptide, the cleavable substrate can be added to the assay sample. However, preferably, the cleavable substrate is added simultaneously with the reducing agent (e.g., added simultaneously with the reducing agent). Thus, preferably, the assay sample is a combination comprising the dissociated L-chain polypeptide, the capture substrate, the complex of the Clostridial neurotoxin HCC domain (such as the HC domain), and the cleavable substrate. The cleavable substrate can be present in the assay sample for at least 30 minutes, 60 minutes, or 120 minutes, preferably at least 160 minutes. The cleavable substrate can be present in the assay sample for ≤6 hours, ≤5 hours, or ≤4 hours, preferably ≤3 hours. The cleavable substrate can 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 can be incubated at 20 - 45 °C or 30 - 40 °C during the contact, preferably at 35 - 40 °C (e.g., 37 °C).Thus, the cleavable substrate can be present in the assay sample for 2.5 - 3.5 hours, and the assay sample containing the cleavable substrate is incubated at 35 - 40 °C during contact. Preferably, the assay sample containing the cleavable substrate is stirred during contact, for example, by using a plate shaker. The stirring speed can be 100 - 1000 rpm, 400 - 800 rpm, or 500 - 700 rpm, preferably 550 - 650 rpm (e.g., 600 rpm).

[0068] A method in which the L-chain polypeptide does not dissociate from the complex of the capture substrate and the Clostridium neurotoxin HCC domain (such as the HC domain) after contact with a reducing agent may be more practical, easier to perform, and / or may have lower variability (assay noise). Advantageously, the increased sensitivity (e.g., measured by the EC50 value) may enable testing of compositions containing extremely small amounts of Clostridium neurotoxin polypeptides. Such compositions can be pharmaceutical compositions produced by diluting a drug substance (obtained after purification of the Clostridium neurotoxin polypeptide). Any of these advantages may be apparent when compared to an equivalent method in which the L-chain polypeptide is separated from the complex of the capture substrate and the Clostridium neurotoxin HCC domain (such as the HC domain) (e.g., and transferred to a separate vial / well of a microplate) after contact with a reducing agent.

[0069] The capture substrate described herein (e.g., in the context of the methods of the invention, the use of the capture substrate, or the isolated capture substrate) can be part of a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain or HC domain). The complexation can occur after the contact step described herein. The complex can comprise the capture substrate and a full-length Clostridium neurotoxin polypeptide comprising or consisting of the light and heavy chains of the Clostridium neurotoxin. The complex preferably comprises the capture substrate and the heavy chain of the Clostridium neurotoxin (e.g., comprising or consisting of the translocation domain [HN domain] and the HC domain). When a reducing agent is added to the complex comprising the capture substrate and the Clostridium neurotoxin polypeptide, the complex preferably lacks the light chain of the Clostridium neurotoxin. In the complex, the HC domain or HCC domain of the heavy chain preferably binds to the capture substrate (e.g., by non-covalent interactions).

[0070] Thus, the assay sample described herein preferably comprises dissociated L-chain polypeptide (e.g., if present) and a complex comprising the capture substrate and the heavy chain of the Clostridium neurotoxin. The heavy chain preferably comprises or consists of the translocation domain (HN domain) and the HC domain of the Clostridium neurotoxin.

[0071] The cleavable substrate can be used at a concentration of 1 - 1000 nM, such as 10 - 500 nM, preferably 50 - 150 nM (e.g., 100 nM) in the methods of the invention (e.g., present in the assay sample).

[0072] The cleavable substrate is preferably not directly or indirectly immobilized on a solid support (e.g., on a plate, such as in a well of a plate). By avoiding such immobilization (e.g., by providing the free cleavable substrate in solution), the sensitivity can be increased. In other words, directly or indirectly immobilizing the cleavable substrate on a solid support (e.g., on a plate, such as in a well of a plate) may reduce the sensitivity. The reduction in sensitivity may be due to a decrease in the total number of available binding sites on the solid support (e.g., on a plate, such as in a well of a plate).

[0073] The capture substrate can be any substrate capable of binding to a Clostridium neurotoxin. Suitably, the capture substrate for use in the methods of the invention is selected according to the Clostridium neurotoxin being assayed and / or the HCC domain of the Clostridium neurotoxin (e.g., the HCC domain). The capture substrate can comprise a Clostridium neurotoxin receptor polypeptide or a ganglioside to which the Clostridium neurotoxin binds. The methods of the invention can employ a combination of a capture substrate comprising a Clostridium neurotoxin receptor polypeptide and a capture substrate comprising a ganglioside. The capture substrate comprising a Clostridium neurotoxin receptor polypeptide and the capture substrate comprising a ganglioside can be complexed (e.g., by non-covalent interactions). Such complexing can occur before, during, or after contact and / or binding with the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the HC or HCC domain). In some cases, the capture substrate comprising a Clostridium neurotoxin receptor polypeptide can be directly or indirectly immobilized on a solid support, and the capture substrate comprising a ganglioside can be added thereto. Then, the capture substrate comprising a ganglioside can form a complex with the immobilized capture substrate comprising a Clostridium neurotoxin receptor polypeptide. However, preferably the capture substrate comprises a Clostridium neurotoxin receptor polypeptide. Thus, in some embodiments, it is preferred that the method does not use a capture substrate comprising (or consisting of) a ganglioside (e.g., GT1b). In fact, when the Clostridium neurotoxin receptor polypeptide used (e.g., the extracellular portion of the neuronal Clostridium neurotoxin receptor polypeptide) is SV2c and the ganglioside is GT1b, the combined use of a capture substrate comprising a Clostridium neurotoxin receptor polypeptide (e.g., comprising the extracellular portion of the neuronal Clostridium neurotoxin receptor polypeptide) and a capture substrate comprising a ganglioside has been shown to reduce the sensitivity of the method. In contrast, when the Clostridium neurotoxin receptor polypeptide used (e.g., the extracellular portion of the neuronal Clostridium neurotoxin receptor polypeptide) is SYT-I, the combined use of a capture substrate comprising a Clostridium neurotoxin receptor polypeptide (e.g., comprising the extracellular portion of the neuronal Clostridium neurotoxin receptor polypeptide) and a capture substrate comprising a ganglioside has been shown to increase the sensitivity of the method. Thus, preferably, when the capture substrate comprises SYT-I (e.g., its extracellular portion), a capture substrate comprising a ganglioside (preferably GT1b) can also be used. The capture substrate may be particularly advantageous when the Clostridium neurotoxin comprises a modified BoNT / B HCC domain as described herein.

[0074] In some instances herein, "capture substrates" are mentioned. However, this is to indicate that there is more than one capture substrate molecule when implementing the method. This does not necessarily mean that there are two or more different types of capture substrates, although this is also included. Thus, the capture substrate can be of one type (e.g., all capture substrates contain a receptor polypeptide or ganglioside for the first Clostridium neurotoxin) or multiple types (e.g., a portion of the capture substrates contain a receptor polypeptide for the first type of Clostridium neurotoxin, while a portion of the capture substrates contain a receptor polypeptide for a second different Clostridium neurotoxin). Preferably, the capture substrate is of one type.

[0075] Gangliosides are oligosaccharide-based ceramides derived from lactosylceramide and contain sialic acid residues such as N-acetylneuraminic acid ('NANA' or 'SA' or 'Neu5Ac' or 'NeuAc'). In some embodiments, the sialic acid moiety is N-glycolylneuraminic acid (Neu5Gc), or a Neu5Ac analogue in which the amine group is replaced by OH (3-deoxy-D-glycero-D-galacto-nonulosonic acid, abbreviated as 'KDN'). Gangliosides are defined by the nomenclature system proposed by Svennerholm, where M, D, T, and Q refer to mono-, di-, tri-, and tetra-sialic acid gangliosides, respectively, and the numbers 1, 2, 3, etc. refer to the migration order of the gangliosides on thin layer chromatography. For example, the migration order of monosialic acid gangliosides is GM3 > GM2 > GM1. To denote variations within the basic structure, additional terms are added, such as GM1a, GD1b, etc. Glycosphingolipids with 0, 1, 2, and 3 sialic acid residues linked to the internal galactose unit are called asialogangliosides (or 0-), a-, b-, and c-series gangliosides, respectively, while gangliosides with sialic acid residues linked to the internal N-galactosamine residue are classified as α-series gangliosides. The biosynthetic pathways of 0-, a-, b-, and c-series gangliosides involve the sequential activities of sialyltransferases and glycosyltransferases, as described by Ledeen et al. in 2015 (Ledeen, Robert W., and Gusheng Wu. "The multi-tasked life of GM1 ganglioside, a true factotum of nature." Trends in biochemical sciences 40.7 (2015): 407-418). Each series is further sialylated at different positions in the carbohydrate chain, which can give rise to increasingly complex and diverse products, such as α-series gangliosides with sialic acid residues linked to the internal N-acetylgalactosamine residue.

[0076] In the cellular environment, gangliosides are transferred to the outer leaflet of the plasma 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 plasma membrane and the oligosaccharide located on the extracellular surface, where they provide recognition sites for extracellular molecules or the surfaces of adjacent cells. The sialoglycan components of gangliosides extend from the cell surface and can participate in intermolecular interactions. They function by recognizing specific molecules on the cell surface and regulating the activity of proteins in the plasma membrane. Gangliosides also specifically bind to viruses and bacterial toxins, such as botulinum, tetanus, and cholera toxins. For example, the specific cell surface receptor for cholera toxin is ganglioside GM1 (or GM1a): Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer.

[0077] BoNTs have two independent binding regions in the HCC domain for binding gangliosides and neuronal protein receptors, respectively. BoNT / A, / B, / E, / F, and / G have a conserved ganglioside binding site in the HCC domain, which is composed of the "E(Q)…H(K)…SXWY…G" motif, while BoNT / C, / D, and / DC show 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 only bind to gangliosides with a 2,3-linked N-acetylneuraminic acid residue (designated as Sia5) linked to Gal4 in the oligosaccharide core, while the corresponding ganglioside binding pocket on tetanus toxin (TeNT) can also bind GM1a, a ganglioside lacking the Sia5 sugar residue. It has been shown that introducing the H1241K mutation into recombinant BoNT / F can confer its GM1 binding ability (Benson, Marc A., et al. "Unique ganglioside recognition strategies for clostridial neurotoxins." Journal of Biological Chemistry 286.39 (2011): 34015-34022). BoNT / D has been found to bind 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).

[0078] The ganglioside can be GM1 (such as GM1a or GM1b), GM2, GM3 (such as NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1b, GQ1b, GD2, or GD3.

[0079] Combined with data from ganglioside-deficient mice and biochemical analysis, BoNT / A, E, F, and G show a preference for the terminal NAcGal-Gal-NAcNeu moiety present in GD1a and GT1b, while BoNT / B, C, D, and TeNT require the disialic acid motif found in GD1b, GT1b, and GQ1b.

[0080] Thus, gangliosides can contain the terminal NAcGal-Gal-NAcNeu moiety or the disialic acid motif. 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.

[0081] Suitable gangliosides can be selected from those described in WO 2018 / 060351.

[0082] The capture substrate containing ganglioside can be used at a concentration of 0.1 - 500 μM, such as 1 - 250 μM, 1 - 100 μM, 10 - 50 μM, or 20 - 30 μM, such as 24.1 μM.

[0083] The capture substrate preferably contains a Clostridial neurotoxin receptor polypeptide. The term "Clostridial neurotoxin receptor polypeptide" can encompass the full-length Clostridial neurotoxin receptor polypeptide or a portion thereof. The Clostridial neurotoxin receptor polypeptide can be a neuronal Clostridial neurotoxin receptor polypeptide, such as the 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 the neuronal Clostridial neurotoxin receptor, more preferably comprises (or consists of) the extracellular portion of the neuronal botulinum neurotoxin receptor.

[0084] Advantageously, by using a capture substrate containing a Clostridial neurotoxin receptor polypeptide (and optionally a capture substrate containing ganglioside), the method of the present invention can be improved. In particular, the method can better determine whether a composition has properties that alter activity (such as unwanted polypeptide modifications and / or degradation (such as oxidation)). For example, compared to an antibody capture substrate, a capture substrate containing a Clostridial neurotoxin receptor polypeptide (and optionally a capture substrate containing ganglioside) may be more sensitive to properties of Clostridial neurotoxins that alter activity (such as unwanted polypeptide modifications and / or degradation (such as oxidation)). Antibodies may not be able to detect or distinguish Clostridial neurotoxins that contain or do not contain properties that alter activity (such as unwanted polypeptide modifications and / or degradation (such as oxidation)).

[0085] The capture substrate may comprise non - extracellular portions of the Clostridium botulinum neurotoxin receptor, but typically only those non - extracellular portions that are soluble in aqueous solution. In fact, it is preferred that no non - extracellular portions are present, for example to improve solubility and thus facilitate recombinant production and / or handling.

[0086] The Clostridium botulinum neurotoxin receptor polypeptide may be a human Clostridium botulinum neurotoxin receptor polypeptide (such as its extracellular portion). The Clostridium botulinum 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 Clostridium botulinum 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 Clostridium botulinum neurotoxin receptor polypeptide (such as its extracellular portion) may be the same as the wild - type (such as human) polypeptide sequence. In another embodiment, the polypeptide of the Clostridium botulinum neurotoxin receptor polypeptide (such as its extracellular portion) may comprise one or more modifications compared to the wild - type (such as human) polypeptide sequence. Preferably, when the Clostridium botulinum neurotoxin receptor polypeptide (such as its extracellular portion) is human SYT - II, the polypeptide sequence comprises a substitution of leucine 51 to phenylalanine as shown in SEQ ID NO:21 (L51F).

[0087] When the Clostridium botulinum neurotoxin composition comprises a Clostridium botulinum neurotoxin containing the BoNT / A HCC (preferably HC) domain, the capture substrate may comprise the extracellular portion of SV2c, SV2a or SV2b, preferably SV2c. There are three SV2 isoforms found in humans, namely SV2a, SV2b and SV2c, however BoNT / A has the highest affinity for SV2c. BoNT / A can specifically bind to the luminal domain 4 of SV2 (SV2 - LD4) through direct backbone - backbone interactions between the β - strand of SV2 - LD4 and the β - strand of BoNT HC, as well as through interactions with the glycan linked to N559.

[0088] When the Clostridium botulinum neurotoxin composition comprises a Clostridium botulinum neurotoxin containing the BoNT / B HCC (preferably HC) domain, the capture substrate may comprise the extracellular portion of SYT - II or SYT - I, preferably SYT - II. Although BoNT / B binds to both SYT - I and SYT - II, SYT - II is believed to be more abundant on human neuronal cells. Thus, SYT - II may be the preferred Clostridium botulinum neurotoxin receptor polypeptide for the present invention.

[0089] When the clostridial neurotoxin composition comprises a clostridial neurotoxin containing a BoNT / D HCC (preferably HC) domain, the capture substrate can comprise the extracellular portion of SV2c, SV2a or SV2b.

[0090] When the clostridial neurotoxin composition comprises a clostridial neurotoxin containing a BoNT / E HCC (preferably HC) domain, the capture substrate can comprise the extracellular portion of SV2b or SV2c.

[0091] When the clostridial neurotoxin composition comprises a clostridial neurotoxin containing a BoNT / F HCC (preferably HC) domain, the capture substrate can comprise the extracellular portion of SV2c, SV2a or SV2b.

[0092] When the clostridial neurotoxin composition comprises a clostridial neurotoxin containing a BoNT / G HCC (preferably HC) domain, the capture substrate can comprise the extracellular portion of SYT-II or SYT-I, preferably SYT-II.

[0093] The extracellular portion of SYT-II can 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.

[0094] The extracellular portion of human SYT-II can refer to amino acid residues 1-61 of full-length human SYT-II. The residue positions can be determined by alignment with the amino acid sequence of SEQ ID NO:23.

[0095] Full-length human SYT-II can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:23. In one embodiment, full-length human SYT-II can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:23. Preferably, full-length human SYT-II can comprise SEQ ID NO:23. Full-length human SYT-II can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:23. In one embodiment, full-length human SYT-II can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:23. Preferably, full-length human SYT-II can consist of SEQ ID NO:23.

[0096] The extracellular portion of mouse SYT-II can refer to amino acid residues 1-64 of full-length mouse SYT-II. The residue positions can be determined by alignment with the amino acid sequence of SEQ ID NO:24.

[0097] Full-length murine SYT-II can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:24. In one embodiment, full-length murine SYT-II can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:24. Preferably, full-length murine SYT-II can comprise SEQ ID NO:24. Full-length murine SYT-II can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:24. In one embodiment, full-length murine SYT-II can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:24. Preferably, full-length murine SYT-II can consist of SEQ ID NO:24.

[0098] Thus, a capture substrate comprising the extracellular portion of murine SYT-II can comprise a polypeptide sequence having 70% sequence identity with SEQ ID NO:22. In one embodiment, a capture substrate comprising the extracellular portion of murine SYT-II can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:22. Preferably, a capture substrate comprising the extracellular portion of murine SYT-II can comprise SEQ ID NO:22. Thus, a capture substrate comprising the extracellular portion of murine SYT-II can consist of a polypeptide sequence having 70% sequence identity with SEQ ID NO:22. In one embodiment, a capture substrate comprising the extracellular portion of murine SYT-II can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:22. Preferably, a capture substrate comprising the extracellular portion of murine SYT-II can consist of SEQ ID NO:22.

[0099] SYT-II is preferably human SYT-II.

[0100] The extracellular portion of human SYT-II can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:19. In one embodiment, the extracellular portion of human SYT-II can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:19. Preferably, the extracellular portion of human SYT-II can comprise SEQ ID NO:19. The extracellular portion of human SYT-II can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:19. In one embodiment, the extracellular portion of human SYT-II can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:19. Preferably, the extracellular portion of human SYT-II can consist of SEQ ID NO:19.

[0101] Accordingly, a capture substrate comprising the extracellular portion of human SYT-II can comprise a polypeptide sequence having 70% sequence identity with SEQ ID NO:18. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-II can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:18. Preferably, a capture substrate comprising the extracellular portion of human SYT-II can comprise SEQ ID NO:18. Accordingly, a capture substrate comprising the extracellular portion of human SYT-II can consist of a polypeptide sequence having 70% sequence identity with SEQ ID NO:18. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-II can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:18. Preferably, a capture substrate comprising the extracellular portion of human SYT-II can consist of SEQ ID NO:18.

[0102] As noted above, preferably when SYT-II is human SYT-II, the polypeptide sequence contains a substitution of leucine 51 to phenylalanine (L51F). In other words, human SYT-II is preferably a modified human SYT-II. The location of residue 51 can be determined by alignment with the amino acid sequence of SEQ ID NO:19. The extracellular portion of the modified human SYT-II can contain the L51F substitution and a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:21. In one embodiment, the extracellular portion of the modified human SYT-II can contain the L51F substitution and a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:21. Preferably, the extracellular portion of the modified human SYT-II can contain SEQ ID NO:21. The extracellular portion of the modified human SYT-II can contain the L51F substitution and consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:21. In one embodiment, the extracellular portion of the modified human SYT-II can contain the L51F substitution and consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:21. Preferably, the extracellular portion of the modified human SYT-II can contain the L51F substitution and consist of SEQ ID NO:21. The extracellular portion of the modified human SYT-II can contain the L51F substitution and be encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:50. In one embodiment, the extracellular portion of the modified human SYT-II can contain the L51F substitution and be encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:50. Preferably, the extracellular portion of the modified human SYT-II can contain the L51F substitution and be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO:50.

[0103] Thus, a capture substrate comprising a modified human SYT-II extracellular portion can 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 a modified human SYT-II extracellular portion can 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 a modified human SYT-II extracellular portion can comprise SEQ ID NO:20. Thus, a capture substrate comprising a modified human SYT-II extracellular portion can comprise an L51F substitution and consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:20. In one embodiment, a capture substrate comprising a modified human SYT-II extracellular portion can comprise an L51F substitution and 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 a modified human SYT-II extracellular portion can comprise an L51F substitution and consist of SEQ ID NO:20.

[0104] The extracellular portion of SYT-I can comprise the extracellular portion of amino acid residues 1-57 (e.g., amino acid residues 33-54) of full-length SYT-I.

[0105] The extracellular portion of murine SYT-I can comprise the extracellular portion of amino acid residues 1-59 of full-length murine SYT-I. The extracellular portion of murine SYT-I can refer to amino acid residues 1-59 of full-length murine SYT-I. The residue positions can be determined by alignment with the amino acid sequence of SEQ ID NO:82.

[0106] The extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-59 of SEQ ID NO:82. In one embodiment, the extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with amino acid residues 1-59 of SEQ ID NO:82. Preferably, the extracellular portion of murine SYT-I can comprise amino acid residues 1-59 of SEQ ID NO:82. The extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-59 of SEQ ID NO:82. In one embodiment, the extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with amino acid residues 1-59 of SEQ ID NO:82. Preferably, the extracellular portion of murine SYT-I can consist of amino acid residues 1-59 of SEQ ID NO:82.

[0107] Full-length murine SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:82. In one embodiment, full-length murine SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:82. Preferably, full-length murine SYT-I can comprise SEQ ID NO:82. Full-length murine SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:82. In one embodiment, full-length murine SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:82. Preferably, full-length murine SYT-I can consist of SEQ ID NO:82.

[0108] The extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:83. In one embodiment, the extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:83. Preferably, the extracellular portion of murine SYT-I can comprise SEQ ID NO:83. The extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:83. In one embodiment, the extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:83. Preferably, the extracellular portion of murine SYT-I can consist of SEQ ID NO:83.

[0109] Accordingly, a capture substrate comprising the extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:84. In one embodiment, a capture substrate comprising the extracellular portion of murine SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:84. Preferably, a capture substrate comprising the extracellular portion of murine SYT-I can comprise SEQ ID NO:84. Accordingly, a capture substrate comprising the extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:84. In one embodiment, a capture substrate comprising the extracellular portion of murine SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:84. Preferably, a capture substrate comprising the extracellular portion of murine SYT-I can consist of SEQ ID NO:84.

[0110] SYT-I is preferably human SYT-I. The extracellular portion of human SYT-I can refer to amino acid residues 1-57 (e.g., amino acid residues 33-54) of full-length human SYT-I. Accordingly, the extracellular portion of human SYT-I can comprise amino acid residues 1-57 (e.g., amino acid residues 33-54) of full-length human SYT-I. The location of the residues can be determined by alignment with the amino acid sequence of SEQ ID NO:28.

[0111] The extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54). In one embodiment, the extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54). Preferably, the extracellular portion of human SYT-I can comprise amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54). The extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54). 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 with amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54). Preferably, the extracellular portion of human SYT-I can consist of amino acid residues 1-57 of SEQ ID NO:28 (e.g., amino acid residues 33-54).

[0112] Preferably, the extracellular portion of SYT-I can 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 can refer to amino acid residues 1-60 (e.g., amino acid residues 33-54) of full-length human SYT-I. The location of the residues can be determined by alignment with the amino acid sequence of SEQ ID NO:28.

[0113] The extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54). In one embodiment, the extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54). Preferably, the extracellular portion of human SYT-I can comprise amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54). The extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54). 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 with amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54). Preferably, the extracellular portion of human SYT-I can consist of amino acid residues 1-60 of SEQ ID NO:28 (e.g., amino acid residues 33-54).

