Cellular VAMP cleavage assay

Abstract

A cell-based assay for VAMP-cleaving clostridial neurotoxins based on a gain of signal readout is provided. [Solution] The present invention relates to VAMP epitopes suitable for generating antibodies against VAMP C-terminal neurotoxin cleavage products, their use to generate antibodies against cleaved VAMP, and the use of such antibodies in a cellular VAMP cleavage assay based on signal acquisition.

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 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 heavy chains (H chains), which have a molecular weight of approximately 100 kDa, and light chains (L chains), which have a molecular weight of approximately 50 kDa. The H chains are connected to an N-terminal cartwheel component (H N domain) and the C-terminal targeting component (H C The cleavage site is located in the L and H chains. N Located between domains. H C After binding of the domain to its target neuron and internalization of the bound toxin into the cell by endosomes, H 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 transport 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 is derived from Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-sensitive factor. The acronym SNAP-25 is derived from 25-kilodalton Synaptosome Associated Protein. The acronym VAMP is derived from Vesicle Associated Membrane Protein. SNARE proteins are essential for intracellular vesicle fusion and, therefore, for the secretion of molecules from cells via vesicle transport. 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 protease of clostridial neurotoxins is a non-cytotoxic protease that cleaves SNARE proteins.The L-chain proteases of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT cleave VAMP (also known as 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 consequent nerve paralysis (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 BoNTs enter neurons by binding to their specific receptors (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 protein receptors, all BoNTs require 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 authorities 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 toxin are not standardized. In fact, assays used by various manufacturers of commercially available toxins differ, particularly 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 assessing the degree of SNARE cleavage in target cells, for example, 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 (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, VAMP cleavage products 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 subcellular fraction assays (Hallis, Bassam, B.A. 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 cells 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. used Coomassie blue staining to show that both VAMP cleavage products were present in small synaptic vesicle fractions prepared from rat cerebral cortex treated 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 synaptosome preparations may not fully contain all the proteases present in total cell lysates, but that VAMP products derived from cellular sources may be present.Dong et al. (2004) reported that in PC12 cells expressing YFP-Syb(FL)-CFP, signals from both VAMP products were detectable after cleavage by BoNT / B, and that the YFP-N-cleaved VAMP product dispersed in the cytosol and redistributed itself to the nucleus, whereas the CFP-C-cleaved product remained 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 an unknown cellular process may interfere with antibody recognition of the N-terminal product. Therefore, measuring VAMP cleavage by loss of only the full-length band is standard practice (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 because 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 of this is that the signals from the antibodies must be balanced and on a linear scale in order to detect any differences for normalization purposes. Qualitatively, this may 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, wherein the antigenic polypeptide consists of 10 to 65 amino acid residues, and the VAMP epitope comprises an amino acid sequence that is at least 90% identical to a VAMP sequence that includes at least 8 amino acid residues immediately C-terminal to a clostridial neurotoxin cleavage site in VAMP.

[0012] In another aspect, the present invention relates to a polypeptide comprising an antigenic polypeptide of the present invention, wherein the polypeptide does not comprise 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 use of an antigenic polypeptide or protein of the present invention to generate antibodies against C-terminal VAMP cleavage products. In one embodiment, an epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products. In another embodiment, an epitope of the present invention is used to generate monoclonal antibodies against C-terminal VAMP cleavage products.

[0015] In another aspect, the present invention provides antibodies that bind to an antigenic polypeptide or protein of the present 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 the 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, wherein the first detection antibody is 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 present invention provides a method for determining immunoresistance 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 cells 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 a VAMP-cleaving 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 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) through e) using a negative control sample in place 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 cells 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 INVENTION

[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 signal gain readout.

