Cellular VAMP cleavage assay

Abstract

To provide cellular VAMP cleavage assays based on a gain of signal readout for determining the potency of pharmaceutical BoNT formulations.SOLUTION: The present invention relates to VAMP epitopes suitable for generating antibodies against a VAMP C-terminal neurotoxin cleavage product, their use to generate antibodies against cleaved VAMP and the use of such antibodies in cellular VAMP cleavage assays based on the gain of signals.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell-based assay for VAMP-cleaving Clostridial neurotoxins. [Background technology]

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

[0003] Clostridial neurotoxins act by inhibiting cholinergic transmission in the peripheral nervous system, particularly at the neuromuscular junction. In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. The active form of the toxin is this two-chain form. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal cartwheel position component (H N domain) and the C-terminal targeting component (H C The cleavage sites are located in the L and H chains. N Located between the domains. H C After binding of the H domain to its target neuron and internalization of the bound toxin into the cell by endosomes, NThe domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0004] Non-cytotoxic proteases act by proteolytically cleaving intracellular trafficking proteins known as SNARE proteins (e.g., SNAP-25, VAMP or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE comes from Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-sensitive factor. The acronym SNAP25 comes from Synaptosome Associated Protein of 25 kilodaltons. The acronym VAMP comes from Vesicle Associated Membrane Protein. SNARE proteins are essential for intracellular vesicle fusion and therefore for the secretion of molecules via vesicle transport from cells. The protease activity is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, the non-cytotoxic protease can inhibit cellular secretion from the target cell. The L-chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.The L-chain proteases of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X and TeNT cleave VAMP (also called synaptobrevin), the L-chain proteases of BoNT / A and BoNT / E cleave SNAP25, and the L-chain protease of BoNT / C cleaves SNAP25 and syntaxin, leading to inhibition of neurotransmitter release and resulting neuroparalysis (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549) (Zhang et al., "Identification and characterization of a novel botulinum neurotoxin"; Nature Communications, 2017, 8:14130).

[0005] Clostridial neurotoxins target neurons through their receptor binding domains (H C ) to its specific receptor (Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis, Physiol Rev, 2000, 80, 717-766). Receptor binding determines the efficacy and specificity with which BoNT recognizes neurons. BoNT / B, D-C and G share two homologous synaptic vesicle proteins synaptotagmin I and II (Syt I / II) as their receptors, whereas BoNT / A, E, D, F and TeNT use another synaptic vesicle protein, SV2. In addition to the protein receptor, all BoNTs require the lipid coreceptor gangliosides, which are abundant on the neuronal surface.

[0006] Clostridial neurotoxins are used in therapy to treat motor and autonomic disorders. Several BoNT / A formulations (including Botox®, Dysport®, and Xeomin®) and one BoNT / B formulation (Neurobloc® / Myobloc®) have been approved by regulatory agencies for use in humans.

[0007] Traditionally, the potency of BoNT pharmaceutical preparations has been quantified in MLD50 (Mouse Lethal Dose 50) units, with one unit corresponding to the median lethal intraperitoneal dose in mice. However, the MLD50 units of botulinum toxins are not standardized units. Indeed, the assays used by various manufacturers of commercial toxins differ, especially in the choice of dilution buffer (Straughan, DW, 2006, ATLA 34(3), 305-313; Hambleton and Pickett, Hambleton, P., and AM Pickett., 1994, Journal of the Royal Society of Medicine 87.11: 719). Furthermore, due to ethical issues and recent regulations, it is currently preferable to avoid the use of animal-based potency assays. Cell-based potency assays avoid the need for animal testing and the associated ethical issues. After cellular intoxication, the potency of clostridial neurotoxins can be measured by, for example, evaluating the degree of SNARE cleavage in target cells by Western blotting. Alternatively, SNARE cleavage can be detected and quantified using a sandwich ELISA method.Such methods have worked well for SNAP25 and syntaxin cleavage (see, e.g., Pellett, Sabine, et al. "Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type A potency determination." Journal of pharmacological and toxicological methods 61.3 (2010):304-310; Fernandez-Salas, Ester, et al. "Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay." PLoS One 7.11 (2012): e49516; Kalandakanond S et al. "Cleavage of intracellular substrates of botulinum toxins A, C and D in mammalian target tissue" The Journal of Pharmacology and Experimental Therapeutics (2001):749-755; Peng L et al. "Cytotoxicity of botulinum "neurotoxins reveal a direct role of syntaxin 1 and SNAP25 in neuron survival." Nature Communications (2013): 4:1472). However, to date, cleavage products of VAMP from cell lysates have proven extremely difficult to detect.Indeed, VAMP cleavage-specific antibodies that recognize cleaved VAMP are available and are suitable for detecting VAMP cleavage in extracellular or cell fraction assays (Hallis, Bassam, BA James, and Clifford C. Shone. "Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities." Journal of clinical microbiology 34.8 (1996): 1934-1938; Kegel, B., et al. "An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product." Toxicology in Vitro 21.8 (2007): 1641-1649; Fujita-Yoshigaki, Junko, et al. "Vesicle-associated Membrane Protein 2 Is Essential for cAMP-regulated Exocytosis in Rat Parotid Acinar Cells The Inhibition of cAMP-dependent Amylase Release by Botulinum Neurotoxin B." Journal of Biological Chemistry 271.22 (1996): 13130-13134), these antibodies do not detect cleaved VAMP in cellular studies.

[0008] The general consensus in the field so far has been that the cleaved VAMP products must be degraded very rapidly in the cell and therefore do not contribute to the longevity of BoNT action (Foran, Patrick G., et al. "Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A Basis for Distinct Durations of Inhibition of Exocytosis in Central Neurons." Journal of biological chemistry 278.2 (2003): 1363-1371). However, Schiavo et al., using Coomassie Blue staining, showed that both VAMP cleavage products were present in small synaptic vesicle fractions prepared from rat cerebral cortex upon treatment with BoNT / B and TeNT (Schiavo G., et al (1992), Tetanus and Botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359 p832-835). This suggests that synaptosomal preparations may not contain enough of all the proteases present in total cell lysates, but that VAMP products derived from cellular sources may be present.Dong et al. (2004) describe that in PC12 cells expressing YFP-Syb(FL)-CFP, signals from both VAMP products are detectable after cleavage by BoNT / B and that the YFP-N-terminal cleaved VAMP product is dispersed in the cytosol and redistributes itself to the nucleus, whereas the CFP-C-terminal product remains localized in vesicles (Dong M., et al (2004) Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells. PNAS 101 (41) p14701-14706). This evidence suggests that both VAMP products may be present, but that as yet unknown cellular processes prevent antibody recognition of the N-terminal product. Therefore, it is standard practice to measure VAMP cleavage by loss of only the full-length band (see, e.g., Pellett, Sabine, et al. "A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies." FEBS letters 581.25 (2007): 4803-4808.; Whitemarsh, Regina CM, et al. "Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection Biological Sciences: Applied Biological Sciences." Toxicological Sciences, 2012, 126(2):426-435). However, assays based on loss of signal carry the risk of error, since there can be discrepancies in total protein loading, which in turn leads to either overestimation or underestimation of VAMP loss.Using a housekeeping protein that does not change during BoNT treatment, VAMP disappearance can be normalized to the density of the control protein. The disadvantage is that the signals of the antibodies must be balanced and on a linear scale in order to detect any difference for normalization purposes. Qualitatively, this can be a reasonable indicator of BoNT activity, but it is not suitable for more detailed quantification, especially for determining the efficacy of pharmaceutical BoNT preparations.

[0009] Therefore, there is a need for a cellular VAMP cleavage assay based on a gain of signal readout. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549 [Non-Patent Document 2] Zhang et al., "Identification and characterization of a novel botulinum neurotoxin"; Nature Communications, 2017, 8:14130 Summary of the Invention

[0011] In a first aspect, the present invention provides an antigenic polypeptide comprising a VAMP epitope, the antigenic polypeptide consisting of 10 to 65 amino acid residues, the VAMP epitope comprising an amino acid sequence that is at least 90% identical to a VAMP sequence including at least 8 amino acid residues immediately C-terminal to a clostridial neurotoxin cleavage site in VAMP.

[0012] In another aspect, the invention relates to a polypeptide comprising an antigenic polypeptide of the invention, which does not contain a region of more than 17, preferably 16, more preferably 15, consecutive amino acids having 100% sequence identity to a naturally occurring VAMP amino acid sequence.

[0013] In another aspect, the present invention provides an antigenic protein comprising a polypeptide of the present invention covalently linked to a carrier.

[0014] In another aspect, the present invention provides the use of the antigenic polypeptide or protein of the present invention to generate antibodies against C-terminal VAMP cleavage products.In one embodiment, the epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products.In another embodiment, the epitope of the present invention is used to generate monoclonal antibodies against C-terminal VAMP cleavage products.

[0015] In another aspect, the invention provides an antibody that binds to an antigenic polypeptide or protein of the invention.

[0016] In another aspect, the invention provides the use of an antibody of the invention in a gain of signal cellular assay of VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0017] In another aspect, the invention provides a method for determining cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin in a cell, comprising: a) contacting a cell with a Clostridial neurotoxin under conditions suitable for Clostridial neurotoxin activity; b) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention; c) detecting by suitable means the binding of said first detection antibody to the C-terminal VAMP cleavage product; The present invention provides a method comprising:

[0018] In another aspect, the invention provides a method for determining immunoresistance to a VAMP-cleaving Clostridial neurotoxin in a subject, comprising the steps of: a) adding a VAMP-cleaving Clostridial neurotoxin to a test sample obtained from a subject; b) contacting the cells with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; c) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention; d) detecting by suitable means the binding of the first detection antibody to the C-terminal VAMP cleavage product; e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody; f) repeating steps a) to e) using a negative control sample instead of the test sample; g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (e) compared to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of a neutralizing antibody against a VAMP-cleaving Clostridial neurotoxin. The present invention provides a method comprising:

[0019] In another aspect, the present invention provides a kit comprising a cell susceptible to intoxication by a VAMP-cleaving neurotoxin and a first detection antibody against cleaved VAMP, wherein said first detection antibody is an antibody of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention is based on the discovery by the inventors that it was possible to generate antibodies suitable for use in a cellular VAMP cleavage assay based on a signal gain readout.

[0021] In particular, the inventors have shown that it is important to detect epitopes located C-terminal to the BoNT cleavage site in order to detect VAMP cleavage in vitro. Indeed, the inventors have demonstrated herein the successful detection of VAMP2 cleavage products in neurons by Western blot (WB) using an antibody that binds to an epitope located C-terminal to the BoNT / F and / or BoNT / D and / or BoNT / B cleavage site, adjacent to the BoNT / D and / or BoNT / F and / or BoNT / B cleavage site. In particular, such an antibody can detect both full-length VAMP and cleavage products in cell lysates. This tool allows for the quantitative evaluation of BoNT potency in gain of signal cellular assays by monitoring the appearance of cleaved VAMP products.

[0022] In a first aspect, the present invention provides an antigenic polypeptide comprising a VAMP epitope, the antigenic polypeptide consisting of 10 to 65 amino acid residues, the VAMP epitope comprising an amino acid sequence that is at least 90% identical to a VAMP sequence including at least 8 amino acid residues immediately C-terminal to a clostridial neurotoxin cleavage site in VAMP.

