Cell VAMP cleavage assay

Abstract

The present invention relates to VAMP epitopes suitable for producing antibodies against VAMP C-terminal neurotoxin cleavage products, their use for producing antibodies against cleaved VAMP and the use of these antibodies in cellular VAMP cleavage assays based on signal gain.

Description

[0001] This application is a divisional application of Chinese patent application 201780063220.0 (International Application No. PCT / EP2017 / 076569), whose application date is October 18, 2017 and whose invention name is “Cellular VAMP Cleavage Assay”. Field of the Invention

[0002] The present invention relates to a cell-based assay for VAMP cleavage of Clostridial neurotoxins. background

[0004] Bacteria in the genus Clostridium produce highly potent and specific protein toxins that can toxicize 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 to G), as well as the neurotoxins produced by C. baratii and C. butyricum.

[0005] 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 that are post-translationally modified by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, commonly referred to as the activation site, which is located between the cysteine ​​residues that provide the interchain disulfide bonds. It is this two-chain form that is the active form of the toxin. These two chains are called heavy chains (H chains) (with a molecular weight of approximately 100 kDa) and light chains (L chains) (with a molecular weight of approximately 50 kDa). The H chain contains an N-terminal translocation component (H N domain) and C-terminal targeting component (H C domain). The cleavage site is located between the L chain and the H N Between domains. C After the domain binds to its target neuron and internalizes the bound toxin into the cell via endosomes, H N The domain translocates the L chain across the endosomal membrane and into the cytosol, and the L chain provides the protease function (also known as a non-cytotoxic protease).

[0006] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP25, VAMP, or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The abbreviation SNARE comes from the term soluble NSF attachment receptor, where NSF stands for N-ethylmaleimide-sensitive factor. The acronym SNAP25 comes from the term synaptosome-associated protein of 25 kilodaltons. The acronym VAMP comes from the term vesicle-associated membrane protein. SNARE proteins are components of intracellular vesicle fusion and, therefore, of molecules secreted from cells via vesicle trafficking. The protease function is a zinc-dependent endopeptidase activity that displays high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cells, non-cytotoxic proteases are able to inhibit cellular secretion from target cells. The L chain protease of Clostridial neurotoxin 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 VAMPs (also known as synaptobrevins), the L-chain proteases of BoNT / A and BoNT / E cleave SNAP25, and the L-chain proteases of BoNT / C cleave SNAP25 and syntaxin, leading to inhibition of neurotransmitter release and subsequent nerve paralysis (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural, and mechanistic insight." Nature Reviews Microbiology 12.8 (2014): 535-549) (Zhang et al., "Identification and characterization of a novel botulinum neurotoxin"; Nature Communications, 2017, 8: 14130).

[0007] Clostridial neurotoxins bind to the receptor C) to target and enter neurons by binding to their specific receptors. These domains are well-defined in the literature (Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affected neuroxocytosis, Physiol Rev, 2000, 80, 717-766). Receptor binding determines the efficacy and specificity of BoNT neuronal recognition. BoNT / B, DC, and G share two homologous synaptic vesicle proteins, synaptotagmin I and II (Syt I / II), as their receptors, while BoNT / A, E, D, F, and TeNT use another synaptic vesicle protein, SV2. In addition to protein receptors, all BoNTs require lipid co-receptors, gangliosides, which are abundant on the surface of neurons.

[0008] Clostridial neurotoxins are used in therapies to treat movement and autonomic disorders. Some BoNT / A products (including and ) and a BoNT / B product It has been approved by regulatory agencies for use in humans.

[0009] Traditionally, the potency of BoNT drug products 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 unit for botulinum toxin is not a standardized unit. In fact, the assays used by different manufacturers of commercially available toxins differ significantly in the choice of dilution buffer (Straughan, DW, 2006, ATLA 34(3), 305-313; Hambleton and Pickett, Hambleton, P. and A. Pickett., 1994, Journal of the Royal Society of Medicine 87.11:719). In addition, due to ethical issues and recent regulations, it is now preferred to avoid the use of animal-based potency assays. Cell-based potency assays avoid the requirement for animal testing and the associated ethical issues. Following cell intoxication, the potency of clostridial neurotoxins can be measured by assessing the extent of SNARE cleavage in target cells, for example by Western blotting. Alternatively, a sandwich ELISA method can be used to detect and quantify SNARE cleavage.These methods are applicable to SNAP25 and syntaxin cleavage (see, e.g., Pellett, Sabine et al., "Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type A potency determination." Journal of pharmacological and toxicological methods 61.3 (2010): 304-310; Fernández-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 Dinmammalian 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, VAMP cleavage products from cell lysates have proven extremely difficult to detect to date.In fact, although VAMP cleavage-specific antibodies that recognize cleaved VAMP are available and are suitable for detecting VAMP cleavage in extracellular or cell fraction assays (Hallis, Bassam, 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 did not detect cleaved VAMP in cell studies.

[0010] The general consensus in the field to date is that the cleaved VAMP products must be degraded very quickly in the cell and therefore do not contribute to the duration 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. have shown the presence of two VAMP cleavage products in small synaptic vesicle fractions prepared from rat cerebral cortex when treated with BoNT / B and TeNT using Coomassie blue staining (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 there may be VAMP products from cellular sources, although synaptosomal preparations may not contain all the proteases present in total cell lysates. Dong et al. (2004) described that in PC12 cells expressing YFP-Syb(FL)-CFP, signals from two VAMP products were detectable after cleavage by BoNT / B, and that the YFP-N-terminally cleaved VAMP product dispersed into the cytosol and redistributed itself to the nucleus, while the CFP-C-terminal 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 there may be two VAMP products, but unknown cellular processes prevent the recognition of antibodies to the N-terminal product.Therefore, standard practice is to measure VAMP cleavage solely by the disappearance of the full-length band (see, e.g., Pellett, Sabine et al., "A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies." FEBS letters 581.25(2007):4803-4808.; Whitemarsh, Regina CM, et al., "Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection Biological Sciences: Applied Biological Sciences." Toxicological Sciences, 2012, 126(2):426–435). However, assays based on signal loss carry the risk of error because there may be differences in total protein loading, which will lead to overestimation or underestimation of VAMP disappearance. Housekeeping proteins that are unchanged during BoNT treatment can be used to normalize the density of VAMP disappearance to a control protein. The disadvantage here is that the signals between antibodies need to match and be in a linear scale in order to detect any differences for normalization purposes. While qualitatively this may be a reasonable indication of BoNT activity, it is not suitable for more detailed quantification, particularly for determining the potency of pharmaceutical BoNT formulations.

[0011] There is therefore a need for cellular VAMP cleavage assays based on gain of signal readout. SUMMARY OF THE INVENTION

[0013] 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, wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to a VAMP sequence comprising at least 8 amino acid residues directly C-terminal to the Clostridial neurotoxin cleavage site in the VAMP.

[0014] 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 having 100% sequence identity to the naturally occurring VAMP amino acid sequence of greater than 17, preferably 16, more preferably 15 consecutive amino acids.

[0015] In another aspect, the present invention provides an antigenic protein comprising the polypeptide according to the present invention covalently linked to a carrier.

[0016] In another aspect, the present invention provides the use of an antigenic polypeptide or protein according to the present invention for generating antibodies against a C-terminal VAMP cleavage product. In one embodiment, the epitope of the present invention is used to generate a polyclonal antibody against a C-terminal VAMP cleavage product. In another embodiment, the epitope of the present invention is used to generate a monoclonal antibody against a C-terminal VAMP cleavage product.

[0017] In another aspect, the present invention provides antibodies that bind to the antigenic polypeptide or protein of the present invention.

[0018] In another aspect, the present invention provides the use of an antibody according to the invention in a signalling cell assay for enhancing VAMP cleavage by a VAMP-cleaving Clostridial neurotoxin.

[0019] In another aspect, the present invention provides a method for determining VAMP cleavage in a cell by a clostridial neurotoxin that cleaves VAMP, comprising:

[0020] a) contacting the cells with a clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

[0021] b) contacting the cytoplasmic contents of the cells after cleavage of VAMP by the VAMP-cleaving clostridial neurotoxin with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention; and

[0022] c) detecting the binding of the first detection antibody to the C-terminal VAMP cleavage product by a suitable method.