[0114] Full-length human SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:28. In one embodiment, full-length human SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:28. Preferably, full-length human SYT-I can comprise SEQ ID NO:28. Full-length human SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:28. In one embodiment, full-length human SYT-I can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:28. Preferably, full-length human SYT-I can consist of SEQ ID NO:28.

[0115] The extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:80. In one embodiment, the extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:80. Preferably, the extracellular portion of human SYT-I can comprise SEQ ID NO:80. The extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:80. 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 with SEQ ID NO:80. Preferably, the extracellular portion of human SYT-I can consist of SEQ ID NO:80.

[0116] Thus, a capture substrate comprising the extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:81. In one embodiment, a capture substrate comprising the extracellular portion of human SYT-I can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:81. Preferably, a capture substrate comprising the extracellular portion of human SYT-I can comprise SEQ ID NO:81. Thus, a capture substrate comprising the extracellular portion of human SYT-I can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:81. In one embodiment, a capture substrate comprising 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 with SEQ ID NO:81. Preferably, a capture substrate comprising the extracellular portion of human SYT-I can consist of SEQ ID NO:81.

[0117] The extracellular portion of SV2a can comprise the extracellular portion of full-length SV2a at amino acid residues 469 - 599 (e.g., amino acid residues 469 - 598 or 469 - 595). The extracellular portion of human SV2a can refer to amino acid residues 469 - 599 (e.g., amino acid residues 469 - 598 or 469 - 595) of full-length human SV2a. The positioning of the residues can be determined by alignment with the amino acid sequence of SEQ ID NO:25.

[0118] Full-length human SV2a may comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO:25. Preferably, full-length human SV2a may consist of SEQ ID NO:25.

[0119] The extracellular portion of SV2b may comprise 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 with the amino acid sequence of SEQID NO:26.

[0120] Full-length human SV2b may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:26. In one embodiment, full-length human SV2b may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:26. Preferably, full-length human SV2b may comprise SEQ ID NO:26. Full-length human SV2b may consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:26. In one embodiment, full-length human SV2b may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:26. Preferably, full-length human SV2b may consist of SEQ ID NO:26.

[0121] The extracellular portion of SV2c can comprise the extracellular portion of full-length SV2c of amino acid residues 400-600, 450-590, 460-580, 470-570 or 500-570. The extracellular portion of human SV2c can comprise at least the 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 comprising at least luminal domain 4 can comprise 473-567 of full-length human SV2c. The residue positions can be determined by alignment with the amino acid sequence of SEQ ID NO:27.

[0122] Full-length human SV2c can comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO:27. Preferably, full-length human SV2c can consist of SEQ ID NO:27.

[0123] The extracellular portion of human SV2c can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:30. In one embodiment, the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:30. Preferably, the extracellular portion of human SV2c can comprise SEQ ID NO:30. The extracellular portion of human SV2c can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:30. In one embodiment, 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 with SEQ ID NO:30. Preferably, the extracellular portion of human SV2c can consist of SEQ ID NO:30.

[0124] Thus, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO:29. Preferably, a capture substrate comprising the extracellular portion of human SV2c can consist of SEQ ID NO:29.

[0125] A capture substrate comprising the extracellular portion of human SV2c can be encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:51. In one embodiment, a capture substrate comprising the extracellular portion of human SV2c can be encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with 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.

[0126] Thus, a capture substrate comprising the extracellular portion of human SV2c can comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO:40. Preferably, a capture substrate comprising the extracellular portion of human SV2c can consist of SEQ ID NO:40.

[0127] SEQ ID NO:40 is most preferred in capture substrates comprising the extracellular portion of human SV2c.

[0128] Before performing the method of the present invention, the capture substrate can be immobilized on a solid support. This immobilization can be achieved using any method known in the art, preferably by means of a tag present on the capture substrate (such as a GST tag) and a suitable binding partner present on the solid support (such as glutathione or its derivatives). Other suitable tags are also known in the art, such as His tags. Suitable solid supports are known in the art. Advantageously, this facilitates washing of any capture substrate - Clostridial neurotoxin complex and easy removal of the supernatant while minimizing loss of such complexes. The solid support can be a plastic support, such as the inner surface (in normal use) of a plate (such as a multi-well plate, such as a microtiter plate), a column or a tube (such as a microcentrifuge tube). Preferably, the solid support is the inner surface (in normal use) of a multi-well plate. The multi-well plate facilitates handling of a large number of samples and is suitable for use in high-throughput techniques.

[0129] The capture substrate of the present invention can comprise post-translational modifications, such as glycosylation. The glycosylation is preferably N-linked glycosylation. N-linked glycosylation preferably occurs at asparagine residues of the Clostridial neurotoxin receptor polypeptide comprised in the capture substrate. Preferably, the Clostridial neurotoxin receptor polypeptide comprises the extracellular portion of SV2c glycosylated at N559. The location of the residue can be determined by alignment with the amino acid sequence of SEQ ID NO:27. The glycosylation can comprise Man-5 glycan, G0f glycan, G1f glycan or G2f glycan. The glycosylation preferably further comprises N-acetylglucosamine (GlcNAc), such as G0f-GlcNAc.

[0130] The capture substrate comprising post-translational modifications can be produced by expressing a nucleic acid encoding said capture substrate in a suitable host cell. The capture substrate can then be isolated from the host cell using standard techniques. For example, the host cell can be a mammalian cell (such as from a mammalian cell line), preferably a human host cell, such as a human cell line host cell. These host cells can be particularly advantageous when assaying Clostridial neurotoxin compositions for human therapeutic and / or cosmetic use, since the capture substrate preferably exhibits more similar post-translational modifications to those found in humans compared to capture substrates produced by alternative methods, such as capture substrates produced by prokaryotic (such as E. coli) expression.

[0131] Advantageously, when the capture substrate comprises SV2c with glycosylation (such as its extracellular portion), the capture substrate can better (such as more sensitively) distinguish between oxidized and unoxidized Clostridial neurotoxin polypeptides compared to an equivalent capture substrate that does not comprise such glycosylation.

[0132] Suitable host cells for generating capture substrates containing post-translational modifications can be HEK293 cells, Chinese hamster ovary (CHO) cells, or Drosophila cells, preferably HEK293 cells. In some embodiments, a Drosophila expression system known in the art can be used to express the nucleic acid encoding the capture substrate.

[0133] A nucleic acid can comprise a sequence having at least 70% sequence identity to SEQ ID NO:50. In one embodiment, a nucleic acid can comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:50. Preferably, a nucleic acid can comprise (more preferably consist of) SEQ ID NO:50.

[0134] A nucleic acid can comprise a sequence having at least 70% sequence identity to SEQ ID NO:51. In one embodiment, a nucleic acid can comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO:51. Preferably, a nucleic acid can comprise (more preferably consist of) SEQ ID NO:51.

[0135] The cleavable substrate for use in the present invention is any substrate that can be cleaved by a Clostridium neurotoxin. Suitably, the cleavable substrate is selected according to the Clostridium neurotoxin and / or the L chain of the Clostridium neurotoxin being assayed. The cleavable substrate comprises a Clostridium neurotoxin cleavage site, such as a cleavable bond of a SNARE. Suitable cleavable substrates and their components can be those described in WO 2018 / 075783A2, which is incorporated herein by reference.

[0136] The Clostridium neurotoxin cleavage site can further comprise several amino acids at the N-terminus and / or C-terminus of the cleavable bond in the SNARE, preferably both at the N-terminus and C-terminus. Thus, the Clostridium neurotoxin cleavage site can comprise at least the amino acid residues P3P2P1P1’P2’P3’ of the SNARE, where P1 and P1’ are the residues located at the N-terminus and C-terminus of the cleavable bond (cleavable peptide bond) cleaved by the Clostridium neurotoxin, such as Gln197 and Arg198 of SNAP-25 cleaved by the L chain of BoNT / A.

[0137] Thus, the Clostridial neurotoxin cleavage site may further comprise at least 2 (e.g., the cleavage site comprises at least P3P2P1P1’), 5, 10, 15, 20, 30, 40, 50 or 100 amino acid residues at the N-terminus of the amino acid residues that form a cleavable bond in the SNARE. The Clostridial neurotoxin cleavage site may further comprise at least 2 (e.g., the cleavage site comprises at least P1P1’P2’P3’), 5, 10, 15, 20, 25, 30, 40, 50 or 100 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE. For example, the Clostridial neurotoxin cleavage site may further comprise: up to 5 amino acid residues at the N-terminus and up to 5 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE; up to 10 amino acid residues at the N-terminus and up to 10 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE; up to 25 amino acid residues at the N-terminus and up to 25 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE; up to 50 amino acid residues at the N-terminus and up to 50 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE; or up to 100 amino acid residues at the N-terminus and up to 100 amino acid residues at the C-terminus of the amino acid residues that form a cleavable bond in the SNARE. For the avoidance of doubt, the term “up to” as used in this context includes the indicated number, e.g., “up to 5 amino acid residues” includes “5 amino acid residues”.

[0138] Preferably, the Clostridial neurotoxin cleavage site comprises 8 amino acid residues at the C-terminus and up to 55 amino acid residues at the N-terminus of the amino acid residues that form a cleavable bond in the SNARE (e.g., the Clostridial neurotoxin cleavage site may comprise up to 65 amino acid portions of the SNARE).

[0139] The Clostridial neurotoxin cleavage site containing a SNARE cleavable bond can 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 the SNAREs described herein, preferably at least 55 amino acid residues of the SNAREs described herein. The Clostridial neurotoxin cleavage site containing a SNARE cleavable bond can comprise (or consist of) ≤200, ≤150, ≤100, ≤90, ≤80, ≤70, ≤60, ≤50, ≤40, ≤30, ≤20, ≤10, ≤9, ≤8 or ≤7 amino acid residues of the SNAREs described herein. For example, the Clostridial neurotoxin cleavage site containing a SNARE cleavable bond can comprise (or consist of) 6-200, 10-150, 20-100, 50-85, 55-80 or 60-75 amino acid residues of the SNAREs described herein. Preferably, the Clostridial neurotoxin cleavage site containing a SNARE cleavable bond can comprise (or consist of) 60-70 amino acid residues of the SNAREs described herein.

[0140] Thus, in some embodiments, the Clostridial neurotoxin cleavage site is from a SNARE (e.g., is a fragment of a SNARE). The SNARE can be SNAP-25, Vesicle-Associated Membrane Protein (VAMP) or Syntaxin. Thus, the Clostridial neurotoxin cleavage site can comprise a cleavable bond of SNAP-25, Vesicle-Associated Membrane Protein (VAMP) or Syntaxin.

[0141] The SNARE for use in the present invention is preferably SNAP-25. Examples of SNAP-25 polypeptides include NCBI Gene ID: 6616, NCBI Reference Sequence: NP_570824.1 (human SNAP25b), and NCBI Reference Sequence: NP_112253.1 (rat SNAP25b). Preferably, SNAP-25 is human SNAP-25. SNAP-25 may comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with SEQ ID NO:4. In one embodiment, SNAP-25 may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:4. Preferably, SNAP-25 may consist of SEQ ID NO:4.

[0142] The cleavable bond of SNAP-25 (SEQ ID NO:4) cleaved by the L chain of BoNT / A may be Gln197-Arg198. The cleavable bond of SNAP-25 (SEQ ID NO:4) cleaved by the L chain of BoNT / C1 may be Arg198-Ala199. The cleavable bond of SNAP-25 (SEQ ID NO:4) cleaved by the L chain of BoNT / E may be Arg180-Ile181.

[0143] The Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable 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, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may comprise SEQ ID NO: 4. The Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable 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, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may consist of SEQ ID NO: 4. Preferably, when the Clostridial neurotoxin cleavage site is included in the cleavable substrate of the present invention, the N-terminal methionine residue (residue 1) of SEQ ID NO: 4 is absent.

[0144] The Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable 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, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may comprise SEQ ID NO: 5. The Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, the Clostridial neurotoxin cleavage site containing a SNAP-25 cleavable 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 containing a SNAP-25 cleavable bond may consist of SEQ ID NO: 5.

[0145] SNARE can be VAMP. VAMP can be VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 or YKT6. VAMP can be human VAMP. Exemplary VAMP polypeptide sequences are shown in the table below. Thus, VAMP can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 31 - 36. In one embodiment, VAMP can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity to any one of SEQ ID NOs: 31 - 36. Preferably, VAMP can comprise any one of SEQ ID NOs: 31 - 36. Thus, VAMP can consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 31 - 36. In one embodiment, VAMP can 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: 31 - 36. Preferably, VAMP can consist of any one of SEQ ID NOs: 31 - 36.

[0146] The cleavable bonds at which VAMP is cleaved by the L chain of the indicated Clostridial neurotoxins are shown in the table below:

[0147]

[0148]

[0149] Human VAMP1, VAMP2 and VAMP3 can be cleaved by the L chain of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X and TeNT. VAMP4, VAMP5 and YKT6 can be cleaved by the L chain of BoNT / X.

[0150] SNARE can be Syntaxin. Syntaxin can be Syntaxin 1A or Syntaxin 1B.

[0151] Syntaxin 1A may comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO: 37. Preferably, Syntaxin 1A may consist of SEQ ID NO: 37.

[0152] Syntaxin 1B may comprise a polypeptide sequence having at least 70% sequence identity with 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 with 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 with 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 with SEQ ID NO: 38. Preferably, Syntaxin 1B may consist of SEQ ID NO: 38.

[0153] Syntaxin 1A and Syntaxin 1B may be cleaved by BoNT / C1. The cleavable bond of Syntaxin1A (SEQ ID NO: 37) cleaved by the L chain of BoNT / C1 may be Lys253-Ala254. The cleavable bond of Syntaxin1B (SEQ ID NO: 38) cleaved by the L chain of BoNT / C1 may be Lys252-Ala253.

[0154] In embodiments where the polypeptide sequence at the Clostridial neurotoxin cleavage site differs in sequence identity from the recited SNARE polypeptide sequences (SEQ ID NO), the Clostridial neurotoxin cleavage site still comprises the relevant cleavable bond of the SNARE polypeptide sequence.

[0155] The cleavable substrate may further comprise an element for indicating whether the cleavable substrate is cleaved at the Clostridial neurotoxin cleavage site. The element may comprise two different states, one state indicating the presence of cleavage and the other state 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, wherein the first module is at the N-terminus of the linker comprising (or consisting of) the Clostridial neurotoxin cleavage site and the second module is at the C-terminus of the linker comprising (or consisting of) the Clostridial neurotoxin cleavage site. When in the uncleaved form, the cleavable substrate is preferably a single-chain polypeptide (such as a fusion polypeptide).

[0156] Advantageously, a cleavable substrate comprising an element for indicating the presence or absence of cleavage may exhibit improved properties when used in the methods of the present invention. In contrast, methods using a cleavable substrate and an antibody that binds to the cleaved form of the substrate (but not to the uncleaved form) may have a high level of background signal. This may be due to non-specific antibody binding.

[0157] The element may comprise a detectable label. The detectable label may be a label that can be visually detected by its optical properties. The detectable label may be a fluorescent label. Such a label can be detected using fluorescence techniques, such as fluorescence microscopy. Thus, in a particularly preferred embodiment, the detectable label is a fluorophore (such as a fluorescent dye) or a fluorescent polypeptide. The fluorescent dye may be a HiLyte fluorescent dye (commercially available from AnaSpec), AlexaFluor (commercially available from Thermo Fisher Scientific), Atto (commercially available from Sigma-Aldrich), quantum dots (commercially available from Sigma-Aldrich) or Janelia Fluor dyes (available from Janelia, USA). The fluorescent polypeptide may be cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP) or red fluorescent protein (RFP).

[0158] 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 such that they can 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 the Clostridium neurotoxin, the fluorescent labels are close to each other and FRET occurs, while when the cleavable substrate is cleaved by the Clostridium neurotoxin, the fluorescent labels are no longer close to each other and FRET no longer occurs. Thus, measuring the change in the FRET signal (e.g., the fluorescence characteristics of the cleavable substrate) can indicate whether the cleavable substrate has been cleaved. Suitable cleavable substrates and related FRET methods are taught in EP 2332959 A2, which is incorporated herein by reference.

[0159] In a preferred embodiment, the element comprises a luciferase. As used herein, the term "luciferase" refers to an enzyme that catalyzes a bioluminescence reaction, e.g., by catalyzing the oxidation of luciferin, emitting light and releasing oxyluciferin. The luciferase can be naturally occurring or engineered. As used herein, a "functional" luciferase refers to a luciferase that can catalyze a reaction in the presence of a suitable substrate.

[0160] Examples of luciferases include NanoLuc (SEQ ID NO:1), firefly luciferase (e.g., Photinus pyralis luciferase), bacterial luciferase (e.g., Vibrio fischeri or Vibrio harveyi luciferase), Renilla luciferase (e.g., Renilla reniformis luciferase), dinoflagellate luciferase, Gaussia luciferase, and copepod luciferase. Other suitable luciferases are described in US Patent No. 8,557,970, which is incorporated herein by reference.

[0161] Preferably, the luciferase comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:1. In one embodiment, the luciferase can 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 can 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 can 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 can consist of SEQ ID NO:1.

[0162] The luciferase can exist as two separate modules of an element for indicating whether a cleavable substrate is cleaved at the cleavage site of a Clostridium neurotoxin. The first module of the element can be a first luciferase domain, and the second module of the element can be a second luciferase domain.

[0163] Thus, in a particularly preferred embodiment, the cleavable substrate is a single-chain polypeptide that comprises: (i) a first luciferase domain; (ii) a linker that contains the cleavage site of a Clostridium neurotoxin; and (iii) a second luciferase domain. In one embodiment: (i) when the linker is intact (i.e., where the cleavage site of the Clostridium neurotoxin has not been cleaved by the Clostridium neurotoxin), the linker functionally links the first and second luciferase domains, thereby providing a functional luciferase (i.e., the cleavable substrate has luciferase activity); and (ii) when the linker is cleaved (i.e., where the cleavage site of the Clostridium neurotoxin has been cleaved by the Clostridium neurotoxin), the linker no longer functionally links the first and second luciferase domains, resulting in the loss of luciferase activity (i.e., the cleavable substrate does not have luciferase activity). The term "single-chain" as used in the context of the cleavable substrate can refer to a single polypeptide molecule having a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. In other words, each of the elements of the single-chain polypeptide can be linked to the other elements by peptide bonds.

[0164] "Functionally linked" as used in the context of the first and second luciferase domains means that the first and second luciferase domains are linked in such a way that no intervening sequence prevents the two fragments from forming an active and functional tertiary structure. When functionally linked, the first and second luciferase domains exhibit luciferase activity, for example as if they were without a linker.

[0165] The first luciferase domain is preferably at the N-terminus of a linker that includes a Clostridial neurotoxin cleavage site. Preferably, the first luciferase domain is the most N-terminal element of the cleavable substrate. The first luciferase domain can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:2. In one embodiment, the first luciferase domain can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:2. Preferably, the first luciferase domain can comprise SEQ ID NO:2. The first luciferase domain can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:2. In one embodiment, the first luciferase domain can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:2. Preferably, the first luciferase domain can consist of SEQ ID NO:2.

[0166] The second luciferase domain is preferably at the C-terminus of a linker that includes a Clostridial neurotoxin cleavage site. Preferably, the second luciferase domain is the most C-terminal element of the cleavable substrate. The second luciferase domain can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:3. In one embodiment, the second luciferase domain can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:3. Preferably, the second luciferase domain can comprise SEQ ID NO:3. The second luciferase domain can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:3. In one embodiment, the second luciferase domain can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO:3. Preferably, the second luciferase domain can consist of SEQ ID NO:3.

[0167] In some embodiments, the linker can consist of a Clostridial neurotoxin cleavage site. However, preferably the linker further comprises one or more spacers. Most preferably, the linker comprises (even 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 there is more than one spacer, the spacers can have the same or different polypeptide sequences (preferably the same polypeptide sequence).

[0168] Those skilled in the art are fully capable of selecting appropriate spacer sequences and sizes. The spacer can be of any suitable length, such as 3 - 20, 2 - 15, 5 - 15, or 4 - 8 amino acid lengths. The spacer can comprise (or consist of) glycine and serine residues. In particular, the spacer can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:39. In one embodiment, the spacer can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO:39. Preferably, the spacer can comprise SEQ ID NO:39. In particular, the spacer can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:39. In one embodiment, the spacer can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO:39. Preferably, the spacer can consist of SEQ ID NO:39.

[0169] Preferably, the cleavable substrate of the present invention comprises (from the N - terminus to the 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, the cleavable substrate of the present invention can consist of (from the N - terminus to the 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 these embodiments, the linker is formed by elements (ii), (iii), and (iv). The first and second spacers preferably have the same polypeptide sequence.

[0170] The cleavable substrate can comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:6. In one embodiment, the cleavable substrate can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO:6. Preferably, the cleavable substrate can comprise SEQ ID NO:6.

[0171] The cleavable substrate can consist of a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:6. In one embodiment, the cleavable substrate can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity with SEQ ID NO:6. Preferably, the cleavable substrate can consist of SEQ ID NO:6.

[0172] In some instances herein, "cleavable substrate" is mentioned. However, this is to indicate that there may be more than one "cleavable substrate" when implementing the method. This does not necessarily mean that there are two or more different types of cleavable substrates, although this is also included. Thus, the cleavable substrate can be of one type (e.g., all cleavable substrates comprise: (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site for SNAP-25; and (iii) a second luciferase domain) or of multiple types (e.g., a portion of the cleavable substrates comprise: (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site for SNAP-25; and (iii) a second luciferase domain, while a portion of the cleavable substrates comprise: (i) YFP; (ii) a linker comprising a Clostridium neurotoxin cleavage site for SNAP-25; and (iii) GFP). Preferably, the cleavable substrate is of one type.

[0173] The method can include determining whether the cleavable substrate has been cleaved. In particular, the method can include determining (preferably in the assay sample) the amount of the cleavable substrate cleaved by the L-chain polypeptide, thereby determining the activity of the Clostridium neurotoxin in the composition. The method for making this determination will depend on the specific nature of the cleavable substrate used in the method. For example, when the cleavable substrate comprises a detectable label, differences in the detection properties can indicate whether the cleavable substrate has been cleaved and / or the amount of the cleavable substrate that has been cleaved. The differences can be determined by comparison with a suitable control. For example, determining whether a cleavable substrate comprising a fluorescent label has been cleaved can include comparing the fluorescence of the cleavable substrate with a control and determining whether there is a difference in fluorescence (e.g., loss of a FRET signal).

[0174] A suitable control can be a negative control. The negative control can be provided by performing the method in the same manner, but where the composition being tested does not contain the Clostridium neurotoxin polypeptide. The negative control can be a negative reference standard. The negative reference standard can correspond to a value that has been determined theoretically or experimentally and that represents a negative result in the method of the present invention. This value can be determined before implementing the method of the present invention, or can be determined simultaneously with or after implementing the method of the present invention.

[0175] 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 the L-chain polypeptide (preferably containing the Clostridium neurotoxin polypeptide). A difference in the properties of the cleavable substrate compared to the negative control can also indicate that the composition contains the H-chain polypeptide (or HC or HCC domain polypeptide). In such cases, this can allow determination that the composition contains the Clostridium neurotoxin polypeptide.