[0023] The term "clostridial neurotoxin" as used herein refers to any polypeptide that enters neurons and inhibits neurotransmitter release. This process includes binding of the neurotoxin to low or high affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin to the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "clostridial neurotoxin" includes any polypeptide produced by bacteria of the genus Clostridium that enters neurons and inhibits neurotransmitter release, and such polypeptides produced by recombinant or chemical techniques. The active form of the neurotoxin is a two-chain form. The two chains are referred to as 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. Clostridial neurotoxins include botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). BoNT serotypes A-G can be distinguished based on inactivation by specific neutralizing antisera, and this classification by serotype correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage of amino acid sequence identity.

[0024] An exemplary BoNT / A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt Accession No. A5HZZ9). An exemplary BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt Accession No. B1INP5). An exemplary BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt Accession No. P18640). An exemplary BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt Accession No. P19321). An exemplary BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (NCBI RefSeq Accession No. WP_003372387). An exemplary BoNT / F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt Accession No. Q57236). An exemplary BoNT / G neurotoxin amino acid sequence is provided as SEQ ID NO: 7 (NCBI RefSeq Accession No. WP_039635782). An example of a BoNT / X neurotoxin amino acid sequence is provided as SEQ ID NO: 41 (Genbank Accession No. BAQ12790.1). An example of a TeNT amino acid sequence is provided as SEQ ID NO: 8 (UniProt Accession No. P04958).

[0025] The term “H C By "domain" herein is meant a functionally distinct region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is divided into two structurally distinct subdomains, "H CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C It consists of the C-terminal portion of the domain.

[0026] The term “LH N "Domain" as used herein means C They lack the endopeptidase domain ("L" or "light chain") and the domain involved in translocation of the endopeptidase to the cytoplasm (the H domain of the heavy chain). N It refers to a neurotoxin consisting of the agonist (domain).

[0027] Exemplary L, H N , H CN and H CC The domains are shown in Table 1. [Table 1]

[0028] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on the subserotype, for example US2007 / 0166332 (herein incorporated by reference in its entirety) cites slightly different Clostridium sequences.

[0029] Vesicle-associated membrane proteins (VAMPs) are a family of SNARE proteins with similar structures and involved in vesicle fusion and exocytosis, particularly neurotransmitter release. VAMPs are members of a family of SNARE proteins called the synaptobrevin family, which includes members such as VAMP1, VAMP2 (both known as synaptobrevin), VAMP3 (also known as cellubrevin), VAMP4, VAMP5, VAMP7 (also known as SYBL1 or tetanus-insensitive VAMP), VAMP8 (also known as endobrevin), YKT6, SEC22A, and others. VAMP1, VAMP2, and VAMP3 are cleaved by the light chains of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT. BoNT / X also cleaves VAMP4, VAMP5, and YKT6.

[0030] The term "VAMP epitope" as used herein means the portion of the VAMP protein to which an antibody binds.

[0031] In a preferred embodiment, the antigenic polypeptide of the present invention consists of 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, or 10 to 15 amino acid residues, preferably 10 to 15 amino acid residues. For example, an antigenic polypeptide of the present invention can consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acid residues.

[0032] In a preferred embodiment, the antigenic polypeptide of the present invention comprises or consists of a VAMP epitope comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a VAMP sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acid residues immediately C-terminal to the clostridial neurotoxin cleavage site in VAMP.

[0033] The amino acid sequences of naturally occurring VAMPs, specifically rat and human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and YKT6, and their corresponding clostridial neurotoxin VAMP cleavage sites, are shown in Table 2 and FIG. [Table 2]

[0034] In one embodiment, the VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6.

[0035] In one embodiment, the VAMP is selected from VAMP1, VAMP2 and / or VAMP3.

[0036] In one embodiment, the VAMP is selected from VAMP4, VAMP5 and / or YKT6.

[0037] In a preferred embodiment, the VAMP is human VAMP, more preferably human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6.

[0038] In one embodiment, the VAMP is selected from human VAMP1, VAMP2 and / or VAMP3.

[0039] In one embodiment, the VAMP is selected from human VAMP4, VAMP5 and / or YKT6.

[0040] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a VAMP epitope that is cleaved by BoNT / F, wherein the at least eight amino acid residues are immediately C-terminal to the BoNT / F cleavage site in VAMP.

[0041] Examples of BoNT / F VAMP epitopes, more specifically, BoNT / F VAMP1, VAMP2 and / or VAMP3 epitopes include the following: KLSELDDRADALQ (SEQ ID NO:15) QKLSELDDRADALQ (SEQ ID NO:16) KLSELDDRAD (SEQ ID NO:17) KLSELDDRADALQAGAS (SEQ ID NO:18) DQKLSELDDRADALQ (SEQ ID NO:31).

[0042] In one embodiment, the BoNT / F VAMP epitope, in particular the BoNT / F VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 15 to SEQ ID NO: 18 and SEQ ID NO: 31. In a preferred embodiment, the BoNT / F VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT / F VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

[0043] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / D VAMP epitope, wherein said at least eight amino acid residues are immediately C-terminal to the BoNT / D cleavage site in VAMP.

[0044] Examples of BoNT / D VAMP epitopes, more specifically, BoNT / D VAMP1, VAMP2 and / or VAMP3 epitopes include the following: KLSELDDRADALQ (SEQ ID NO:15) LSELDDRADALQ (SEQ ID NO: 19) LSELDDRADA (SEQ ID NO:20) LSELDDRADALQAGAS (SEQ ID NO:21).

[0045] In one embodiment, the BoNT / D VAMP epitope, in particular the BoNT / D VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 15 and SEQ ID NO: 19 to SEQ ID NO: 21. In a preferred embodiment, the BoNT / D VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT / D VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

[0046] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a VAMP epitope cleaved by BoNT / F5 or BoNT / FA, wherein the at least eight amino acid residues are immediately C-terminal to the BoNT / F5 or BoNT / FA cleavage site in VAMP.

[0047] Examples of BoNT / F5 or BoNT / FA VAMP epitopes, more specifically, BoNT / F5 or BoNT / FA VAMP1, VAMP2 and / or VAMP3 epitopes include the following: ERDQKLSELDDRA (SEQ ID NO:32) LERDQKLSELDDRA (SEQ ID NO:33) VLERDQKLSELDDRA (SEQ ID NO:34).

[0048] In one embodiment, the BoNT / F5 or BoNT / FA VAMP epitope, in particular the BoNT / F5 or BoNT / FA VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 32 to SEQ ID NO: 34. In a preferred embodiment, the BoNT / F5 or BoNT / FA VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to ERDQKLSELDDRA (SEQ ID NO: 32). In a more preferred embodiment, the BoNT / F5 or BoNT / FA VAMP epitope comprises or consists of ERDQKLSELDDRA (SEQ ID NO: 32).

[0049] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / B or TeNT VAMP epitope, wherein said at least eight amino acid residues are immediately C-terminal to a BoNT / B or TeNT cleavage site in VAMP.

[0050] Examples of BoNT / B or TeNT VAMP epitopes, more specifically, BoNT / B or TeNT VAMP1, VAMP2 and / or VAMP3 epitopes include the following: FETSAAKLKRKYW (SEQ ID NO:22) FESSAAKLKRKYW (SEQ ID NO:23) QFETSAAKLKRKYW (SEQ ID NO:24) FETSAAKLKR (SEQ ID NO:25) FETSAAKLKRKYWWKN (SEQ ID NO:26) ETSAAKLKRKYWWK (SEQ ID NO:48) FETSAAKLKRKYWWK (SEQ ID NO:49) QFESSAAKLKRKYW (SEQ ID NO:50) FESSAAKLKR (SEQ ID NO:51) FESSAAKLKRKYWWK (SEQ ID NO:52).

[0051] In one embodiment, the BoNT / B or TeNT VAMP epitope, in particular the BoNT / B or TeNT VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 22 to SEQ ID NO: 26 and SEQ ID NO: 48 to SEQ ID NO: 52. In a preferred embodiment, the BoNT / B or TeNT VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49). In a more preferred embodiment, the BoNT / B or TeNT VAMP epitope comprises or consists of FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49). Surprisingly, antibodies that bind to the latter epitope allow not only the detection of BoNT / B VAMP cleavage, but also the detection of BoNT / F VAMP cleavage.

[0052] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / G VAMP epitope, wherein said at least eight amino acid residues are immediately C-terminal to the BoNT / G cleavage site in VAMP.

[0053] Examples of BoNT / G VAMP epitopes, more specifically, BoNT / G VAMP1, VAMP2 and / or VAMP3 epitopes include the following: AKLKRKYWWKN (SEQ ID NO:27) AAKLKRKYWWKN (SEQ ID NO:28) AKLKRKYWWKNCKM (SEQ ID NO:29) AKLKRKYWWKNLKM (SEQ ID NO:30).

[0054] In one embodiment, the BoNT / G VAMP epitope, in particular the BoNT / G VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 27 to SEQ ID NO: 30. In a preferred embodiment, the BoNT / G VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to AKLKRKYWWKN (SEQ ID NO: 27). In a more preferred embodiment, the BoNT / G VAMP epitope comprises or consists of AKLKRKYWWKN (SEQ ID NO: 27).

[0055] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / X VAMP epitope, wherein said at least eight amino acid residues are immediately C-terminal to the BoNT / X cleavage site in VAMP.

[0056] Examples of BoNT / X VAMP epitopes, more specifically, BoNT / X VAMP1, VAMP2 and / or VAMP3 epitopes include the following: ADALQAGASQF (SEQ ID NO:53) ADALQAGASQ (SEQ ID NO:54) RADALQAGASQF (SEQ ID NO:55) ADALQAGASQFE (SEQ ID NO:56) ADALQAGASVF (SEQ ID NO:57) ADALQAGASV (SEQ ID NO:58) ADALQAGASVFE (SEQ ID NO:59) RADALQAGASVF (SEQ ID NO:60) RADALQAGAS (SEQ ID NO:61).

[0057] In one embodiment, the BoNT / X VAMP epitope, in particular the BoNT / X VAMP1, VAMP2 and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 53 to SEQ ID NO: 61. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to ADALQAGASQF (SEQ ID NO: 53). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of ADALQAGASQF (SEQ ID NO: 53).

[0058] Other examples of BoNT / X VAMP epitopes, and more particularly, BoNT / X VAMP4 epitopes, include the following: SESLSDNATAF (SEQ ID NO:62) SESLSDNATA (SEQ ID NO:63) KSESLSDNATAF (SEQ ID NO:64) SESLSDNATAFS (SEQ ID NO:65).

[0059] In one embodiment, the BoNT / X VAMP epitope, particularly the BoNT / X VAMP4 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 62 to SEQ ID NO: 65. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SESLSDNATAF (SEQ ID NO: 62). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SESLSDNATAF (SEQ ID NO: 62).