[0023] In another aspect, the present invention provides a method for determining immune tolerance to a clostridial neurotoxin that cleaves VAMP in a subject, comprising:

[0024] a) adding a clostridial neurotoxin that cleaves VAMP to a test sample obtained from a subject;

[0025] b) contacting the cells with the test sample of step a) under conditions suitable for clostridial neurotoxin activity;

[0026] c) contacting the cytoplasmic contents of the cells after cleavage of VAMP by the VAMP-cleaving clostridial neurotoxin with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention;

[0027] d) detecting binding of the first detection antibody to the C-terminal VAMP cleavage product by a suitable method;

[0028] e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

[0029] f) repeating steps a) to e) using the negative control sample instead of the test sample; and

[0030] g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein detecting a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (e) relative to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of neutralizing antibodies to the clostridial neurotoxin that cleaves VAMP.

[0031] In another aspect, the present invention provides a kit comprising cells susceptible to neurotoxin intoxication by cleaved VAMP; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is an antibody according to the present invention. Detailed Description of the Invention

[0033] The present invention is based on the inventors' discovery that antibodies suitable for use in cellular VAMP cleavage assays can be generated based on gain of signal readout.

[0034] In particular, the inventors have shown that detecting an epitope located C-terminally to the BoNT cleavage site is crucial for detecting VAMP cleavage in vitro. Indeed, the inventors have demonstrated successful detection of neuronal VAMP2 cleavage products by Western blotting (WB) using an antibody that binds to an epitope located C-terminally to the BoNT / F and / or BoNT / D and / or BoNT / B cleavage sites adjacent to the BoNT / D and / or BoNT / F and / or BoNT / B cleavage sites. In particular, this antibody is able to detect both full-length VAMP and cleavage products in cell lysates. This tool enables quantitative assessment of BoNT potency in cellular assays for signal gain by monitoring the appearance of cleaved VAMP products.

[0035] 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, wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to a VAMP sequence comprising at least 8 amino acid residues directly C-terminal to the clostridial neurotoxin cleavage site in the VAMP.

[0036] As used herein, the term "clostridial neurotoxin" refers to any polypeptide that enters neurons and inhibits neurotransmitter release. This process includes binding of the neurotoxin to low- or high-affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and enzymatic modification of neurotoxin substrates. More specifically, the term "clostridial neurotoxin" includes any polypeptide produced by Clostridium bacteria that enters neurons and inhibits neurotransmitter release, as well as such polypeptides produced by recombinant or chemical techniques. It is a two-chain form and is the active form of the neurotoxin. These two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. Clostridial neurotoxins include botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). BoNT serotypes A to G can be distinguished based on inactivation by specific neutralizing antisera, and this classification of serotypes is correlated with the percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage amino acid sequence identity.

[0037] An example of a BoNT / A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt Accession No. A5HZZ9). An example of a BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt Accession No. B1INP5). An example of a BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt Accession No. P18640). An example of a BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt Accession No. P19321). An example of a BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (NCBI RefSeq Accession No. WP_003372387). An example of a BoNT / F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt Accession No. Q57236). An example of a BoNT / G neurotoxin amino acid sequence is provided as SEQ ID NO: 7 (NCBI RefSeq Accession No. WP_039635782). An example of a BoNT / X neurotoxin amino acid sequence is provided as SEQ ID NO: 41 (Genbank Accession No. BAQ12790.1). An example of a TeNT amino acid sequence is provided as SEQ ID NO: 8 (UniProt Accession No. P04958).

[0038] The term "H" used herein C The "domain" refers to the functionally distinct region of the neurotoxin heavy chain, which has a molecular weight of approximately 50 kDa and enables the neurotoxin to bind to receptors on the surface of target cells. CThe domain consists of two structurally different subdomains, the "H CN Subdomain (H C N-terminal portion of the domain) and "H CC Subdomain (H C The C-terminal part of the subdomain), each subdomain has a molecular weight of approximately 25 kDa.

[0039] The term "LH" as used herein N domain" refers to the lack of H C The neurotoxin is composed of an endopeptidase domain ("L" or "light chain") and a domain responsible for the translocation of the endopeptidase into the cytoplasm (H of the heavy chain). N domain).

[0040] Exemplary L, H N , H CN and H CC The domain structures are shown in Table 1.

[0041] Table 1 - Exemplary L, H N , H CN and H CC domain

[0042]

[0043] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated herein by reference in its entirety) cites slightly different Clostridium sequences.

[0044] Vesicle-associated membrane proteins (VAMPs) are a family of SNARE proteins that share a similar structure and are involved in vesicle fusion and exocytosis, specifically neurotransmitter release. VAMPs are members of the SNARE protein family, also known as the synaptobrevin family, which includes members such as VAMP1, VAMP2 (also known as synaptobrevin), VAMP3 (also known as cellubrevin), VAMP4, VAMP5, VAMP7 (also known as SYBL1, or tetanus-insensitive VAMP), VAMP8 (also known as endobrevin), YKT6, and SEC22A. VAMP1, VAMP2, and VAMP3 are cleaved by the light chain of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT. BoNT / X can also cleave VAMP4, VAMP5, and YKT6.

[0045] As used herein, the term "VAMP epitope" refers to a portion of the VAMP protein to which an antibody binds.

[0046] 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. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acid residues.

[0047] 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 directly C-terminal to the Clostridial neurotoxin cleavage site in said VAMP.

[0048] 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 Figure 1.

[0049] Table 2 - Clostridial neurotoxin VAMP cleavage sites

[0050]

[0051]

[0052]

[0053] In one embodiment, the VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6.

[0054] In one embodiment, the VAMP is selected from VAMP1, VAMP2 and / or VAMP3.

[0055] In one embodiment, the VAMP is selected from VAMP4, VAMP5 and / or YKT6.

[0056] In a preferred embodiment, the VAMP is human VAMP, more preferably human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6.

[0057] In one embodiment, the VAMP is selected from human VAMP1, VAMP2 and / or VAMP3.

[0058] In one embodiment, the VAMP is selected from human VAMP4, VAMP5 and / or YKT6.

[0059] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT / F-cleaved VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / F cleavage site in VAMP.

[0060] Examples of BoNT / F VAMP epitopes, more particularly BoNT / F VAMP1, VAMP2, and / or VAMP3 epitopes, include:

[0061] ·KLSELDDRADALQ (SEQ ID NO: 15)

[0062] ·QKLSELDDRADALQ(SEQ ID NO: 16)

[0063] KLSELDDRAD (SEQ ID NO: 17)

[0064] ·KLSELDDRADALQAGAS (SEQ ID NO: 18)

[0065] • DQKLSELDDRADALQ (SEQ ID NO: 31).

[0066] In one embodiment, a BoNT / F VAMP epitope, particularly a 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 the group consisting of SEQ ID NO: 15 to SEQ ID NO: 18 and SEQ ID NO: 31. In a preferred embodiment, the BoNT / F VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT / F VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

[0067] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / D VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / D cleavage site in VAMP.

[0068] Examples of BoNT / D VAMP epitopes, more particularly BoNT / D VAMP1, VAMP2, and / or VAMP3 epitopes, include:

[0069] ·KLSELDDRADALQ (SEQ ID NO: 15)

[0070] ·LSELDDRADALQ (SEQ ID NO: 19)

[0071] LSELDDRADA (SEQ ID NO: 20)

[0072] ·LSELDDRADALQAGAS (SEQ ID NO: 21).

[0073] In one embodiment, a BoNT / D VAMP epitope, particularly a 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 the group consisting of SEQ ID NO: 15 and SEQ ID NO: 19 to SEQ ID NO: 21. In a preferred embodiment, the BoNT / D VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT / D VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

[0074] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT / F5 or BoNT / FA cleaved VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / F5 or BoNT / FA cleavage site in VAMP.

[0075] Examples of BoNT / F5 or BoNT / FA VAMP epitopes, more particularly BoNT / F5 or BoNT / FA VAMP1, VAMP2 and / or VAMP3 epitopes, include:

[0076] ·ERDQKLSELDDRA (SEQ ID NO: 32)

[0077] ·LERDQKLSELDDRA (SEQ ID NO: 33)

[0078] · VLERDQKLSELDDRA (SEQ ID NO: 34).

[0079] In one embodiment, a BoNT / F5 or BoNT / FA VAMP epitope, particularly a 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 the group consisting of SEQ ID NO: 32 to SEQ ID NO: 34. In a preferred embodiment, a 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 an amino acid sequence of ERDQKLSELDDRA (SEQ ID NO: 32). In a more preferred embodiment, a BoNT / F5 or BoNT / FA VAMP epitope comprises or consists of ERDQKLSELDDRA (SEQ ID NO: 32).