[0021] In particular, we have demonstrated the importance of detecting epitopes located C-terminal to the BoNT cleavage site for detecting VAMP cleavage in vitro. Indeed, we have demonstrated successful detection of VAMP2 cleavage products in neurons by Western blot (WB) using antibodies that bind to epitopes located C-terminal to, and adjacent to, the BoNT / D and / or BoNT / F and / or BoNT / B cleavage sites. Notably, such antibodies can detect both full-length VAMP and cleavage products in cell lysates. This tool allows for quantitative evaluation of BoNT efficacy 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, wherein the antigenic polypeptide consists of 10 to 65 amino acid residues, and the VAMP epitope comprises an amino acid sequence that is at least 90% identical to a VAMP sequence that includes 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 involves 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" encompasses any polypeptide produced by Clostridium bacteria that enters neurons and inhibits neurotransmitter release, as well as such polypeptides produced by recombinant or chemical techniques. The active form of neurotoxin is a two-chain form. The two chains are referred to as a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. 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 number BAQ12790.1). An example of a TeNT amino acid sequence is provided as SEQ ID NO: 8 (UniProt accession number 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" and "H" each with a molecular weight of approximately 25 kDa. CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C It consists of the C-terminal part of the domain.

[0026] The term “LH N "Domain" as used herein means a C The heavy chain lacks the endopeptidase domain ("L" or "light chain") and the domain responsible for translocation of the endopeptidase to the cytoplasm (H of the heavy chain). N It refers to a neurotoxin consisting of the ATP 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 (the disclosure of which is incorporated herein by reference in its entirety) cites slightly different Clostridial sequences.

[0029] Vesicle-associated membrane proteins (VAMPs) are a family of SNARE proteins with similar structures that are involved in vesicle fusion and exocytosis, particularly neurotransmitter release. VAMPs are members of the synaptobrevin family, a family of SNARE proteins that includes members such as VAMP1, VAMP2 (both known as synaptobrevin), VAMP3 (also known as cereubrevin), VAMP4, VAMP5, VAMP7 (also known as SYBL1 or tetanus-insensitive VAMP), VAMP8 (also known as endobrevin), YKT6, and SEC22A. 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, particularly 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. 1. [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, particularly 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 NOs: 15 to 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 the 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, particularly 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 NOs: 19 to 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 particularly 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, particularly 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 NOs: 32 to 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 the at least eight amino acid residues are immediately C-terminal to the BoNT / B or TeNT cleavage site in VAMP.

[0050] Examples of BoNT / B or TeNT VAMP epitopes, more particularly 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, a BoNT / B or TeNT VAMP epitope, particularly a 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, a 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 for the detection of not only BoNT / B VAMP cleavage, but also 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 the 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 particularly 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, particularly 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 NOs: 27 to 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 particularly 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, a BoNT / X VAMP epitope, particularly a BoNT / X VAMP1, VAMP2, and / or VAMP3 epitope, comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 53 to 61. In a preferred embodiment, a BoNT / X VAMP epitope comprises or consists of an amino acid sequence 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, a 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 62 to 65. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence 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, more particularly BoNT / X VAMP5 epitopes, include the following: SDQLLDMSSTF (SEQ ID NO: 66) SDQLLDMSST (SEQ ID NO: 67) RSDQLLDMSSTF (SEQ ID NO: 68) SDQLLDMSSTFN (SEQ ID NO: 69) SDQLLDMSSAF (SEQ ID NO: 70) SDQLLDMSSA (SEQ ID NO: 71) RSDQLLDMSSAF (SEQ ID NO: 72) SDQLLDMSSAFS (SEQ ID NO: 73) RSDQLLDMSS (SEQ ID NO: 74).

[0061] In one embodiment, the BoNT / X VAMP epitope, particularly the BoNT / X VAMP5 epitope, comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 66 to 74. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence 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, 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 75 to 78. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence 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 "sequence identity percentage" 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.Therefore, identity percentage 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.The calculation of sequence identity percentage can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences.The sequence comparison and identity percentage determination between two or more sequences can be carried out using a specific mathematical algorithm well known to those skilled in the art, for example, a global alignment mathematical algorithm (for example, as described by Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972).