[0060] Other examples of BoNT / X VAMP epitopes, and more particularly, BoNT / X VAMP5 epitopes, include the following: SDQLLDMSSTF (sequence number 66) SDQLLDMSST (sequence number 67) RSDQLLDMSSTF (sequence number 68) SDQLLDMSSTFN (sequence number 69) SDQLLDMSSAF (sequence number 70) SDQLLDMSSA (SEQ ID NO:71) RSDQLLDMSSAF (sequence number 72) SDQLLDMSSAFS (sequence number 73) RSDQLLDMSS (sequence number 74).

[0061] In one embodiment, the BoNT / X VAMP epitope, particularly the BoNT / X VAMP5 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 66 to SEQ ID NO: 74. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SDQLLDMSSTF (SEQ ID NO: 66). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SDQLLDMSSTF (SEQ ID NO: 66).

[0062] Other examples of BoNT / X VAMP epitopes, and more preferably BoNT / X YKT6 epitopes, include the following: SEVLGTQSKAF (SEQ ID NO:75) SEVLGTQSKA (SEQ ID NO:76) KSEVLGTQSKAF (SEQ ID NO:77) SEVLGTQSKAFY (SEQ ID NO:78).

[0063] In one embodiment, the BoNT / X VAMP epitope, particularly the BoNT / X YKT6 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 75 to SEQ ID NO: 78. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEVLGTQSKAF (SEQ ID NO: 75). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SEVLGTQSKAF (SEQ ID NO: 75).

[0064] As used herein, the "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical nucleotides or amino acids at the same position shared by the aligned sequences. Thus, percent identity can be calculated as the number of identical nucleotides or amino acids at each position in the alignment divided by the total number of nucleotides or amino acids in the aligned sequences, multiplied by 100. Calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. Comparison of sequences between two or more sequences and determination of percent identity can be performed using certain mathematical algorithms well known to those skilled in the art, such as the global alignment mathematical algorithm (e.g., as described by Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972).

[0065] In another aspect, the invention relates to a polypeptide comprising an antigenic polypeptide of the invention, wherein the polypeptide does not contain a region of more than 17, 16, 15, 14, 13, 12, 11, 10, preferably 16, more preferably 15, consecutive amino acids having 100% sequence identity to a naturally occurring VAMP amino acid sequence. One skilled in the art will readily appreciate that such polypeptides are also antigenic.

[0066] In a preferred embodiment, the polypeptide comprises a covalent linker, preferably at its N-terminus and / or at its C-terminus. Examples of covalent linkers suitable according to the invention are provided below.

[0067] In another aspect, the present invention provides an antigenic protein comprising a polypeptide of the present invention covalently linked to a carrier.

[0068] Preferably, the carrier is a non-immunogenic or weakly immunogenic protein. Examples of suitable carriers include keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP).

[0069] In one embodiment, the antigenic protein comprises a covalent linker between the polypeptide of the invention (which may already comprise a linker as shown above) and the carrier. The linker may be one or more amino acids, natural or unnatural, that can form a covalent bond with other amino acids (of the polypeptide and / or the carrier) due to the presence of reactive groups present at their N-terminus, C-terminus and / or side chain, as is well known in the art. In particular, an amino acid having a primary amine group (-NH2) at the N-terminus and / or in its side chain (such as lysine) can react with an amino acid having a carboxyl (-COOH) group at the C-terminus and / or in its side chain (such as aspartic acid or glutamic acid) to form a covalent bond, and an amino acid having a sulfhydryl (-SH) group at the side chain (such as cysteine ​​or selenocysteine) can react with an amino acid having a sulfhydryl (-SH) group at the side chain (such as cysteine ​​or selenocysteine) to form a covalent bond. For example, the covalent linker may be a cysteine ​​added in the C-terminus or N-terminus of the polypeptide of the invention, said cysteine ​​forming a disulfide bridge with another cysteine ​​added or present in the carrier. The covalent linker may alternatively or additionally be in the form of several amino acids forming a spacer, for example, the linker may be a peptide containing uncharged amino acids with small side chain R groups, such as glycine, alanine, valine, leucine or serine. Examples of suitable spacers of the invention include G-spacers such as GGG, GGGG and GGGGS or A-spacers such as AAA, AAAA and AAAAV. In one embodiment, the linker consists of about 1 to about 30 amino acid residues, preferably about 2 to about 25 amino acid residues, more preferably about 3 to about 20 amino acid residues, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

[0070] In another aspect, the present invention provides the use of the antigenic polypeptide or protein of the present invention to generate antibodies against C-terminal VAMP cleavage products.In one embodiment, the epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products.In another embodiment, the epitope of the present invention is used to generate monoclonal antibodies against C-terminal VAMP cleavage products.

[0071] Methods for producing antibodies are well known in the art, see, e.g., Greenfield, Edward A., ed. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, 2014; Leenaars, Marlies, and Coenraad FM Hendriksen. "Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations." Ilar Journal 46.3 (2005): 269-279.

[0072] Polyclonal antibodies that bind to the VAMP epitope as described herein can be produced by injecting an animal, e.g., a mammal such as a rabbit, goat, mouse, hamster or monkey, or an egg such as a chicken egg, with an antigenic polypeptide or protein of the present invention. Polyclonal antibodies against a VAMP epitope as disclosed herein can be isolated from the animal (e.g., from the blood) or from the egg and further purified by protein affinity chromatography to obtain an IgG fraction or by affinity purification against the VAMP epitope used to produce the antibody. Some contract research organizations offer custom antibody production services, for example, Eurogentec offers a "Speedy 28-day programme" with an immunization on day 0, followed by three booster injections on days 7, 10 and 18. A middle bleed on day 21 and a final bleed on day 28. This is an example of a common technique for polyclonal antibody production that is well known in the art.

[0073] Monoclonal antibodies that bind to VAMP epitopes as described herein can be produced using hybridoma techniques. See, for example, Chapter 7, Greenfield, Edward A., ed. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, 2014. Briefly, a host animal, e.g., a mammal such as a rabbit, goat, mouse, hamster or monkey, is exposed to one or more injections of an antigenic polypeptide or protein of the present invention to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to cleaved VAMP. Antibody titers in immunized animals can be monitored over time by standard techniques, such as using ELISA (enzyme-linked immunosorbent assay). Alternatively, lymphocytes can be immunized in vitro using a suitable cell culture line. At an appropriate time after immunization, e.g., when antibody titers are at their highest, antibody-producing cells are isolated from the animal. Generally, peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The isolated antibody-producing cells are fused with an immortal cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine and human origin. Usually, a mouse myeloma cell line is fused with spleen cells harvested from an appropriately immunized mouse to produce a hybridoma. The preferred immortal cell line is a mouse myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT). Any of several myeloma cell lines can be used as fusion partners according to standard techniques (e.g., P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines). The hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and non-productively fused myeloma cells (unfused splenocytes are not transformed and will die after a few days in culture).The culture medium in which the hybridoma cells are grown can be assayed for the presence of monoclonal antibodies that bind to VAMP epitopes as described herein. For example, the hybridoma supernatants can be screened using cleaved VAMP positive media in immunoprecipitation assays, in vitro binding assays (such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA)), or cell-based activity assays. The binding affinity of the monoclonal antibodies can also be determined, for example, by Scatchard analysis. See, for example, Peter J. Munson and David Rodbard, Ligand: A Versatile Computerized Approach For Characterization of Ligand-Binding Systems, 107(1) Anal. Biochem. 220-239 (1980). After the desired hybridoma cells are identified, limiting dilution procedures are used to isolate clones of single cell origin until a clonal cell line expressing the desired monoclonal antibody is obtained. Alternatively, monoclonal antibodies that bind to the VAMP epitopes as described herein can be produced by screening recombinant combinatorial immunoglobulin libraries, such as antibody phage display libraries, with the antigenic polypeptides, proteins or peptides of the invention. Kits for generating and screening phage display libraries are commercially available, for example, Recombinant Phage Antibody System (Amersham GE Healthcare, Piscataway, NJ) and SurfZAP. TMPhage Display kit (Stratagene, La Jolla, CA), etc. Further, examples of methods and reagents useful in generating and screening antibody display libraries can be found, for example, in Ladner et al. US Patent 5,223,409, Borrebaeck et al. US Patent 5,712,089, Griffiths et al. US Patent 5,885,793, Griffiths et al. US Patent 5,962,255, McCafferty et al. US Patent 5,969,108, Griffiths et al. US Patent 6,010,884, Jespers et al. US Patent 6,017,732, Borrebaeck et al. US Patent 6,027,930, Johnson et al. US Patent 6,140,471, and McCafferty et al. US Patent 6,172,197, each of which is incorporated herein by reference in its entirety.

[0074] In another aspect, the invention provides an antibody that binds to an antigenic polypeptide or protein of the invention.

[0075] In one embodiment, the antibody is a polyclonal antibody.

[0076] In one embodiment, the antibody is a monoclonal antibody.

[0077] The binding affinity between an antibody and an antigenic polypeptide or protein is determined by the equilibrium dissociation constant (K), which measures the rate at which new antibody-antigen complexes form that equals the rate at which antibody-antigen complexes dissociate at equilibrium. D The equilibrium dissociation constant is expressed as M and is defined by the ratio Kd / Ka at equilibrium, where Ka is the association rate constant of the antibody and Kd is the dissociation rate constant of the antibody. K D= [Ab] x [Ag] / [Ab + Ag], where [Ab] is the molar concentration of antibody, [Ag] is the molar concentration of antigen, and [Ab + Ag] is the molar concentration of antibody-antigen complex, all concentrations being those of such components when the system is at equilibrium. The smaller the equilibrium dissociation constant, the more tightly an antibody is bound to its antigen, or the higher the binding affinity between an antibody and an antigen.

[0078] In one embodiment, the K between an antibody of the invention and an antigenic polypeptide or protein epitope D is 10 -6 In a preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -7 In a more preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -8 In a more preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -9 In a more preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -10 In a more preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -11 In a more preferred embodiment, the K between the antibody of the invention and the antigenic polypeptide or protein is less than M. D is 10 -12 Less than M.

[0079] In another aspect, the invention provides the use of an antibody of the invention in a gain of signal cellular assay for VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0080] In one embodiment, the use is an in vitro or ex vivo use.

[0081] In another aspect, the invention provides a method for determining cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin in a cell, comprising: a) contacting a cell with a Clostridial neurotoxin under conditions suitable for Clostridial neurotoxin activity; b) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention; c) detecting by suitable means the binding of said first detection antibody to the C-terminal VAMP cleavage product; The present invention provides a method comprising:

[0082] In one embodiment, the method of the invention further comprises the step of d) quantifying by suitable means the amount of C-terminal VAMP cleavage product bound to said first detection antibody.

[0083] In one embodiment of the method of the present invention, step b) comprises contacting the cytoplasmic contents of the cell with a second detection antibody against full-length VAMP under conditions suitable for binding of the second detection antibody to full-length VAMP, step c) comprises detecting binding of the second detection antibody to full-length VAMP by suitable means, and step d) comprises quantifying the amount of full-length VAMP bound to the second detection antibody by suitable means.

[0084] In one embodiment, the method is an in vitro or ex vivo method.