[0080] In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a BoNT / B or TeNT VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / B or TeNT cleavage site in VAMP.

[0081] Examples of BoNT / B or TeNT VAMP epitopes, more particularly BoNT / B or TeNT VAMP1, VAMP2 and / or VAMP3 epitopes, include:

[0082] ·FETSAAKLKRKYW(SEQ ID NO:22)

[0083] ·FESSAAKLKRKYW(SEQ ID NO: 23)

[0084] ·QFETSAAKLKRKYW(SEQ ID NO: 24)

[0085] FETSAAKLKR (SEQ ID NO: 25)

[0086] ·FETSAAKLKRKYWWKN(SEQ ID NO: 26)

[0087] ·ETSAAKLKRKYWWK(SEQ ID NO: 48)

[0088] ·FETSAAKLKRKYWWK(SEQ ID NO: 49)

[0089] ·QFESSAAKLKRKYW(SEQ ID NO: 50)

[0090] FESSAAKLKR (SEQ ID NO: 51)

[0091] · FESSAAKLKRKYWWK (SEQ ID NO: 52).

[0092] 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 the group consisting of 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 binding to the latter epitope allow detection of not only BoNT / B VAMP cleavage, but also BoNT / F VAMP cleavage.

[0093] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT / G VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / G cleavage site in VAMP.

[0094] Examples of BoNT / G VAMP epitopes, more particularly BoNT / G, VAMP1, VAMP2, and / or VAMP3 epitopes, include:

[0095] AKLKRKYWWKN (SEQ ID NO: 27)

[0096] ·AAKLKRKYWWKN(SEQ ID NO: 28)

[0097] ·AKLKRKYWWKNCKM (SEQ ID NO: 29)

[0098] ·AKLKRKYWWKNLKM (SEQ ID NO: 30).

[0099] In one embodiment, a BoNT / G VAMP epitope, particularly a BoNT / G VAMP1, VAMP2, and / or VAMP3 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NO: 27 to SEQ ID NO: 30. In a preferred embodiment, the BoNT / G VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AKLKRKYWWKN (SEQ ID NO: 27). In a more preferred embodiment, the BoNT / G VAMP epitope comprises or consists of AKLKRKYWWKN (SEQ ID NO: 27).

[0100] In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT / X VAMP epitope, wherein at least 8 amino acid residues are located directly C-terminal to the BoNT / X cleavage site in VAMP.

[0101] Examples of BoNT / X VAMP epitopes, more particularly BoNT / X VAMP1, VAMP2 and / or VAMP3 epitopes, include:

[0102] ADALQAGASQF (SEQ ID NO: 53)

[0103] ADALQAGASQ (SEQ ID NO: 54)

[0104] ·RADALQAGASQF (SEQ ID NO: 55)

[0105] ·ADALQAGASQFE(SEQ ID NO:56)

[0106] ADALQAGASVF (SEQ ID NO: 57)

[0107] ADALQAGASV (SEQ ID NO: 58)

[0108] ·ADALQAGASVFE(SEQ ID NO:59)

[0109] ·RADALQAGASVF (SEQ ID NO: 60)

[0110] · RADALQAGAS (SEQ ID NO: 61).

[0111] 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 53 to SEQ ID NO: 61. In a preferred embodiment, a BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ADALQAGASQF (SEQ ID NO: 53). In a more preferred embodiment, a BoNT / X VAMP epitope comprises or consists of ADALQAGASQF (SEQ ID NO: 53).

[0112] Other examples of BoNT / X VAMP epitopes, more particularly BoNT / X VAMP4 epitopes, include:

[0113] SESLSDNATAF (SEQ ID NO: 62)

[0114] SESLSDNATA (SEQ ID NO: 63)

[0115] ·KSESLSDNATAF (SEQ ID NO: 64)

[0116] · SESLSDNATAFS (SEQ ID NO: 65).

[0117] In one embodiment, a BoNT / X VAMP epitope, particularly a BoNT / X VAMP4 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NO: 62 to SEQ ID NO: 65. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SESLSDNATAF (SEQ ID NO: 62). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SESLSDNATAF (SEQ ID NO: 62).

[0118] Other examples of BoNT / X VAMP epitopes, more particularly BoNT / X VAMP5 epitopes, include:

[0119] SDQLLDMSSTF (SEQ ID NO: 66)

[0120] SDQLLDMSST (SEQ ID NO: 67)

[0121] ·RSDQLLDMSSTF (SEQ ID NO: 68)

[0122] ·SDQLLDMSSTFN(SEQ ID NO: 69)

[0123] SDQLLDMSSAF (SEQ ID NO: 70)

[0124] SDQLLDMSSA (SEQ ID NO: 71)

[0125] ·RSDQLLDMSSAF(SEQ ID NO:72)

[0126] ·SDQLLDMSSAFS(SEQ ID NO:73)

[0127] · RSDQLLDMSS (SEQ ID NO: 74).

[0128] In one embodiment, a BoNT / X VAMP epitope, particularly a BoNT / X VAMP5 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 66 to SEQ ID NO: 74. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SDQLLDMSSTF (SEQ ID NO: 66). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SDQLLDMSSTF (SEQ ID NO: 66).

[0129] Other examples of BoNT / X VAMP epitopes, more particularly BoNT / X YKT6 epitopes, include:

[0130] SEVLGTQSKAF (SEQ ID NO: 75)

[0131] SEVLGTQSKA (SEQ ID NO: 76)

[0132] ·KSEVLGTQSKAF(SEQ ID NO:77)

[0133] · SEVLGTQSKAFY (SEQ ID NO: 78).

[0134] In one embodiment, a BoNT / X VAMP epitope, particularly a BoNT / X YKT6 epitope, comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 75 to SEQ ID NO: 78. In a preferred embodiment, the BoNT / X VAMP epitope comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEVLGTQSKAF (SEQ ID NO: 75). In a more preferred embodiment, the BoNT / X VAMP epitope comprises or consists of SEVLGTQSKAF (SEQ ID NO: 75).

[0135] As used herein, "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical nucleotides or amino acids at the same position shared by the aligned sequences. Thus, % identity can be calculated as the number of identical nucleotides or amino acids at each position in the alignment divided by the total number of nucleotides or amino acids in the aligned sequences, multiplied by 100. The calculation of % sequence identity can also take into account the number of rooms, and the length of each room that needs to be introduced for optimizing the alignment of two or more sequences. The comparison of sequences between two or more sequences and the determination of percent identity can be performed using a specific mathematical algorithm familiar to those skilled in the art, such as a global alignment mathematical algorithm (e.g., described by Needleman and Wunsch, J. Mol. Biol. 48 (3), 443-453, 1972).

[0136] In another aspect, the present invention relates to a polypeptide comprising an antigenic polypeptide according to the present invention, wherein the polypeptide does not comprise more than 17, 16, 15, 14, 13, 12, 11, 10, preferably 16, more preferably 15 consecutive amino acids having 100% sequence identity with the naturally occurring VAMP amino acid sequence. As will be readily understood by those skilled in the art, such polypeptides are also antigenic.

[0137] In a preferred embodiment, the polypeptide comprises a covalent linker, preferably at its N-terminus and / or at the C-terminus.Examples of covalent linkers suitable according to the invention are provided below.

[0138] In another aspect, the present invention provides an antigenic protein comprising the polypeptide of the present invention covalently linked to a carrier.

[0139] 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 multi-attachment peptide (MAP).

[0140] In one embodiment, the antigenic protein comprises a covalent linker between a polypeptide of the present invention (which may already contain a linker as described above) and a carrier. The linker can be one or more natural or non-natural amino acids, as is well known in the art, and due to the presence of active groups at their N-terminus, C-terminus and / or side chains, the amino acids can form covalent bonds with other amino acids (of the polypeptide and / or carrier). It is worth noting that amino acids having a primary amine group (-NH2) at the N-terminus and / or side chain (e.g., lysine) can react with amino acids having a carboxyl group (-COOH) at the C-terminus and / or side chain (e.g., aspartic acid or glutamic acid) to form a covalent bond; amino acids having a sulfhydryl group (-SH) on the side chain (e.g., cysteine ​​or selenocysteine) can react with amino acids having a sulfhydryl group (-SH) on the side chain (e.g., cysteine ​​or selenocysteine) to form a covalent bond. For example, the covalent linker can be a cysteine ​​added to the C-terminus or N-terminus of the polypeptide of the present invention, which forms a disulfide bridge with another cysteine ​​added or present in the carrier. Alternatively or additionally, the covalent linker can be in the form of several amino acids forming a spacer, for example, a linker can be a peptide comprising a non-charged amino acid with a small side chain R group (e.g., 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, more preferably about 3 to about 20 amino acid residues, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

[0141] In another aspect, the present invention provides the use of an antigenic polypeptide or protein according to the present invention for generating antibodies against a C-terminal VAMP cleavage product. In one embodiment, the epitope of the present invention is used to generate a polyclonal antibody against a C-terminal VAMP cleavage product. In another embodiment, the epitope of the present invention is used to generate a monoclonal antibody against a C-terminal VAMP cleavage product.