[0065] In another aspect, the present invention relates to polypeptides 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 that have 100% sequence identity to a naturally occurring VAMP amino acid sequence. Those 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 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 described above) and the carrier. The linker may be one or more naturally occurring or unnatural amino acids that can form covalent bonds with other amino acids (of the polypeptide and / or the carrier) due to the presence of reactive groups at their N-terminus, C-terminus, and / or side chains, as is well known in the art. In particular, amino acids having a primary amine group (-NH) at their N-terminus and / or in their side chains (e.g., lysine) can react with amino acids having a carboxyl (-COOH) group at their C-terminus and / or in their side chains (e.g., aspartic acid or glutamic acid) to form covalent bonds, and amino acids having a sulfhydryl (-SH) group in their side chains (e.g., cysteine ​​or selenocysteine) can react with amino acids having a sulfhydryl (-SH) group in their side chains (e.g., cysteine ​​or selenocysteine) to form covalent bonds. For example, a covalent linker can be a cysteine ​​added to the C- or N-terminus of a polypeptide of the present invention, which forms a disulfide bridge with another cysteine ​​added or present in the carrier. Alternatively, or in addition, the covalent linker can be in the form of several amino acids forming a spacer. For example, the linker can 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 present 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, and more preferably about 3 to about 20 amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues.

[0070] In another aspect, the present invention provides use of an antigenic polypeptide or protein of the present invention to generate antibodies against C-terminal VAMP cleavage products. In one embodiment, an epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products. In another embodiment, an 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 antigen polypeptide or protein of the present invention into animals, e.g., mammals such as rabbits, goats, mice, hamsters, or monkeys, or eggs, such as chicken eggs. Polyclonal antibodies against the VAMP epitope as disclosed herein can be isolated from animals (e.g., from blood) or eggs 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 program" with immunization on day 0, followed by three booster injections on days 7, 10, and 18. An intermediate bleed on day 21 and a final bleed on day 28. This is an example of a common technique for polyclonal antibody production 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. The antibody titer in the immunized animal can be monitored over time by standard techniques, such as 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 the antibody titer is at its 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. Isolated antibody-producing cells are fused with an immortal cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Typically, mouse myeloma cell lines are fused with splenocytes collected from appropriately immunized mice to produce hybridomas. Preferred immortal cell lines are mouse myeloma cell lines that are 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 the VAMP epitope as described herein. For example, hybridoma supernatants can be screened using cleaved VAMP-positive media in immunoprecipitation assays, in vitro binding assays (e.g., radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA)), or cell-based activity assays. The binding affinity of 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 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, the disclosures of each of which are incorporated herein by reference in their entirety.

[0074] In another aspect, the present invention provides antibodies that bind to an antigenic polypeptide or protein of the present 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 equal to the rate at which antibody-antigen complexes dissociate at equilibrium. D The equilibrium dissociation constant, denoted M, 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 the 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 present 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 It is 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 the cell with a clostridial neurotoxin under conditions suitable for 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 a VAMP-cleaving 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 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 the 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 skilled 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 the C-terminal VAMP cleavage product and to full-length VAMP.

[0087] In an alternative embodiment, the second detection antibody, unlike the first detection antibody, 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).

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

[0089] It will be apparent to one skilled 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 the cell with a clostridial neurotoxin under conditions suitable for 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 a VAMP-cleaving 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 of the invention that binds to the C-terminal VAMP cleavage product and full-length VAMP; c) by suitable 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 the 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 suitable 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 and quantifying the Includes:

[0091] It will be apparent to one skilled 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 the 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 cells 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 a VAMP-cleaving 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 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) through e) using a negative control sample in place 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 against a VAMP-cleaving clostridial neurotoxin" means any antibody that binds to a region of a VAMP-cleaving clostridial neurotoxin in such a way as to reduce or prevent 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 lymph 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 the subject who is resistant to treatment with VAMP-cleaving clostridial neurotoxin.

[0098] As used herein, the term "control sample" refers to any sample whose presence or absence of a test sample is known, and includes 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, a sample from the same individual who provides the test sample but collected before undergoing treatment with a VAMP-cleaving clostridial neurotoxin, a sample collected from a different individual who has never been exposed to a VAMP-cleaving clostridial neurotoxin, or a pooled sample collected from multiple different individuals who have never been exposed to a VAMP-cleaving clostridial neurotoxin.

[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 the 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 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).