[0085] It will be apparent to one of skill in the art that an increase in the amount of C-terminal VAMP cleavage product bound to the first antibody and / or a decrease in the amount of full-length VAMP bound to the second detection antibody indicates increased VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0086] In one embodiment, the second detection antibody is identical to the first detection antibody and binds to a C-terminal VAMP cleavage product and to full-length VAMP.

[0087] In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP but not to C-terminal VAMP cleavage products.Optionally, the second detection antibody binds to a VAMP epitope that is N-terminal to the clostridial neurotoxin cleavage site.Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

[0088] In certain embodiments, a method for determining cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin in a cell comprises: a) contacting a cell with a Clostridial neurotoxin under conditions suitable for Clostridial neurotoxin activity; b) removing the cytoplasmic contents of said cells; contacting, after cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin, with a first detection antibody against a C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; and with a second detection antibody that binds full length VAMP but not C-terminal VAMP cleavage products; c) By appropriate means. binding of the first antibody to a C-terminal VAMP cleavage product and to full-length VAMP; and Binding of the second detection antibody to full-length VAMP and detecting d) By appropriate means, the combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody, and The amount of full-length VAMP bound to the second detection antibody Quantifying the Includes.

[0089] It will be apparent to one of skill in the art that a decrease in the amount of full-length VAMP bound to the second detection antibody and no change in the combined amount of full-length and C-terminal VAMP cleavage products bound to the first detection antibody indicates VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0090] In another particular embodiment, the method for determining cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin in a cell comprises: a) contacting a cell with a Clostridial neurotoxin under conditions suitable for Clostridial neurotoxin activity; b) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; c) By appropriate means, binding of the first antibody to the C-terminal VAMP cleavage product; and Binding of the first detection antibody to full-length VAMP detecting a signal resulting from binding of the first detection antibody to a C-terminal VAMP cleavage product can be distinguished from a signal resulting from binding of the first detection antibody to full-length VAMP; d) By appropriate means, the amount of C-terminal VAMP cleavage product bound to the first detection antibody, and The amount of full-length VAMP bound to the first detection antibody Quantifying the Includes.

[0091] It will be apparent to one of skill in the art that an increase in the amount of C-terminal VAMP cleavage product bound to the first antibody and a decrease in the amount of full-length VAMP bound to the first detection antibody indicates increased VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0092] In another aspect, the present invention provides a method for determining immune resistance to a VAMP-cleaving Clostridial neurotoxin in a subject, comprising: a) adding a VAMP-cleaving Clostridial neurotoxin to a test sample obtained from a subject; b) contacting the cell with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; c) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention; d) detecting by suitable means the binding of the first detection antibody to the C-terminal VAMP cleavage product; e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody; f) repeating steps a) to e) using a negative control sample instead of the test sample; g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (e) compared to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of a neutralizing antibody against a VAMP-cleaving Clostridial neurotoxin. The present invention provides a method comprising:

[0093] In one embodiment, step f) further comprises repeating steps a) through e) with a positive control sample.

[0094] As used herein, the term "neutralizing antibody to a VAMP-cleaving Clostridial neurotoxin" means any antibody that binds to a region of a VAMP-cleaving Clostridial neurotoxin in a manner that reduces or prevents the VAMP-cleaving Clostridial neurotoxin from exerting its therapeutic effect in a subject under physiological conditions.

[0095] In one embodiment, the subject is a mammal, hi a preferred embodiment, the subject is a human.

[0096] In one embodiment, the sample is selected from blood, plasma, serum and lymphatic fluid obtained from a subject.

[0097] The test sample can be obtained from the subject before exposure to VAMP-cleaving Clostridial neurotoxin, after a single treatment with VAMP-cleaving Clostridial neurotoxin, or after multiple treatments with VAMP-cleaving Clostridial neurotoxin. In certain embodiments, the test sample is derived from a subject that is resistant to treatment with VAMP-cleaving Clostridial neurotoxin.

[0098] As used herein, the term "control sample" refers to any sample that is known to be free of the test sample, including both negative and positive control samples. With respect to neutralizing antibodies against VAMP-cleaving Clostridial neurotoxins, negative control samples can be obtained from individuals who have never been exposed to VAMP-cleaving Clostridial neurotoxins, and can include, but are not limited to, samples from the same individuals who provide the test sample but taken before undergoing treatment with VAMP-cleaving Clostridial neurotoxins, samples taken from different individuals who have never been exposed to VAMP-cleaving Clostridial neurotoxins, and pooled samples taken from multiple different individuals who have never been exposed to VAMP-cleaving Clostridial neurotoxins.

[0099] With respect to neutralizing antibodies against a VAMP-cleaving Clostridial neurotoxin, positive control samples can be obtained from individuals who exhibit immune resistance to a VAMP-cleaving Clostridial neurotoxin, including, but not limited to, individuals who test positive in a patient-based test assay, individuals who test positive in an in vivo bioassay, and individuals who exhibit hyperimmunity, e.g., subjects vaccinated against a VAMP-cleaving Clostridial neurotoxin.

[0100] In one embodiment, the method is an in vitro or ex vivo method.

[0101] In one embodiment of the method for determining immune resistance, step c) comprises contacting the cytoplasmic contents of the cells with a second detection antibody against full-length VAMP under conditions suitable for binding of the second detection antibody to full-length VAMP, step d) comprises detecting binding of the second detection antibody to full-length VAMP by suitable means, and step e) comprises quantifying the amount of full-length VAMP bound to the second detection antibody.

[0102] In one embodiment, the second detection antibody is identical to the first detection antibody and binds to a C-terminal VAMP cleavage product and to full-length VAMP.

[0103] In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP but not to C-terminal VAMP cleavage products. Optionally, the second detection antibody binds to a VAMP epitope that is N-terminal to the Clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

[0104] In certain embodiments, a method for determining immune resistance to a VAMP-cleaving Clostridial neurotoxin in a subject comprises: a) adding a VAMP-cleaving Clostridial neurotoxin to a test sample obtained from a subject; b) contacting the cell with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; c) removing the cytoplasmic contents of said cells; contacting, after cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin, with a first detection antibody against a C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; and with a second detection antibody that binds to full-length VAMP but not to C-terminal VAMP cleavage products; d) By appropriate means, binding of the first antibody to a C-terminal VAMP cleavage product and to full-length VAMP; and Binding of the second detection antibody to full-length VAMP and detecting e) the combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody, and The amount of full-length VAMP bound to the second detection antibody Quantifying the f) repeating steps a) through e) using a negative control sample instead of the test sample; g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody in step (e) compared to the corresponding amount in step (f) and / or a higher combined amount of full-length VAMP bound to the second detection antibody in step (e) compared to the corresponding amount in step (f) indicates the presence of neutralizing antibodies against a VAMP-cleaving Clostridial neurotoxin. Includes.

[0105] In another particular embodiment, the method for determining immune resistance to a VAMP-cleaving Clostridial neurotoxin in a subject comprises: a) adding a VAMP-cleaving Clostridial neurotoxin to a test sample obtained from a subject; b) contacting the cell with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; c) contacting the cytoplasmic contents of said cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, said first detection antibody being an antibody of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; d) By appropriate means, binding of the first antibody to a C-terminal VAMP cleavage product and to full-length VAMP; and Binding of the first detection antibody to full-length VAMP detecting a signal resulting from binding of the first detection antibody to a C-terminal VAMP cleavage product can be distinguished from a signal resulting from binding of the first detection antibody to full-length VAMP; e) the amount of C-terminal VAMP cleavage product bound to the first detection antibody, and The amount of full-length VAMP bound to the first detection antibody Quantifying the f) repeating steps a) through e) using a negative control sample instead of the test sample; g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (e) compared to the corresponding amount in step (f) and / or a higher amount of full-length VAMP bound to the first detection antibody in step (e) compared to the corresponding amount in step (f) indicates the presence of a neutralizing antibody against a VAMP-cleaving Clostridial neurotoxin. Includes.

[0106] As used herein, "VAMP-cleaving Clostridial neurotoxin" refers to a Clostridial neurotoxin that binds to a receptor on a target cell and translocates the Clostridial light chain (L) into the cytosol, which in turn proteolytically cleaves VAMP and disrupts secretion of the molecule by the cell via vesicular transport.

[0107] Preferably, in the methods or uses of the invention, the VAMP-cleaving Clostridial neurotoxin comprises BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or a TeNT light chain. Optionally, the BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT light chain is - amino acid residues 1 to 441 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - amino acid residues 1 to 442 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - amino acid residues 1 to 439 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - amino acid residues 1 to 446 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - amino acid residues 1 to 439 of SEQ ID NO: 41 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - amino acid residues 1 to 456 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; The present invention includes a sequence selected from the following:

[0108] It is understood that a BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT light chain as described herein has the ability to cleave VAMP.

[0109] In one embodiment of the method or use of the invention, the VAMP-cleaving Clostridial neurotoxin is selected from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X and TeNT. Optionally, BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT is - SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - SEQ ID NO: 41 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, - SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto; The present invention includes a sequence selected from the following:

[0110] It is understood that a BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT clostridial neurotoxin as described herein has the ability to bind to a receptor on a target cell, translocate a clostridial light chain into the cytosol, and cleave VAMP.

[0111] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is a mosaic neurotoxin. As used in this context, the term "mosaic neurotoxin" refers to a naturally occurring Clostridial neurotoxin that contains at least one functional domain from different types of Clostridial neurotoxins (e.g., different serotypes of Clostridial neurotoxins) that are not normally included. Examples of naturally occurring VAMP-cleaving mosaic neurotoxins include BoNT / DC and BoNT / FA. BoNT / DC is a serotype D L chain and H chain. N Domain and H of serotype C C BoNT / FA contains the D / C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan. Vet. Microbiol. (2010): 140:147-154., BoNT / FA is a subtype of BoNT / A1 H C H closely related to the domain N domain, and the BoNT / F5 light chain (Pellett, Sabine, et al. "Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain." mSphere 1.1 (2016): e00100-15).

[0112] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is a mosaic neurotoxin selected from BoNT / DC and BoNT / FA.

[0113] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is a chimeric neurotoxin. The term "chimeric neurotoxin" as used herein means a neurotoxin that contains one or more domains originating from a first neurotoxin and one or more domains originating from a second neurotoxin. For example, a chimeric neurotoxin may contain an LH domain originating from a first neurotoxin. N H originating from domain and second neurotoxin C Another example of a chimeric neurotoxin may include an LH domain derived from a first neurotoxin. N H CN H originating from domain and second neurotoxin CC There are neurotoxins that contain the domain. Examples of chimeric neurotoxins are provided in GB1607901.4 (not yet published), which is incorporated herein by reference.

[0114] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is - a light chain (L) derived from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT, - H derived from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or TeNT N domain, - H derived from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or TeNT CN domain, - H derived from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or TeNT CC domain wherein at least two of the domains are derived from different Clostridial neurotoxins.

[0115] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is - LH derived from a first clostridial neurotoxin selected from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT N domain, - H derived from a second clostridial neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or TeNT CN H CC domain wherein the first and second Clostridial neurotoxins are different.

[0116] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is - LH derived from a first clostridial neurotoxin selected from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT N H CN domain, - H derived from a second clostridial neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or TeNT CC domain wherein the first and second Clostridial neurotoxins are different.