[0176] In one embodiment, when the control is a negative control, no difference in the properties of the cleavable substrate as compared to the negative control indicates that the test composition does not contain an L-chain polypeptide (preferably does not contain a Clostridium neurotoxin polypeptide). In one embodiment, when the control is a negative control, no difference in the properties of the cleavable substrate as compared to the negative control indicates that the test composition does not contain an L-chain polypeptide and / or does not contain an H-chain polypeptide (or HC or HCC domain polypeptide) (preferably does not contain a Clostridium neurotoxin polypeptide). In such a case, this can allow determination that the composition does not contain a Clostridium neurotoxin polypeptide.

[0177] In one embodiment, when the control is a negative control, a difference in the properties of the cleavable substrate as compared to the negative control indicates that the test composition contains an active L-chain polypeptide (preferably contains an active Clostridium neurotoxin polypeptide). The degree of the difference can be quantified to indicate the amount of the active L-chain polypeptide (e.g., the amount of the active Clostridium neurotoxin polypeptide). A difference in the properties of the cleavable substrate as compared to the negative control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide) that does not have a property of reduced activity. The degree of the difference can be quantified to indicate the amount of the H-chain polypeptide (or HC or HCC domain polypeptide) that does not have a property of reduced activity (e.g., the amount of the active Clostridium neurotoxin polypeptide). The degree of the difference can be quantified to indicate the activity level of the composition containing the Clostridium neurotoxin polypeptide. Preferably, the greater the difference as compared to the negative control, the higher the amount of the active L-chain polypeptide (e.g., the amount of the active Clostridium neurotoxin polypeptide). Preferably, the greater the difference as compared to the negative control, the higher the amount of the H-chain polypeptide (or HC or HCC domain polypeptide) that does not have a property of reduced activity. Preferably, the greater the difference as compared to the negative control, the higher the activity level of the composition containing the Clostridium neurotoxin polypeptide.

[0178] In one embodiment, when the control is a negative control, no difference in the properties of the cleavable substrate as compared to the negative control indicates that the test composition contains an inactive L-chain polypeptide (preferably contains an inactive Clostridium neurotoxin polypeptide). In one embodiment, when the control is a negative control, no difference in the properties of the cleavable substrate as compared to the negative control indicates that the test composition contains an inactive L-chain polypeptide and / or contains an H-chain polypeptide (or HC or HCC domain polypeptide) that has a property of reduced activity. Such a composition can be considered an inactive composition.

[0179] In one embodiment, when the control is a negative control, a difference in the properties of the cleavable substrate as compared to the negative control can be used to indicate the activity level of the composition. The difference can be used to indicate the amount of the active L-chain polypeptide (e.g., the active Clostridium neurotoxin polypeptide) present in the composition. The difference can be used to indicate the amount of the H-chain polypeptide (or HC or HCC domain polypeptide) that does not have a property of reduced activity present in the composition.

[0180] In some embodiments, the difference in the properties of the cleavable substrate can be quantified to indicate the activity level of the composition (e.g., the amount of active Clostridial neurotoxin polypeptide present in the composition) compared to a control.

[0181] A suitable control can be a positive control. The positive control can be provided by performing the method in the same manner, but where the composition contains a Clostridial neurotoxin polypeptide, preferably having a known activity level. The positive control can be a positive reference standard. The positive reference standard can correspond to a value that has been determined theoretically or experimentally and that represents a positive result in the method of the present invention. The positive result can represent an ideal activity level of a composition containing a Clostridial neurotoxin polypeptide. Such a value can be used for quality control purposes. The value can be determined before performing the method of the present invention, or can be determined simultaneously with or after performing the method of the present invention. Thus, the "properties of the cleavable substrate" or "luminescence" (e.g., luminescence level) as used herein when referring to a control can refer to the "properties of the cleavable substrate" or "luminescence" (e.g., luminescence level) represented by the control.

[0182] In one embodiment, when the control is a positive control, no difference in the properties of the cleavable substrate compared to the positive control indicates that the tested composition contains an L-chain polypeptide (preferably containing a Clostridial neurotoxin polypeptide). No difference in the properties of the cleavable substrate compared to the positive control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide). In such cases, this can allow for the determination that the composition contains a Clostridial neurotoxin polypeptide.

[0183] In one embodiment, when the control is a positive control, no difference in the properties of the cleavable substrate compared to the positive control indicates that the tested composition contains an active L-chain polypeptide (preferably containing an active Clostridial neurotoxin polypeptide). No difference in the properties of the cleavable substrate compared to the positive control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide) that does not have the property of reducing activity. Such a composition can be considered an active composition.

[0184] 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 amount of active L-chain polypeptide (e.g., active Clostridial neurotoxin polypeptide) present in the composition. The difference can be used to indicate the amount of H-chain polypeptide (or HC or HCC domain polypeptide) present in the composition that does not have the property of reducing activity.

[0185] In one embodiment, when the control is a positive control, no difference in the properties of the cleavable substrate compared to the positive control can be used to indicate that the composition has the same activity level as the positive control. This can be used to indicate the amount of active L-chain polypeptide (such as an active Clostridium neurotoxin polypeptide) present in the composition. This can be used to indicate the amount of H-chain polypeptide (or HC or HCC domain polypeptide) present in the composition that does not have properties of reduced activity.

[0186] When the cleavable substrate comprises (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site; and (iii) a second luciferase domain, determining whether the cleavable substrate has been cleaved can be evaluated by measuring luciferase activity. This can be achieved by adding a suitable luciferase substrate (preferably adding the luciferase substrate to the assay sample). Preferably, the luciferase substrate is furazine (2-furylmethyl-deoxy-coelenterazine). In this case, determining whether the cleavable substrate has been cleaved can be evaluated by measuring luminescence. For example, the cleavable substrate can be contacted with furazine in the assay sample at a concentration of 1 - 100 μM, preferably 10 - 50 μM (such as 37.5 μM). Any suitable technique can be used to measure luminescence, such as an automated microplate reader (such as the BMG Labtech CLARIOstar microplate reader).

[0187] Thus, in a preferred embodiment, the property of the cleavable substrate can be luciferase activity. The difference in the property of the cleavable substrate can be the difference in the luciferase activity level. As pointed out above, this can be determined by measuring luminescence.

[0188] Thus, in one embodiment, when the control is a negative control, a lower luminescence level compared to the negative control indicates that the tested composition contains an L-chain polypeptide (preferably containing a Clostridium neurotoxin polypeptide). A lower luminescence level compared to the negative control can also indicate that the tested composition further contains an H-chain polypeptide (or HC or HCC domain polypeptide). In this case, this can allow determination that the composition contains a Clostridium neurotoxin polypeptide.

[0189] In one embodiment, when the control is a negative control, the same luminescence level compared to the negative control indicates that the tested composition does not contain an L-chain polypeptide (preferably does not contain a Clostridium neurotoxin polypeptide). In one embodiment, when the control is a negative control, the same luminescence level compared to the negative control indicates that the tested composition does not contain an L-chain polypeptide and / or does not contain an H-chain polypeptide (or HC or HCC domain polypeptide) (preferably does not contain a Clostridium neurotoxin polypeptide). In this case, this can allow determination that the composition does not contain a Clostridium neurotoxin polypeptide.

[0190] In one embodiment, when the control is a negative control, a lower luminescence level compared to the negative control indicates that the tested composition contains an active L-chain polypeptide (preferably an active Clostridium neurotoxin polypeptide). The degree of reduction in luminescence level can be quantified to indicate the amount of the active L-chain polypeptide (e.g., the amount of the active Clostridium neurotoxin polypeptide). A lower luminescence level compared to the negative control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide) that does not have activity-reducing properties. The degree of reduction in luminescence level can be quantified to indicate the activity level of the composition containing the Clostridium neurotoxin polypeptide. Preferably, the lower the luminescence level compared to the negative control, the higher the amount of the active L-chain polypeptide (e.g., the amount of the active Clostridium neurotoxin polypeptide). Preferably, the lower the luminescence level compared to the negative control, the higher the amount of the H-chain polypeptide (or HC or HCC domain polypeptide) that does not have activity-reducing properties. Preferably, the lower the luminescence level compared to the negative control, the higher the activity level of the composition containing the Clostridium neurotoxin polypeptide.

[0191] In one embodiment, when the control is a negative control, the same luminescence level compared to the negative control indicates that the tested composition contains an inactive L-chain polypeptide (preferably an inactive Clostridium neurotoxin polypeptide). In one embodiment, when the control is a negative control, the same luminescence level compared to the negative control indicates that the tested composition contains an inactive L-chain polypeptide and / or contains an H-chain polypeptide (or HC or HCC domain polypeptide) that has activity-reducing properties.

[0192] Thus, in one embodiment, when the control is a positive control, the same (or lower) luminescence level compared to the positive control indicates that the tested composition contains an L-chain polypeptide (preferably a Clostridium neurotoxin polypeptide). The same (or lower) luminescence level compared to the positive control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide). In this case, this can allow for the determination that the composition contains a Clostridium neurotoxin polypeptide.

[0193] In one embodiment, when the control is a positive control, the same (or lower) luminescence level compared to the positive control indicates that the tested composition contains an active L-chain polypeptide (preferably an active Clostridium neurotoxin polypeptide). The same (or lower) luminescence level compared to the positive control can also indicate that the composition contains an H-chain polypeptide (or HC or HCC domain polypeptide) that does not have activity-reducing properties.

[0194] The same luminescence level compared to the positive control indicates that the tested composition contains the same number of active L-chain polypeptides (such as active clostridial neurotoxin polypeptides) as the positive control. The same luminescence level compared to the positive control indicates that the tested composition contains the same number of H-chain polypeptides (or HC or HCC domain polypeptides) that do not have the property of reduced activity as the positive control. The same luminescence level compared to the positive control indicates that the composition has the same activity level as the positive control.

[0195] A lower luminescence level compared to the positive control indicates that the tested composition contains a greater number of active L-chain polypeptides (such as active clostridial neurotoxin polypeptides) than the positive control. A lower luminescence level compared to the positive control indicates that the tested composition contains a greater number of H-chain polypeptides (or HC or HCC domain polypeptides) that do not have the property of reduced activity and / or contains H-chain polypeptides (or HC or HCC domain polypeptides) with the property of increased activity compared to the positive control. A lower luminescence level compared to the positive control indicates that the composition has a higher activity level than the positive control. The degree of reduction in luminescence level can be quantified to indicate the activity level of the composition containing clostridial neurotoxin polypeptides. Preferably, the lower the luminescence level compared to the positive control, the higher the number of active L-chain polypeptides (such as the number of active clostridial neurotoxin polypeptides). Preferably, the lower the luminescence level compared to the positive control, the higher the number of H-chain polypeptides (or HC or HCC domain polypeptides) that do not have the property of reduced activity. Preferably, the lower the luminescence level compared to the positive control, the greater the effect of the increased activity property of the H-chain polypeptides (or HC or HCC domain polypeptides). Preferably, the lower the luminescence level compared to the positive control, the higher the activity level of the composition containing clostridial neurotoxin polypeptides.

[0196] In one embodiment, when the control is a positive control, a higher luminescence level compared to the positive control indicates that the tested composition does not contain L-chain polypeptides (preferably contains clostridial neurotoxin polypeptides). In one embodiment, when the control is a positive control, a higher luminescence level compared to the positive control indicates that the tested composition does not contain L-chain polypeptides (preferably contains clostridial neurotoxin polypeptides) and / or does not contain H-chain polypeptides (or HC or HCC domain polypeptides). In this case, this can allow determination that the composition does not contain clostridial neurotoxin polypeptides. For a more definitive determination, comparison with a negative control can also be made as described herein.

[0197] In one embodiment, when the control is a positive control, a higher luminescence level compared to the positive control indicates that the tested composition contains an inactive L-chain polypeptide (preferably containing an active clostridial neurotoxin polypeptide). In one embodiment, when the control is a positive control, a higher luminescence level compared to the positive control indicates that the tested composition contains an inactive L-chain polypeptide (preferably containing an active clostridial neurotoxin polypeptide) and / or contains an H-chain polypeptide (or HC or HCC domain polypeptide) having reduced activity properties.

[0198] A higher luminescence level compared to the positive control indicates that the tested composition contains a lower number of active L-chain polypeptides (such as an active clostridial neurotoxin polypeptide) than the positive control. A higher luminescence level compared to the positive control indicates that the tested composition contains a lower number of H-chain polypeptides (or HC or HCC domain polypeptides) that do not have reduced activity properties than the positive control. A higher luminescence level compared to the positive control indicates that the composition has a lower activity level than the positive control. The degree of increase in the luminescence level can be quantified to indicate the activity level of the composition containing the clostridial neurotoxin polypeptide. Preferably, the higher the luminescence level compared to the positive control, the lower the number of active L-chain polypeptides (such as the number of active clostridial neurotoxin polypeptides). Preferably, the higher the luminescence level compared to the positive control, the lower the number of H-chain polypeptides (or HC or HCC domain polypeptides) that do not have reduced activity properties. Preferably, the higher the luminescence level compared to the positive control, the lower the activity level of the composition containing the clostridial neurotoxin polypeptide.

[0199] As used herein, "different" (and related terms such as "variation", "change", "difference" and their synonyms) can refer to a significant difference compared to a comparator (e.g., a control as described herein). A difference (and related terms such as "variation", "change", "difference" and their synonyms) can refer to a statistically significant difference when compared to a comparator (e.g., a control as described herein). A "significant difference" can be a difference of at least 5%, 10%, 15%, 20%, 25% or 30% compared to a comparator (e.g., a control as described herein). The term "no difference" (and related terms such as "unchanged" and "same" and their synonyms) can refer to no significant difference compared to a comparator (e.g., a control as described herein). No difference (and related terms such as "unchanged" and "same" and their synonyms) can refer to no statistically significant difference when compared to a comparator (e.g., a control as described herein). As used herein, "lower" (and related terms such as "less than") can refer to at least 10%, 25%, 20%, 50%, 75%, 100%, 150% or 200% lower compared to a comparator (e.g., a control as described herein). The term "lower" (and related terms such as "less than") can refer to statistically significantly lower when compared to a comparator (e.g., a control as described herein). As used herein, "higher" (and related terms such as "greater than") can refer to at least 10%, 25%, 20%, 50%, 75%, 100%, 150% or 200% higher compared to a comparator (e.g., a control as described herein). The term "higher" (and related terms such as "greater than") can refer to statistically significantly higher when compared to a comparator (e.g., a control as described herein).

[0200] The last step of the method of the invention can include assigning a measured activity value. The measured activity value can be a luciferase activity value (e.g., determined 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 method of the invention can be used to determine the effective value (EC50) of a composition comprising a Clostridium neurotoxin polypeptide. To obtain the effective value, the method can be performed using at least a second, third, and / or fourth composition comprising different concentrations of the Clostridium neurotoxin polypeptide, and determining the activity values of the at least second, third, and / or fourth compositions, and determining the effective value (EC50) of the composition by comparing the measured activity value (of the first composition) to the measured luciferase activity values of the at least second, third, and / or fourth compositions.

[0201] "EC50" can refer to the amount or concentration of a clostridial neurotoxin (e.g., in a composition) that induces a response halfway between the baseline and maximum after a specific exposure time. It is commonly used to measure potency. The EC50 of a graded dose-response curve can represent the concentration at which 50% of the maximum effect of the clostridial neurotoxin is observed.

[0202] In some instances herein, "clostridial neurotoxin polypeptide" and "light chain polypeptide" are mentioned separately. However, this is to indicate that there may be more than one clostridial neurotoxin polypeptide or light chain polypeptide present (e.g., in a composition) when implementing the method. This does not necessarily mean that there are two or more different types of clostridial neurotoxin polypeptides or light chain polypeptides, although this is also included. Thus, the clostridial neurotoxin polypeptide or light chain polypeptide can be of one type (e.g., all clostridial neurotoxin polypeptides are BoNT / A polypeptides, or all light chain polypeptides are BoNT / A light chain polypeptides) or multiple types (e.g., some of the clostridial neurotoxin polypeptides are BoNT / B polypeptides and some are BoNT / A polypeptides; or some of the light chain polypeptides are BoNT / A light chain polypeptides and some are BoNT / D light chain polypeptides). Preferably, the clostridial neurotoxin polypeptide is of one type. Preferably, the light chain polypeptide is of one type. The above similarly applies to some cases herein where "botulinum neurotoxin polypeptide" is mentioned.

[0203] The clostridial neurotoxin according to the present invention can comprise the HCC domain of botulinum neurotoxin or tetanus neurotoxin (TeNT). The clostridial neurotoxin of the present invention can comprise the BoNT / A HCC domain, BoNT / B HCC domain, BoNT / C1 HCC domain, BoNT / D HCC domain, BoNT / E HCC domain, BoNT / F HCC domain, BoNT / G HCC domain, BoNT / X HCC domain or TeNT HCC domain. Preferably, the clostridial neurotoxin of the present invention comprises the BoNT / B HCC domain or the BoNT / A HCC domain, more preferably the BoNT / B HCC domain.

[0204] The clostridial neurotoxin according to the present invention can comprise the HC domain of botulinum neurotoxin or tetanus neurotoxin (TeNT). The clostridial neurotoxin of the present invention can comprise the BoNT / A HC domain, BoNT / B HC domain, BoNT / C1 HC domain, BoNT / D HC domain, BoNT / E HC domain, BoNT / F HC domain, BoNT / G HC domain, BoNT / X HC domain or TeNT HC domain. Preferably, the clostridial neurotoxin of the present invention comprises the BoNT / B HC domain or the BoNT / A HC domain, more preferably the BoNT / B HC domain.

[0205] The term "clostridial neurotoxin" encompasses toxins produced by Clostridium botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (botulinum neurotoxin serotype E), and Clostridium baratii (botulinum neurotoxin serotype F). The reference BoNT / A sequence is shown as SEQ ID NO:12. The reference BoNT / B sequence is shown as SEQ ID NO:13. The reference BoNT / C1 (also referred to herein as BoNT / C) sequence is shown as SEQ ID NO:41. The reference BoNT / D sequence is shown as SEQ ID NO:42. The reference BoNT / E sequence is shown as SEQ ID NO:43. The reference BoNT / F sequence is shown as SEQ ID NO:44. The reference BoNT / G sequence is shown as SEQ ID NO:45. The reference TeNT sequence is shown as SEQ ID NO:46. The reference BoNT / X sequence is shown as SEQ ID NO:47. The term "clostridial neurotoxin" can also encompass newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as an Enterococcus-encoded toxin with the closest sequence identity to BoNT / X, a Weissella paramesenteroides-encoded toxin named BoNT / Wo (NCBI Ref Seq:WP_027699549.1) that cleaves VAMP2 at W89-W90, an Enterococcus faecium-encoded toxin (GenBank:OTO22244.1 that cleaves VAMP2 and SNAP25), and a Flavobacterium piperi-encoded toxin (NCBI Ref.Seq:WP_034687872.1).

[0206] Thus, the clostridial neurotoxin can be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Thus, the composition of the present invention can comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT. Thus, the clostridial neurotoxin polypeptide can 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. Thus, the composition of the present invention can comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X. Thus, the clostridial neurotoxin polypeptide can 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.

[0207] The clostridial neurotoxin is formed by two polypeptide chains, with the heavy chain (H chain) having a molecular weight of approximately 100 kDa and the light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor-binding domain or HC domain) and an N-terminal translocation component (HN domain). Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum in the form of a large protein complex, which is composed of the BoNT itself complexed with some 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 have similar structures and modes of action. Different BoNT serotypes can be distinguished by the inactivating action of specific neutralizing antisera, and this serotype-based classification is related to the percentage of sequence identity at the amino acid level. The BoNT proteins of a given serotype are further divided into different subtypes based on the percentage amino acid sequence identity.

[0208] BoNTs are absorbed in the gastrointestinal tract and, after entering the systemic circulation, bind to the presynaptic membrane of cholinergic nerve endings, preventing the release of their neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E cleave synaptosomal-associated protein of 25 kDa (SNAP-25); BoNT / C1 also cleaves Syntaxin. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and Syntaxin 1. Tetanus toxin is produced by Clostridium tetani in a single serotype. Clostridium butyricum produces BoNT / E, while Clostridium baratii produces BoNT / F.

[0209] Examples of light chain reference sequences include:

[0210] - Botulinum neurotoxin type A: amino acid residues 1-448

[0211] - Botulinum neurotoxin type B: amino acid residues 1-440

[0212] - Botulinum neurotoxin type C1: amino acid residues 1-441

[0213] - Botulinum neurotoxin type D: amino acid residues 1-445

[0214] - Botulinum neurotoxin type E: amino acid residues 1-422

[0215] - Botulinum neurotoxin type F: amino acid residues 1-439

[0216] - Botulinum neurotoxin type G: amino acid residues 1-441

[0217] - Tetanus neurotoxin: amino acid residues 1-457

[0218] For the recently discovered BoNT / X, its L chain is reported to correspond to amino acids 1-439 therein, and the L chain boundary may vary by approximately 25 amino acids (e.g., 1-414 or 1-464).

[0219] The L chain of BoNT / A 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 L chain of BoNT / B 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 L chain of BoNT / C1 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 L chain of BoNT / D 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 L chain of BoNT / E 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 L chain of BoNT / F 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 L chain of BoNT / G 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 L chain of BoNT / X 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 L chain of TeNT 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.

[0220] The reference sequences identified above should be considered as a guide, as minor variations may occur depending on the subtype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites slightly different Clostridium sequences:

[0221] - Clostridium botulinum neurotoxin type A: amino acid residues M1-K448

[0222] - Clostridium botulinum neurotoxin type B: amino acid residues M1-K441

[0223] - Clostridial neurotoxin type C1: Amino acid residues M1 - K449

[0224] - Clostridial neurotoxin type D: Amino acid residues M1 - R445

[0225] - Clostridial neurotoxin type E: Amino acid residues M1 - R422

[0226] - Clostridial neurotoxin type F: Amino acid residues M1 - K439

[0227] - Clostridial neurotoxin type G: Amino acid residues M1 - K446

[0228] - Tetanus neurotoxin: Amino acid residues M1 - A457

[0229] The translocation domain is a fragment of the H chain of the clostridial neurotoxin, roughly corresponding to the N-terminal half of the H chain, or the domain in the intact H chain corresponding to this fragment. In one embodiment, the HC function of the H chain can be removed by deleting the HC amino acid sequence (at the DNA synthesis level, or after synthesis by nuclease or protease treatment). Alternatively, the HC function can be inactivated by chemical or biological treatment. Thus, in some embodiments, the H chain cannot bind to the binding site on the target cell to which the native clostridial neurotoxin (i.e., the intact toxin) binds.

[0230] Examples of suitable (reference) translocation domains include:

[0231] - Clostridial neurotoxin type A - Amino acid residues (449 - 871)

[0232] - Clostridial neurotoxin type B - Amino acid residues (441 - 858)

[0233] - Clostridial neurotoxin type C - Amino acid residues (442 - 866)

[0234] - Clostridial neurotoxin type D - Amino acid residues (446 - 862)

[0235] - Clostridial neurotoxin type E - Amino acid residues (423 - 845)

[0236] - Clostridial neurotoxin type F - Amino acid residues (440 - 864)

[0237] - Clostridial neurotoxin type G - Amino acid residues (442 - 863)

[0238] - Tetanus neurotoxin - Amino acid residues (458 - 879)

[0239] The HN domain of BoNT / A may 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. The HN domain of BoNT / B may 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. The HN domain of BoNT / C1 may 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. The HN domain of BoNT / D may 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. The HN domain of BoNT / E may 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. The HN domain of BoNT / F may 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. The HN domain of BoNT / G may 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. The HN domain of BoNT / X may 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. The HN domain of TeNT 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.