[0142] Methods for producing antibodies are well known in the art, see, for example, 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.

[0143] Polyclonal antibodies in conjunction with VAMP epitopes as described herein can be produced by injecting animals, for example, mammals, such as rabbits, goats, mice, hamsters or monkeys, or eggs, such as eggs, with antigenic polypeptides or proteins of the present invention. Polyclonal antibodies to VAMP epitopes disclosed herein can be separated from animals (e.g., from blood) or eggs, and further purified by well-known techniques, such as protein affinity chromatography to obtain IgG fractions, or by affinity purification for the VAMP epitopes used to produce antibodies. Some contract research organizations provide customized antibody generation services, such as Eurogentec provides a "Rapid 28-Day Program," in which they immunize on day 0, then perform three booster immunization injections on day 7, day 10, and day 18, with blood drawn in the middle of day 21 and finally on day 28. This is an example of the general technology for polyclonal antibody production well known in the art.

[0144] The hybridoma method can be used to produce monoclonal antibodies that bind to VAMP epitopes as described herein. See, for example, Chapter 7, Greenfield, Edward A., ed., Antibodies: alaboratory manual. Cold Spring Harbor Laboratory Press, 2014. In short, a host animal, such as a mammal such as a rabbit, goat, mouse, hamster or monkey, is exposed to one or more injections of the antigenic polypeptide or protein of the present invention to elicit lymphocytes that produce or can produce antibodies that specifically bind to the cleaved VAMP. The antibody titer in the immune animal can be monitored over time by standard techniques, such as using ELISA (enzyme-linked immunosorbent assay). Alternatively, a suitable cell culture system can be used to immunize lymphocytes in vitro. At the appropriate time after immunization, such as when the antibody titer is the highest, cells that produce antibodies are isolated from the animal. Typically, if human cells are needed, peripheral blood lymphocytes are used, or if non-human mammalian sources are needed, spleen cells or lymph node cells are used. The antibody-producing cells separated are fused with an immortalized cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. Immortal cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, a mouse myeloma cell line is fused with spleen cells harvested from an appropriately immunized mouse to produce a hybridoma. A preferred immortal cell line is a mouse myeloma cell line that is sensitive to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT). According to standard techniques, any of a number of myeloma cell lines can be used as a fusion partner, such as P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines. HAT culture medium is then used to select the hybridoma cells produced by the fusion, which kills unfused and non-productive fused myeloma cells (unfused spleen cells die after several days of culture because they are not transformed). The presence of a monoclonal antibody in the culture medium where the hybridoma cells are grown can then be determined for binding to a VAMP epitope as described herein. For example, the VAMP-positive culture medium of cutting can be used in immunoprecipitation assay, in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or in cell-based activity assay, screening hybridoma supernatant.The binding affinity of monoclonal antibodies can also be determined by, for example, Scatchard analysis.See, e.g., 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, clones derived from individual cells are isolated using a limiting dilution procedure until a clonal cell line expressing the desired monoclonal antibody is obtained. Alternatively, monoclonal antibodies that bind to a VAMP epitope as described herein can be generated by screening a recombinant combinatorial immunoglobulin library, e.g., an antibody phage display library, with the antigenic polypeptides, proteins, or peptides of the invention. Kits for generating and screening phage display libraries are commercially available, e.g., the recombinant phage antibody system (Amersham GE Healthcare, Piscataway, NJ); and. Phage display kit (Stratagene, La Jolla, CA). In addition, examples of methods and reagents for generating and screening antibody display libraries can be found in, for example, Ladner et al., U.S. Patent 5,223,409; Borrebaeck et al., U.S. Patent 5,712,089; Griffiths et al., U.S. Patent 5,885,793; Griffiths et al., U.S. Patent 5,962,255; McCafferty et al., U.S. Patent 5,969,108; Griffiths et al., U.S. Patent 6,010,884; Jespers et al., U.S. Patent 6,017,732; Borrebaeck et al., U.S. Patent 6,027,930; Johnson et al., U.S. Patent 6,140,471; McCafferty et al., U.S. Patent 6,172,197, each of which is incorporated herein by reference in its entirety.

[0145] In another aspect, the present invention provides antibodies that bind to the antigenic polypeptide or protein of the present invention.

[0146] In one embodiment, the antibody is a polyclonal antibody.

[0147] In one embodiment, the antibody is a monoclonal antibody.

[0148] The binding affinity between an antibody and an antigenic peptide or protein can be determined by measuring the equilibrium dissociation constant (K DThe equilibrium dissociation constant (Kd) is a measure of the rate at which new antibody-antigen complexes form, equal to the rate at which the antibody-antigen complexes dissociate at equilibrium. The equilibrium dissociation constant is expressed as M and is defined by the Kd / Ka ratio at equilibrium, where Ka is the association rate constant and Kd is the dissociation rate constant for the antibody. D =[Ab]x[Ag] / [Ab+Ag], where [Ab] is the molar concentration of the antibody, [Ag] is the molar concentration of the antigen, and [Ab+Ag] is the molar concentration of the antibody-antigen complex, where all concentrations are the concentrations of these components when the system is in equilibrium. The smaller the equilibrium dissociation constant, the tighter the binding of the antibody to its antigen, or the higher the binding affinity between the antibody and the antigen.

[0149] In one embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein epitope is D Less than 10 -6 In a preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -7 In a more preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -8 In a more preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -9 In a more preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -10 In a more preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -11 In a more preferred embodiment, the K between the antibody of the present invention and the antigenic polypeptide or protein is D Less than 10 -12 M.

[0150] In another aspect, the present invention provides the use of an antibody according to the invention in a cellular assay for gain of signaling by VAMP-cleaving Clostridial neurotoxin.

[0151] In one embodiment, the use is in vitro or ex vivo.

[0152] In another aspect, the present invention provides a method for determining VAMP cleavage in a cell by a clostridial neurotoxin that cleaves VAMP, comprising:

[0153] a) contacting the cells with a clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

[0154] b) contacting the cytoplasmic contents of the cells after cleavage of VAMP by the VAMP-cleaving clostridial neurotoxin with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention; and

[0155] c) detecting the binding of the first detection antibody to the C-terminal VAMP cleavage product by a suitable method.

[0156] In one embodiment, the method according to the present invention further comprises d) quantifying the amount of the C-terminal VAMP cleavage product bound to the first detection antibody by a suitable method.

[0157] 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 directed against full-length VAMP under conditions suitable for binding of the second detection antibody to full-length VAMP; step c) comprises detecting binding of the second detection antibody to full-length VAMP by a suitable method, and step d) comprises quantifying the amount of full-length VAMP bound to the second detection antibody by a suitable method.

[0158] In one embodiment, the method is an in vitro or ex vivo method.

[0159] It is clear to the skilled person 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 cleavage of VAMP by a clostridial neurotoxin that cleaves VAMP.

[0160] In one embodiment, the second detection antibody is the same as the first detection antibody and binds to both the C-terminal VAMP cleavage product and full-length VAMP.

[0161] In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP but not to C-terminal VAMP cleavage products. Suitably, the second detection antibody binds to a VAMP epitope at the N-terminal end of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies, such as ab3347 (Abeam) or ab181869 (Abeam).