[0117] The VAMP-cleaving Clostridial neurotoxin may be a modified neurotoxin or a derivative thereof, including but not limited to those described below. The modified neurotoxin or derivative may contain one or more amino acids that are modified compared to the natural (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the natural (unmodified) form of the toxin. As an example, the modified Clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the natural (unmodified) Clostridial neurotoxin sequence. Such modifications may modify the functional aspects of the neurotoxin, such as biological activity or persistence.

[0118] The modified VAMP-cleaving Clostridial neurotoxins as described herein retain the ability to bind to a receptor on a target cell, translocate the light chain to the cytoplasm of the cell, and cleave VAMP.

[0119] A modified VAMP-cleaving Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (modified H C domain, the modified heavy chain binds to the target neuronal cell with higher or lower affinity than the native (unmodified) neurotoxin. C Such modifications in the H domain alter binding to ganglioside receptors and / or protein receptors on target neurons. CC The modified neurotoxins may include modified residues in the ganglioside binding site or in the protein receptor binding site of the domain. Examples of such modified neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety. For example, the H CC The domain is the natural BoNT / BH CC Compared with the sequence, human Syt II BoNT / BH CC BoNT / BH domain contains at least one amino acid residue substitution, addition or deletion that has the effect of increasing the binding affinity of the domain. CC Suitable amino acid residue substitutions, additions or deletions in the subdomains are disclosed in WO2013 / 180799 and the yet to be disclosed PCT / US2016 / 024211 (both of which are incorporated herein by reference). CC Suitable amino acid residue substitutions, additions or deletions in the subdomains 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.

[0120] In one embodiment, the VAMP-cleaving clostridial neurotoxin is a retargeted neurotoxin. The term "retargeted neurotoxin" (also called "targeted secretion inhibitor", "TSI", "TVEMP" or "TEM"), as used herein, refers to a clostridial neurotoxin that contains a targeting moiety (TM) that binds to a non-clostridial receptor. The TM is the H of the clostridial neurotoxin heavy chain. C or H CC Examples of retargeted neurotoxins are described in WO96 / 33273, WO98 / 07864, WO00 / 10598, WO01 / 21213, WO01 / 53336, WO02 / 07759, WO2005 / 023309, WO2006 / 026780, WO2006 / 099590, WO2006 / 056093, WO2006 / 059105, WO2006 / 059113, WO 2007 / 138339, WO2007 / 106115, WO2007 / 106799, WO2009 / 150469, WO2009 / 150470, WO2010 / 055358, W O2010 / 020811, WO2010 / 138379, WO2010 / 138395, WO2010 / 138382, WO2011 / 020052, WO2011 / 020056, No. 5,393,991, all of which are incorporated herein by reference.

[0121] Examples of cells suitable for use in the methods or uses of the invention include prokaryotic cells, such as E. coli cells, yeast cells, insect cells, animal cells, mammalian cells, human cells, mouse cells, primate cells and / or neuronal cells. Preferably, the cells are neuronal cells, in particular cells that are highly sensitive to BoNT.

[0122] The cell with high sensitivity to BoNT is a cell that is sensitive to BoNT poisoning.In some embodiments, the cell with high sensitivity to BoNT is a cell that is sensitive to BoNT poisoning, for example, about 500pM or less, about 400pM or less, about 300pM or less, about 200pM or less, about 100pM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about 10pM or less, about 9pM or less, about 8pM or less, about 7pM or less, about 6pM or less, about 5pM or less, The cells are sensitive to about 4 pM or less, about 3 pM or less, about 2 pM or less, about 1 pM or less, about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM, about 0.1 pM or less, about 90 fM or less, about 80 fM or less, about 70 fM or less, about 60 fM or less, about 50 fM or less, about 40 fM or less, about 30 fM or less, about 20 fM or less, or about 10 fM or less.

[0123] Preferably, the cells have high sensitivity (as defined above) to a VAMP-cleaving BoNT.

[0124] In one embodiment, the cells are primary neuronal cells that are highly sensitive to BoNT, such as cortical, hippocampal and / or spinal cord neurons. For example, the cells are rat cortical neurons.

[0125] In one embodiment, the cells are derived from a neuronal cell line that is highly sensitive to BoNT, such as, for example, BE(2)-M17, Kelly, LA1-55n, N1 E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SY5Y, SiMa, and / or SK-N-BE(2)-C.

[0126] In one embodiment, the cells are stem cell derived, in particular induced pluripotent stem cell (iPS cell) derived neuronal cells, such as i-Cell® Neurons, i-Cell® DopaNeurons, iCell Glutamatergic Neurons, iCell MotoNeurons (Cellular dynamics Inc), cerebral cortical neurons, neural stem cells (Axol Biosciences), Peri.4U neurons, CNS.4U neurons, Dopa.4U neurons (Axiogenesis), MNP cells (Lonza), cortical neurons, motor neurons (iStem) and / or iPSC derived neuronal cells (MTI-GlobalStem).

[0127] In one embodiment, cells may be recombinantly modified to express high levels of VAMP, e.g., VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6, more preferably, VAMP1, VAMP2 and / or VAMP3.

[0128] In one embodiment, where the VAMP-cleaving neurotoxin is BoNT / B, BoNT / DC or BoNT / G, the cells express high levels of synaptotagmin I and / or synaptotagmin II (Syt I / Syt II). In one embodiment, where the VAMP-cleaving neurotoxin is BoNT / B, BoNT / DC or BoNT / G, the cells are recombinantly modified to express high levels of synaptotagmin I and / or synaptotagmin II (Syt I / Syt II).

[0129] In one embodiment, where the VAMP-cleaving neurotoxin is BoNT / FA, BoNT / F, BoNT / D or TeNT, the cells express high levels of synaptic vesicle protein (SV2). In one embodiment, where the VAMP-cleaving neurotoxin is BoNT / FA, BoNT / F, BoNT / D or TeNT, the cells are recombinantly modified to express high levels of synaptic vesicle protein (SV2).

[0130] As used herein, "conditions suitable for clostridial neurotoxin activity" refers to conditions (e.g., temperature, pH, cofactors, etc.) under which a clostridial neurotoxin can bind to a clostridial neurotoxin receptor present on a cell membrane, translocate the clostridial neurotoxin light chain into the cytoplasm of the cell, and cleave VAMP.

[0131] In one embodiment of the method of the invention, the conditions suitable for Clostridial neurotoxin activity may include incubation at about 37° C. for a period of about 1 hour to about 48 hours. In one embodiment of the method of the invention, the conditions suitable for Clostridial neurotoxin activity may include incubation at about 37° C. for a period of about 2 hours to about 36 hours. In one embodiment of the method of the invention, the conditions suitable for Clostridial neurotoxin activity may include incubation at about 37° C. for a period of about 4 hours to about 24 hours.

[0132] For example, conditions suitable for clostridial neurotoxin activity may include incubation at 37° C. for 24 hours.

[0133] As used herein, "conditions suitable for binding of a first detection antibody to cleaved VAMP" and "conditions suitable for binding of a second detection antibody to full-length VAMP" refer to conditions (e.g., temperature, pH, cofactors, etc.) under which the first and / or second detection antibody can bind to cleaved VAMP and / or full-length VAMP.

[0134] In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 4° C. for a period of about 8 hours to about 48 hours. In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 4° C. for a period of about 10 hours to about 24 hours. In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 4° C. for a period of about 12 hours to about 16 hours.

[0135] In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 25° C. for a period of about 30 minutes to about 8 hours. In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 25° C. for a period of about 1 hour to about 4 hours. In one embodiment of the method of the invention, the conditions suitable for antibody binding may include incubation at about 25° C. for a period of about 1.5 hours to about 3 hours.

[0136] Suitable means for detecting and quantifying the binding of detection antibody to cleaved or full-length VAMP are well known in the art.For example, the binding of detection antibody to cleaved or full-length VAMP can be detected and quantified by Western blotting.Since each protein runs at a specific molecular weight by SDS-PAGE, cleaved VAMP is detected at a lower molecular weight than full-length VAMP.The analysis of bands by densitometry allows the cleavage percentage reading using both full-length band and cleaved band in the same lane on gel.Alternatively, VAMP cleavage can be detected and quantified using enzyme-linked immunosorbent assay (ELISA), for example, sandwich ELISA.

[0137] In one embodiment of the method of the invention, the first detection antibody is a polyclonal antibody and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified by enzyme-linked immunosorbent assay.

[0138] In one embodiment of the method of the invention, the first detection antibody is a polyclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

[0139] In one embodiment of the method of the invention, the first detection antibody is a monoclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified by enzyme-linked immunosorbent assay.

[0140] In one embodiment of the method of the invention, the first detection antibody is a monoclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

[0141] In one embodiment of the method of the invention, the cells are lysed and then the cytoplasmic contents are contacted with the detection antibody(ies).

[0142] In an alternative embodiment of the method of the invention, the cells are permeabilized and then their cytoplasmic contents are contacted with the detection antibody(ies).

[0143] In another aspect, the present invention provides a kit comprising a cell susceptible to intoxication by a VAMP-cleaving neurotoxin and a first detection antibody against cleaved VAMP, said first detection antibody being an antibody of the present invention.

[0144] In one embodiment, the kit further comprises a second detection antibody that binds to full-length VAMP but not to the C-terminal VAMP cleavage product. Optionally, the second detection antibody binds to a VAMP epitope that is N-terminal to the Clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

[0145] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in carrying out or testing the embodiments of the present disclosure. Numerical ranges include the numbers that define the range. Unless otherwise specified, each nucleic acid sequence is written from left to right in the 5' to 3' direction, and amino acid sequence is written from left to right in the amino to carboxy direction.

[0146] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range is encompassed within the present disclosure, subject to any specifically excluded limit in the stated range, whether or not both limits are included in the smaller range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the present disclosure.