[0240] The reference sequences determined above should be considered as a guide, as minor variations may occur depending on the subtype. For example, US2007 / 0166332 (incorporated herein by reference) cites slightly different Clostridium sequences:

[0241] - Clostridium botulinum neurotoxin type A - amino acid residues (A449 - K871)

[0242] - Botulinum neurotoxin type B - Amino acid residues (A442 - S858)

[0243] - Botulinum neurotoxin type C - Amino acid residues (T450 - N866)

[0244] - Botulinum neurotoxin type D - Amino acid residues (D446 - N862)

[0245] - Botulinum neurotoxin type E - Amino acid residues (K423 - K845)

[0246] - Botulinum neurotoxin type F - Amino acid residues (A440 - K864)

[0247] - Botulinum neurotoxin type G - Amino acid residues (S447 - S863)

[0248] - Tetanus neurotoxin - Amino acid residues (S458 - V879)

[0249] In the context of the present invention, various Clostridial neurotoxin HN regions containing the translocation domain may be useful in various aspects of the present invention. The HN region of the heavy chain of Clostridial neurotoxins is approximately 410 - 430 amino acids in length and contains the translocation domain. Studies have shown that the entire HN region of the heavy chain of Clostridial neurotoxins is not essential for the translocation activity of the translocation domain. Thus, aspects of this embodiment may include Clostridial neurotoxin HN regions containing a translocation domain that is, for example, at least 350 amino acids in length, at least 375 amino acids in length, at least 400 amino acids in length, and at least 425 amino acids in length. Other aspects of this embodiment may include Clostridial neurotoxin HN regions containing a translocation domain that is, for example, at most 350 amino acids in length, at most 375 amino acids in length, at most 400 amino acids in length, and at most 425 amino acids in length.

[0250] For more details on the genetic basis of toxin production in Clostridium botulinum and Clostridium tetani, see the article by Henderson et al. (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0251] The term HN includes the naturally occurring neurotoxin HN portion, as well as modified HN portions having amino acid sequences and / or synthetic amino acid residues that do not exist in nature. In one embodiment, the modified HN portion still exhibits the above-described translocation function.

[0252] The reference sequences for the receptor-binding domains (HC) of Clostridial neurotoxins include:

[0253] BoNT / A-N872-L1296

[0254] BoNT / B-E859-E1291

[0255] BoNT / C1-N867-E1291

[0256] BoNT / D-S863-E1276

[0257] BoNT / E-R846-K1252

[0258] BoNT / F-K865-E1274

[0259] BoNT / G-N864-E1297

[0260] TeNT-I880-D1315

[0261] For the recently discovered BoNT / X, its HC domain is reported to correspond to amino acids 893 - 1306, and the domain boundaries may vary by approximately 25 amino acids (e.g., 868 - 1306 or 918 - 1306).

[0262] The HC domain of BoNT / A may 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. The HC domain of BoNT / B may 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. The HC domain of BoNT / C1 may 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. The HC domain of BoNT / D may 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. The HC domain of BoNT / E may 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. The HC domain of BoNT / F may 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. The HC domain of BoNT / G may 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. The HC domain of BoNT / X 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. The HC domain of TeNT 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:46.

[0263] The heavy chain of the clostridial neurotoxin (e.g., the heavy chain C-terminal domain portion) may further comprise a translocation promoting domain (or a fragment thereof may be a translocation promoting domain fragment). This domain facilitates the delivery of the light chain into the cytoplasm of the target cell and is described, for example, in WO 08 / 008803 and WO 08 / 008805, the contents of both of which are incorporated herein by reference.

[0264] For example, the translocation promoting domain may comprise a Clostridial neurotoxin HCN domain or a fragment or variant thereof. More specifically, the length of the Clostridial neurotoxin HCN translocation promoting domain can be at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, the length of the Clostridial neurotoxin HCN translocation promoting domain is preferably at most 200 amino acids, at most 225 amino acids, at most 250 amino acids or at most 275 amino acids. Specific (reference) examples include:

[0265] - Botulinum neurotoxin type A - amino acid residues (872 - 1110)

[0266] - Botulinum neurotoxin type B - amino acid residues (859 - 1097)

[0267] - Botulinum neurotoxin type C - amino acid residues (867 - 1111)

[0268] - Botulinum neurotoxin type D - amino acid residues (863 - 1098)

[0269] - Botulinum neurotoxin type E - amino acid residues (846 - 1085)

[0270] - Botulinum neurotoxin type F - amino acid residues (865 - 1105)

[0271] - Botulinum neurotoxin type G - amino acid residues (864 - 1105)

[0272] - Tetanus neurotoxin - amino acid residues (880 - 1127)

[0273] The above sequence positions may vary slightly depending on the serotype / subtype. More examples of suitable (reference) Clostridial neurotoxin HCN domains include:

[0274] - Botulinum neurotoxin type A - amino acid residues (874 - 1110)

[0275] - Botulinum neurotoxin type B - amino acid residues (861 - 1097)

[0276] - Botulinum neurotoxin type C - amino acid residues (869 - 1111)

[0277] - Botulinum neurotoxin type D - amino acid residues (865 - 1098)

[0278] - Botulinum neurotoxin type E - amino acid residues (848 - 1085)

[0279] - Botulinum neurotoxin type F - amino acid residues (867 - 1105)

[0280] -Botulinum neurotoxin type G - Amino acid residues (866 - 1105)

[0281] -Tetanus neurotoxin - Amino acid residues (882 - 1127)

[0282] Any of the above translocation - facilitating domains can be combined with any of the translocation - domain peptides described above and applicable to the present invention. Thus, for example, a non - Clostridial translocation - facilitating domain can be combined with a non - Clostridial translocation - domain peptide or a Clostridial translocation - domain peptide. Alternatively, the Clostridial neurotoxin HCN translocation - facilitating domain can be combined with a non - Clostridial translocation - domain peptide. Alternatively, the Clostridial neurotoxin HCN translocation - facilitating domain can be combined with a Clostridial translocation - domain peptide, and examples thereof include:

[0283] -Botulinum neurotoxin type A - Amino acid residues (449 - 1110)

[0284] -Botulinum neurotoxin type B - Amino acid residues (442 - 1097)

[0285] -Botulinum neurotoxin type C - Amino acid residues (450 - 1111)

[0286] -Botulinum neurotoxin type D - Amino acid residues (446 - 1098)

[0287] -Botulinum neurotoxin type E - Amino acid residues (423 - 1085)

[0288] -Botulinum neurotoxin type F - Amino acid residues (440 - 1105)

[0289] -Botulinum neurotoxin type G - Amino acid residues (447 - 1105)

[0290] -Tetanus neurotoxin - Amino acid residues (458 - 1127)

[0291] The C-terminal peptide of the heavy chain (HC peptide) of a native Clostridial neurotoxin contains approximately 400 - 440 amino acid residues and consists of two functionally distinct domains, each of approximately 25 kDa, namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C-terminal region (commonly referred to as the HCC peptide or domain). This fact has been confirmed by the following publications, the contents of which are hereby incorporated by reference in their entirety: Umland TC (1997) Nature Structural Biology 4:788 - 792; Herreros J (2000) Journal of Biological Chemistry 347:199 - 204; Halpern J (1993) Journal of Biological Chemistry 268:15, pp. 11188 - 11192; Rummel A (2007) Proceedings of the National Academy of Sciences of the United States of America 104:359 - 364; Lacey DB (1998) Nature Structural Biology 5:898 - 902; Knapp (1998) Abstracts of Papers of the American Crystallographic Association 25:90; Swaminathan and Eswaramoorthy (2000) Nature Structural Biology 7:1751 - 1759; and Rummel A (2004) Molecular Microbiology 51(3), 631 - 643. Additionally, it has been well documented that the C-terminal region (HCC) consisting of the C-terminal 160 - 200 amino acid residues is responsible for the binding of the Clostridial neurotoxin to its native cell receptor, i.e., binding to the nerve endings at the neuromuscular junction - a fact also confirmed by the aforementioned publications. Thus, in this specification, reference to a Clostridial heavy chain lacking a functional heavy chain HC peptide (or domain) such that the heavy chain cannot bind to the cell surface receptor to which the native Clostridial neurotoxin binds means that the Clostridial heavy chain simply lacks a functional HCC peptide. In other words, the HCC peptide region can be partially or completely absent, or otherwise modified (e.g., by conventional chemical or proteolytic treatment) to reduce its native binding ability to the nerve endings at the neuromuscular junction.

[0292] The HCC reference sequences are as follows:

[0293] -Botulinum neurotoxin type A - amino acid residues (Y1111 - L1296)

[0294] -Botulinum neurotoxin type B - amino acid residues (Y1098 - E1291)

[0295] -Botulinum neurotoxin type C - amino acid residues (Y1112 - E1291)

[0296] -Botulinum neurotoxin type D - amino acid residues (Y1099 - E1276)

[0297] -Botulinum neurotoxin type E - Amino acid residues (Y1086 - K1252)

[0298] -Botulinum neurotoxin type F - Amino acid residues (Y1106 - E1274)

[0299] -Botulinum neurotoxin type G - Amino acid residues (Y1106 - E1297)

[0300] -Tetanus neurotoxin - Amino acid residues (Y1128 - D1315).

[0301] The reference sequences identified above should be considered as a guide, as minor variations may occur according to the sub - serotype.

[0302] The HCC domain of a botulinum neurotoxin type A (BoNT / A) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1111-1296 of SEQ ID NO:12. The HCC domain of a botulinum neurotoxin type B (BoNT / B) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1098-1291 of SEQ ID NO:13. The HCC domain of a botulinum neurotoxin type C1 (BoNT / C1) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1112-1291 of SEQ ID NO:41. The HCC domain of a botulinum neurotoxin type D (BoNT / D) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1099-1276 of SEQ ID NO:42. The HCC domain of a botulinum neurotoxin type E (BoNT / E) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1086-1252 of SEQ ID NO:43. The HCC domain of a botulinum neurotoxin type F (BoNT / F) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1106-1274 of SEQ ID NO:44. The HCC domain of a botulinum neurotoxin type G (BoNT / G) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1106-1297 of SEQ ID NO:45. The HCC domain of a tetanus toxin (TeNT) may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% identity to amino acid residues 1128-1315 of SEQ ID NO:46.

[0303] The term "clostridial neurotoxin" is also intended to cover modified clostridial neurotoxins and their derivatives, including but not limited to those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that have been modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. For example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications can alter functional aspects of the toxin, such as biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.

[0304] A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., the modified HC domain), wherein the modified heavy chain has a higher or lower binding affinity for the target nerve cell than the native (unmodified) clostridial neurotoxin. These modifications in the HC domain can include modifying residues at the ganglioside binding site or the protein (SV2 or synaptotagmin) binding site of the HC domain, which alter the binding to the ganglioside receptor and / or protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0305] Thus, the HCC domain of BoNT / A is preferably a modified BoNT / A HCC domain, more preferably a modified BoNT / A HC domain. Thus, preferably, the clostridial neurotoxin according to the invention is a modified BoNT / A. Preferably, the modified clostridial neurotoxin contains one or more modifications that result in an increase in the isoelectric point of the clostridial neurotoxin compared to an equivalent unmodified clostridial neurotoxin lacking the one or more modifications. Suitable modified clostridial neurotoxins are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 14-17 described herein (preferably SEQ ID NO: 14-mrBoNT / A).

[0306] The modified BoNT / A can be BoNT / A modified at one or more amino acid residues (such as selected from ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277). Compared with the use of known BoNT / A, such modified BoNT / A can exhibit reduced or no side effects. The modified BoNT / A can exhibit increased tissue retention properties, thereby providing increased potency and / or duration of action, and can allow the use of lower doses (or increased doses without any additional adverse effects) than known Clostridial toxin therapeutics, thereby providing further advantages.

[0307] The modification can be a modification compared to BoNT / A shown in SEQ ID NO:12, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:12. Since the presence of a methionine residue at position 1 of SEQ ID NO:12 (and the corresponding SEQ ID NOs of the modified BoNT / A polypeptides described herein) is optional, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:12 includes methionine, the position numbering will be defined as above (e.g., ASN 886 will be ASN 886 of SEQ ID NO:12). Alternatively, if methionine is absent in SEQ ID NO:12, the amino acid residue numbering should be modified by minus 1 (e.g., ASN 886 will be ASN 885 of SEQ ID NO:12). Similar considerations apply when methionine is present / absent at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using conventional techniques in the art.

[0308] The alignment for determining amino acid residue numbering described herein can be performed using any method for determining sequence homology and / or % sequence identity described herein.

[0309] The amino acid residues indicated above for modification are surface-exposed amino acid residues.

[0310] Modified BoNT / A can be modified at one or more amino acid residues (such as selected from ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277).

[0311] When using "one or more amino acid residues" in the context of modified BoNT / A, it preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residues. Thus, modified BoNT / A can be modified at the indicated amino acid residues at least 2, 3, 4, 5, 6, or 7 (preferably 7) times. Modified BoNT / A can be subjected to 1 - 30, 3 - 20, or 5 - 10 amino acid modifications. More preferably, when using "one or more amino acid residues" in the context of modified BoNT / A, it means all of the indicated amino acid residues.

[0312] Preferably, in addition to one or more amino acid modifications at the indicated amino acid residues, modified BoNT / A does not contain any further amino acid modifications compared to SEQ ID NO:12.

[0313] The modifications can be selected from:

[0314] i. Replacing acidic surface - exposed amino acid residues with basic amino acid residues;

[0315] ii. Replacing acidic surface - exposed amino acid residues with uncharged amino acid residues;

[0316] iii. Replacing uncharged surface - exposed amino acid residues with basic amino acid residues;

[0317] iv. Inserting basic amino acid residues;

[0318] v. Deleting acidic surface - exposed amino acid residues.

[0319] The modifications as described above result in modified BoNT / A having an increased positive surface charge and an increased isoelectric point compared to the corresponding unmodified BoNT / A (such as SEQ ID NO:12). Without wishing to be bound by theory, it is believed that the increased net positive charge promotes the electrostatic interaction between the polypeptide and anionic extracellular components, thus promoting the binding of the polypeptide to the cell surface and therefore increasing the retention at the administration site and / or the duration of action.

[0320] The isoelectric point (pI) is a specific property of a given protein. As is well known, proteins are composed of a specific sequence of amino acids (also known as amino acid residues in the protein). Each of the standard twenty amino acids has a different side chain (or R-group), which means that each amino acid residue in 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 characteristics of a protein will depend on the sum of these various factors.

[0321] Certain amino acid residues (detailed below) have ionizable side chains that may exhibit a charge depending on the surrounding pH. At a given pH, whether these side chains are charged depends on the pKa of the relevant ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) that specifies the dissociation of a proton from the conjugate base.

[0322] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Thus, these side chains exhibit a negative charge at pH 7.4 (commonly referred to as "physiological pH"). At low pH values, these side chains will be protonated and lose their charge.

[0323] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with a pKa value of approximately 10 - 12. Thus, these side chains exhibit a positive charge at pH 7.4. These side chains will be deprotonated and lose their charge at high pH values.

[0324] 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. Changing the surrounding pH will change the overall charge on the protein. Thus, for each protein, there is a specific pH value at which the number of positive and negative charges is equal and the protein exhibits a zero net charge. This point is called the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry and should be familiar to those skilled in the art.

[0325] The isoelectric point (pI) is thus 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. Thus, an increase in pI represents an increase in the net positive charge of the protein at a given pH value. Conversely, a decrease in pI means that a lower pH value is required for the protein to exhibit a zero net charge. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH value.

[0326] Methods for determining the pI of a protein are known in the art and should be familiar to those skilled in the art. For example, the pI of a protein can be calculated based on 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 of ExPASy (https: / / web.expasy.org / compute_pi / ), which is the preferred method for calculating pI according to the present invention. Comparison of pI values between different molecules should use the same calculation technique / program.

[0327] In appropriate cases, isoelectric focusing techniques ("observed pI") can be used to experimentally confirm the calculated pI of a protein. This technique uses electrophoresis to separate proteins according to their isoelectric points. 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 the pH value at which its net charge is zero, which is the pI of the protein. The results of isoelectric focusing are generally of relatively low resolution, and thus the inventors believe that the results of calculated pI as described above are more suitable for use.

[0328] Throughout this specification, "pI" refers to "calculated pI" unless otherwise specified.

[0329] The pI of a protein can be increased or decreased by changing the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be provided by reducing the number of acidic residues or increasing the number of basic residues.

[0330] The modified BoNT / A of the present invention can have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than that of BoNT / A (e.g., SEQ ID NO:12). Preferably, the modified BoNT / A can have a pI of at least 6.6, such as at least 6.8.

[0331] The properties of the twenty standard amino acids are shown in the following table:

[0332]

[0333]

[0334] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0335] 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 referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

[0336] The following amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0337] The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0338] In amino acid insertion, an additional amino acid residue (an amino acid that is not normally present) is incorporated into the BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in the sequence. In amino acid deletion, an amino acid residue is removed from the clostridial toxin amino acid sequence, thereby reducing the total number of amino acid residues in the sequence.

[0339] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A. In 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 replacing amino acid residue can be one of the twenty standard amino acids described above. Alternatively, the replacing amino acid in amino acid substitution can be a non-standard amino acid (an amino acid that is not part of the twenty standard amino acids described above). For example, the replacing amino acid can 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 Escherichia coli auxotrophic expression hosts.

[0340] In one embodiment, the substitution is selected from: replacing an acidic amino acid residue with a basic amino acid residue, replacing an acidic amino acid residue with an uncharged amino acid residue, and replacing an uncharged amino acid residue with a basic amino acid residue. In one embodiment, when the substitution is replacing an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced with asparagine and glutamic acid is replaced with glutamine).

[0341] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is carried out with lysine or arginine. Most preferably, the modification is carried out with lysine.

[0342] After modification according to the present invention, the modified BoNT / A preferably can bind to the target cell receptor to which unmodified BoNT / A (such as SEQ ID NO: 12) binds.

[0343] Preferably, the modified BoNT / A for use in the present invention comprises 4 to 40 amino acid modifications located in the HCN domain of the clostridial toxin. The modified BoNT / A preferably also has a pI of at least 6.6. The modified BoNT / A preferably comprises at least 4 amino acid modifications selected from ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026 and ASN 1052, wherein the modification comprises substituting an amino acid with a lysine residue or an arginine residue. For example, the modified BoNT / A can comprise at least 5 amino acid modifications selected from ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052 and GLN 1229, wherein the modification comprises substituting an amino acid with a lysine residue or an arginine residue.

[0344] Methods for modifying amino acids of a protein by substitution, insertion or deletion are known in the art. For example, amino acid modification can be achieved by modifying the DNA sequence encoding the polypeptide (such as encoding unmodified BoNT / A or a fragment thereof). This can be done using standard molecular cloning techniques, such as site-directed mutagenesis, where a short DNA strand (oligonucleotide) encoding the desired amino acid is used and a polymerase enzyme is used to replace the original coding sequence, or by inserting / deleting parts of the gene using various enzymes (such as ligases and restriction endonucleases). Alternatively, the modified gene sequence can be prepared by chemical synthesis.

[0345] The modified BoNT / A can 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 can 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 can comprise any one of SEQ ID NOs: 14-17. The modified BoNT / A can 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 can 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 can consist of any one of SEQ ID NOs: 14-17. Among the said SEQ ID NOs, SEQ ID NO: 14 is the most preferred. Those skilled in the art will understand that if there is a % sequence identity difference in the polypeptide sequence of the modified BoNT / A compared to a given SEQ ID NO, at least one modification (e.g., a modification that increases the pI) still exists (e.g., unmodified) in the variant modified BoNT / A.

[0346] Thus, the compositions of the present invention preferably comprise a modified BoNT / A, such as the modified BoNT / A described above. In some embodiments, the Clostridium neurotoxin polypeptide is a modified BoNT / A polypeptide, the L chain is a BoNT / A L chain, the HN domain is a BoNT / A HN domain, and the HCC domain (e.g., the HC domain) is a modified BoNT / A HCC domain (e.g., the modified BoNT / A HCC domain).

[0347] 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 (more preferably consists of) the nucleotide sequence of SEQ ID NO: 49. Those skilled in the art will understand that if there is a % sequence identity difference in the nucleotide sequence encoding the modified BoNT / A compared to a given SEQ ID NO, the encoded modified BoNT / A still comprises at least one modification (e.g., a modification that increases the pI), and thus the relevant region of the nucleotide sequence encoding the at least one modification still exists (e.g., unmodified) in the variant nucleotide sequence.

[0348] Modified BoNT / A can comprise 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) positions 930, 955, 991, 1026, 1052, 1229, and 886. Preferably, modified BoNT / A comprises lysine or arginine (more preferably lysine) at one or more positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, modified BoNT / A comprises lysine or arginine (more preferably lysine) at at least two, three, four, five, six, or all positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, modified BoNT / A comprises lysine or arginine (more preferably lysine) at all positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0349] Clostridial neurotoxins can comprise (or consist of) hybrid or chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of the light chain from one clostridial neurotoxin or its subtype, and at least a portion of the heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin can comprise the entire light chain of the light chain of one clostridial neurotoxin subtype and the heavy chain of another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin can comprise a portion of the heavy chain (e.g., the binding domain) from one clostridial neurotoxin subtype and another portion of the heavy chain from another clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element can comprise a portion of the light chain from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of these clostridial neurotoxins to subjects that are immunoresistant to a given clostridial neurotoxin subtype, subjects with a below-average concentration of receptors for a given clostridial neurotoxin heavy chain binding domain, or subjects with protease-resistant variants of membrane or vesicle toxin substrates (e.g., SNAP-25, VAMP, and Syntaxin). Hybrid and chimeric clostridial neurotoxins are described in US 8,071,110, which is incorporated herein by reference in its entirety.

[0350] The Clostridial neurotoxin of the present invention can be a Clostridial neurotoxin comprising a BoNT / B HCC domain (e.g., a chimeric Clostridial neurotoxin comprising a BoNT / B HC domain), preferably a chimeric Clostridial neurotoxin comprising a BoNT / B HC domain. Thus, in a particularly preferred embodiment, the Clostridial neurotoxin of the present invention can be a chimeric Clostridial neurotoxin comprising (preferably consisting of) a BoNT / A light chain and a translocation domain (LHN domain) and a BoNT / B receptor-binding domain (HC domain). Most preferably, the BoNT / B HC domain comprises the following substitutions E1191M and S1199Y. Suitable chimeric Clostridial neurotoxins can be those taught in WO 2017 / 191315 A1, which is incorporated herein by reference. Such preferred sequences include SEQ ID NOs: 7-11, where SEQ ID NO: 7 is the most preferred.

[0351] The LHN domain of BoNT / A can be covalently linked to the HC domain of BoNT / B. The chimeric BoNT / A is also referred to as "BoNT / AB" or "BoNT / AB chimera".