[0162] In a specific embodiment, a method for VAMP cleavage by determining a clostridial neurotoxin that cleaves VAMP in a cell comprises:

[0163] a) contacting the cells with a clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

[0164] b) subjecting the cytoplasmic contents of said cells to:

[0165] following cleavage of VAMP by a clostridial neurotoxin that cleaves VAMP, contacting with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention that binds to both the C-terminal VAMP cleavage product and full-length VAMP; and

[0166] contacting with a second detection antibody that binds to full-length VAMP but not to C-terminal VAMP cleavage products;

[0167] c) Detection by appropriate methods

[0168] Binding of the primary antibody to C-terminal VAMP cleavage products and full-length VAMP; and

[0169] Binding of the second detection antibody to full-length VAMP; and

[0170] d) Quantify by appropriate means:

[0171] The combined amount of C-terminal VAMP cleavage products and full-length VAMP bound to the first detection antibody; and

[0172] • The amount of full-length VAMP bound to the secondary detection antibody.

[0173] It is clear 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 clostridial neurotoxin that cleaves VAMP.

[0174] In another specific embodiment, a method of determining VAMP cleavage by a Clostridial neurotoxin in a cell by cleaving VAMP comprises:

[0175] a) contacting the cells with a clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

[0176] b) after cleavage of VAMP by the clostridial neurotoxin that cleaves VAMP, contacting the cytoplasmic contents of the cells with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to C-terminal VAMP, wherein the first detection antibody is an antibody according to the present invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP;

[0177] c) Detection by appropriate methods

[0178] Binding of the primary antibody to the C-terminal VAMP cleavage product; and

[0179] Binding of the primary detection antibody to full-length VAMP;

[0180] wherein the signal generated by the binding of the first detection antibody to the C-terminal VAMP cleavage product is distinguishable from the signal generated by the binding of the first detection antibody to the full-length VAMP; and

[0181] d) Quantify by appropriate means:

[0182] The amount of C-terminal VAMP cleavage product bound to the primary detection antibody; and

[0183] • The amount of full-length VAMP bound to the primary detection antibody.

[0184] It is clear to the skilled person 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 an increase in VAMP cleavage by the clostridial neurotoxin which cleaves VAMP.

[0185] In another aspect, the present invention provides a method of determining immune tolerance to a clostridial neurotoxin that cleaves VAMP in a subject, comprising:

[0186] a) adding a clostridial neurotoxin that cleaves VAMP to a test sample obtained from a subject;

[0187] b) contacting the cells with the test sample of step a) under conditions suitable for clostridial neurotoxin activity;

[0188] c) contacting the cytoplasmic contents of the cells after cleavage of VAMP by the VAMP-cleaving clostridial neurotoxin with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention;

[0189] d) detecting binding of the first detection antibody to the C-terminal VAMP cleavage product by a suitable method;

[0190] e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

[0191] f) repeating steps a) to e) using the negative control sample instead of the test sample; and

[0192] 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 is detected in step (e) relative to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f), indicating the presence of neutralizing antibodies to the clostridial neurotoxin that cleaves VAMP.

[0193] In one embodiment, step f) further comprises repeating steps a) to e) using a positive control sample.

[0194] As used herein, the term "neutralizing antibody to a clostridial neurotoxin that cleaves VAMP" refers to any antibody that, under physiological conditions, will bind to a clostridial neurotoxin that cleaves VAMP in a manner that reduces or prevents the clostridial neurotoxin from exerting its therapeutic effect in a subject.

[0195] In one embodiment, the subject is a mammal. In a preferred embodiment, the subject is a human.

[0196] In one embodiment, the sample is selected from the group consisting of blood, plasma, serum, and lymph fluid obtained from a subject.

[0197] A test sample can be obtained from a subject prior to exposure to a clostridial neurotoxin that cleaves VAMP, after a single treatment with a clostridial neurotoxin that cleaves VAMP, or after multiple treatments with a clostridial neurotoxin that cleaves VAMP. In a specific embodiment, the test sample is from a subject that is resistant to treatment with a clostridial neurotoxin that cleaves VAMP.

[0198] As used herein, the term "control sample" refers to any sample in which the presence or absence of a test sample is known, and includes negative and positive control samples. With respect to neutralizing antibodies to clostridial neurotoxin that cleaves VAMP, a negative control sample can be obtained from an individual who has not been exposed to clostridial neurotoxin that cleaves VAMP, and can include, but is not limited to, a sample from the same individual who provided the test sample but prior to receiving clostridial neurotoxin that cleaves VAMP treatment; a sample from a different individual who has not been exposed to clostridial neurotoxin that cleaves VAMP; and a pooled sample from multiple different individuals who have not been exposed to clostridial neurotoxin that cleaves VAMP.

[0199] With respect to neutralizing antibodies to clostridial neurotoxin that cleaves VAMP, positive control samples can be obtained from individuals who exhibit immunological resistance to clostridial neurotoxin that cleaves VAMP, and include, but are not limited to, individuals who test positive in patient-based testing assays; individuals who test positive in in vivo bioassays; and individuals who exhibit hyperimmunity, such as subjects vaccinated against clostridial neurotoxin that cleaves VAMP.

[0200] In one embodiment, the method is an in vitro or ex vivo method.

[0201] In one embodiment of the method for determining immunoresistance, step c) comprises contacting the cytoplasmic contents of the cell with a second detection antibody directed 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 a suitable method, and step e) comprises quantifying the amount of full-length VAMP bound to the second detection antibody.

[0202] In one embodiment, the second detection antibody is the same as the first detection antibody and binds to both the C-terminal VAMP cleavage product and full-length VAMP.

[0203] In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP but not to C-terminal VAMP cleavage products. Suitably, the second detection antibody binds to a VAMP epitope at the N-terminal end of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies, such as ab3347 (Abeam) or ab181869 (Abeam).

[0204] In a specific embodiment, a method of determining immune tolerance to a clostridial neurotoxin that cleaves VAMP in a subject comprises:

[0205] a) adding a clostridial neurotoxin that cleaves VAMP to a test sample obtained from a subject;

[0206] b) contacting the cells with the test sample of step a) under conditions suitable for clostridial neurotoxin activity;

[0207] c) subjecting the cytoplasmic contents of the cells to

[0208] following cleavage of VAMP by the clostridial neurotoxin that cleaves VAMP, contacting with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention that binds to both the C-terminal VAMP cleavage product and full-length VAMP; and

[0209] contacting with a second detection antibody that binds to full-length VAMP but not to C-terminal VAMP cleavage products;

[0210] d) Detection by appropriate methods

[0211] Binding of the primary antibody to C-terminal VAMP cleavage products and full-length VAMP; and

[0212] Binding of the secondary detection antibody to full-length VAMP;

[0213] e) Quantification

[0214] The combined amount of C-terminal VAMP cleavage products and full-length VAMP bound to the first detection antibody; and

[0215] The amount of full-length VAMP bound to the secondary detection antibody;

[0216] f) repeating steps a) to e) using the negative control sample instead of the test sample; and

[0217] g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein detecting a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody and full-length VAMP and / or a higher amount of full-length VAMP bound to the second detection antibody in step (e) relative to the corresponding amounts in step (f) indicates the presence of neutralizing antibodies to the clostridial neurotoxin that cleaves VAMP.

[0218] In another specific embodiment, a method of determining immune tolerance to a Clostridial neurotoxin that cleaves VAMP in a subject comprises:

[0219] a) adding a clostridial neurotoxin that cleaves VAMP to a test sample obtained from a subject;

[0220] b) contacting the cells with the test sample of step a) under conditions suitable for clostridial neurotoxin activity;

[0221] c) following cleavage of VAMP by the clostridial neurotoxin that cleaves VAMP, contacting the cytoplasmic contents of the cell with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention that binds to the C-terminal VAMP cleavage product and to full-length VAMP;

[0222] d) Detection by appropriate means

[0223] Binding of the primary antibody to C-terminal VAMP cleavage products and full-length VAMP; and

[0224] Binding of the primary detection antibody to full-length VAMP;

[0225] wherein the signal generated by the binding of the first detection antibody to the C-terminal VAMP cleavage product can be distinguished from the signal generated by the binding of the first detection antibody to the full-length VAMP;

[0226] e) Quantification

[0227] The amount of C-terminal VAMP cleavage product bound to the primary detection antibody; and

[0228] The amount of full-length VAMP bound to the primary detection antibody;

[0229] f) repeating steps a) to e) using the negative control sample instead of the test sample;

[0230] 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 and / or a higher amount of full-length VAMP bound to the first detection antibody in step (e) relative to the corresponding amount in step (f) indicates the presence of neutralizing antibodies to the clostridial neurotoxin that cleaves VAMP.