[0147] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a Clostridial neurotoxin" includes a plurality of such candidate agents, reference to "the Clostridial neurotoxin" includes reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0148] The invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0149] [Figure 1] Figure 1 shows VAMP sequences with clostridial neurotoxin cleavage sites: (A) Human and rat VAMP1, VAMP2 and VAMP3 sequences with BoNT / F5 and BoNT / FA, BoNT / F, BoNT / D and BoNT / DC, BoNT / B, BoNT / G, TeNT and BoNT / X cleavage sites; (B) Human and rat VAMP4, VAMP5 and YKT6 sequences with BoNT / X cleavage sites. [Diagram 2] FIG. 2 is a diagram showing VAMP sequences with Ab epitopes (i.e., immunogenic epitope regions) and BoNT / F, BoNT / D and BoN / B cleavage sites. The sequences of human and rat VAMP1, VAMP2 and VAMP3 are shown for comparison. The rat and human VAMP2 sequences are identical in selected epitope regions. The cleavage sites are indicated by arrows. The VAMP2 cleavage points of BoNT / F and BoNT / D are located at adjacent amino acids, Q58-K59 and K59-L60, respectively, and the cleavage point of BoNT / B is located at amino acids Q76-F77 (based on the human VAMP2 sequence amino acid positions). [Diagram 3] Figure 3 shows cell-free cleavage of recombinant VAMP2-GFP with MBP-LF and LHND. Recombinant VAMP2-GFP was incubated with 0.01 μg / μL LHND or MBP-LF for 1 h at 37°C. Equal volumes of sample buffer were added and 0.5 μg (Coomassie) and 0.3 μg (blot) protein was run by SDS-PAGE and stained with Coomassie or blotted with various anti-VAMP2 antibodies. Schematic diagram shows the location of antibody epitopes. Recombinant protein representation and epitope line lengths are not to scale. 1-BSA, 2-VAMP2-GFP, 3-truncated VAMP2-GFP (aa59 / 60-end), 4-truncated VAMP2-GFP (aa1-58 / 59). [Figure 4]Figure 4 shows in vitro VAMP cleavage after BoNT / F and BoNT / D treatment. Rat cortical neurons grown in 96-well plates from DIV18 to 21 were treated for 24 h with either BoNT / F (A) or BoNT / D (B). Lysates were run by SDS-PAGE and blotted with custom made anti-VAMP2 antibodies, i.e., anti-Pep1, anti-Pep2 or anti-Pep3, or with commercial antibody ab181869. 1-full length VAMP2, 2-cleaved VAMP2. Anti-pep 2 data show dose-dependent disappearance of full length VAMP2 and appearance of smaller molecular weight cleavage fragments. Both band signals were used to quantify the dose-dependent percentage of VAMP2 cleavage by BoNT / F (C) and BoNT / D (D). [Diagram 5] FIG. 5. (A) Rat cortical neurons were treated with native BoNT / F1 (□), native BoNT / A1 (●), or recombinant BoNT / FA (△) for 24 h. Cells were lysed, run on SDS-PAGE, and blotted for VAMP-2 or SNAP-25 cleavage. Percent SNARE cleavage was determined from the ratio of full-length to cleaved protein by densitometric analysis. Data were fitted using a four-parameter logistic equation to determine the concentration of BoNT required for 50% maximal SNARE cleavage (pEC50) (B). Data are means ± sem (n=3 (BoNT / F1 and BoNT / A1) or 4 (BoNT / FA) triplicate independent experiments). [Figure 6] Figure 6 shows in vitro VAMP cleavage after BoNT / B and BoNT / F treatment. Rat cortical neurons grown to DIV18-21 were treated with either BoNT / F or BoNT / B for 24 h. Lysates were run by SDS-PAGE and blotted with a new custom-made anti-VAMP2 antibody (anti-Pep4), a BoNT / B cleavage-specific anti-VAMP2 antibody or anti-Pep1, anti-Pep2 or anti-Pep3 antibodies. EXAMPLES

[0150] [Example 1] Detection of VAMP protein cleavage by BoNT / D and BoNT / F

[0151] A-Method

[0152] 1. Antibody Preparation

[0153] Antibodies were generated by Eurogentec using their Speedy 28-day program (https: / / secure.eurogentec.com / product / research-anti-protein-28-day-speedy-polyclonal-packages.html?country=gbr). The following peptides were used to immunize two rabbits per peptide: - VAMP PEP1: H2N- SNR RLQ QTQ AQV DEC -CONH2 (SEQ ID NO: 39), - VAMP PEP2: AcNH - KLS ELD DRA DAL Q - CONH2 (SEQ ID NO: 15) or - VAMP PEP3:H2N - CLQ AGA SQ - CONH2 (SEQ ID NO: 40).

[0154] Animals received an initial immunization and three subsequent boosts. Pre-immunization, mid-bleeds and a terminal bleed were taken.

[0155] 2. Recombinant Protein Cleavage

[0156] Active constructs containing the light chain and translocation domain of BoNT / D or the equivalent BoNT / F domain fused to maltose binding protein (MPB) were generated as previously described (Masuyer et al., "Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B. J Struct Biol", 174, p52-57, 2011; Sutton et al., "Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications. Protein Expression and Purification 40:31-41, 2005). Briefly, LH N Either MBP-LF (SEQ ID NO: 35) or a fusion protein called MBP-LF (SEQ ID NO: 36) (the latter is a fusion of MBP with the light chain of BoNT / F1 and a C-terminal 6-histidine motif. MPB and the 6-histidine motif are well-known affinity tags) was diluted to 0.01 μg / μL in assay buffer (50 mM HEPES pH 7.2, 200 μM ZnCl2, 1 μg / μL BSA, 10 mM DTT). VAMP2-GFP (SEQ ID NO: 37) (a fusion protein of amino acids 2-94 of human VAMP2 and the detectable marker green fluorescent protein (GFP)) was diluted to 8 μM in assay buffer (50 mM HEPES pH 7.2, 200 μM ZnCl2, 1 μg / μL BSA, 10 mM DTT). An equal volume of LH N D or MBP-LF and VAMP2-GFP (SEQ ID NO:37) (8 μM) were combined and incubated for 1 hour at 37° C. The reaction was stopped by adding 2× reducing sample buffer (NuPage LDS sample buffer, 100 mM DTT).

[0157] 3. Rat cortical neuron cell culture

[0158] Rat cortical neurons were prepared from E17-E18 CD rat embryos. Dissected cortical tissues were collected in ice-cold Hank's Balanced Salt Solution (HBSS) (without Ca2+ or Mg2+) and then dissociated in papain solution at 37°C for 40 min according to the manufacturer's instructions (Worthington Biochemical, NJ, USA). Cortical cells were plated at a density of 20,000 cells / well in 125 μL of Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin in a poly-L-ornithine (PLO)-coated 96-well plate. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (days in vitro) 4, an additional 125 μL of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added. Cells were maintained by replacing half the medium twice a week. At DIV 11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the medium to prevent proliferation of non-neuronal cells.

[0159] 4. BoNT Treatment

[0160] Rat cortical neurons at DIV 18–21 were treated with a concentration range of native BoNT / F1 (Metabiologics, USA) (1 nM–0.1 pM) or BoNT / D (Metabiologics, USA) (10 nM–1 pM) in triplicate wells for 24 h at 37 °C. The medium was removed and cells were washed once with PBS. Cells were lysed in 40 μL of LDS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 benzonase) for 10 min at room temperature.

[0161] 5. SDS-PAGE and Western Blot

[0162] Neuronal lysates were boiled at 90°C for 5 min. 15 μL of lysates per lane were loaded onto a 12% Bis-Tris gel and run at 200V for 50 min in MES buffer. Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using the low MW program. Membranes were blocked with 5% low-fat milk / PBS-Tween for 1 h at room temperature and then incubated overnight at 4°C with custom-made anti-Pep1, anti-Pep2 or anti-Pep3 anti-VAMP2 primary antibodies or with commercial anti-VAMP2 antibodies (Abcam ab3347 and ab181869). Membranes were washed three times with PBS-Tween and incubated with anti-rabbit-HRP secondary antibody for 1 h at room temperature. Membranes were washed 3×5 min with PBS-Tween and then developed with SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

[0163] B-Results

[0164] Assessment of recombinant protein detection

[0165] The regions of the three selected peptide epitopes from VAMP2 relative to the BoNT cleavage site are shown in Figure 2. The sequences of human and rat VAMP1, VAMP2 and VAMP3 are shown for comparison. The rat and human VAMP2 sequences are identical in the selected epitope regions. The cleavage sites of BoNT / B and BoNT / D are located adjacent amino acids.

[0166] The antibodies were first tested in a cell-free assay using recombinant VAMP2-GFP. BoNT / F and BoNT / D substitutes containing the enzymatic light chain domain of the toxin (MBP-LF and LH ND) was used to cleave the VAMP protein (Figure 3). In addition, two other commercially available VAMP2 antibodies were used for comparison: ab3347 (epitope aa1-18) and ab181869 (epitope within aa1-100). Figure 3 shows that anti-Pep1 antibody detected the full-length VAMP2 and the N-terminal cleaved portion (aa1-58 / 59) with a significantly reduced signal. As expected, the C-terminal cleavage product was not detected by this antibody because its epitope is not located in this portion. Anti-Pep2 and anti-Pep3 antibodies detected both the full-length protein and the C-terminal cleavage product of VAMP2-GFP. Ab3347 only detected the full-length VAMP2 and did not detect the N-terminal cleavage fragment, whereas ab181869 detected both.

[0167] These first results show that the antibodies were able to detect full-length and predicted cleavage products of recombinant VAMP2, with the exception of ab3347, which detected only full-length VAMP2 and not the N-terminal cleavage fragments.

[0168] Assessment of endogenous protein detection

[0169] The next question was whether these antibodies could detect any cleavage products in neuronal cell assays where endogenous proteases would be present. Rat primary cortical neurons were treated with either BoNT / F or BoNT / D and lysed for WB analysis (Fig. 4). Anti-Pep1 antibody recognized only the full-length protein and no detectable cleavage products. Anti-Pep2 antibody detected both full-length and C-terminal cleavage products. Anti-Pep3 antibody showed very poor affinity with a weak signal for monomeric VAMP in cell lysates, detecting high molecular weight species that are probably dimers and other proteins (data not shown). The full-length monomer signal was very small, but there were bands for the C-terminal products cleaved by BoNT / F and BoNT / D. In other words, anti-Pep3 did not detect full-length VAMP, but weakly detected the C-terminal fragments cleaved by BoNT / F and BoNT / D. This was in contrast to previous results in a cell-free system that showed strong signals from full-length and cleaved recombinant VAMP. Commercial antibody Ab3347 was not tested in vitro due to lack of detection of the truncated protein in cell-free assays. Commercial antibody ab181869 detected full-length VAMP2 in cortical lysates but did not detect the truncated fragment in cortical lysates, despite positive binding to the N-terminal truncated recombinant fragment in cell-free assays. Pep2 data were used to quantify dose-dependent cleavage of VAMP2 by BoNT / F (Figure 4C) and BoNT / D (Figure 4D).

[0170] We are the first to show that both recombinant VAMP cleavage products can be detected in a cell-free system. However, we also show that when transferred to cell lysates, the N-terminal product is not detectable, but that an as yet unknown mechanism other than degradation may be involved. In contrast, we show that the C-terminal VAMP fragment that remains associated with the vesicle membrane is not degraded or altered in a way that prevents detection by antibody binding or Western blot. The Pep2 epitope is adjacent to the BoNT / D and BoNT / F cleavage sites, and antibodies raised against this peptide detect both full-length VAMP and the cleavage products. In contrast, anti-Pep3 antibodies raised against a shorter epitope further away from the BoNT F / D cleavage site also detect the cleavage products, although weakly.

[0171] [Example 2] Detection of VAMP protein cleavage by BoNT / FA and BoNT / F1 in rat cortical neurons

[0172] A-Method

[0173] 1. Rat cortical neuron cell culture

[0174] Rat cortical neurons were prepared as detailed in Example 1.

[0175] 2. BoNT Treatment

[0176] Rat cortical neurons at DIV 18–21 were treated with a range of concentrations (1 pM–1 fM) of recombinant BoNT / FA (SEQ ID NO: 38) or a range of concentrations (1 nM–1 pM) of native BoNT / F1 (Metabiologics, USA) or a range of concentrations (1 nM–1 fM) of native BoNT / A1 (List Biological Laboratories Inc., USA) in triplicate wells for 24 h at 37 °C. The medium was removed and cells were washed once with PBS. Cells were lysed in 40 μL of LDS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 benzonase) for 10 min at room temperature.