[0352] In the BoNT / AB chimera, the C-terminal amino acid residue of the LHN domain corresponds to the 8th N-terminal amino acid residue of the 310 helix between the LHN and HC domains of BoNT / A, and the N-terminal amino acid residue of the HC domain corresponds to the 7th N-terminal amino acid residue of the 310 helix between the LHN and HC domains of BoNT / B.

[0353] As used herein, the "1st N-terminal residue of the 310 helix between the LHN and HC domains of BoNT / A" refers to the N-terminal residue of the 310 helix that separates the LHN and HC domains.

[0354] As used herein, the "2nd N-terminal residue of the 310 helix between the LHN and HC domains of BoNT / B" refers to the amino acid residue located after the N-terminal residue of the 310 helix that separates the LHN and HC domains.

[0355] "310 helix" is a secondary structure found in proteins and polypeptides, together with α-helices, β-sheets, and reverse turns. The amino acids in a 310 helix are arranged in a right-handed helical structure, where each turn consists of three residues and ten atoms that separate the intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn of the helix), and is translated along the helix axis (= 0.2 nm), and there are 10 atoms in the ring forming the hydrogen bond. Most importantly, the N-H group of the amino acid forms a hydrogen bond with the C=O group of the amino acid three residues earlier; this repeating i+3→i hydrogen bond defines the 310 helix. The 310 helix is a standard concept in structural biology and should be familiar to those skilled in the art.

[0356] This 310 helix that separates the LHN and HC domains corresponds to four residues forming the actual helix and two cap (or transition) residues, one at each end. The term "310 helix that separates the LHN and HC domains" includes these six residues herein.

[0357] By performing structural analysis and sequence alignment, the 310 helix that separates the LHN and HC domains was determined. This 310 helix is surrounded by an α-helix at its N-terminus (i.e., in the C-terminal part of the LHN domain) and by a β-strand at its C-terminus (i.e., in the N-terminal part of the HC domain). The 1st (N-terminal) residue of the 310 helix that separates the LHN and HC domains also corresponds to the C-terminal residue of this α-helix.

[0358] The 310 helix that separates the LHN and HC domains can be determined, for example, from publicly available crystal structures of botulinum neurotoxins. For example, 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) correspond to botulinum neurotoxin A1 and B1, respectively.

[0359] Publicly available computer simulation and alignment tools can also be used to determine the positions of the 310 helices in the LHN and HC domains isolated from other neurotoxins, such as the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY 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 Clustal Omega (http: / / www.clustal.org / omega / ), and many other tools / services listed in "Internet Resources for Molecular and Cell Biologists" (http: / / molbiol-tools.ca / ). In particular, the region around the "HN / HCN" linker region is highly conserved structurally, making it an ideal region for superposing different serotypes.

[0360] For example, the following methods can be used to determine the sequence of this 310 helix in other neurotoxins:

[0361] 1. Use the structural homology modeling tool LOOP (http: / / loopp.org) to obtain the predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb);

[0362] 2. Edit the resulting structure (pdb) file to include only the N-terminus of the HCN domain and approximately 80 residues preceding it (which are part of the HN domain), thus retaining the highly conserved structurally "HN / HCN" region;

[0363] 3. Use the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) to superpose each serotype onto the 3BTA.pdb structure;

[0364] 4. Examine the superposed pdb files to determine the 310 helix at the start of the HC domain of BoNT / A1 and identify the corresponding residues in other serotypes;

[0365] 5. Align other BoNT serotype sequences with Clustal Omega to check whether the corresponding residues are correct. The following are examples of the LHN, HC, and 310 helix domains determined by this method:

[0366]

[0367]

[0368]

[0369] Through structural analysis and sequence alignment, it was found that the β-strand after the 310 helix separating the LHN and HC domains is a conserved structure in all botulinum and tetanus neurotoxins, and starts from the 1st residue of the 310 helix separating the LHN and HC domains (for example, residue 879 for BoNT / A1).

[0370] The BoNT / AB chimera can contain the LHN domain of BoNT / A covalently linked to the HC domain of BoNT / B,

[0371] · where the C-terminal amino acid residue of the LHN domain corresponds to the 8th N-terminal amino acid residue of the 310 helix between the LHN and HC domains of BoNT / A, and

[0372] · where the N-terminal amino acid residue of the HC domain corresponds to the 7th N-terminal amino acid residue of the 310 helix between the LHN and HC domains of BoNT / B.

[0373] The BoNT / AB chimera can contain the LHN domain of BoNT / A covalently linked to the HC domain of BoNT / B,

[0374] · where the C-terminal amino acid residue of the LHN domain corresponds to the C-terminal amino acid residue of the LHN domain of BoNT / A, and

[0375] · where the N-terminal amino acid residue of the HC domain corresponds to the amino acid residue located after the C-terminal amino acid residue of the LHN domain of BoNT / A.

[0376] The basic principle of the BoNT / AB chimera design process is to try to ensure that the secondary structure is not affected, thereby minimizing any changes to the tertiary structure. Without wishing to be bound by theory, it is assumed that the best conformation of the chimeric neurotoxin is ensured by not disrupting the four central amino acid residues of the 310 helix in the BoNT / AB chimera.

[0377] The LHN domain of BoNT / A can correspond to amino acid residues 1 to 872 of SEQ ID NO:12, or a polypeptide sequence having at least 70% sequence identity. The LHN domain of BoNT / A can correspond to amino acid residues 1 to 872 of SEQ ID NO:12, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity. Preferably, the LHN domain of BoNT / A corresponds to amino acid residues 1 to 872 of SEQ ID NO:12.

[0378] The HC domain of BoNT / B can correspond to amino acid residues 860 to 1291 of SEQ ID NO:13, or a polypeptide sequence having at least 70% sequence identity. The HC domain of BoNT / B can correspond to amino acid residues 860 to 1291 of SEQ ID NO:13, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity. Preferably, the HC domain of BoNT / B corresponds to amino acid residues 860 to 1291 of SEQ ID NO:13.

[0379] Preferably, the LHN domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO:12), and the HC domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO:13).

[0380] Preferably, the HC domain of BoNT / B further comprises at least one amino acid residue substitution, addition or deletion in the HCC domain (such as a subdomain), which has the effect of increasing the binding affinity of the BoNT / B neurotoxin for human SYT II, compared with the native BoNT / B sequence. Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC domain have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both are incorporated herein by reference).

[0381] Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC 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.

[0382] Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC domain also 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.

[0383] Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC domain also include a combination of three substitution mutations, namely E1191M, S1199W and W1178Q.

[0384] Preferably, suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC domain include a combination of two substitution mutations, namely E1191M and S1199Y.

[0385] The modification can be a modification compared to unmodified BoNT / B (such as SEQ ID NO:13), wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:13. Since the presence of a methionine residue at position 1 of SEQ ID NO:13 is optional, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:13 includes methionine, the position numbering will be defined as above (e.g., E1191 will be E1191 of SEQ ID NO:13). Alternatively, if methionine is absent in SEQ ID NO:13, the amino acid residue numbering should be modified by minus 1 (e.g., E1191 will be E1190 of SEQ ID NO:13). Similar considerations apply when methionine is present / absent at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using conventional techniques in the art.

[0386] The chimeric Clostridium neurotoxin can comprise a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NOs: 7-11. In one embodiment, the chimeric Clostridium neurotoxin can comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with any one of SEQ ID NOs: 7-11. Preferably, the chimeric Clostridium neurotoxin can comprise any one of SEQ ID NOs: 7-11. The chimeric Clostridium neurotoxin can consist of a polypeptide sequence having at least 70% sequence identity with any one of SEQ ID NOs: 7-11. In one embodiment, the chimeric Clostridium neurotoxin can consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with any one of SEQ ID NOs: 7-11. Preferably, the chimeric Clostridium neurotoxin can consist of any one of SEQ ID NOs: 7-11. Among the said SEQ ID NOs, SEQ ID NO: 7 is most preferred. Those skilled in the art will understand that if there is a % sequence identity difference in the polypeptide sequence of the chimeric Clostridium neurotoxin comprising at least one BoNT / B HCC domain mutation compared to a given SEQ ID NO, then at least one BoNT / B HCC domain mutation (preferably E1191M and S1199Y) is present (e.g., unmodified) in the variant chimeric Clostridium neurotoxin.

[0387] Thus, the compositions of the present invention most preferably comprise a chimeric Clostridium neurotoxin, such as the chimeric Clostridium neurotoxin described above. In some embodiments, the Clostridium neurotoxin polypeptide is a chimeric Clostridium neurotoxin polypeptide, the L chain is a BoNT / A L chain, the HN domain is a BoNT / A HN domain, and the HCC domain (e.g., HC domain) is a BoNT / B HCC domain (e.g., BoNT / B HCC domain).

[0388] In another embodiment, the Clostridium neurotoxin of the present invention can be a chimeric or hybrid Clostridium neurotoxin comprising a BoNT / X light chain and a translocation domain (LHN domain) and a receptor-binding domain (HC domain) or a portion thereof from a different (i.e., non-BoNT / X) Clostridium neurotoxin. Suitable chimeric and / or hybrid Clostridium neurotoxins can be those taught in WO2020 / 065336A1, which is incorporated herein by reference.

[0389] In embodiments where the Clostridium neurotoxin described herein has a tag (e.g., His tag) and / or linker for purification, the tag and / or linker is optional.

[0390] The Clostridium neurotoxin of the present invention can be free of the complexing proteins present in the naturally occurring Clostridium neurotoxin complex.

[0391] 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.

[0392] In one embodiment, there is provided a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a Clostridial neurotoxin. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0393] In a preferred embodiment, the vector has a promoter selected from:

[0394]

[0395] In another preferred embodiment, the vector has a promoter selected from:

[0396]

[0397] The nucleic acid molecule can be prepared using any suitable method known in the art. Thus, the nucleic acid molecule can be prepared using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be prepared using molecular biology techniques.

[0398] The DNA constructs of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0399] The above nucleic acid sequence information can be codon-biased modified according to the host cell (e.g., E. coli) expression system to be ultimately used.

[0400] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably herein. Preferably, the nucleotide sequence is a DNA sequence.

[0401] The Clostridial neurotoxins of the present invention preferably exist in the form of a disulfide-linked light chain and heavy chain, wherein the light chain is linked to the heavy chain (or a component thereof, such as the HN domain) by a disulfide bond. Thus, the Clostridial neurotoxins of the present invention can be any Clostridial neurotoxin or variant (expressed by the percentage sequence identity with a given SEQ ID NO) herein that has been cleaved by a protease at its activation loop (one or more sites).

[0402] The Clostridial neurotoxin of the present invention preferably comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising at least 70% sequence identity with SEQ ID NO: 14 at one or more sites in its activation loop with a protease. In one embodiment, the Clostridial neurotoxin of the present invention comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO: 14 at one or more sites in its activation loop with a protease. Preferably, the Clostridial neurotoxin of the present invention comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising SEQ ID NO: 14 at one or more sites in its activation loop with a protease.

[0403] The most preferred Clostridial neurotoxin of the present invention comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising at least 70% sequence identity with SEQ ID NO: 7 at one or more sites in its activation loop with a protease. In one embodiment, the Clostridial neurotoxin of the present invention comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity with SEQ ID NO: 7 at one or more sites in its activation loop with a protease. Preferably, the Clostridial neurotoxin of the present invention comprises an L chain and an H chain, wherein the L chain and the H chain are linked by a disulfide bond and can be obtained (for example, by) cleaving a polypeptide comprising SEQ ID NO: 7 at one or more sites in its activation loop with a protease.

[0404] In a particularly preferred embodiment, the disulfide-linked 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 with 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 with SEQ ID NO:79, wherein the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the disulfide-linked 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 chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the disulfide-linked 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 chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by a cysteine residue at position 429 of SEQ ID NO:77 or 78 and a cysteine residue at position 6 of SEQ ID NO:79. The disulfide-linked Clostridial neurotoxin may correspond to the disulfide form of SEQ ID NO:7.

[0405] The protease for cleaving the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce a disulfide-linked Clostridial neurotoxin are taught in WO 2014 / 080206, WO 2014 / 079495 and EP2677029A2, which are incorporated herein by reference.

[0406] Suitable activation loop sequences are shown in the following table:

[0407]

[0408]

[0409]

[0410] Lys-C can cleave the loop C-terminal to one or more lysine residues in the activation loop. If Lys-C cleaves the activation loop multiple times, those skilled in the art will understand that the small peptides of the activation loop of the disulfide-linked Clostridial neurotoxin may not be present compared to the SEQ ID NOs shown herein. For example, SEQ ID NO:75 or 76 may not be present.

[0411] 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 the nucleic acid described herein in an expression host, lysing the host cells to provide a host cell homogenate comprising the single-chain Clostridial neurotoxin, and isolating the single-chain Clostridial neurotoxin. In one aspect, the present invention provides a method for proteolytically processing the Clostridial neurotoxin described herein, the method comprising contacting the Clostridial neurotoxin with a protease that hydrolyzes a peptide bond in the activation loop of the Clostridial neurotoxin, thereby converting the (single-chain) Clostridial neurotoxin into the corresponding disulfide-linked Clostridial neurotoxin (e.g., wherein the light chain and the heavy chain are linked by a disulfide bond).

[0412] Accordingly, the present invention provides a disulfide-linked Clostridial neurotoxin obtained by the method of the present invention.

[0413] If an initial methionine amino acid residue or the corresponding initial codon is indicated in any of the SEQ ID NOs disclosed herein, the residue / codon is optional. Preferably, the initial methionine amino acid residue or the corresponding initial codon is absent.

[0414] The composition for use in the method of the present invention may be a first Clostridial neurotoxin preparation comprising one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

[0415] The method may further comprise determining the Clostridial neurotoxin activity of at least a second Clostridial neurotoxin preparation, wherein the at least second Clostridial neurotoxin preparation comprises the same amount of the same Clostridial neurotoxin as in the first Clostridial neurotoxin preparation 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, propellants, and / or salts present in the first Clostridial neurotoxin preparation.

[0416] The method may comprise comparing the Clostridial neurotoxin activity of the first Clostridial neurotoxin preparation and the at least second Clostridial neurotoxin preparation and selecting one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts when the Clostridial neurotoxin preparations comprising the same show the highest activity.

[0417] The above method may allow the selection of preferred pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts for formulating the Clostridial neurotoxin.

[0418] The method can further include determining the Clostridial neurotoxin activity of at least a second Clostridial neurotoxin preparation, wherein the at least second Clostridial neurotoxin preparation comprises the same amount of the same Clostridial neurotoxin as in the first Clostridial neurotoxin preparation 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 the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts present in the first Clostridial neurotoxin preparation, but are present in different amounts (e.g., different concentrations).

[0419] The method can include comparing the Clostridial neurotoxin activity of the first Clostridial neurotoxin preparation and the at least second Clostridial neurotoxin preparation, and selecting the amounts of the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts when the Clostridial neurotoxin preparations containing the same show the highest activity.

[0420] The above method can allow for the selection of the preferred amounts of pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts for formulating Clostridial neurotoxins.

[0421] In one aspect, the present invention provides a method for producing a Clostridial neurotoxin composition for therapeutic or cosmetic use, the method comprising:

[0422] (a) obtaining the results of the method according to the present invention, and formulating and / or packaging the composition for therapeutic or cosmetic use when the Clostridial neurotoxin activity (e.g., activity level) is the same as or higher than that of the positive control; or

[0423] (b) further purifying the composition when the Clostridial neurotoxin activity is lower than that of the positive control, and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

[0424] In one aspect, there is provided a Clostridial neurotoxin composition for therapeutic or cosmetic use obtained by the method of the present invention, optionally wherein the Clostridial neurotoxin composition for therapeutic or cosmetic use has been packaged.

[0425] As used herein, the term "obtainable" also includes the term "obtained".

[0426] 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, the portion comprising amino acid modifications and / or post-translational modifications. The isolated capture substrate can be complexed with a Clostridial neurotoxin, wherein the HCC domain (e.g., HC domain) of the Clostridial neurotoxin binds to the extracellular portion of the Clostridial neurotoxin receptor polypeptide. The isolated capture substrate can be complexed with a Clostridial neurotoxin receptor binding domain (HCC domain or HC domain). Preferably, the isolated capture substrate comprises an extracellular portion of SYT-II having an L51F substitution, as described herein. Alternatively, the isolated capture substrate can comprise an extracellular portion of SV2c having a post-translational modification (preferably glycosylation) as described herein. An isolated capture substrate refers to a capture substrate that has been isolated from a cell. Such a capture substrate can be produced recombinantly and isolated using standard techniques. Thus, in some embodiments, the term "isolated capture substrate" is intended to include a capture substrate in an in vitro environment. Preferably, the isolated capture substrate, such as the capture substrate described herein, is generally immobilized on a solid support.

[0427] In one aspect, the present invention provides the use of an isolated capture substrate for a Clostridial neurotoxin to determine whether a Clostridial neurotoxin polypeptide contained in a composition has a property of altered activity, wherein the isolated capture substrate comprises an extracellular portion of a Clostridial neurotoxin receptor polypeptide, the portion comprising amino acid modifications and / or post-translational modifications.

[0428] The property that modifies activity can be a property of increased activity or decreased activity, preferably a property of decreased activity. In one embodiment, the property that modifies activity can be any property that modifies the activity of a Clostridial neurotoxin polypeptide compared to an otherwise identical Clostridial neurotoxin (preferably an otherwise identical disulfide-linked Clostridial neurotoxin) lacking said property. In one embodiment, the property that modifies activity can be any property that modifies the activity of a portion of a Clostridial neurotoxin (such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) compared to an otherwise identical portion of a Clostridial neurotoxin lacking said property. Thus, the property that modifies activity can be a topological change and / or a structural change. The property that modifies activity can be a modification (such as a post-translational modification), such as a covalent modification and / or a non-covalent modification. In one embodiment, the property that modifies activity is deamidation, cleavage (such as truncation of a Clostridial neurotoxin polypeptide, such as resulting in the loss of a domain), aggregation, oxidation (such as of a methionine residue or a tryptophan), glycosylation (such as of a lysine residue), lactosylation, incorrect disulfide bond formation (such as resulting in the loss of the L chain or the H chain), incorrect charge (such as incorrect charge distribution), and / or an intact activation loop (such as the composition may be contaminated with a single-chain Clostridial neurotoxin polypeptide). Such a property that modifies activity is preferably a property of decreased activity. Preferably, the property of decreased activity is oxidation.

[0429] An example of a property that modifies activity of the L chain includes an intact activation loop, resulting in an inactive L chain polypeptide. This can occur in the case of failure and / or inefficiency of proteolytic cleavage of a single-chain Clostridial neurotoxin into an active disulfide-linked form. Other properties that modify activity of the L chain can include topological changes, structural changes, deamidation, cleavage, aggregation, oxidation (such as of a methionine residue or a tryptophan), glycosylation (such as of a lysine residue), lactosylation, incorrect disulfide bond formation, and / or incorrect charge (such as incorrect charge distribution).

[0430] An example of a property that modifies activity of the H chain (such as the HC domain) includes topological changes, structural changes, deamidation, cleavage, aggregation, oxidation (such as of a methionine residue or a tryptophan), glycosylation (such as of a lysine residue), lactosylation, and / or incorrect charge (such as incorrect charge distribution).

[0431] Amino acids that can be oxidized include methionine, cysteine, histidine, tryptophan, tyrosine, and / or phenylalanine (Torosantucci et al., 2014, Drug Research 31, 541 - 553).

[0432] The property that alters activity can be a property that alters the activity of a Clostridial neurotoxin (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) by at least 1%, 2%, 5%, 10%, 25%, 50% or 100%, compared to an otherwise identical Clostridial neurotoxin (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) lacking said property. The property that increases activity can be a property that increases the activity of a Clostridial neurotoxin polypeptide (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) by at least 1%, 2%, 5%, 10%, 25%, 50% or 100%, compared to an otherwise identical Clostridial neurotoxin (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) lacking said property. The property that decreases activity can be a property that decreases the activity of a Clostridial neurotoxin polypeptide (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) by at least 1%, 2%, 5%, 10%, 25%, 50% or 100%, compared to an otherwise identical Clostridial neurotoxin (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) lacking said property.

[0433] The property that alters activity is preferably a property that alters the activity of the H chain (such as a modification that alters the activity of the H chain), more preferably a property that decreases the activity of the HC domain, such as a modification that decreases activity present in the HC domain.

[0434] The inventors have found that by combining the method of the present invention with one or more additional methods, valuable insights into the properties of Clostridial neurotoxin compositions can be obtained (see, for example, Example 3). This can allow interrogation of the quality and / or efficacy of such compositions in a domain-specific manner. Advantageously, this represents a significant improvement over cell-based assays, which do not provide mechanistic insights into the specific causes of potency variations.

[0435] Thus, in one embodiment, the method of the present invention can further comprise obtaining the results of a cell-free substrate cleavage assay. In one embodiment, the method of the present invention can further comprise obtaining the results of a heavy chain binding assay. Preferably, the method of the present invention can further comprise obtaining the results of a cell-free substrate cleavage assay and a heavy chain binding assay.

[0436] In one embodiment, the results can be obtained prior to performing the method of the present invention. In some embodiments, negative results or results below the control determined by the cell-free substrate cleavage assay and / or the heavy chain binding assay may require performing the method of the present invention.

[0437] The results of cell-free substrate cleavage assays (such as endopeptidase assays) and / or the results of heavy chain binding assays can be compared with the results of the present invention (such as the determined Clostridium neurotoxin activity of a composition). Such comparison can allow determination of whether the Clostridium neurotoxin polypeptide (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) contained in the composition contains a property that alters activity. Such comparison can allow determination of the amount of Clostridium neurotoxin polypeptide (or a portion thereof, such as the L chain or the H chain or a portion thereof, such as the HC or HCC domain) having a property that alters activity contained in the composition. Preferably, the results of the cell-free substrate cleavage assay and the results of the heavy chain binding assay are compared with the results of the present invention (such as the determined Clostridium neurotoxin activity of a composition).

[0438] The heavy chain binding control and / or (preferably and) the substrate cleavage assay control can be a negative control. The heavy chain binding control and / or (preferably and) the substrate cleavage assay control is preferably a positive control. Suitable positive controls are described herein. Preferably, the heavy chain binding positive control represents a composition comprising a Clostridium neurotoxin polypeptide that does not have a property that alters the activity of the Clostridium neurotoxin H chain, preferably a Clostridium neurotoxin polypeptide that does not have a property that alters the activity of the HC domain. Preferably, the substrate cleavage positive control represents a composition comprising a Clostridium neurotoxin polypeptide that does not have a property that alters the activity of the L chain.

[0439] If the result of the heavy chain binding assay represents lower heavy chain binding of the composition compared to the heavy chain binding positive control, the result of the cell-free substrate cleavage assay represents the same L chain activity of the composition compared to the substrate cleavage positive control, and the result of the method of the present invention represents lower Clostridium neurotoxin activity of the composition compared to a positive control (such as a positive reference standard), then it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has a property of reduced activity of the H chain, preferably a property of reduced activity of the HC domain.

[0440] If the result of the heavy chain binding assay represents higher heavy chain binding of the composition compared to the heavy chain binding positive control, the result of the cell-free substrate cleavage assay represents the same L chain activity of the composition compared to the substrate cleavage positive control, and the result of the method of the present invention represents higher Clostridium neurotoxin activity of the composition compared to a positive control (such as a positive reference standard), then it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has a property of increased activity of the H chain, preferably a property of increased activity of the HC domain.