[0231] As used herein, "VAMP-cleaving Clostridial neurotoxins" refer to Clostridial neurotoxins that bind to receptors on target cells, translocate the Clostridial light chain (L) into the cytosol, and then proteolytically cleave VAMP, thereby disrupting secretion of the molecule by trafficking through cellular vesicles.

[0232] Preferably, in the methods or uses of the present invention, the VAMP-cleaving Clostridial neurotoxin comprises a BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT light chain. Suitably, the BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT light chain comprises a sequence selected from the group consisting of:

[0233] - 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,

[0234] - 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,

[0235] - 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,

[0236] - 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,

[0237] - 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, and

[0238] - 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.

[0239] 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.

[0240] In one embodiment of the methods or uses of the invention, the VAMP-cleaving Clostridial neurotoxin is selected from the group consisting of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT. Suitably, BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, or TeNT comprises a sequence selected from the group consisting of:

[0241] - 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,

[0242] - 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,

[0243] - 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,

[0244] - 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,

[0245] - 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, and

[0246] - 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.

[0247] 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 receptors on target cells, translocate the clostridial light chain to the cytoplasm, and cleave VAMP.

[0248] In one embodiment, the VAMP-cleaving clostridial neurotoxin is a mosaic neurotoxin. As used herein, the term "mosaic neurotoxin" refers to a naturally occurring clostridial neurotoxin that contains at least one functional domain from another clostridial neurotoxin (e.g., a clostridial neurotoxin of a different serotype) that does not normally contain at least one functional domain. Examples of naturally occurring mosaic neurotoxins that cleave VAMP are BoNT / DC and BoNT / FA. BoNT / DC contains the L chain of serotype D and the H chain of N domain and serotype C H C domain, Nakamura K et al., "Characterization of the D / C mosaic neurotoxin produced by Clostridiumbotulinum associated with bovine botulism in Japan." Vet.Microbiol. (2010): 140: 147–154, while BoNT / FA consists of BoNT / F5 light chain, H N Domains and BoNT / A1 H C Domain composition (Pellett, Sabine et al., "Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain." mSphere 1.1(2016):e00100-15).

[0249] In one embodiment, the VAMP-cleaving Clostridial neurotoxin is a mosaic neurotoxin selected from the group consisting of BoNT / DC and BoNT / FA.

[0250] In one embodiment, the clostridial neurotoxin that cleaves VAMP is a chimeric neurotoxin. As used herein, the term "chimeric neurotoxin" refers to a neurotoxin that comprises one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may comprise an LH derived from a first neurotoxin. Ndomain and H from a second neurotoxin C Another example of a chimeric neurotoxin is a neurotoxin comprising an LH derived from a first neurotoxin. N H CN domain and H from a second neurotoxin CC Domains. Examples of chimeric neurotoxins are provided in GB 1607901.4 (not yet published), which is incorporated herein by reference.

[0251] In one embodiment, the Clostridial neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

[0252] - a light chain (L) from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X or TeNT,

[0253] - H from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT N domain,

[0254] - H from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT CN domain,

[0255] - H from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT CC domain,

[0256] At least two of the domains are derived from different Clostridial neurotoxins.

[0257] In one embodiment, the Clostridial neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

[0258] - LH from a first clostridial neurotoxin selected from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, or TeNT N domain,

[0259] - H 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,

[0260] wherein the first and second clostridial neurotoxins are different.

[0261] In one embodiment, the Clostridial neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

[0262] - LH from a first clostridial neurotoxin selected from BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, or TeNT N H CN domain,

[0263] - H 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,

[0264] wherein the first and second clostridial neurotoxins are different.

[0265] The clostridial neurotoxin that cuts VAMP can be a modified neurotoxin or a derivative thereof, including but not limited to those described below. The modified neurotoxin or derivative can contain one or more amino acids that have been modified compared to the natural (unmodified) form of the neurotoxin, or can contain one or more inserted amino acids that are not present in the natural (unmodified) form of the toxin. For example, a modified clostridial neurotoxin can have a modified amino acid sequence in one or more domains relative to the natural (unmodified) clostridial neurotoxin sequence. Such modifications can modify the functional aspects of the neurotoxin, such as biological activity or persistence.

[0266] The modified VAMP-cleaving Clostridial neurotoxins described herein retain the ability to bind to receptors on target cells, translocate the light chain into the cytoplasm, and cleave VAMP.

[0267] Modified VAMP-cleaving Clostridial neurotoxins may have one or more modifications in the amino acid sequence of the heavy chain (e.g., a modified H C domain), wherein the modified heavy chain binds to target neural cells with a higher or lower affinity than the native (unmodified) neurotoxin. C Such modifications in the domain may include modification of the ganglioside binding site or H CC The present invention also provides a modification of the present invention by modifying residues in the protein receptor binding site of the structural domain of the neurotoxin that alter binding to ganglioside receptors and / or protein receptors of target neural cells. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entireties. For example, H from BoNT / B neurotoxin CCThe domain contains at least one amino acid residue substitution, addition or deletion that has a similar structure to that of a native BoNT / BH CC Increased BoNT / BH compared to the sequence CC Effect of the structural domain on the binding affinity of human Syt II. BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains are disclosed in WO 2013 / 180799 and unpublished PCT / US2016 / 024211 (both incorporated herein by reference). CC Suitable amino acid residue substitutions, additions or deletions in the subdomain 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.

[0268] In one embodiment, a clostridial neurotoxin that cleaves VAMP is a retargeted neurotoxin. As used herein, the term "retargeted neurotoxin" (also referred to as a "targeted secretion inhibitor," "TSI," "TVEMP," or "TEM") refers to a clostridial neurotoxin that comprises a targeting moiety (TM) that binds to a non-clostridial receptor. The TM can replace part or all of the H heavy chain of the clostridial neurotoxin. C or H CCExamples of retargeted neurotoxins are disclosed 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, WO2007 / 138339, WO2007 / 106115, WO2007 / 106799, WO2009 / 150469, WO2009 / 150470, WO2010 / 055358. , WO2010 / 020811, WO2010 / 138379, WO2010 / 138395, WO2010 / 138382, WO2011 / 020052, WO2011 / 020 056, WO2011 / 020114, WO2011 / 020117, WO2011 / 20119, WO2012 / 156743, WO2012 / 134900, WO2012 / 1 34897, 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.

[0269] Examples of cells suitable for 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 cell is a neuronal cell, particularly a cell that is highly sensitive to BoNT.

[0270] A cell with a high sensitivity to BoNTs is a cell susceptible to BoNT intoxication. In some embodiments, a cell with a high sensitivity to BoNTs is a cell susceptible to BoNT intoxication. For example, a cell with a high sensitivity to BoNTs is resistant to BoNTs by 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, about 4 pM or less. In some embodiments, the amount of BoNT intoxication-susceptible cells is about 10 fM 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.

[0271] Preferably, the cells have a high sensitivity (as defined above) to a BoNT that cleaves VAMP.

[0272] In one embodiment, the cell is a primary neuronal cell that is highly sensitive to BoNT, such as a cortical neuron, a hippocampal neuron, and / or a spinal cord neuron. For example, the cell is a rat cortical neuron.

[0273] In one embodiment, the cell is from a neuronal cell line with high sensitivity 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.

[0274] In one embodiment, the cell is a neuronal cell derived from a stem cell, in particular a neuronal cell from an induced pluripotent stem cell (iPS cell), e.g. neurons, DopaNeurons iCell glutamatergic neurons, iCell MotoNeurons (Cell Dynamics), 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 neural cells (MTI-GlobalStem).

[0275] In one embodiment, cells can be modified by recombinant techniques to express high levels of VAMP, such as VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6, more preferably VAMP1, VAMP2 and / or VAMP3.

[0276] In one embodiment wherein 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 wherein the VAMP-cleaving neurotoxin is BoNT / B, BoNT / DC, or BoNT / G, the cells are modified by recombinant techniques to express high levels of synaptotagmin I and / or synaptotagmin II (Syt I / Syt II).

[0277] In one embodiment wherein the VAMP-cleaving neurotoxin is BoNT / FA, BoNT / F, BoNT / D, or TeNT, the cells express high levels of synaptobrevin (SV2). In one embodiment wherein the VAMP-cleaving neurotoxin is BoNT / FA, BoNT / F, BoNT / D, or TeNT, the cells are modified by recombinant techniques to express high levels of synaptobrevin (SV2).