[0104] In certain embodiments, 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 cells with the test sample of step a) under conditions suitable for clostridial neurotoxin activity; c) removing the cytoplasmic contents of said cells; after cleavage of VAMP by a VAMP-cleaving Clostridial neurotoxin, contacting the C-terminal VAMP cleavage product with a first detection antibody against the C-terminal VAMP cleavage product 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 of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; and contacting with a second detection antibody that binds to full-length VAMP but not to C-terminal VAMP cleavage products; d) by suitable means, binding of the first antibody to the 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 and quantifying the f) repeating steps a) through e) using a negative control sample in place of the test sample; g) comparing the amounts of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower detected amount of the combined 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 detected 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 a neutralizing antibody against a VAMP-cleaving Clostridial neurotoxin. Includes:

[0105] In another specific 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 cells 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 a VAMP-cleaving 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 of the invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP; d) by suitable means, binding of the first antibody to the 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 the 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 in place of the test sample; g) comparing the amounts of C-terminal VAMP cleavage products bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage products 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 neutralizing antibodies against VAMP-cleaving Clostridial neurotoxins. 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 the 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 sequence includes a sequence selected from:

[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 sequence includes a sequence selected from:

[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 a different type of clostridial neurotoxin (e.g., a different serotype of clostridial neurotoxin) that is 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 an H chain. N Domain and H of serotype C C The BoNT / FA contains the D / C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan. (2010): 140:147-154.) 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 refers to 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 is a chimeric neurotoxin derived from a first neurotoxin. N H CN H originating from domain and second neurotoxin CC 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 native (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the toxin. For example, the modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify 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] The 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 target neurons 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 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, H from BoNT / B neurotoxin CC The domain is the same as that of natural BoNT / BH CC Compared with the sequence, human Syt II BoNT / BH CC Contains at least one amino acid residue substitution, addition, or deletion that has the effect of increasing the binding affinity of the BoNT / BH 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 referred to as a "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 located on the H of the clostridial neurotoxin heavy chain. C or H CC Some or all of the domains may be replaced. Examples of retargeted neurotoxins are those 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, and WO2011 / 020114, WO2011 / 020117, WO2011 / 20119, WO2012 / 156743, WO2012 / 134900, WO2012 / 134897, WO2012 / 134904, WO2012 / 134902, WO2012 / 135343, WO2012 / 135448, WO2012 / 135304, WO2012 / 134902, WO2014 / 033441, WO2014 / 128497, WO2014 / 053651, WO2015 / 004464, all of which are incorporated herein by reference.

[0121] Examples of cells suitable for use in the methods or uses of the present 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 that is highly sensitive to BoNT is the cell that is sensitive to BoNT poisoning.In some embodiments, the cell that is highly sensitive to BoNT is the cell that is sensitive to BoNT poisoning, for example, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less, about 10 pM or less, about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM 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 VAMP-cleaving BoNTs.

[0124] In one embodiment, the cells are primary neuronal cells that are highly sensitive to BoNT, such as cortical neurons, hippocampal neurons, 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 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)-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 the 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 present invention, the conditions suitable for clostridial neurotoxin activity may comprise incubation at about 37° C. for a period of about 1 hour to about 48 hours. In one embodiment of the method of the present invention, the conditions suitable for clostridial neurotoxin activity may comprise incubation at about 37° C. for a period of about 2 hours to about 36 hours. In one embodiment of the method of the present invention, the conditions suitable for clostridial neurotoxin activity may comprise 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 the binding of a first detection antibody to cleaved VAMP" and "conditions suitable for the 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, 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, 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, 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, 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, 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, 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 flows at a specific molecular weight by SDS-PAGE, cleaved VAMP is detected at a molecular weight lower than that of full-length VAMP.By analyzing the bands by densitometry, both full-length band and cleaved band in the same lane on the gel can be used to read the cleavage percentage.Alternatively, VAMP cleavage can be detected and quantified by using enzyme-linked immunosorbent assay (ELISA), for example, sandwich ELISA.

[0137] In one embodiment of the method of the present 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 present 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 present 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 present 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 present 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 present 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 cells 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.

[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 practicing or testing embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise specified, any nucleic acid sequence is written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxy direction, respectively.