[0441] If the result of the heavy chain binding assay represents that the heavy chain binding of the composition is the same as that of the heavy chain binding positive control, the result of the cell-free substrate cleavage assay represents that the L-chain activity of the composition is lower than that of the substrate cleavage positive control, and the result of the method of the present invention represents that the Clostridium neurotoxin activity of the composition is lower than that of the positive control (e.g., positive reference standard), it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has the characteristic of reduced L-chain activity.

[0442] If the result of the heavy chain binding assay represents that the heavy chain binding of the composition is the same as that of the heavy chain binding positive control, the result of the cell-free substrate cleavage assay represents that the L-chain activity of the composition is higher than that of the substrate cleavage positive control, and the result of the method of the present invention represents that the Clostridium neurotoxin activity of the composition is higher than that of the positive control (e.g., positive reference standard), it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has the characteristic of increased L-chain activity.

[0443] Those skilled in the art will understand that any differences can be quantified to determine the amount of the polypeptide with a given altered activity characteristic contained in the composition.

[0444] The heavy chain binding assay may include:

[0445] (a) Providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0446] (b) Contacting the capture substrate with a composition containing the Clostridium neurotoxin polypeptide so that the Clostridium neurotoxin polypeptide binds to the capture substrate;

[0447] (c) Removing the unbound Clostridium neurotoxin polypeptide; and

[0448] (d) Determining the amount of the Clostridium neurotoxin polypeptide bound to the capture substrate.

[0449] Thus, in one aspect, the present invention provides a method (cell-free) for detecting the heavy chain binding of a Clostridium neurotoxin polypeptide, the method comprising:

[0450] (a) Providing a capture substrate for the Clostridium neurotoxin polypeptide;

[0451] (b) Contacting the capture substrate with a composition containing the Clostridium neurotoxin polypeptide or a portion thereof (at least comprising the HC or HCC domain of the Clostridium neurotoxin polypeptide) so that the Clostridium neurotoxin polypeptide or a portion thereof binds to the capture substrate;

[0452] (c) Removing the unbound Clostridium neurotoxin polypeptide or a portion thereof; and

[0453] (d) Determining the amount of the Clostridium neurotoxin polypeptide or a portion thereof bound to the capture substrate.

[0454] The amount of Clostridial neurotoxin polypeptide (or a portion thereof) bound to a capture substrate can 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) can be used. A second antibody that specifically binds to the said antibody (suitably a polyclonal antibody) can be used to detect the antibody, and the second antibody comprises a suitable detection means, such as a conjugated peroxidase (e.g., horseradish peroxidase), which can be detected by incubation with a chromogenic (e.g., DAB, TMB, OPD), fluorescent (e.g., ADHP) or chemiluminescent (e.g., ECL) substrate. Changes in chromogenic, fluorescent and / or chemiluminescent signals (indicating the catalytic action of peroxidase) can 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).

[0455] Heavy chain binding assays can be ELISA, such as those known in the art. Suitable methods are provided in Example 3 herein. Another heavy chain binding assay technique can be or can include biolayer interferometry. Such techniques can use an Octet Red 96e system.

[0456] The term "specifically" as used herein in the context of an antibody specifically binding to an antigen (e.g., a Clostridial neurotoxin polypeptide) can mean that the antibody binds to the antigen with a higher specificity and affinity than it binds to non-antigen. In some embodiments, the binding can be at least 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold stronger than the binding to non-antigen.

[0457] The cell-free substrate cleavage assay preferably includes using a cleavable substrate as described herein. In one embodiment, the cell-free substrate cleavage assay can be performed in the same manner as the method of the present invention, but omitting the capture substrate, contacting it with the composition, and removing unbound Clostridial neurotoxin polypeptide. The cell-free substrate cleavage assay can include:

[0458] (a) contacting a composition comprising a Clostridial neurotoxin polypeptide and a reducing agent with a cleavable substrate; and

[0459] (b) determining the amount of cleavage of the cleavable substrate by the L chain polypeptide, thereby determining the L chain activity of the composition.

[0460] Thus, in one aspect, the present invention provides a method for cell-free detection of substrate cleavage, comprising:

[0461] (a) contacting a composition comprising a Clostridial neurotoxin polypeptide and a reducing agent with a cleavable substrate; or

[0462] (b) contacting a composition comprising at least the Clostridial neurotoxin L chain with a cleavable substrate; and

[0463] (b) Determine the amount of cleavage of a cleavable substrate by an L-chain polypeptide (such as the L-chain of a Clostridium neurotoxin polypeptide) to determine the L-chain activity of the composition.

[0464] Due to the presence of a reducing agent, the composition may contain an L-chain polypeptide dissociated from the H-chain polypeptide of the corresponding Clostridium neurotoxin polypeptide. Determining the amount of cleavage of the L-chain polypeptide on the cleavable substrate can be appropriately carried out using any method described herein. Preferably, the same cleavable substrate is used and the same method as in the method of the present invention is used.

[0465] A suitable cell-free substrate cleavage assay method is provided in Example 3 herein.

[0466] In one aspect, the present invention provides the use of an isolated capture substrate for Clostridium neurotoxin to determine whether a Clostridium neurotoxin polypeptide contained in a composition has a reduced activity due to oxidation of the heavy chain (H-chain, such as the HC or HCC domain).

[0467] In one aspect, the present invention provides a cell-free method for determining whether a Clostridium neurotoxin polypeptide or a portion thereof (such as the H-chain, such as the HC or HCC domain) contained in a composition has a property that alters its activity, the method comprising:

[0468] (a) Providing a capture substrate for a Clostridium neurotoxin polypeptide or a portion thereof (such as the H-chain, such as the HC or HCC domain);

[0469] (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide or a portion thereof to the capture substrate;

[0470] (c) Removing the unbound Clostridium neurotoxin polypeptide or a portion thereof;

[0471] (d) Determining the amount of the Clostridium neurotoxin polypeptide or a portion thereof bound to the capture substrate;

[0472] (e) Comparing the amount of the Clostridium neurotoxin polypeptide or a portion thereof bound to the capture substrate with a control; and

[0473] (f) Determining whether the Clostridium neurotoxin polypeptide or a portion thereof (such as the H-chain, such as the HC or HCC domain) contained in the composition has a property that alters its activity (such as an altered activity property of the H-chain) based on the comparison result.

[0474] Preferably, the present invention provides a cell-free method for determining whether a Clostridium neurotoxin polypeptide contained in a composition has a property that alters its activity, the method comprising:

[0475] (a) Providing a capture substrate for a Clostridium neurotoxin polypeptide;

[0476] (b) Contact the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate;

[0477] (c) Remove unbound Clostridium neurotoxin polypeptide;

[0478] (d) Determine the amount of Clostridium neurotoxin polypeptide bound to the capture substrate;

[0479] (e) Compare the amount of Clostridium neurotoxin polypeptide bound to the capture substrate with a control; and

[0480] (f) Determine whether the Clostridium neurotoxin polypeptide contained in the composition has a property that alters activity (e.g., an altered activity property of the H chain) based on the comparison result.

[0481] A suitable control can be any control described herein. A negative control can represent a composition containing a Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) having a property of reduced activity. In such an embodiment, when the amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) bound to the capture substrate is higher than the negative control, it can be determined that the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) contained in the composition does not have the property of reduced activity of the H chain. In such an embodiment, when the amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) bound to the capture substrate is the same as or lower than the negative control, it can be determined that the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) contained in the composition has the property of reduced activity of the H chain.

[0482] The control is preferably a positive control, more preferably the heavy chain-binding positive control described herein.

[0483] In one embodiment, when the amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) bound to the capture substrate is higher than the heavy chain-binding positive control, it can be determined that the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) contained in the composition has the property of increased activity of the H chain.

[0484] In one embodiment, when the amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) bound to the capture substrate is lower than the heavy chain-binding positive control, it can be determined that the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the H chain, e.g., the HC or HCC domain) contained in the composition has the property of reduced activity of the H chain.

[0485] In one embodiment, when the amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the heavy chain, e.g., the HC or HCC domain) bound to the capture substrate is the same as that of the heavy chain binding positive control, it can be determined that the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the heavy chain, e.g., the HC or HCC domain) contained in the composition does not have an altered activity characteristic of the heavy chain.

[0486] The amount of the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the heavy chain, e.g., the HC or HCC domain) bound to the capture substrate can be determined using any suitable method, as described above.

[0487] Any method described herein can further determine whether the Clostridium neurotoxin polypeptide or a portion thereof (e.g., the heavy chain, e.g., the HC or HCC domain) contained in the composition has an altered activity characteristic.

[0488] The method can further include: (i) adding a reducing agent to dissociate the light chain (L chain) of the bound Clostridium neurotoxin polypeptide; and (ii) determining the amount of cleavage of the cleavable substrate by the L chain polypeptide, thereby determining the Clostridium neurotoxin activity of the composition.

[0489] The method can further include: (i) adding a reducing agent to dissociate the light chain (L chain) of the bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L chain polypeptide and a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, e.g., the HC domain); and (ii) determining the amount of cleavage of the cleavable substrate by the L chain polypeptide in the assay sample, thereby determining the Clostridium neurotoxin activity of the composition.

[0490] In one embodiment, when the method includes using a capture substrate (e.g., a cell extracellular portion comprising modified human SYT-II), the method can include comparing the amount of cleavage of the cleavable substrate by the L chain polypeptide with a control.

[0491] A suitable control can be any control described herein. A negative control can represent a composition comprising a Clostridium neurotoxin polypeptide having a reduced activity characteristic.

[0492] The control is preferably a positive control, more preferably the substrate cleavage positive control described herein.

[0493] In one embodiment, when the Clostridium neurotoxin activity of the composition is lower than the substrate cleavage positive control, it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has a reduced activity characteristic of the L chain.

[0494] In one embodiment, when the Clostridium neurotoxin activity of the composition is higher than the substrate cleavage positive control, it can be determined that the Clostridium neurotoxin polypeptide contained in the composition has an increased activity characteristic of the L chain.

[0495] In one embodiment, when the Clostridium neurotoxin activity of the composition is the same as that of the substrate cleavage positive control, it can be determined that the Clostridium neurotoxin polypeptide contained in the composition does not have the altered activity characteristic of the L chain.

[0496] In one aspect, the present invention provides a method for producing a Clostridium neurotoxin composition for therapeutic or cosmetic use, the method comprising:

[0497] (a) obtaining the result of the method of the present invention; and

[0498] (b) formulating and / or packaging the composition for therapeutic or cosmetic use when the Clostridium neurotoxin polypeptide contained in the composition does not have the characteristic of altered activity; or

[0499] (c) further purifying the composition when the Clostridium neurotoxin polypeptide contained in the composition has the characteristic of altered activity; and

[0500] (d) formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

[0501] In one embodiment, when referring to "obtaining the result of the method of the present invention" or "obtaining the result of the method of the present invention" herein, the method carried out to obtain the said result can be carried out as part of the method of the present invention.

[0502] In one aspect, the present invention provides a kit, comprising:

[0503] (a) the isolated capture substrate described herein; and

[0504] (b) optionally, means for detecting the binding of botulinum neurotoxin to the capture substrate; and / or

[0505] (c) optionally, instructions for using the kit.

[0506] Embodiments related to the various methods of the present invention are intended to be equally applicable to alternative methods, products, and / or uses, and vice versa.

[0507] Sequence homology

[0508] A variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybridization methods, such as segmental methods. Protocols for determining percent identity are routine procedures well known to those of skill in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by accumulating scores for 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 common to all input sequences.Non-limiting methods include, for example, Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, “Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences”, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C.E. Lawrence et al., “Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment”, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Wallé et al., “Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences”, 20(9) Bioinformatics: 1428-1435 (2004).

[0509] 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

[0510] 89:10915-19, 1992”. Briefly, two amino acid sequences are aligned using a method with a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix shown by Henikoff and Henikoff (supra) to optimize the alignment score, and this method is generally used to align sequences with the SEQ ID NOs described herein to define amino acid position numbers.

[0511] The "percent sequence identity" between two or more nucleic acid or amino acid sequences depends on the number of identical positions shared by these sequences. Thus, the percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. When calculating the percent sequence identity, the number of gaps, as well as the length of each gap introduced to optimize the alignment of two or more sequences, may also be considered. The sequence comparison and determination of the percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which should be familiar to those skilled in the art.

[0512] Alignment scores are used to determine sequence identity

[0513]

[0514] The percent identity is calculated as:

[0515] Total number of identical matches

[0516] __________________________________________x 100

[0517] Length of the longer sequence plus the total number of gaps introduced to align the two sequences

[0518] Essentially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, typically 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.

[0519] Conservative amino acid substitutions

[0520] Basic: Arginine

[0521] Lysine

[0522] Histidine

[0523] Acidic: Glutamic acid

[0524] Aspartic acid

[0525] Polar: Glutamine

[0526] Asparagine

[0527] Hydrophobic: Leucine

[0528] Isoleucine

[0529] Valine

[0530] Aromatic: phenylalanine

[0531] Tryptophan

[0532] Tyrosine

[0533] Small: glycine

[0534] Alanine

[0535] Serine

[0536] Threonine

[0537] Methionine

[0538] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace the polypeptide amino acid residues. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0539] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. There are several methods available for introducing non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed, in which chemically aminoacylated suppressor tRNAs are used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system containing Escherichia coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, Robertson et al., Journal of the American Chemical Society 113:2722, 1991; Ellman et al., Methods in Enzymology 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proceedings of the National Academy of Sciences of the United States of America 90:10145-9, 1993. A second method is translation in Xenopus oocytes by microinjecting mutant mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., Journal of Biological Chemistry 271:19991-8, 1996). A third method is to culture Escherichia coli cells in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid incorporates into the polypeptide in place of its natural counterpart. See Koide et al., Biochemistry 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-natural species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Science 2:395-403, 1993).

[0540] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0541] The essential amino acids in the polypeptides of the present 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, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutagenesis of amino acids at putative contact sites. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., Journal of Molecular Biology 224:899-904, 1992; Wlodaver et al., FEBS Letters 309:59-64, 1992. The identity of essential amino acids can also be inferred from homology analysis with related components of the polypeptides of the present invention (such as translocation or protease components).

[0542] 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 (Proceedings of the National Academy of Sciences of the United States of America 86:2152-6, 1989). Briefly, these authors disclosed methods of simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochemistry 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).

[0543] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology, 20th Edition, John Wiley and Sons, New York (1994), and Hale & Marham, Harper Collins Dictionary of Biology, Harper Perennial, New York (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in the present invention.

[0544] The present invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction.

[0545] The headings provided herein are not limitations on the various aspects or embodiments of the present invention.

[0546] As used herein, amino acids are referred to by their name, three-letter abbreviation, or single-letter abbreviation. The term "protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the specification and claims of the present invention, the conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids conform to the definitions of the Joint Commission on Biochemical Nomenclature (JCBN) of the International Union of Pure and Applied Chemistry (IUPACIUB). It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by multiple nucleotide sequences.

[0547] Other definitions of terms may occur throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and thus may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present invention will be defined only by the appended claims.

[0548] When a numerical range is provided, it should be understood that unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of that range to one-tenth of the lower limit unit is also specifically disclosed. Each smaller range between any two specified values or intermediate values within the stated range is included within the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range, and each range where either, neither, or both of the limits are included is also included within the present invention, subject to any specific exclusionary limitations within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included within the present invention.

[0549] It must be noted that, as used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "a Clostridial neurotoxin" includes a plurality of such candidate agents, a reference to "the Clostridial neurotoxin" includes a reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0550] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the appended claims. Brief Description of the Drawings

[0552] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings and examples.

[0553] Figure 1 A schematic diagram of an embodiment detection is shown. Botulinum neurotoxin (BoNT, three - quarters circle) binds to a capture substrate (triangle). After appropriate washing steps, dithiothreitol (DTT) is added to reduce the disulfide bond between the heavy and light chains of BoNT, thereby releasing the light chain. The light chain polypeptide is transferred to a separate vial (e.g., well) and incubated with a cleavable substrate that contains synaptosomal - associated protein 25 (SNAP - 25) flanked by cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Activity is determined by measuring the CFP / YFP ratio.

[0554] Figure 2 Toxin binding, as reflected by an increase in the optical density (OD) value, is shown when using casein or 1% bovine serum albumin (BSA) as the carrier protein blocking buffer.

[0555] Figure 3 Binding of recombinant modified botulinum neurotoxin A (mrBoNT / A) to either the SV2c capture substrate alone or SV2c and GT1b is shown.

[0556] Figure 4 Shown is in accordance with Figure 1 In the assay of, the amount of cleaved SNAP - 25 (represented by a decrease in the CFP / YFP ratio) when the test composition contains full - length mrBoNT / A (circles) or only the light chain (squares).

[0557] Figure 5 Verification of the detection of mrBoNT / A (A) and mrBoNT / AB (B) is shown, demonstrating linearity, accuracy, and precision between the 150% and 50% levels.

[0558] Figure 6Shows the percentage of activity of the mrBoNT / A composition after forced oxidation as a function of time shown on the x-axis when tested in a cell-based assay (circles), an endopeptidase assay (triangles), an enzyme-linked immunosorbent assay (ELISA, inverted triangles), or a binding and cleavage assay (diamonds) as shown in Figure 1 Also shown is the amount of oxidized mrBoNT / A peptide (specifically, the percentage unmodified / unoxidized) as a function of time, which peptide encompasses at least part of the C-terminal domain of the heavy chain (HC domain, squares).

[0559] Figure 7 Shows a summary of the biolayer interferometry (BLI) binding kinetics of unoxidized (control) or oxidized (48 hours, 0.01% HO) mrBoNT / A to a capture substrate containing the extracellular portion of non-glycosylated GST-SV2c expressed in E. coli (upper panel), or to a capture substrate containing the extracellular portion of glycosylated GST-SV2c expressed in human embryonic kidney 293 (HEK293) cells (lower panel).

[0560] Figure 8 Shows the percentage of activity of the mrBoNT / AB composition when tested in a cell-based assay (triangles) or in an ELISA using a capture substrate containing the extracellular portion of human synaptotagmin II (SYTII) with the L51F mutation (circles). Also shown is the amount of oxidized (specifically, the percentage unchanged / unoxidized) mrBoNT / AB peptide (squares) that encompasses at least part of the HC domain.

[0561] Figure 9 Shows a summary of the biolayer interferometry (BLI) binding kinetics of unoxidized (control) or oxidized (72 hours, 0.001% HO) mrBoNT / AB to a capture substrate containing the extracellular portion of human SYTII with the L51F mutation.

[0562] Figure 10 Shows the correlation between the BoNT activity results of a cell-free assay and a cell-based assay according to Figure 1 .

[0563] Figure 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; (B) A schematic of an exemplary assay. BoNT (three-quarters circle) binds to a capture substrate (triangle), and after appropriate washing steps, dithiothreitol (DTT) and the cleavable substrate are added to reduce the disulfide bond between the heavy and light chains of BoNT, thereby releasing the light chain. The light chain is not removed. Subsequently, a luciferase substrate is added and the luminescence intensity is measured to calculate the BoNT activity of the composition.Figure 12 Shown: (A) Using Figure 1 the cleavable substrate shown (comprising CFP and YFP) to evaluate the BoNT activity of the composition; (B) Using Figure 11 the cleavable substrate shown in A (comprising the first and second luciferase domains) to evaluate the BoNT activity of the same composition, but after reduction with DTT, the light chain polypeptide is transferred to a separate vial (e.g., well) and incubated with the cleavable substrate; (C) Using Figure 11 the method shown in B (i.e., the light chain is not removed, and Figure 11 the cleavable substrate shown in A is added to the vial (e.g., well)) to evaluate the BoNT activity of the same composition.

[0564] Figure 13 Shown Figure 11 is the detection verification of the mrBoNT / AB method shown in B, demonstrating linearity, accuracy, and precision between the 150% and 50% levels.

[0565] Figure 14 Shown: (A) A reference curve generated by evaluating as Figure 1 performed but using Figure 11 the cleavable substrate shown in A (comprising the first and second luciferase domains); (B) The target recovery of 5 ng / mL mrBoNT / AB in drug buffer reconstituted incrementally in simulated saline.

[0566] Figure 15 Shown is the linearity and parallelism of drug assay variation between the 130% and 75% levels.

[0567] Figure 16 Shown are the combined results of various assay formats of the forced degradation samples of mrBoNT / A (A) and mrBoNT / AB (48 hours, 0.001% HO) (B), compared to cell-based assay data (control).

[0568] Figure 17 Shown is the percentage of activity of oxidized mrBoNT / AB in a modified binding and cleavage assay (see Example 2) using human SYT-I (squares) or human SYT-II (L51F modified - circles) capture substrates. This activity is shown corresponding to the percentage of the clostridial neurotoxin polypeptide oxidized at the reference amino acid residue in the composition.

[0569] Sequence Listing

[0570] When the starting methionine (Met) amino acid residue or the corresponding start codon is indicated in any of the following SEQ ID NOs, the residue / codon is optional. Preferably, the starting methionine amino acid residue or the corresponding start codon is absent.