[0278] As used herein, "conditions suitable for clostridial neurotoxin activity" refer to conditions (e.g., temperature, pH, cofactors, etc.) under which clostridial neurotoxins can bind to clostridial neurotoxin receptors present on cell membranes, transfer clostridial neurotoxin light chains to the cytoplasm, and cleave VAMP.

[0279] In one embodiment of the methods of the invention, conditions suitable for clostridial neurotoxin activity may comprise incubation at about 37° C. for a time of about 1 hour to about 48 hours. In one embodiment of the methods of the invention, conditions suitable for clostridial neurotoxin activity may comprise incubation at about 37° C. for a time of about 2 hours to about 36 hours. In one embodiment of the methods of the invention, conditions suitable for clostridial neurotoxin activity may comprise incubation at about 37° C. for a time of about 4 hours to about 24 hours.

[0280] For example, conditions suitable for Clostridial neurotoxin activity may include incubation at 37°C for 24 hours.

[0281] As used herein, “conditions suitable for binding of the first detection antibody to cleaved VAMP” and “conditions suitable for binding of the second detection antibody to full-length VAMP” refer to conditions (e.g., temperature, pH, cofactors, etc.) under which the first and / or second detection antibodies can bind to cleaved VAMP and / or full-length VAMP.

[0282] In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 4° C. for a time 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 time 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 time of about 12 hours to about 16 hours.

[0283] In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25° C. for a time of about 30 minutes to about 8 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25° C. for a time of about 1 hour to about 4 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25° C. for a time of about 1.5 hours to about 3 hours.

[0284] The method that is applicable to detection and quantitative detection antibody is combined with cutting or full-length VAMP is well known in the art.For example, can detect and quantitative detection antibody and cutting or the combination of full-length VAMP by western blot.When every kind of protein is run with specific molecular weight by SDS-PAGE, will detect the VAMP of cutting with molecular weight lower than full-length VAMP.By densitometry, the analysis of band is allowed to use the full-length band in the same swimming lane on gel and the percentage cutting reading of cutting band.Or, can use enzyme-linked immunosorbent assay (ELISA), for example sandwich ELISA detects and quantitative VAMP cutting.

[0285] In one embodiment of the method of the invention, the first detection antibody is a polyclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in an enzyme-linked immunosorbent assay.

[0286] In one embodiment of the method of the invention, the first detection antibody is a polyclonal antibody and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

[0287] In one embodiment of the method of the invention, the first detection antibody is a monoclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in an enzyme-linked immunosorbent assay.

[0288] In one embodiment of the method of the invention, the first detection antibody is a monoclonal antibody, and binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

[0289] In one embodiment of the methods of the invention, the cells are lysed prior to contacting the cytoplasmic contents with the detection antibody.

[0290] In an alternative embodiment of the method of the invention, the cells are permeabilized before their cytoplasmic contents are contacted with the detection antibody.

[0291] In another aspect, the present invention provides a kit comprising cells susceptible to intoxication by a neurotoxin that cleaves VAMP; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is an antibody of the present invention.

[0292] In one embodiment, the kit further comprises a second detection antibody that binds to full-length VAMP but does not bind to C-terminal VAMP cleavage products. Suitably, the second detection antibody binds to a VAMP epitope at the N-terminal end of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies, such as ab3347 (Abeam) or ab181869 (Abeam).

[0293] 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 to practice or test embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

[0294] Where a range of values ​​is provided, it is to be understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range is also specifically disclosed, to the tenth of the unit of the lower limit. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from that range, and each range that includes either, neither, or both limits within that smaller range is also encompassed within this disclosure, subject to any specifically excluded limits in that range. Where the stated range includes one or both limits, ranges that exclude either or both of those included limits are also encompassed within this disclosure.

[0295] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents and 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.

[0296] The invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0297] Attached photos

[0298] Figure 1 - VAMP sequences with cleavage sites for Clostridial neurotoxins. (A) Human and rat VAMP1, VAMP2, and VAMP3 sequences with cleavage sites for BoNT / F5, BoNT / FA, BoNT / F, BoNT / D, BoNT / DC, BoNT / B, BoNT / G, TeNT, and BoNT / X. (B) Human and rat VAMP4, VAMP5, and YKT6 sequences with cleavage sites for BoNT / X.

[0299] Figure 2 - VAMP sequences with Ab epitopes (i.e., immunogenic epitope regions) and cleavage sites for BoNT / F, BoNT / D, and BoNT / B. Sequences of human and rat VAMP1, VAMP2, and VAMP3 are shown for comparison. Rat and human VAMP2 sequences are identical in selected epitope regions. Cleavage sites are indicated by arrows: the VAMP2 cleavage sites for BoNT / F and BoNT / D are located at adjacent amino acids Q58-K59 and K59-L60, respectively, while the BoNT / B cleavage site is located at amino acids Q76-F77 (based on the amino acid positions of the human VAMP2 sequence).

[0300] Figure 3 -Use MBP-LF and LH N D. Cell-free cleavage of recombinant VAMP2-GFP. Recombinant VAMP2-GFP was mixed with 0.01 μg / μl LH N D or MBP-LF were incubated 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 on SDS-PAGE and stained with Coomassie or blotted with various anti-VAMP2 antibodies. The sketch represents the location of the antibody epitopes. The representation of the recombinant proteins and the line lengths of the epitopes are not drawn to scale. 1 - BSA, 2 - VAMP2-GFP, 3 - cleaved VAMP2-GFP (aa59 / 60-terminal), 4 - cleaved VAMP2-GFP (aa1-58 / 59).

[0301] Figure 4 In vitro VAMP cleavage after BoNT / F and BoNT / D treatment. Rat cortical neurons grown in 96-well plates until DIV 18-21 were treated with BoNT / F (A) or BoNT / D (B) for 24 hours. Lysates were electrophoresed by SDS-PAGE and blotted with custom anti-VAMP2 antibodies: anti-Pep1, anti-Pep2, or anti-Pep3, or with the commercial antibody ab1818691 for full-length VAMP2 and cleaved VAMP2. Anti-Pep2 data showed a dose-dependent disappearance of full-length VAMP2 and the appearance of a low-molecular-weight cleaved fragment. Two band signals were used to quantify the dose-dependent percentage of VAMP2 cleavage by BoNT / F (C) and BoNT / D (D).

[0302] Figure 5 Rat cortical neurons were treated with native BoNT / F1 (□), native BoNT / A1 (●), or recombinant BoNT / FA (Δ) for 24 hours. Cells were lysed, electrophoresed 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, and the BoNT concentration required for 50% maximal SNARE cleavage (pEC50) was determined. Data are mean ± sem (n = 3 (BoNT / F1 and BoNT / A1) or 4 (BoNT / FA) independent experiments, performed in triplicate).

[0303] Figure 6 In vitro VAMP cleavage after BoNT / B and BoNT / F treatment. Rat cortical neurons grown to DIV 18-21 were treated with BoNT / F or BoNT / B for 24 hours. Lysates were electrophoresed by SDS-PAGE and blotted with a 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

[0304] Example 1: Detection of VAMP proteolytic cleavage by BoNT / D and BoNT / F

[0305] A-Method

[0306] 1. Antibody Generation

[0307] Eurogentec generated antibodies using their Speedy 28-day program (https: / / secure.eurogentec.com / product / research-anti-protein-28-day-speedy-polyclonal-packages.html?country=gbr). Two rabbits were immunized with the following peptides:

[0308] -VAMP PEP1: H2N-SNR RLQ QTQ AQV DEC-CONH2 (SEQ ID NO: 39);

[0309] - VAMP PEP2: AcNH-KLS ELD DRA DAL Q-CONH2 (SEQ ID NO: 15); or

[0310] -VAMP PEP3: H2N-CLQ AGA SQ-CONH2 (SEQ ID NO: 40).

[0311] Animals received a first immunization and three subsequent booster immunizations, and were bled before, during, and at the end of each immunization.