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

[0147] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a Clostridial neurotoxin" includes a plurality of such candidate agents, reference to "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 explanation 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. [Figure 2] Figure 2 shows VAMP sequences with Ab epitopes (i.e., immunogenic epitope regions) and BoNT / F, BoNT / D, and BoNT / 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. Cleavage sites are indicated by arrows. The VAMP2 cleavage points for BoNT / F and BoNT / D are located adjacent amino acids Q58-K59 and K59-L60, respectively, and the cleavage point for BoNT / B is located at amino acids Q76-F77 (based on the amino acid positions in the human VAMP2 sequence). [Figure 3] Figure 3 shows the cell-free cleavage of recombinant VAMP2-GFP using MBP-LF and LHND. Recombinant VAMP2-GFP was incubated with 0.01 μg / μL LHND or MBP-LF at 37°C for 1 hour. An equal volume of sample buffer was added, and 0.5 μg (Coomassie) and 0.3 μg (blot) of protein were run by SDS-PAGE and stained with Coomassie or blotted with various anti-VAMP2 antibodies. Schematic diagrams show the location of antibody epitopes. Recombinant protein designations and epitope line lengths are not to scale. 1 - BSA, 2 - VAMP2-GFP, 3 - cleaved VAMP2-GFP (aa59 / 60-end), 4 - cleaved 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 DIV 18 to DIV 21 were treated with either BoNT / F (A) or BoNT / D (B) for 24 h. Lysates were run on SDS-PAGE and blotted with custom-made anti-VAMP2 antibodies, i.e., anti-Pep1, anti-Pep2, or anti-Pep3, or with the commercially available antibody ab181869. 1 - full-length VAMP2; 2 - cleaved VAMP2. Anti-Pep2 data show the dose-dependent disappearance of full-length VAMP2 and the 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). [Figure 5] Figure 5. (A) Rat cortical neurons were treated with native BoNT / F1 (□), native BoNT / A1 (●), or recombinant BoNT / FA (△) for 24 hours. 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 from DIV 18 to 21 were treated with either BoNT / F or BoNT / B for 24 hours. 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. [Example]

[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-immune, mid-bleed, and final bleeds 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-D (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. MBP 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 ZnCl, 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 ZnCl, 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 tissue was collected in ice-cold Hank's Balanced Salt Solution (HBSS) (without Ca2+ or Mg2+) according to the manufacturer's instructions (Worthington Biochemical, NJ, USA) and then dissociated in papain solution at 37°C for 40 minutes. 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. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (day in vitro) 4, an additional 125 μL of Neurobasal medium containing 2% B27 and 0.5 mM GlutaMAX was added. Cells were maintained by replacing half of 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 18–21 days postincubation (DIV) were treated in triplicate wells with a range of concentrations of native BoNT / F1 (Metabiologics, USA) (1 nM–0.1 pM) or BoNT / D (Metabiologics, USA) (10 nM–1 pM) 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 minutes. 15 μL of lysate per lane was loaded onto a 12% Bis-Tris gel and run at 200V for 50 minutes in MES buffer. Proteins were transferred to nitrocellulose membranes using a Transblot Turbo (Biorad) with a low MW program. The membranes were blocked with 5% low-fat milk / PBS-Tween for 1 hour 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 commercially available anti-VAMP2 antibodies (Abcam ab3347 and ab181869). The membranes were washed three times with PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. The membrane was washed 3 x 5 min with PBS-Tween, 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 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) were also tested. NThe VAMP protein was cleaved using ab3347 (epitope aa 1-18) and ab181869 (epitope within aa 1-100) (Figure 3). Additionally, two other commercially available VAMP2 antibodies were used for comparison: ab3347 (epitope aa 1-18) and ab181869 (epitope within aa 1-100). Figure 3 shows that the anti-Pep1 antibody detected both the full-length VAMP2 and the N-terminal cleaved portion (aa 1-58 / 59) with significantly reduced signal. As expected, this antibody did not detect the C-terminal cleavage product because its epitope is not located in this region. The anti-Pep2 and anti-Pep3 antibodies detected both the full-length VAMP2 protein and the C-terminal cleavage product. Ab3347 only detected the full-length VAMP2 and not the N-terminal cleavage fragment, whereas ab181869 detected both.