[0571] SEQ ID NO: 1 - Polypeptide Sequence of Luciferase

[0572] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL

[0573] SEQ ID NO: 2 - Polypeptide Sequence of Luciferase Domain 1

[0574] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS

[0575] SEQ ID NO: 3 - Polypeptide Sequence of Luciferase Domain 2

[0576] VTGYRLFEEIL

[0577] SEQ ID NO: 4 - Polypeptide Sequence of Full - Length Synaptosomal - Associated Protein 25 (SNAP - 25)

[0578] MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

[0579] SEQ ID NO: 5 - Polypeptide Sequence of 65 - Amino - Acid Synaptosomal - Associated Protein 25 (SNAP - 25)

[0580] RENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

[0581] SEQ ID NO: 6 - Polypeptide Sequence of Luciferase Cleavable Substrate

[0582] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINSSGGGGSRENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGSGGGGSVTGYRLFEEIL

[0583] SEQ ID NO: 7 - Polypeptide Sequence of Recombinant Modified Botulinum Neurotoxin AB (mrBoNT / AB)

[0584]

[0585] SEQ ID NO: 8 - Polypeptide Sequence of Botulinum Neurotoxin AB Variant 2 (BoNT / AB Variant 2)

[0586]

[0587] SEQ ID NO: 9 - Polypeptide Sequence of Botulinum Neurotoxin AB Variant 3

[0588]

[0589] SEQ ID NO: 10 - Polypeptide Sequence of Botulinum Neurotoxin AB Variant 4

[0590]

[0591] SEQ ID NO: 11 - Polypeptide Sequence of Botulinum Neurotoxin AB Variant 5

[0592]

[0593] SEQ ID NO: 12 - Polypeptide Sequence of Native Botulinum Neurotoxin A (BoNT / A)

[0594]

[0595] SEQ ID NO: 13 - Polypeptide Sequence of Botulinum Neurotoxin B

[0596]

[0597] SEQ ID NO: 14 - Polypeptide Sequence of Recombinant Modified Botulinum Neurotoxin A (mrBoNT / A)

[0598]

[0599] SEQ ID NO: 15 - Polypeptide Sequence of Cationic Botulinum Neurotoxin A Variant 2

[0600]

[0601] SEQ ID NO: 16 - Polypeptide Sequence of Cationic Botulinum Neurotoxin A Variant 3

[0602]

[0603] SEQ ID NO: 17 - Polypeptide Sequence of Cationic Botulinum Neurotoxin A Variant 4

[0604]

[0605] SEQ ID NO: 18 - Polypeptide Sequence of Capture Substrate Comprising the Extracellular Portion of Human Wild - Type Synaptotagmin II (Amino Acids 1 - 61) Polypeptide Sequence

[0606] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLP

[0607] SEQ ID NO: 19 - Polypeptide Sequence of the Extracellular Portion of Human Wild - Type Synaptotagmin II (Amino Acids 1 - 61)

[0608] MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLP

[0609] SEQ ID NO: 20 - Polypeptide Sequence of Capture Substrate Comprising the Extracellular Portion of Human Modified Synaptotagmin II (L51F) (Amino Acids 1 - 61) Polypeptide Sequence

[0610] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKFFNEINKIPLP

[0611] SEQ ID NO: 21 - Polypeptide Sequence of the Extracellular Portion of Human Modified Synaptotagmin II (L51F) (Amino Acids 1 - 61)

[0612] MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKFFNEINKIPLP

[0613] SEQ ID NO: 22 - Polypeptide Sequence of Capture Substrate Comprising the Extracellular Portion of Mouse Synaptotagmin II (Amino Acids 1 - 64) Polypeptide Sequence

[0614] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMRNIFKRNGEPNVAPATTTATMPLAPVAPADNSTESTGPGESQEDMFAKLKEKFFNEINKIPLP

[0615] SEQ ID NO: 23 - Polypeptide Sequence of Full - Length Human Wild - Type Synaptotagmin II

[0616] MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLPPWALIAIAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDDDDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVKVFLLPDKKKKYETKVHRKTLNPAFNETFTFKVPYQELGGKTLVMAIYDFDRFSKHDIIGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVVTVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGKNK

[0617] SEQ ID NO: 24 - Polypeptide Sequence of Full - Length Mouse Wild - Type Synaptotagmin II

[0618] MRNIFKRNQEPNVAPATTTATMPLAPVAPADNSTESTGPGESQEDMFAKLKEKFFNEINKIPLPPWALIAMAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDDDDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVKVFLLPDKKKKYETKVHRKTLNPAFNETFTFKVPYQELAGKTLVMAIYDFDRFSKHDIIGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVVTVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGKNK

[0619] SEQ ID NO: 25 - Polypeptide Sequence of Human Full - Length Synaptophysin 2a (SV2a)

[0620] MEEGFRDRAAFIRGAKDIAKEVKKHAAKKVVKGLDRVQDEYSRRSYSRFEEEDDDDDFPAPSDGYYRGEGTQDEEEGGASSDATEGHDEDDEIYEGEYQGIPRAESGGKGERMADGAPLAGVRGGLSDGEGPPGGRGEAQRRKEREELAQQYEAILRECGHGRFQWTLYFVLGLALMADGVEVFVVGFVLPSAEKDMCLSDSNKGMLGLIVYLGMMVGAFLWGGLADRLGRRQCLLISLSVNSVFAFFSSFVQGYGTFLFCRLLSGVGIGGSIPIVFSYFSEFLAQEKRGEHLSWLCMFWMIGGVYAAAMAWAIIPHYGWSFQMGSAYQFHSWRVFVLVCAFPSVFAIGALTTQPESPRFFLENGKHDEAWMVLKQVHDTNMRAKGHPERVFSVTHIKTIHQEDELIEIQSDTGTWYQRWGVRALSLGGQVWGNFLSCFGPEYRRITLMMMGVWFTMSFSYYGLTVWFPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAYMVYFVSFLGTLAVLPGNIVSALLMDKIGRLRMLAGSSVMSCVSCFFLSFGNSESAMIALLCLFGGVSIASWNALDVLTVELYPSDKRTTAFGFLNALCKLAAVLGISIFTSFVGITKAAPILFASAALALGSSLALKLPETRGQVLQ

[0621] SEQ ID NO: 26 - Polypeptide Sequence of Human Full - Length Synaptophysin SV2b

[0622] MDDYKYQDNYGGYAPSDGYYRGNESNPEEDAQSDVTEGHDEEDEIYEGEYQGIPHPDDVKAKQAKMAPSRMDSLRGQTDLMAERLEDEEQLAHQYETIMDECGHGRFQWILFFVLGLALMADGVEVFVVSFALPSAEKDMCLSSSKKGMLGMIVYLGMMAGAFILGGLADKLGRKRVLSMSLAVNASFASLSSFVQGYGAFLFCRLISGIGIGGALPIVFAYFSEFLSREKRGEHLSWLGIFWMTGGLYASAMAWSIIPHYGWGFSMGTNYHFHSWRVFVIVCALPCTVSMVALKFMPESPRFLLEMGKHDEAWMILKQVHDTNMRAKGTPEKVFTVSNIKTPKQMDEFIEIQSSTGTWYQRWLVRFKTIFKQVWDNALYCVMGPYRMNTLILAVVWFAMAFSYYGLTVWFPDMIRYFQDEEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDNDFLIYLVSFLGSLSVLPGNIISALLMDRIGRLKMIGGSMLISAVCCFFLFFGNSESAMIGWQCLFCGTSIAAWNALDVITVELYPTNQRATAFGILNGLCKFGAILGNTIFASFVGITKVVPILLAAASLVGGGLIALRLPETREQVLM

[0623] SEQ ID NO: 27 - Polypeptide Sequence of Human Full - Length Synaptophysin SV2c

[0624] MEDSYKDRTSLMKGAKDIAREVKKQTVKKVNQAVDRAQDEYTQRSYSRFQDEEDDDDYYPAGETYNGEANDDEGSSEATEGHDEDDEIYEGEYQGIPSMNQAKDSIVSVGQPKGDEYKDRRELESERRADEEELAQQYELIIQECGHGRFQWALFFVLGMALMADGVEVFVVGFVLPSAETDLCIPNSGSGWLGSIVYLGMMVGAFFWGGLADKVGRKQSLLICMSVNGFFAFLSSFVQGYGFFLFCRLLSGFGIGGAIPTVFSYFAEVLAREKRGEHLSWLCMFWMIGGIYASAMAWAIIPHYGWSFSMGSAYQFHSWRVFVIVCALPCVSSVVALTFMPESPRFLLEVGKHDEAWMILKLIHDTNMRARGQPEKVFTVNKIKTPKQIDELIEIESDTGTWYRRCFVRIRTELYGIWLTFMRCFNYPVRDNTIKLTIVWFTLSFGYYGLSVWFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAYWIYFVNFLGTLAVLPGNIVSALLMDRIGRLTMLGGSMVLSGISCFFLWFGTSESMMIGMLCLYNGLTISAWNSLDVVTVELYPTDRRATGFGFLNALCKAAAVLGNLIFGSLVSITKSIPILLASTVLVCGGLVGLCLPDTRTQVLM

[0625] SEQ ID NO: 28 - Polypeptide Sequence of Human Full - Length Synaptotagmin SYT - I

[0626] MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPWALIAIAIVAVLLVLTCCFCICKKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLAVKK

[0627] SEQ ID NO: 29 - Polypeptide Sequence of Capture Substrate Comprising the Extracellular Portion of Synaptophysin SV2c (Amino Acids 473 to 567) Polypeptide Sequence

[0628] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLSSGLEVLFQGPVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0629] SEQ ID NO: 30 - Polypeptide Sequence of the Extracellular Portion of Synaptophysin SV2c (Amino Acids 473 to 567)

[0630] VERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0631] Polypeptide sequence of SEQ ID NO: 31 - human vesicle-associated membrane protein 1 (VAMP1_human, P23763)

[0632] MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT

[0633] Polypeptide sequence of SEQ ID NO: 32 - human vesicle-associated membrane protein 2 (VAMP2_human, P63027)

[0634] MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

[0635] Polypeptide sequence of SEQ ID NO: 33 - human vesicle-associated membrane protein 3 (VAMP3_human, Q15836) MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

[0636] Polypeptide sequence of SEQ ID NO: 34 - human vesicle-associated membrane protein 4 (VAMP4_human, O75379)

[0637] MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

[0638] Polypeptide sequence of SEQ ID NO: 35 - human vesicle-associated membrane protein 5 (VAMP5_human, O95183) MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN

[0639] Polypeptide sequence of SEQ ID NO: 36 - human YKT6 (O15498)

[0640] MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

[0641] Polypeptide sequence of SEQ ID NO: 37 - synaptotagmin 1A

[0642] MKDRTQELRTAKDSDDDDDVAVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEEVKRKHSAILASPNPDEKTKEELEELMSDIKKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGRTTTSEELEDMLESGNPAIFASGIIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVILGIVIASTVGGIFA

[0643] Polypeptide sequence of SEQ ID NO: 38 - synaptotagmin 1B

[0644] MKDRTQELRSAKDSDDEEEVVHVDRDHFMDEFFEQVEEIRGCIEKLSEDVEQVKKQHSAILAAPNPDEKTKQELEDLTADIKKTANKVRSKLKAIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMTEYNATQSKYRDRCKDRIQRQLEITGRTTTNEELEDMLESGKLAIFTDDIKMDSQMTKQALNEIETRHNEIIKLETSIRELHDMFVDMAMLVESQGEMIDRIEYNVEHSVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVVLGVVLASSIGGTLGL

[0645] Polypeptide sequence of SEQ ID NO: 39 - spacer region

[0646] SGGGGS

[0647] Polypeptide of a capture substrate comprising the extracellular portion (amino acids 473 to 567) of synaptotagmin SV2c Sequence MGWSCIILFLVATATGVHSGGGGSSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLSSGLEVLFQGPVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT

[0648] Polypeptide sequence of SEQ ID NO: 41 - botulinum neurotoxin BoNT / C - Universal Protein Resource (UniProt) accession number P18640

[0649] Polypeptide sequence of SEQ ID NO: 42 - botulinum neurotoxin BoNT / D - Universal Protein Resource (UniProt) accession number P19321

[0650]

[0651] Polypeptide sequence of SEQ ID NO: 43 - botulinum neurotoxin BoNT / E - Universal Protein Resource (UniProt) accession number Q00496

[0652]

[0653] Polypeptide sequence of SEQ ID NO: 44 - botulinum neurotoxin BoNT / F - Universal Protein Resource (UniProt) accession number A7GBG3

[0654]

[0655] Polypeptide sequence of SEQ ID NO: 45 - botulinum neurotoxin BoNT / G - Universal Protein Resource (UniProt) accession number Q60393

[0656]

[0657] Polypeptide sequence of SEQ ID NO: 46 - tetanus neurotoxin (TeNT) - Universal Protein Resource (UniProt) accession number P04958

[0658] Polypeptide sequence of SEQ ID NO: 47 - botulinum neurotoxin BoNT / X

[0659]

[0660] Polypeptide sequence of SEQ ID NO: 48 - His - TEV sequence

[0661] MHHHHHHDDDDK

[0662] Nucleotide sequence encoding recombinant modified botulinum neurotoxin A (mrBoNT / A)

[0663]

[0664] Nucleotide sequence encoding the extracellular portion (amino acids 1 - 61) of human modified synaptotagmin SYTII (L51F) Nucleotide sequence encoding the SV2c capture substrate

[0665] ATGCGTAACATCTTCAAACGTAACCAAGAGCCGATTGTTGCGCCGGCGACCACCACCGCGACCATGCCGATTGGTCCGGTTGACAACAGCACCGAAAGCGGTGGCGCGGGTGAAAGCCAAGAAGATATGTTTGCGAAGCTGAAAGAGAAGTTCTTTAACGAAATCAACAAGATTCCGCTGCCG

[0666] Polypeptide sequence of SEQ ID NO: 75 - C-terminal light chain fragment

[0667]

[0668] Polypeptide sequence of SEQ ID NO: 76 - C-terminal light chain fragment 2

[0669] TKSLDKGYNK

[0670] Polypeptide sequence of SEQ ID NO: 77 - double-stranded light chain 1

[0671] SLDKGYNK

[0672] Polypeptide sequence of SEQ ID NO: 78 - double-stranded light chain 2

[0673] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0674] Polypeptide sequence of SEQ ID NO: 79 - double-stranded heavy chain

[0675] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0676] Polypeptide sequence of SEQ ID NO: 80 - extracellular portion (amino acids 1 - 60) of human synaptotagmin SYT-I

[0677] ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0678] Polypeptide of a capture substrate comprising the extracellular portion (amino acids 1 - 60) of human synaptotagmin SYT-I

[0679] MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPWA

[0680] Sequence Polypeptide sequence of SEQ ID NO: 82 - full-length mouse synaptotagmin SYT-I

[0681] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPPWA

[0682] Polypeptide sequence of SEQ ID NO: 83 - extracellular portion (amino acids 1 - 59) of mouse synaptotagmin SYT-I

[0683] MVSASRPEALAAPVTTVATLVPHNATEPASPGEGKEDAFSKLKQKFMNELHKIPLPPWALIAIAIVAVLLVVTCCFCVCKKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLAVKK

[0684] ​

[0685] MVSASRPEALAAPVTTVATLVPHNATEPASPGEGKEDAFSKLKQKFMNELHKIPLPPWA

[0686] Serial number 84 - polypeptide sequence of a capture substrate containing the extracellular portion of murine synaptotagmin SYT-I (amino acids 1 - 59) Peptide sequence

[0687] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSGTENLYFQGMVSASRPEALAAPVTTVATLVPHNA Example

[0688] Example 1

[0689] Materials and methods

[0690] The relevant capture substrate for botulinum neurotoxin serotype was fixed on a transparent Maxisorp 96-well microtiter plate at a concentration of 5 μg / mL in Dulbecco's PBS at 37°C and 600 rpm for 1 hour. Subsequently, it was blocked with 150 μL of Dulbecco's PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20. Then, the relevant botulinum neurotoxin test samples were serially diluted in Dulbecco's PBS containing 1% BSA and 0.05% Tween 20 and added to the plate. After shaking at 37°C and 600 rpm for 1 hour, the unbound botulinum neurotoxin on the plate was removed by washing once with Dulbecco's PBS containing 1% BSA and 0.05% Tween 20. Next, it was shaken at 37°C and 600 rpm for 15 minutes in a solution containing 50 mM Hepes-NaOH (pH 7.1), 5 mM sodium chloride, 0.1% Tween-20, 10 μM zinc chloride, and 5 mM dithiothreitol (DTT) to release the bound toxin from the capture substrate. Then the supernatant was transferred to a black opaque microtiter plate containing 2 μM of the synaptosome-associated protein 25 (SNAP25) substrate labeled with cyan fluorescent protein (CFP) / yellow fluorescent protein (YFP) (see Figure 1) Incubate at 37 °C and 600 revolutions per minute for 3 hours, and then measure fluorescence at excitation / emission wavelengths of 434 / 526 (fluorescence resonance energy transfer (FRET) signal) and 434 / 470 (CFP signal).

[0691] Results

[0692] A novel plate-based detection platform was developed to evaluate the affinity of Clostridium neurotoxins for their receptors and their endopeptidase activity ( Figure 1 ). This assay utilizes the commercially available BoTest endopeptidase reporter system (BioSentinel), combined with a custom recombinant expressed receptor substrate (capture substrate), to measure the masses of the toxin light and heavy chains, respectively. The aims of this method are: (i) to provide accurate sample titer prediction to reduce the need for cell-based assays; (ii) to characterize and understand the mechanism of action during toxin sample degradation.

[0693] Substrate

[0694] For the binding experiment of recombinant modified botulinum neurotoxin A (mrBoNT / A, SEQ ID NO: 14, converted to a double-chain toxin by incubation with lysine-C (Lys-C), see WO2014 / 080206, which is incorporated herein by reference), a toxin-binding region containing SV2c (519-563) was employed. Recombinant glutathione-S-transferase (GST)-SV2c (amino acids 473-567) as shown in SEQ ID NO: 29 was expressed in Escherichia coli.

[0695] To obtain a similar material with eukaryotic peptide glycosylation, the GST-SV2c (473-567) substrate (SEQ ID NO: 40) was also prepared in human embryonic kidney 293 (HEK293) mammalian cells. Mass spectrometry analysis was performed, and the results showed that when expressed in mammalian HEK293 cells, the GST-SV2c (473-567) substrate was a mixture of G0F, G1F, and G2F glycans, indicating that glycosylation was consistent but heterogeneous (see the table below).

[0696]

[0697] Specifically, glycosylation was observed at the physiologically relevant amino acid residue N559.

[0698] For the binding experiment of mrBoNT / AB (SEQ ID NO:7, converted to a double-chain toxin by incubation with Lys-C), the synaptotagmin II (SYTII) substrate, i.e., GST-tagged SYTII(1-61), was prepared in E. coli. This SYTII is human-derived and has an L51F amino acid mutation (SEQ ID NO:20).

[0699] Binding characteristics of the capture substrate

[0700] After serial dilution of the toxin and addition to the plate, the captured toxin was probed with an anti-botulinum neurotoxin antibody, followed by secondary detection with a horseradish peroxidase (HRP)-conjugated anti-species antibody. The level of binding to the captured receptor is proportional to the affinity of the neurotoxin for the captured substrate. The effect of the carrier protein blocking buffer on the detection sensitivity was evaluated. Surprisingly, using 1% BSA as the blocking agent improved the binding compared to casein ( Figure 2 ).

[0701] To increase toxin binding, an attempt was made to co-immobilize ganglioside GT1b with the capture substrate containing the extracellular portion of SV2c on the plate. Neither pre-coating GT1b before immobilizing the SV2c portion capture substrate nor co-immobilizing GT1b with the SV2c portion capture substrate improved toxin capture better than using the SV2c portion capture substrate alone. In fact, surprisingly, co-immobilization with GT1b actually decreased the detection sensitivity ( Figure 3 ). Therefore, to improve sensitivity, it is considered advantageous not to use gangliosides (such as GT1b) in the binding and cleavage assays.

[0702] Binding and cleavage detection

[0703] After characterizing the binding properties, the binding and cleavage assays of the complete capture substrate were characterized. The assays were performed as described above. Briefly, the capture substrate was immobilized in the wells of a microplate before incubation with mrBoNT / A or mrBoNT / AB, respectively. The botulinum neurotoxin-capture substrate complex was washed, and then a buffer containing 50 mM HEPES-NaOH (pH 7.1), 5 mM sodium chloride, 0.1% Tween-20, 10 μM zinc chloride, and 5 mM DTT was added to release the light chain from the bound toxin. Then the supernatant was transferred to another assay plate containing a CFP / YFP FRET-based cleavable substrate ( Figure 1 ). This enabled the measurement of the endopeptidase activity of the light chain of the toxin specifically bound to its corresponding capture substrate, thus excluding the influence of free light chain polypeptides that may arise from toxin degradation and impurities.

[0704] To evaluate the effect of any non-specific endopeptidase activity in the toxin binding step, a control containing only the recombinant light chain was used. Figure 4 It was shown that no endopeptidase activity was detected in the sample containing only the recombinant light chain, indicating that there was no residual neurotoxin not bound to its specific receptor. Thus, the assay showed little / no non-specific background.

[0705] Detection verification

[0706] To evaluate accuracy, precision and reproducibility, binding and cleavage assays were performed in triplicate at five levels from 50% to 150% for mrBoNT / A and mrBoNT / AB, respectively. As Figure 5 shown in A, the assay showed high accuracy and precision at the five levels. The standard deviation of triplicates at the five levels was on average less than 6%, and the deviation of the average recovery from the target value was less than 5%. This indicates that the assay has good linearity, accuracy and precision between 50% and 150%. As Figure 5 shown in B, when the assay was performed using a composition containing mrBoNT / AB, the assay composition also showed comparable accuracy, linearity and precision. The following table summarizes the assays performed using mrBoNT / A or mrBoNT / AB compositions.

[0707]

[0708]

[0709] Determination of the stability of the Clostridial neurotoxin composition

[0710] To test the stability indicating, comparability and potency prediction properties of the assay, degraded samples of mrBoNT / A and mrBoNT / A were tested and compared with orthogonal binding methods, mass spectrometry and cell-based methods. Oxidized mrBoNT / A

[0711] Figure 6 showed that the binding and cleavage assay was highly comparable to existing cell-based methods, and the measured activity levels were correlated with the amount of oxidized mrBoNT / A present in the composition. When tested using the standard BoTest, forced oxidation of mrBoNT / A did not appear to affect the light chain activity alone; however, when tested in the binding and cleavage assay, the potency estimate was closely correlated with the total physiological potency determined by the cell-based assay.

[0712] mrBoNT / A samples from the same oxidation study were also evaluated by biolayer interferometry (BLI) using the Octet Red 96e system, using glycosylated and non-glycosylated capture substrates containing the extracellular portion of GST-tagged SV2c.

[0713] As Figure 7 shown, the non-glycosylated capture substrate bound to mrBoNT / A faster than the glycosylated form. However, the dissociation rate of the glycosylated capture substrate from the toxin was significantly slower. Oxidized mrBoNT / A showed reduced binding to both glycosylated and non-glycosylated forms of the capture substrate. The dissociation rate of the toxin from both types (glycosylated and non-glycosylated) of capture substrate was also prolonged by oxidized mrBoNT / A.

[0714] Thus, combining Figure 6 and Figure 7 results, binding and cleavage assays were able to detect differences in the activity of oxidized botulinum neurotoxin, which may be due to reduced binding of the capture substrate.

[0715] Oxidized mrBoNT / AB

[0716] Next, the forced-oxidized mrBoNT / AB samples were analyzed by the above ELISA method using a capture substrate containing the extracellular portion of human SYTII with the L51F mutation. Figure 8 It was shown that oxidation of the heavy chain (HC) domain of mrBoNT / AB was associated with reduced activity as evaluated by cell-based assays, and its receptor binding was also reduced by ELISA assays.

[0717] Similarly, for mrBoNT / A, a BLI tandem assay was performed on the forced-oxidized samples ( Figure 9 ). The binding affinity of oxidized mrBoNT / AB for the SYTII capture substrate containing the L51F mutation was reduced.

[0718] Therefore, these results indicate that oxidized mrBoNT / AB has a different receptor binding profile from unoxidized equivalent mrBoNT / AB. Thus, binding and cleavage assays have the potential to distinguish the activity levels of oxidized and unoxidized mrBoNT / AB. In addition, binding and cleavage assays have the potential to attribute the loss of activity to specific parts of the toxin mechanism of action (e.g., binding and / or proteolytic activity), thus providing mechanistic insights into the root cause of the loss of activity of the composition.

[0719] Correlation with cell-based activity results

[0720] Because the binding and cleavage assay simultaneously measures the heavy chain affinity of the toxin composition and the endopeptidase activity of the toxin that has successfully bound to its corresponding receptor, samples that generate a signal in this assay are highly likely to be biologically active. To confirm this, a given composition was tested using the binding and cleavage assay, and the same composition was tested in parallel using a cell-based assay. Figure 10 showed a highly significant correlation (P = 0.0001) between the activity data obtained using the binding and cleavage assay and the cell-based assay. This data set included 27 samples, including mrBoNT / AB or mrBoNT / A samples that had been subjected to forced degradation (including oxidation, glycosylation, and heat stress), or samples obtained from different stages of the mrBoNT / AB production process (including samples at different purity levels, or samples expressed or isolated under different conditions).