[0177] 3. SDS Page and Western Blot of Rat Cortical Neurons

[0178] Rat cortical neurons were lysed in 40 μL of lysis buffer (NuPage LDS sample buffer, 1 mM DTT and 1:500 benzonase) for 10 min at room temperature. Samples were boiled at 90° C. for 5 min and 15 μL of lysate was loaded per lane on a 12% Bis-Tris gel and run either in MOPS buffer at 200 V for 80 min (SNAP-25) or in MES buffer at 200 V for 50 min (VAMP2). Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using the mixed MW (SNAP25) or low MW (VAMP2) program. The membranes were blocked with 5% low fat milk / PBS-Tween for 1 hour at room temperature, then incubated with either anti-SNAP25 antibody (Sigma S9684 1:4000) or anti-Pep2 (1:500), custom-made anti-VAMP2 (Eurogentec) antibody as described in Example 1, with each primary antibody incubated overnight at 4°C. The membranes were washed 3 times with PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. The membranes were washed 3x5 minutes with PBS-Tween, then developed with SuperSignal West Dura or West Femto chemiluminescent substrates and visualized using a Syngene PXi system. Band densitometry was analyzed using Genetools software, and the ratio of full-length protein to cleavage product was used to determine the % protein cleavage for both SNAP-25 and VAMP2.

[0179] B-Results

[0180] After 24 h treatment with BoNT / F1, BoNT / A1 or BoNT / FA, rat cortical neurons were lysed, run on SDS-PAGE and Western blotted for VAMP-2 (BoNT / F1 and BoNT / FA) or SNAP-25 (BoNT / A1). Percent SNARE cleavage was determined from the ratio of full-length to cleaved protein by densitometric analysis.

[0181] The results are shown in Figure 5. Recombinant BoNT / FA cleaved VAMP-2 with a potency of pEC50 = 12.75 ± 0.14, n = 4. Native BoNT / F1 cleaved VAMP-2 with a potency of pEC50 = 10.77 ± 0.12, n = 3. Native BoNT / A1 cleaved SNAP-25 with a potency of pEC50 = 12.38 ± 0.14, n = 3.

[0182] [Example 3] Detection of VAMP protein cleavage by BoNT / B in rat cortical neurons

[0183] A-Method

[0184] 1. Antibody Preparation

[0185] Monoclonal antibodies were generated by Abeam using rabbits immunized with the peptide Pep4: FETSAAKLKRKYWWK (SEQ ID NO: 49).

[0186] For comparative studies, a BoNT / B cleavage-specific anti-VAMP2 antibody (Kegel et al., Toxicology in Vitro; 2007, 21: p1641-1649) was used.

[0187] 2. Rat cortical neuron cell culture

[0188] Rat cortical neurons were prepared from E17-E18 CD rat embryos. Dissected cortical tissues were collected in ice-cold Hank's Balanced Salt Solution (HBSS) (without Ca2+ or Mg2+) and then dissociated in papain solution at 37°C for 40 min according to the manufacturer's instructions (Worthington Biochemical, NJ, USA). Cortical cells were plated at a density of 20,000 cells / well in 125 μL of Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS) and 100 U / ml penicillin / streptomycin in poly-L-ornithine (PLO)-coated 96-well plates. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (days in vitro) 4, an additional 125 μL of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added. Cells were maintained by replacing half the medium twice a week. At DIV 11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the medium to prevent proliferation of non-neuronal cells.

[0189] 3. BoNT Treatment

[0190] Rat cortical neurons were cultured in T25 flasks and treated with 1 nM and 10 pM BoNT / B (provided and obtained by List Biological Laboratories, Inc.) (SEQ ID NO: 2) for 24 hours at 37°C. The medium was removed and the cells were washed once with PBS. The cells were lysed in 1.5 ml NuPage sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 benzonase) for 10 minutes at room temperature.

[0191] 4. SDS-PAGE and Western Blot

[0192] Neuronal lysates were boiled at 90°C for 5 min. 15 μL of lysates per lane were loaded onto a 12% Bis-Tris gel and run at 200V for 50 min in MES buffer. Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using the low MW program. Membranes were blocked with 5% low-fat milk / PBS-Tween for 1 h at room temperature and then incubated overnight at 4°C with custom-made anti-Pep1, anti-Pep2, anti-Pep3 or anti-Pep4 antibodies or with BoNT-B cleavage specific antibodies. Membranes were washed 3 times with PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 h at room temperature. Membranes were washed 3×5 min with PBS-Tween and then developed with SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

[0193] B-Results

[0194] Based on the results obtained in Examples 1 and 2 above, which suggested that the location of the epitope is important for detecting cleaved VAMP in vitro, a novel monoclonal antibody was generated against an epitope adjacent to the BoNT / B cleavage site located on the C-terminal side.

[0195] This antibody was tested in the same rat cortex assay after BoNT / B and BoNT / F treatment and compared with anti-Pep1, anti-Pep2, anti-Pep3 and BoNT / B cleavage-specific antibodies (Figure 6). The epitope regions of all comparative antibodies were located N-terminal to the BoNT / B cleavage site. Figure 6 shows that the location of the epitope of the new antibody against Pep4 allowed the detection of full-length VAMP2 and the cleavage products of both BoNT / B and BoNT / F treatments. In contrast, anti-Pep2 and anti-Pep3 antibodies detected only BoNT / F cleavage products, but not BoNT / B cleavage products. Anti-Pep1 antibody did not detect any cleavage products as expected. BoNT / B cleavage-specific antibodies also did not detect any BoNT / B cleavage products in these cell lysates.

[0196] Overall, the present data show that an important consideration for detecting cleaved VAMP is the location of the antibody epitope. Only antibodies raised against epitopes located on membrane-bound VAMP fragments after cleavage were able to detect the fragments. By placing the monoclonal antibody epitope at the C-terminus of VAMP, it was hypothesized that this region should be present in the VAMP fragments produced by VAMP-cleaving neurotoxin serotypes B, D and F. This turned out to be correct, making it possible to generate a single antibody (anti-Pep4 Mab) that provided positive results for BoNT / B and BoNT / F treated neurons. Furthermore, since TeNT shares an identical cleavage site with the BoNT / B cleavage site and BoNT / D is in close proximity to the BoNT / F cleavage site, it is predicted that this antibody will also be applicable to TeNT and BoNT / D cleavage.

[0197] An additional advantage of the Pep4 epitope region is that antibodies against this region can detect both full-length and cleaved VAMP with similar sensitivity. The ability to simultaneously detect both protein forms in the same sample provides a robust tool for normalization without the need to blot for additional housekeeping proteins. This provides a highly useful and straightforward signal acquisition Western blot assay for quantification of BoNT potency in cell models.

[0198] The present data show the difference in VAMP detection between cell-free recombinant protein assays and whole cell models. It was this failure to detect cellular cleaved VAMP that was the basis for the hypothesis that VAMP degradation occurred very rapidly in cells (Foran et al., "Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E and F compared with the long-lasting type A". J. Biol Chem 278 (2) pp1363 - 1371 2003). In contrast to the antibodies of the present invention, the majority of commercially available VAMP antibodies are directed against epitopes within the N-terminal region of the protein, and therefore N-terminal VAMP fragments were the focus of their early work. Herein, it is shown that the smaller C-terminal VAMP fragments are not degraded in cells, but the larger N-terminal fragments are not detected either. Interestingly, however, our results in the cell-free system show that not all commercial antibodies are able to detect the expected N-terminal fragment, even when it is present in a cell-free system that does not contain any proteases. From this data, we can conclude that the VAMP degradation hypothesis is most likely related only to the N-terminal fragment, and that the C-terminal VAMP fragment is not degraded and remains bound to the vesicle membrane.

[0199] [Sequence information]

[0200] SEQ ID NO:1 - BoNT / A1 - UniProtKB accession number P10845 (Clostridium botulinum) SEQ ID NO:2 - BoNT / B1 - UniProtKB accession number P10844 (Clostridium botulinum) SEQ ID NO:3 - BoNT / C1 - UniProtKB accession number P18640 (Clostridium botulinum) SEQ ID NO:4 - BoNT / D - UniProtKB accession number P19321 (Clostridium botulinum) SEQ ID NO:5 - BoNT / E - Accession number WP_003372387 (Clostridium botulinum) SEQ ID NO:6 - BoNT / F - UniProtKB accession number YP_001390123 (Clostridium botulinum) SEQ ID NO:7 - BoNT / G - UniProtKB accession number WP_039635782 (Clostridium botulinum) SEQ ID NO:8 - TeNT - UniProtKB accession number P04958 (Clostridium tetani) SEQ ID NO:9 - VAMP1_Rat (Q63666) MSAPAQPPAEGTEGAAPGGGPPGPPPNTTSNRRLQQTQAQVEEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYIFT SEQ ID NO:10 - VAMP1_human (P23763) MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT Sequence number 11 - VAMP2_Rat (P63045) MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST SEQ ID NO:12 - VAMP2_human (P63027) MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST Sequence number 13 - VAMP3_Rat (P63025) MSTGVPSGSSAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGISVLVIIVIIIIVWCVS SEQ ID NO:14 - VAMP3_human (Q15836) MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS SEQ ID NO:15 - VAMP epitope KLSELDDRADALQ SEQ ID NO: 16 - VAMP epitope QKLSELDDRADALQ SEQ ID NO:17 - VAMP epitope KLSELDDRAD SEQ ID NO:18 - VAMP epitope KLSELDDRADALQAGAS SEQ ID NO:19 - VAMP epitope LSELDDRADALQ SEQ ID NO:20 - VAMP epitope LSELDDRADA SEQ ID NO:21 - VAMP epitope LSELDDRADALQAGAS SEQ ID NO:22 - VAMP epitope FETSAAKLKRKYW SEQ ID NO:23 - VAMP epitope FESSAAKLKRKYW SEQ ID NO:24 - VAMP epitope QFETSAAKLKRKYW SEQ ID NO:25 - VAMP epitope FETSAAKLKR SEQ ID NO:26 - VAMP epitope FETSAAKLKRKYWWKN SEQ ID NO:27 - VAMP epitope AKLKRKYWWKN SEQ ID NO:28 - VAMP epitope AAKLKRKYWWKN SEQ ID NO:29 - VAMP epitope AKLKRKYWWKNCKM SEQ ID NO:30 - VAMP epitope AKLKRKYWWKNLKM SEQ ID NO:31 - VAMP epitope DQKLSELDDRADALQ SEQ ID NO:32 - VAMP epitope ERDQKLSELDDRA SEQ ID NO:33 - VAMP epitope LERDQKLSELDDRA SEQ ID NO:34 - VAMP epitope VLERDQKLSELDDRA Sequence number 35- LH N D MGSMTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIILFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIF CMDPVIALMHELTHSLHQLYGINIPSDKRIRRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKAlgHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKS EEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDDFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNNFNQAFATAGVA FLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEAHHHHHHHHHHH · sequence no. 36 - MBP-LF MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPVAINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDSIPDKGLVEKIVKFAVDKLAAALEHHHHHH · SEQ ID NO: 37 (Recombinant VAMP2-GFP) GPLGSSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKLENVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK · SEQ ID NO: 38 - Recombinant BoNT / FA SEQ ID NO:39 - Pep1 SNRRLQQTQAQVDEC Sequence number 40 - Pep3 CLQAGASQ SEQ ID NO: 41 - BoNT / X Genbank accession number BAQ12790 (Clostridium botulinum) Sequence number 42 - VAMP4_Rat (D4A560) MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALAAAILLLMIITQIILHLKK SEQ ID NO:43 - VAMP4_human (O75379) MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT Sequence number 44 - VAMP5_Rat (Q9Z2J5) MAGKELERCQRQADQVTEIMLNNFDKVLERDGKLSELQQRSDQLLDMSSAFSKTTKTLAQQKRWENIRCRVYLGLAVAGGLLLILVVLLVIFLPSGEDSSKP SEQ ID NO:45 - VAMP5_human (O95183) MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN Sequence number 46 - YKT6_Rat (Q5EGY4) MKLYSLSVFYKGEPKAVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSAKGSRASVKEQEYLCHVYVRSDSLAGVVIADSEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYTALDGHLSRYQNPREADPMSKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM SEQ ID NO:47 -YKT6_human (O15498) MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM SEQ ID NO: 48 - VAMP epitope ETSAAKLKRKYWWK SEQ ID NO: 49 - VAMP epitope FETSAAKLKRKYWWK SEQ ID NO:50 - VAMP epitope QFESSAAKLKRKYW SEQ ID NO:51 - VAMP epitope FESSAAKLKR SEQ ID NO:52 - VAMP epitope FESSAAKLKRKYWWK SEQ ID NO:53 - VAMP epitope ADALQAGASQF SEQ ID NO:54 - VAMP epitope ADALQAGASQ SEQ ID NO:55 - VAMP epitope RADALQAGASQF SEQ ID NO:56 - VAMP epitope ADALQAGASQFE SEQ ID NO:57 - VAMP epitope ADALQAGASVF SEQ ID NO:58 - VAMP epitope ADALQAGASV SEQ ID NO:59 - VAMP epitope ADALQAGASVFE SEQ ID NO:60 - VAMP epitope RADALQAGASVF SEQ ID NO:61 - VAMP epitope RADALQAGAS SEQ ID NO:62 - VAMP epitope SESLSDNATAF SEQ ID NO:63 - VAMP epitope SESLSDNATA SEQ ID NO:64 - VAMP epitope KSELSDNATAF SEQ ID NO:65 - VAMP epitope SESLSDNATAFS SEQ ID NO:66 - VAMP epitope SDQLLDMSSTF SEQ ID NO:67 - VAMP epitope SDQLLDMSST SEQ ID NO:68 - VAMP epitope RSDQLLDMSSTF SEQ ID NO:69 - VAMP epitope SDQLLDMSSTFN SEQ ID NO: 70 - VAMP epitope SDQLLDMSSAF SEQ ID NO: 71 - VAMP epitope SDQLLDMSSA SEQ ID NO:72 - VAMP epitope RSDQLLDMSSAF SEQ ID NO:73 - VAMP epitope SDQLLDMSSAFS SEQ ID NO:74 - VAMP epitope RSDQLLDMSS SEQ ID NO:75 - VAMP epitope SEVLGTQSKAF SEQ ID NO:76 - VAMP epitope SEVLGTQSKA SEQ ID NO:77 - VAMP epitope KSEVLGTQSKAF SEQ ID NO:78 - VAMP epitope SEVLGTQSKAFY [Explanation of symbols]