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

[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 are likely present. Rat primary cortical neurons were treated with either BoNT / F or BoNT / D and lysed for WB analysis (Fig. 4). The anti-Pep1 antibody recognized only the full-length protein, with no detectable cleavage products. The anti-Pep2 antibody detected both the full-length and C-terminal cleavage products. The anti-Pep3 antibody showed very poor affinity for monomeric VAMP in cell lysates, with a weak signal, and detected high-molecular-weight species, likely dimers, and other proteins (data not shown). Although the full-length monomer signal was very small, there were bands representing the C-terminal products cleaved by BoNT / F and BoNT / D. In other words, anti-Pep3 did not detect full-length VAMP, but it weakly detected the C-terminal fragments cleaved by BoNT / F and BoNT / D. This contrasts with previous results in a cell-free system, which showed strong signals from both full-length and cleaved recombinant VAMP. The commercially available antibody Ab3347 was not tested in vitro due to the lack of detection of the cleaved protein in cell-free assays. The commercially available antibody ab181869 detected full-length VAMP2 in cortical lysates but not the cleaved fragment, despite positive binding to the N-terminal truncated recombinant fragment in cell-free assays. The Pep2 data were used to quantify the dose-dependent cleavage of VAMP2 by BoNT / F (Figure 4C) and BoNT / D (Figure 4D).

[0170] We first demonstrated that both recombinant VAMP cleavage products could be detected in a cell-free system. However, we also showed that when transferred to cell lysates, the N-terminal product was not detectable, although an as yet unknown mechanism other than degradation may be involved. In contrast, we showed that the C-terminal VAMP fragment, which remains associated with the vesicle membrane, is not degraded or altered in a manner that precludes 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, albeit 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 18-21 days of incubation were treated in triplicate wells with a range of concentrations (1 pM-1 fM) of recombinant BoNT / FA (SEQ ID NO: 38), 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) 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) at room temperature for 10 min. Samples were boiled at 90°C for 5 min, and 15 μL of lysate was loaded per lane onto a 12% Bis-Tris gel and run at 200 V for 80 min in MOPS buffer (SNAP-25) or 200 V for 50 min in MES buffer (VAMP2). Proteins were transferred to nitrocellulose membranes using a Transblot Turbo (Biorad) gel with a 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 and then incubated with either anti-SNAP25 antibody (Sigma S9684 1:4000), anti-Pep2 (1:500), or a custom-made anti-VAMP2 (Eurogentec) antibody, as described in Example 1. Each primary antibody was incubated overnight at 4°C. The membranes were washed three times with PBS-Tween and incubated with anti-rabbit HRP secondary antibody at room temperature for 1 hour. The membranes were washed three times with PBS-Tween for 5 minutes and then developed with SuperSignal West Dura or West Femto chemiluminescent substrate 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 hours of 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 tissue was collected in ice-cold Hank's balanced salt solution (HBSS) (without Ca2+ or Mg2+) according to the manufacturer's instructions (Worthington Biochemical, NJ, USA) and then dissociated in papain solution at 37°C for 40 minutes. 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. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (day in vitro) 4, an additional 125 μL of Neurobasal medium containing 2% B27 and 0.5 mM GlutaMAX was added. Cells were maintained by replacing half of 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 of NuPage sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 benzonase) at room temperature for 10 minutes.

[0191] 4. SDS-PAGE and Western Blot

[0192] Neuronal lysates were boiled at 90°C for 5 minutes. 15 μL of lysate per lane was loaded onto a 12% Bis-Tris gel and run at 200V for 50 minutes in MES buffer. Proteins were transferred to nitrocellulose membranes using a Transblot Turbo (Biorad) with a low MW program. The membranes were blocked with 5% low-fat milk / PBS-Tween for 1 hour 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. The membranes were washed three times with PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. The membranes were washed 3 times with PBS-Tween for 5 minutes, then developed with SuperSignal West Femto chemiluminescent substrate and visualized using a 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 C-terminal BoNT / B cleavage site.