[0721] Conclusion

[0722] In summary, the binding and cleavage assay can reliably predict the biological activity of Clostridium neurotoxin compositions. Because the data is highly correlated with the results of cell-based assays, the binding and cleavage assay can also be used as a high-throughput screening tool to classify large numbers of compositions prior to cell-based testing. By testing samples, this assay has proven to be a stability indicator for several covalent post-translational modifications of toxins, such as oxidation and glycosylation, which can be attributed to specific domains. This assay has also proven to be a stability indicator for changes in the toxin manufacturing process (with unknown structural / biological differences). The results in this example show that the loss of neurotoxin potency can be predicted by a combinatorial approach and specifically attributed to domains of the toxin sample (see Example 3).

[0723] Example 2

[0724] Materials and methods

[0725] The relevant capture substrate for botulinum neurotoxin serotype was fixed on a white Maxisorp 96-well microtiter plate at a concentration of 5 μg / mL in Dulbecco’s PBS at 37 °C and 600 rpm for 1 hour. Subsequently, it was blocked with 150 μL of Dulbecco’s PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20. Then, the relevant botulinum neurotoxin test samples were serially diluted in Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20 and added to the plate. After shaking at 37 °C and 600 rpm for 1 hour, the unbound botulinum neurotoxin on the plate was removed by washing once with Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20. Next, the bound toxin was released from the capture substrate in a solution containing 50 mM Hepes-NaOH (pH 7.1), 0.1 mg / mL BSA, 0.1% Tween-20, 50 μM zinc chloride, 5 mM DTT, and 0.1 μM cleavage substrate (comprising a first luciferase domain, a second luciferase domain, and a SNAP-25 cleavage site (SEQ ID NO:6 plus an N-terminal His-TEV tag [SEQ ID NO:48])). Then the detection plate was shaken at 37 °C and 600 rpm for 3 hours. Subsequently, 50 μL of furimazine diluted to 37.5 μM in 50 mM HEPES-NaOH, 0.1% Tween 20, 50 μM zinc chloride, and 5 mM DTT was added to all wells. Subsequently, a luminometric signal was captured using a microplate reader-based method.

[0726] Results

[0727] Cleavable substrate

[0728] A new cleavable substrate (comprising SEQ ID NO:6) was prepared, which comprised a first luciferase domain (SEQ ID NO:2) at the N-terminus and a second luciferase domain (SEQ ID NO:3) at the C-terminus, connected by a linker containing a SNAP-25 cleavage site with two spacer regions on both sides of the linker ( Figure 11 A).

[0729] Improved binding and cleavage detection

[0730] Binding and cleavage detection were performed as in Example 1, but with Figure 11The substrate shown in A replaces the CFP / YFP cleavage substrate (and luminescence is measured in white Maxisorp 96-well microtiter plates). However, it was found that the sensitivity of this assay was much lower compared to the same assay using the same mrBoNT / AB composition and CFP / YFP cleavage substrate ( Figure 12 B).

[0731] As part of the assay characterization procedure, the assay was performed as shown in Figure 11 B and as 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-capture substrate complex) and incubated while adding DTT. Subsequently, the luciferase substrate (furazine [Carbosynth ZEC04024]) was added and luminescence was evaluated.

[0732] Prior to conducting the experiment, it was expected that non-specific background binding would be significant and the specificity of the assay would be negatively affected. Unexpectedly, this was not the case. It was found that using this method not only significantly improved the sensitivity of the assay ( Figure 12 C), but also achieved this without increasing non-specific background, as shown by the control assay on the assay plate where the capture substrate was not immobilized, and the non-specific background was minimal ( Figure 12 C, square). In fact, compared to the assay using the CFP / YFP cleavage substrate in Example 1, the sensitivity was significantly improved ( Figure 12 A).

[0733] Given this finding, it is advantageous that the number of plates required for a given assay can be reduced. This means that the improved binding and cleavage assay reduces waste and cost and is more suitable for high-throughput testing.

[0734] To further explore the accuracy, precision, and reproducibility of the assay, the improved binding and cleavage assay was performed in triplicate at 5 levels from 50% to 150% for mrBoNT / AB. As Figure 13 shown, the assay had high accuracy and precision at the 5 levels. The standard deviation of the triplicates at the 5 levels averaged 8.6%, and the deviation of the average recovery from the target value was less than 0.4%. This indicates that the assay had good linearity, accuracy, and precision between 50% and 150%.

[0735] Characterization of Clostridial neurotoxin preparations

[0736] Figure 14 A shows the use of the assay method in Example 1, but using Figure 11Reference curve of the mrBoNT / AB composition prepared with a cleavable substrate (comprising a first and a second luciferase domain and measuring luminescence in a white Maxisorp 96-well microtiter plate) as shown in A. When evaluating different clostridial neurotoxin preparations for cosmetic or therapeutic use, various excipients and saline reconstitution buffers may interfere with the assay. The assay in Example 1 lacked the sensitivity required to achieve a parallel linear potency assay.

[0737] In contrast, due to the increased sensitivity, Figure 11 the modified binding and cleavage assay shown in B exhibited linearity and was able to detect very small differences in activity ( Figure 15 ). As Figure 14 shown in B, the modified binding and cleavage assay was not significantly affected by increased saline reconstitution (changing excipient and buffer concentrations). Thus, the modified binding and cleavage assay is particularly suitable for characterizing / screening suitable excipients and / or excipient concentrations for the preparation of clostridial neurotoxin compositions for therapeutic and / or cosmetic use.

[0738] Example 3

[0739] Materials and methods

[0740] Heavy chain receptor affinity enzyme-linked immunosorbent assay (ELISA)

[0741] The relevant capture substrate for the neurotoxin serotype was dissolved in Dulbecco’s PBS at a concentration of 5 μg / mL and fixed on a transparent Maxisorp 96-well microtiter plate at 37 °C and 600 rpm for 1 hour. Subsequently, it was blocked with 150 μL of Dulbecco’s PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20. Then, the relevant neurotoxin test samples were serially diluted in Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20 and added to the above plate. After shaking at 37 °C and 600 rpm for 1 hour, it was washed once with Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20 to remove the unbound neurotoxin on the plate. The captured toxin was fixed with 2% paraformaldehyde at room temperature for 10 minutes and then washed once again with Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20. Then, an anti-botulinum neurotoxin (BoNT) antibody diluted to 2 μg / mL with Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20 was added and incubated at 37 °C and 600 rpm for 1 hour. Next, the plate was washed once with Dulbecco’s PBS containing 1% BSA and 0.05% Tween 20, and then a relevant anti-species horseradish peroxidase (HRP)-conjugated antibody with a concentration of 0.4 μg / mL was added. After the final 1-hour incubation at 37 °C and 600 rpm, the plate was washed twice with Dulbecco’s PBS, and then the pre-warmed 3,3',5,5'-tetramethylbenzidine (TMB, ThermoFisher N301) substrate was added to react for 5 minutes, and finally the reaction was terminated with an ELISA stop solution (0.16 M sulfuric acid, TMB stop solution, ThermoFisher N600).

[0742] Endopeptidase activity assay only

[0743] Serial dilutions of the relevant BoNT neurotoxin preparation were made in 50 mM Hepes-NaOH, 50 μM zinc chloride (ZnCl), 0.1% Tween 20, 5 mM dithiothreitol (DTT). The prepared serial dilutions of the neurotoxin were added to a white 96-well microtiter plate pre-loaded with 10 μL of 1 μM cleavage substrate, which contained a first luciferase domain, a second luciferase domain, and a synaptosome-associated protein 25 (SNAP-25) cleavage site (containing SEQ ID NO:6), and was dissolved in 50 mM Hepes-NaOH, 50 μM zinc chloride, 0.1% Tween 20, 5 mM DTT. The plate was then incubated at 37 °C and 600 revolutions per minute for 3 hours. Subsequently, 50 μL of furazoline diluted to 37.5 μM in 50 mM Hepes-NaOH, 50 μM zinc chloride, 0.1% Tween 20, 5 mM DTT was added to all wells. Thereafter, luminescence was detected using a microplate reader-based method.

[0744] Results

[0745] The mrBoNT / AB and mrBoNT / A samples were subjected to forced degradation treatment and tested by heavy chain receptor affinity ELISA, in vitro endopeptidase assay, or the binding and cleavage assay described herein.

[0746] Figure 16 It was shown that the combination of (i) heavy chain receptor affinity ELISA, (ii) in vitro endopeptidase assay, and (iii) the binding and cleavage assay in Example 2 enabled the quality and efficacy of the neurotoxin preparation to be explored in a domain-specific manner. The reduction in the receptor affinity of mrBoNT / A ( Figure 16 A) or mrBoNT / AB ( Figure 16 B) could be easily detected using the receptor capture sandwich ELISA method. When the endopeptidase activity was comparable to the control (such as a reference standard), but a loss of potency was observed in the binding and cleavage assay (confirmed by traditional cell assays), it could be attributed to topological changes or post-translational covalent modifications of the heavy chain of the neurotoxin bound to the relevant receptor ( Figure 16 B). Conversely, when the heavy chain receptor affinity was comparable, but the endopeptidase activity was reduced ( Figure 16 A), the loss of potency detected in the binding and cleavage assay (confirmed by traditional cell assays) could be attributed to the loss of the kinetic activity of the endopeptidase.

[0747] Conclusion

[0748] In traditional neurotoxin analysis techniques that focus on the potency of formulations, such as mouse median lethal dose (LD50) assays and more recent cell-based assays, the specific causes of potency variations are not easily determined. The functions of the individual domains of neurotoxins have been well reported and characterized, and they each have specific functions in the mode of action, which are crucial for the extremely high potency and clinical efficacy of clostridial neurotoxin products. A reduced affinity for the relevant neurotoxin receptor will lead to decreased cell binding, which in turn affects cell penetration and hinders the therapeutic response. Conversely, a reduced catalytic activity of the neurotoxin formulation will result in slower or no cleavage of SNARE proteins within the cell cytoplasm. In either case, neither the mouse LD50 assay nor the cell-based assay can diagnose the cause of reduced or increased potency.

[0749] Cell-free binding and cleavage assays can predict physiological potency by exploring the major functions of neurotoxin domains and are comparable to cell-based assays in determining potency. In addition, when combined with heavy chain receptor affinity ELISA and in vitro endopeptidase assays, decreases or increases in activity can be easily detected. Thus, unlike cell-based assays, potency variations can be attributed to specific functions of the neurotoxin subunits.

[0750] Example 4

[0751] Materials and methods

[0752] To test the binding to the synaptotagmin-I (SYT-I) receptor substrate, mrBoNT / AB (SEQ ID NO:14 converted to a double-chain toxin by incubation with lysine-C (Lys-C)) or wild-type BoNT / B (purchased from Metabiologics, Madison, WI, USA) was evaluated using an Octet Red 96e system by biolayer interferometry (BLI). The SYT-I receptor substrates of mouse (SEQID NO:84) and human sequences (SEQ ID NO:81) were immobilized on His or glutathione-S-transferase (GST) tag capture biosensor probes and exposed to dilution series of mrBoNT / AB (starting concentration of 300 μg / ml, with half-log dilution series) or wild-type BoNT / B (starting concentration of 500 μg / ml). All presented data are the mean of 3n, and the data were fit in the Octet software using a 1:1 binding model, with the goodness of fit R 2 acceptance criterion of >0.95 (except for the GT1b assay, which had a lower goodness of fit for the data R 2 fit).

[0753] The GT1b ganglioside preparation was purchased from Biosynth (UK) (product catalogue number OO16323). When using GT1b, a fixed concentration of 50 μg / ml (24.1 μM) was used, and blank subtraction was performed with a control containing only the assay buffer. GT1b was added to the experimental protocol both in the pre-binding baseline step and in the binding / dissociation phase. This ensured that the receptor-ganglioside complex was formed before toxin binding and that the signal would return to the baseline level of the receptor-ganglioside complex during the dissociation phase.

[0754] Results

[0755] The following table shows that mrBoNT / AB is able to bind to human and murine SYT-I. The affinity of mrBoNT / AB for human and murine SYT-I is further increased by the simultaneous use of GT1b compared to SYT-I alone. This appears to be due to a slower dissociation rate (Kdis) of the SYT-I-GT1b complex compared to SYT-I alone.

[0756]

[0757] The following table shows that BoNT / B is able to bind to human and murine SYT-I.

[0758] Capture substrate <![CDATA[BoNT / B(K D in nM)]]> SYT-I - human 1202.0 SYT-I - murine 435.7

[0759] Thus, SYT-I, optionally together with GT1b, has been shown to be a capture substrate suitable for the present invention.

[0760] To further confirm, mrBoNT / AB was subjected to forced oxidation and tested using human SYT-I or human SYT-II (L51F modified) capture substrates in a modified binding and cleavage assay (see Example 2). Briefly, 0.05% hydrogen peroxide (H2O2) was added to mrBoNT / AB and wrapped in aluminium foil at room temperature for different time points up to 96 hours, then quenched with methionine to destroy any residual peroxide. The percentage of oxidation at different time points was detected by mass spectrometry. The activity was tested at each time point relative to the control / T = 0 time point. Figure 17 It was shown that the assays using the two capture substrates gave very similar activity results, with a decrease in activity as the percentage of oxidation of the Clostridial neurotoxin polypeptide in the composition increased at the reference amino acid residue.

[0761] All publications mentioned in the above specification are hereby incorporated by reference. It will be obvious to those skilled in the art that various modifications and variations can be made to the methods and systems described herein without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described embodiments that are obvious to those skilled in the fields of biochemistry, biotechnology, or related fields are intended to fall within the scope of the following claims.

Claims

1. A cell-free method for determining the Clostridial neurotoxin activity of a composition comprising a Clostridial neurotoxin polypeptide, the method comprises: (a) providing a capture substrate for the 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 polypeptide; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound Clostridial neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L chain polypeptide and a complex comprising the capture substrate and the Clostridial neurotoxin receptor-binding domain (HCC domain, e.g., HC domain); and (e) determining the amount of a cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby determining the Clostridial neurotoxin activity of the composition.

2. The method according to claim 1, wherein the method comprises: (a) providing a capture substrate for the Clostridial neurotoxin polypeptide, wherein the capture substrate comprises a Clostridial neurotoxin receptor polypeptide or a ganglioside that binds to Clostridial neurotoxin; (b) contacting the capture substrate with the composition to bind the Clostridial neurotoxin polypeptide to the capture substrate; (c) removing unbound Clostridial neurotoxin polypeptide; (d) adding a reducing agent to dissociate the light chain (L chain) polypeptide of the bound Clostridial neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L chain polypeptide and a complex comprising the capture substrate and the Clostridial neurotoxin receptor-binding domain (HCC domain, e.g., HC domain); and (e) determining the amount of a cleavable substrate cleaved by the L chain polypeptide in the assay sample, thereby determining the Clostridial neurotoxin activity of the composition, wherein each cleavable substrate comprises 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 functionally links the first and second luciferase domains to form a functional luciferase.

3. A cell-free method for determining the Clostridial neurotoxin activity of a composition comprising a Clostridial neurotoxin polypeptide, the method comprises: (a) providing a capture substrate for the 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 polypeptide; (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 a cleavable substrate cleaved by the L chain polypeptide, thereby determining the Clostridial neurotoxin activity of the composition.

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

5. A cell-free method for determining whether a composition comprises a Clostridium neurotoxin polypeptide, the method comprising: (a) Providing a capture substrate for the Clostridium neurotoxin polypeptide; (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate; (c) Removing any unbound Clostridium neurotoxin polypeptide; (d) Adding a reducing agent to dissociate the light chain (L chain) polypeptide of any bound Clostridium neurotoxin polypeptide; and (e) Determining that a cleavable substrate has been cleaved, thereby determining that the composition comprises a Clostridium neurotoxin polypeptide, or determining that the cleavable substrate has not been cleaved, thereby determining that the composition does not comprise a Clostridium neurotoxin polypeptide.

6. The method according to claim 5, wherein the method comprises: (a) Providing a capture substrate for the Clostridium neurotoxin polypeptide, wherein the capture substrate comprises a Clostridium neurotoxin receptor polypeptide or a ganglioside that binds to Clostridium neurotoxin; (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate; (c) Removing any unbound Clostridium neurotoxin polypeptide; (d) Adding a reducing agent to dissociate the light chain (L chain) polypeptide of any bound Clostridium neurotoxin polypeptide; and (e) Determining that a cleavable substrate has been cleaved, thereby determining that the composition comprises a Clostridium neurotoxin polypeptide, or determining that the cleavable substrate has not been cleaved, thereby determining that the composition does not comprise a Clostridium neurotoxin polypeptide, wherein each cleavable substrate comprises a single-chain polypeptide that comprises: (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally links the first and second luciferase domains to form a functional luciferase.

7. The method according to claim 5, wherein the method comprises: (a) Providing a capture substrate for the Clostridium neurotoxin polypeptide; (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate; (c) Removing any unbound Clostridium neurotoxin polypeptide; (d) Adding a reducing agent to dissociate the light chain (L chain) polypeptide of any bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising any dissociated L chain polypeptide and any complex comprising the capture substrate and the Clostridium neurotoxin receptor binding domain (HCC domain, e.g., HC domain); and (e) Determine that the cleavable substrate in the assay sample has been cleaved, thereby determining that the composition contains a Clostridium neurotoxin polypeptide, or determine that the cleavable substrate in the assay sample has not been cleaved, thereby determining that the composition does not contain a Clostridium neurotoxin polypeptide.

8. The method according to claim 5 or 7, wherein the method comprises: (a) Providing a capture substrate for a Clostridium neurotoxin polypeptide, wherein the capture substrate comprises a Clostridium neurotoxin receptor polypeptide or a ganglioside that binds to a Clostridium neurotoxin; (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide to the capture substrate; (c) Removing unbound Clostridium neurotoxin polypeptide; (d) Adding a reducing agent to dissociate the L-chain polypeptide of the bound Clostridium neurotoxin polypeptide, thereby providing an assay sample comprising the dissociated L-chain polypeptide and a complex comprising the capture substrate and the Clostridium neurotoxin receptor-binding domain (HCC domain, such as the HC domain); and (e) Determine that the cleavable substrate in the assay sample has been cleaved, thereby determining that the composition contains a Clostridium neurotoxin polypeptide, wherein each cleavable substrate is a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally links the first and second luciferase domains to form a functional luciferase.

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

10. The method according to any one of the preceding claims, wherein the cleavable substrate comprises (preferably) a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a Clostridium neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally links the first and second luciferase domains to form a functional luciferase, preferably, wherein: (i) When the linker is cleaved, the construct cannot exhibit luciferase activity; and (ii) When the linker is intact, the construct can exhibit luciferase activity.

11. A cell-free method for determining whether a Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain therein) contained in a composition has an altered activity property, the method comprises: (a) Providing a capture substrate for a Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain); (b) Contacting the capture substrate with the composition to bind the Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain) to the capture substrate; (c) Removing unbound Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain); (d) Determining the amount of Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain) bound to the capture substrate; (e) The bound Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or the amount of the HCC domain) is compared with a control; and (f) based on the comparison, determining whether the Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain therein) contained in the composition has an activity-altering property (such as the activity-altering property of the heavy chain, such as the HC or HCC domain).

12. 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. The method according to any one of the preceding claims, wherein the method employs a combination of a capture substrate comprising a Clostridium neurotoxin receptor polypeptide and a capture substrate comprising a ganglioside.

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

15. The method according to any one of the preceding claims, 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 according to claim 15, wherein the extracellular portion of the neuronal BoNT receptor polypeptide comprises amino acid modifications and / or post-translational modifications.

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

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

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

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

21. The method according to any one of claims 14-20, wherein each BoNT polypeptide comprises a BoNT / B HCC domain.

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

23. The method according to claims 15-16 or 22, wherein the extracellular portion of the BoNT receptor polypeptide is the extracellular portion of SV2c, preferably, wherein the extracellular portion comprises the luminal domain 4 of SV2c (such as amino acid residues 519-563 of SV2c).

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

25. The method according to any one of claims 1-16, 23 or 24, wherein the extracellular portion of the neuronal BoNT receptor polypeptide is the extracellular portion of SV2c having glycosylation at N559.

26. The method according to any one of claims 22-25, 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. The method according to any one of the preceding claims, wherein the capture substrate has been recombinantly produced in mammalian cells, preferably in human cells.

28. The method according to any one of claims 14 - 16 or 22 - 27, wherein each BoNT polypeptide comprises a BoNT / A HCC domain.

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

30. The method according to any one of the preceding claims, wherein the composition is a first Clostridial neurotoxin preparation, which comprises one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

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

32. The method according to claim 31, wherein the Clostridial neurotoxin activities of the first Clostridial neurotoxin preparation and the at least second Clostridial neurotoxin preparation are compared, and when the Clostridial neurotoxin preparation containing the same Clostridial neurotoxin exhibits the highest activity, the one or more pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts or their amounts are selected.

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

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

35. The method according to claim 34, wherein the comparison allows determination of whether the Clostridial neurotoxin polypeptide (or a part thereof, e.g., the L chain or the H chain or parts thereof, such as the HC or HCC domain) comprised in the composition has an activity - altering property, or determination of the amount of the Clostridial neurotoxin polypeptide (or a part thereof, e.g., the L chain or the H chain or parts thereof, such as the HC or HCC domain) having an activity - altering property comprised in the composition.

36. Use of an isolated Clostridium neurotoxin capture substrate for determining whether a Clostridium neurotoxin polypeptide or a portion thereof (such as the heavy chain, such as the HC or HCC domain therein) contained in a composition has an altered activity property, wherein the isolated capture substrate comprises an extracellular portion of a Clostridium neurotoxin receptor polypeptide, and the extracellular portion comprises amino acid modifications and / or post-translational modifications.

37. The use according to claim 36, wherein the altered activity property is a heavy chain modification that reduces activity, more preferably a heavy chain oxidation that reduces activity.

38. A method for preparing a therapeutic or cosmetic Clostridium neurotoxin composition, the method comprising: (a) obtaining the result of the method according to any one of claims 1-4 or 9-10 or 12-35, and when the Clostridium neurotoxin activity is the same as or higher than a positive control (such as a positive reference standard), formulating and / or packaging the composition for therapeutic or cosmetic use; or (b) when the Clostridium neurotoxin activity is lower than the positive control (such as a positive reference standard), further purifying the composition and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

39. A method for preparing a therapeutic or cosmetic Clostridium neurotoxin composition, the method comprising: (a) obtaining the result of the method according to any one of claims 11-35; and (b) when the Clostridium neurotoxin polypeptide contained in the composition does not have an altered activity property, formulating and / or packaging the composition for therapeutic or cosmetic use; or (c) when the Clostridium neurotoxin polypeptide contained in the composition does have an altered activity property, further purifying the composition; and (d) formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

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

41. An isolated Clostridium neurotoxin capture substrate, wherein the capture substrate comprises an extracellular portion of a Clostridium neurotoxin receptor polypeptide, and the extracellular portion comprises amino acid modifications and / or post-translational modifications.

42. A kit, comprising: (a) the isolated capture substrate according to claim 41; and (b) optionally, a tool for detecting the binding of a Clostridium neurotoxin (such as botulinum neurotoxin) to the capture substrate; and / or (c) optionally, instructions for its use.

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