[0201] [Figure 1AB, Figure 2] SEQ ID NO: Sequence number motif: motif Rat: Human: [Figure 3, Figure 4] Coomassie: Coomassie C-term: C-terminal side N-term: N-terminal side Anti-Pep1: Anti-Pep1 Anti-Pep2: Anti-Pep2 Anti-Pep3: Anti-Pep3 [Figure 4] Untr: Unprocessed % VAMP2 cleavage: VAMP2 cleavage% [Figure 5] % SNARE cleavage: % SNARE cleavage Un-Tr: Unprocessed Toxin: toxin [Figure 6] UnTreated: Untreated Anti-Pep4: Anti-Pep4 Cleavage-specific: Cleavage-specific Anti-Pep1: Anti-Pep1 Anti-Pep2: Anti-Pep2 Anti-Pep3: Anti-Pep3

Claims

1. A method for determining cleavage of VAMP by a Clostridial neurotoxin containing a botulinum neurotoxin serotype X (BoNT / X) light chain in a cell, comprising: a) contacting a cell with a clostridial neurotoxin under conditions suitable for said clostridial neurotoxin activity; b) contacting the cytoplasmic contents of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody binds to: i. an antigenic polypeptide; or ii. a polypeptide comprising the antigenic polypeptide, the polypeptide not including a region of more than 17 consecutive amino acids having 100% sequence identity to a naturally occurring VAMP amino acid sequence; or iii. an antigenic protein comprising said antigenic polypeptide, or a polypeptide comprising said antigenic polypeptide, covalently linked to a carrier; wherein the antigenic polypeptide is an antigenic polypeptide comprising a VAMP epitope, the antigenic polypeptide being composed of 10 to 65 amino acid residues, the VAMP epitope comprising an amino acid sequence that is at least 90% identical to a VAMP sequence comprising at least 8 amino acid residues immediately C-terminal to a BoNT / X cleavage site in the VAMP. c) detecting by suitable means the binding of said first detection antibody to said C-terminal VAMP cleavage product; The method includes:

2. The method of claim 1, further comprising the step of d) quantifying by suitable means the amount of the C-terminal VAMP cleavage product bound to the first detection antibody.

3. A method for determining immune resistance to a Clostridial neurotoxin containing a botulinum neurotoxin serotype X (BoNT / X) light chain in a subject, comprising: a) adding to a test sample obtained from a subject a Clostridial neurotoxin comprising a botulinum neurotoxin serotype X (BoNT / X) light chain; b) contacting the cell with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; c) contacting the cytoplasmic contents of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody binds to: i. an antigenic polypeptide; or ii. a polypeptide comprising the antigenic polypeptide, the polypeptide not including a region of more than 17 consecutive amino acids having 100% sequence identity to a naturally occurring VAMP amino acid sequence; or iii. an antigenic protein comprising said antigenic polypeptide, or a polypeptide comprising said antigenic polypeptide, covalently linked to a carrier; wherein the antigenic polypeptide is an antigenic polypeptide comprising a VAMP epitope, the antigenic polypeptide being composed of 10 to 65 amino acid residues, the VAMP epitope comprising an amino acid sequence that is at least 90% identical to a VAMP sequence comprising at least 8 amino acid residues immediately C-terminal to a BoNT / X cleavage site in the VAMP. d) detecting by suitable means the binding of a first detection antibody to said C-terminal VAMP cleavage product; e) quantifying the amount of the C-terminal VAMP cleavage product bound to the first detection antibody; f) repeating steps a) through e) using a negative control sample in place of the test sample; g) comparing the amount of the C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of the C-terminal VAMP cleavage product bound to the first detection antibody in step (e) compared to the amount of the C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of a neutralizing antibody against the Clostridial neurotoxin. The method includes:

4. An antigenic polypeptide comprising a VAMP epitope, said antigenic polypeptide consisting of 10 to 17 amino acid residues, said VAMP epitope comprising a VAMP sequence including at least 8 amino acid residues immediately C-terminal to a botulinum neurotoxin serotype B (BoNT / B), botulinum neurotoxin serotype G (BoNT / G), or tetanus neurotoxin (TeNT) cleavage site in said VAMP.

5. The antigen polypeptide of claim 4, wherein the polypeptide consists of 10 to 17 amino acid residues.

6. The antigen polypeptide of claim 4, wherein the polypeptide consists of 10 to 16 amino acid residues.

7. The antigen polypeptide of claim 4, wherein the polypeptide consists of 10 to 15 amino acid residues.

8. An antigen polypeptide according to any one of claims 4 to 7, wherein the VAMP is selected from VAMP1, VAMP2, and / or VAMP3.

9. An antigen polypeptide described in any one of claims 4 to 8, wherein the VAMP epitope comprises or consists of a VAMP sequence selected from SEQ ID NO: 22 to SEQ ID NO: 30, and SEQ ID NO: 49 to SEQ ID NO:

52.

10. An antigenic polypeptide comprising a VAMP epitope, said antigenic polypeptide consisting of 10 to 65 amino acid residues, said VAMP epitope consisting of a VAMP sequence selected from SEQ ID NOs: 15-31, 33, 34 or 48-78.

11. A polypeptide comprising an antigenic polypeptide described in any one of claims 4 to 10, which does not contain a region of more than 17 consecutive amino acids having 100% sequence identity to the naturally occurring VAMP amino acid sequence.

12. An antigenic protein comprising an antigenic polypeptide described in any one of claims 4 to 10 or a polypeptide described in claim 11, covalently linked to a carrier.

13. Use of an antigenic polypeptide described in any one of claims 4 to 10, or a polypeptide described in claim 11, or an antigenic protein described in claim 12, for producing an antibody against a C-terminal VAMP cleavage product.

14. An antibody that binds to an antigen polypeptide described in any one of claims 4 to 10, or that binds to a polypeptide described in claim 11, or that binds to an antigen protein described in claim 12.

15. The antibody described in claim 14, which is a polyclonal antibody.

16. The antibody described in claim 14, which is a monoclonal antibody.

17. An antibody described in any one of claims 14 to 16, wherein the KD between the antibody and the epitope is less than 10 -7 M.

18. Use of an antibody according to any one of claims 14 to 17 in a gain of signal cellular assay for VAMP cleavage by a clostridial neurotoxin.

19. A method for determining cleavage of VAMP by a Clostridial neurotoxin in a cell, comprising: a) contacting a cell with a clostridial neurotoxin under conditions suitable for said clostridial neurotoxin activity; b) contacting the cytoplasmic contents of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to any one of claims 14 to 17; c) detecting by suitable means the binding of said first detection antibody to said C-terminal VAMP cleavage product; The method includes:

20. The method of claim 19, further comprising the step of d) quantifying by suitable means the amount of the C-terminal VAMP cleavage product bound to the first detection antibody.

21. A method for determining immune resistance to a Clostridial neurotoxin in a subject, comprising: a) adding a Clostridial neurotoxin to a test sample obtained from a subject; b) contacting the cells with the test sample of step a) under conditions suitable for Clostridial neurotoxin activity; A step of touching; c) contacting the cytoplasmic contents of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the Clostridial neurotoxin under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to any one of claims 14 to 17; d) detecting by suitable means the binding of a first detection antibody to said C-terminal VAMP cleavage product; e) quantifying the amount of the C-terminal VAMP cleavage product bound to the first detection antibody; f) repeating steps a) through e) using a negative control sample in place of the test sample; g) comparing the amount of the C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of the C-terminal VAMP cleavage product bound to the first detection antibody in step (e) compared to the amount of the C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of a neutralizing antibody against the Clostridial neurotoxin. The method includes:

22. A kit comprising a cell susceptible to intoxication by a neurotoxin and a first detection antibody against cleaved VAMP, wherein the first detection antibody is an antibody described in any one of claims 14 to 17.

Images