[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 (Fig. 6). The epitope regions of all comparative antibodies were located N-terminal to the BoNT / B cleavage site. Fig. 6 shows that the location of the epitope of the novel antibody against Pep4 enabled detection of both 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. As expected, the anti-Pep1 antibody did not detect any cleavage products. The BoNT / B cleavage-specific antibody also did not detect any BoNT / B cleavage products in these cell lysates.

[0196] Overall, the present data indicate 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 proved to be correct, allowing us to generate a single antibody (anti-Pep4 Mab) that provided positive results for BoNT / B- and BoNT / F-treated neurons. Furthermore, because TeNT shares the same cleavage site as BoNT / B and BoNT / D is close to the BoNT / F cleavage site, we predict 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 demonstrate the difference in VAMP detection between cell-free recombinant protein assays and whole-cell models. It was this inability to detect cellular cleaved VAMP that formed the basis for the hypothesis that VAMP degradation occurred extremely rapidly in cells (Foran et al., "Evaluation of the therapeutic usefulness of botulinum neurotoxins B, C1, E, and F compared with the long-lasting type A", J. Biol Chem 278 (2) pp. 1363-1371, 2003). In contrast to the antibodies of the present invention, the majority of commercially available VAMP antibodies were directed against epitopes within the N-terminal region of the protein, and thus, N-terminal VAMP fragments were the focus of their early studies. Herein, we show that the smaller C-terminal VAMP fragments are not degraded in cells, but the larger N-terminal fragments are also not detected. Interestingly, however, our results in a cell-free system indicate that not all commercially available antibodies can 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 most likely relates 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 MGSMTWPVKDFNYSDPVNNDDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNSPEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIF CMDPVIALMHELTHSLHQLYGINIPSDKRIRRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKAlgHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKS EEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDDFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNNFNQAFATAGVA FLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEAHHHHHHHHHHH · sequence number 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) SEQ ID NO: 42 - VAMP4_Rat (D4A560) MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALAAAILLLMIITQIILHLKK Sequence number 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 KSESLSDNATAF 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: Rat Human: 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. An antigenic polypeptide comprising a VAMP epitope at its N-terminus, the antigenic polypeptide consisting of 10 to 15 amino acid residues, the VAMP epitope being at least 8 amino acid residues of the VAMP sequence immediately C-terminal to a botulinum neurotoxin serotype X (BoNT / X) cleavage site in the VAMP. An antigenic polypeptide (excluding those containing the amino acid sequence SESLSDNATAFSNR).

2. 2. Use of the antigen polypeptide according to claim 1 for producing an antibody against a C-terminal VAMP cleavage product produced by cleavage of VAMP by the BoNT / X light chain.

3. An antibody against a C-terminal VAMP cleavage product produced by cleavage of VAMP by the BoNT / X light chain, which binds to the antigen polypeptide described in claim 1, and which is capable of binding to full-length VAMP and the C-terminal VAMP cleavage product in a cell lysate.

4. The antibody of claim 3, which is a polyclonal antibody.

5. The antibody of claim 3, which is a monoclonal antibody.

6. K between the antibody and the epitope D But, 10 -7 The antibody of any one of claims 3 to 5, having a molecular weight of less than M.

7. 7. Use of an antibody according to any one of claims 3 to 6 in a gain of signal cellular assay for VAMP cleavage by the BoNT / X light chain.

8. 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 Clostridial neurotoxin activity; (b) contacting the cytoplasmic content of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the BoNT / X light chain 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 3 to 6; (c) detecting by suitable means the binding of said first detection antibody to the C-terminal VAMP cleavage product; The method includes:

9. The method of claim 8, further comprising the step of: (d) quantifying by suitable means the amount of C-terminal VAMP cleavage product bound to the first detection antibody.

10. 1. 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 that contains a 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 content of the cells with a first detection antibody against a C-terminal VAMP cleavage product following cleavage of VAMP by the BoNT / X light chain 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 3 to 6; (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)-(e) using a negative control sample in place 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 Clostridial neurotoxin containing a BoNT / X light chain. The method includes:

11. A kit comprising a cell susceptible to intoxication by a Clostridial neurotoxin containing a botulinum neurotoxin serotype X (BoNT / X) light chain and a first detection antibody against cleaved VAMP, wherein the first detection antibody is an antibody described in any one of claims 3 to 6.

Images