[0312] 2. Recombinant Protein Cleavage

[0313] 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 BJ 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 ND (SEQ ID NO: 35) or a fusion protein called MBP-LF (SEQ ID NO: 36) (the latter is a fusion of MBP with the BoNT / F1 light chain and a C-terminal 6-histidine motif, and MPB and 6-histidine motifs are commonly known affinity tags) was diluted to 0.01 μg / μl in assay buffer (50 mM HEPES pH 7.2, 200 μM ZnCl2, 1 μg / μl BSA, 10 mM DTT). VAMP2-GFP (SEQ ID NO: 37) (a fusion protein of amino acids 2-94 of human VAMP2 and detectably labeled green fluorescent protein (GFP)) was diluted to 8 μM in assay buffer (50 mM HEPES pH 7.2, 200 μM ZnCl2, 1 μg / μl BSA, 10 mM DTT). An equal volume of LH N D or MBP-LF and VAMP2-GFP (SEQ ID NO: 37) (8 μM) were combined and incubated for 1 hour at 37° C. The reaction was terminated by adding 2× reducing sample buffer (NuPage LDS sample buffer, 100 mM DTT).

[0314] 3. Rat Cortical Neuron Cell Culture

[0315] Rat cortical neurons were prepared from E17-E18 CD rat embryos. Dissected cortical tissue was collected into ice-cold Hank's balanced salt solution (HBSS) w / o Ca2+ or Mg2+ and then dissociated in papain solution at 37°C for 40 minutes according to the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded at a density of 20,000 cells / well on poly-L-ornithine (PLO)-coated 96-well plates 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. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (days in vitro) 4, another 125 μl of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added. The cells were maintained by replacing half of the medium twice a week. At DIV11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the culture medium to prevent the proliferation of non-neuronal cells.

[0316] 4. BoNT treatment

[0317] Rat cortical neurons from DIV 18 to 21 were treated with a range of concentrations of native BoNT / F1 (Metabiologics, US) (1 nM to 0.1 pM) or BoNT / D (Metabiologics, US) (10 nM to 1 pM) in triplicate wells at 37°C for 24 hours. The culture medium was removed and the cells were washed once with PBS. The cells were lysed in 40 μl of LRS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 Benzonase) at room temperature for 10 minutes.

[0318] 5. SDS-PAGE and Western Blotting

[0319] Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysate was loaded onto a 12% Bis-Tris gel per lane and run in MES buffer at 200 μV for 50 min. Proteins were transferred to nitrocellulose membranes using a low MW program using a Transblot Turbo (Biorad). The membranes were blocked with 5% low-fat milk / PBS-Tween for 1 hour at room temperature and then incubated with custom anti-Pep1, anti-Pep2, or anti-Pep3 anti-VAMP2 primary antibodies or with commercial anti-VAMP2 antibodies (Abcam ab3347 and ab181869) at 4°C overnight. The membranes were washed three times in PBS-Tween and incubated with anti-rabbit-HRP secondary antibodies for 1 hour at room temperature. The membranes were washed 3×5 minutes in PBS-Tween and then developed with SuperSignal West Femto chemiluminescent substrate and visualized using a Syngene PXi system.

[0320] B-Results

[0321] Evaluation of recombinant protein detection

[0322] Figure 2 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 for BoNT / B and BoNT / D are located at adjacent amino acids.

[0323] Initially, the antibodies were tested in a cell-free assay using recombinant VAMP2-GFP. BoNT / F and BoNT / D surrogates containing the enzymatic light chain domains of the toxins (MBP-LF and LH N D) used to cleave VAMP protein ( Figure 3In addition, two other commercially available VAMP2 antibodies, ab3347 (epitope aa1-18) and ab181869 (epitope within aa1-100), were used for comparison. Figure 3 The anti-Pep1 antibody detected both full-length VAMP2 and the N-terminal cleavage fragment (aa1-58 / 59) with a significantly reduced signal. As expected, this antibody did not detect the C-terminal cleavage product, as its epitope is not located on this fragment. The anti-Pep2 and anti-Pep3 antibodies detected both the full-length protein and the C-terminal cleavage product of VAMP2-GFP. Ab3347 detected only full-length VAMP2 and not the N-terminal cleavage fragment, while ab181869 detected both.

[0324] These first results indicate that the antibodies are able to detect both full-length and the expected cleavage products of recombinant VAMP2. The exception is ab3347, which only detects full-length VAMP2 and not the N-terminal cleavage fragment.

[0325] Evaluation of endogenous protein detection

[0326] The next question was whether these antibodies could detect any cleavage products in a neuronal cell assay where endogenous proteases would be present. Rat primary cortical neurons were treated with BoNT / F or BoNT / D and lysed for WB analysis ( Figure 4 ). The anti-Pep1 antibody only recognized the full-length protein and had no detectable cleavage products. The anti-Pep 2 antibody detected both full-length and C-terminally cleaved products. The anti-Pep3 antibody showed a weak signal in the cell lysate with very poor affinity for monomeric VAMP and detected higher molecular weight species that were most likely dimers and other proteins (data not shown). The full-length monomer signal was very low, but there were bands for the C-terminal products of BoNT / F and BoNT / D cleavage. In other words, anti-Pep3 did not detect full-length VAMP, but weakly detected the C-terminal fragments cleaved by BoNT / F and BoNT / D. This contrasts with earlier cell-free results, which showed strong signals from full-length and cleaved recombinant VAMP. The commercial antibody Ab3347 was not tested in vitro due to the absence of cleaved protein detection in the cell-free assay. Despite positive binding to the N-terminally cleaved recombinant fragment in the cell-free assay, the commercial antibody ab181869 detected full-length VAMP2 in cortical lysates, but no cleaved fragments were detected in cortical lysates. Pep 2 data were used to quantify BoNT / F( Figure 4 C) and BoNT / D( Figure 4 D) Dose-dependent cleavage of VAMP2.

[0327] The inventors initially demonstrated that both recombinant VAMP cleavage products could be detected in a cell-free system. However, when transferred to cell lysates, the inventors also showed that the N-terminal product was undetectable, but other, as yet unknown, mechanisms other than degradation may be involved. In contrast, the inventors have shown that the C-terminal VAMP fragment, which remains bound to the vesicle membrane, is not degraded or altered in a manner that prevents antibody binding and detection by 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 from the BoNT F / D cleavage site also detect the cleavage product, albeit weakly.

[0328] Example 2: Detection of VAMP proteolytic cleavage of BoNT / FA and BoNT / F1 in rat cortical neurons

[0329] A-Method

[0330] 1. Rat Cortical Neuron Cell Culture

[0331] Rat cortical neurons were prepared as detailed in Example 1.

[0332] 2. BoNT treatment

[0333] In triplicate wells, rat cortical neurons from DIV 18-21 were treated with recombinant BoNT / FA (SEQ ID NO: 38) at a concentration range of 1 pM to 1 fM, or native BoNT / F1 (Metabiologics, US) at a concentration range of 1 nM to 1 pM, or native BoNT / A1 (List Biological Laboratories Inc., US) at a concentration range of 1 nM to 1 fM at 37°C for 24 hours. The culture medium was removed and the cells were washed once with PBS. The cells were lysed in 40 μl of LRS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 Benzonase) at room temperature for 10 minutes.

[0334] 3. SDS-PAGE and Western Blotting of Rat Cortical Neurons

[0335] Rat cortical neurons were lysed in 40 μl of lysis buffer (NuPage LDS sample buffer, 1 mM DTT, and 1:500 Benzonase) for 10 minutes at room temperature. Samples were boiled at 90°C for 5 minutes, and 15 μl of lysate was loaded onto a 12% Bis-Tris gel per lane and run in MOPS buffer at 200 V for 80 minutes (SNAP-25) or MES buffer at 200 V for 50 minutes (VAMP2). Proteins were transferred to nitrocellulose membranes using a Transblot Turbo (Biorad) using either the mixed MW (SNAP-25) or low MW (VAMP2) program. The membrane was blocked with 5% low-fat milk / PBS-Tween for 1 hour at room temperature and then incubated with anti-SNAP25 antibody (Sigma S9684, 1:4000) or anti-Pep2 (1:500), a custom antibody developed as described in Example 1, and VAMP2 (Eurogentec); each primary antibody was incubated overnight at 4°C. The membrane was washed three times in PBS-Tween and incubated with anti-rabbit-HRP secondary antibody for 1 hour at room temperature. The membrane was washed 3 times in PBS-Tween for 5 minutes and then developed with SuperSignal West Dura or West Femto chemiluminescent substrates and visualized using the Syngene Pxi system. Band densitometry was analyzed using Genetools software, and the ratio of full-length protein to cleavage product for SNAP-25 and VAMP2 was used to determine the percentage of protein cleavage.

[0336] B-Results

[0337] After 24 hours of treatment with BoNT / F1, BoNT / A1, or BoNT / FA, rat cortical neurons were lysed, electrophoresed on SDS-PAGE, and Western blotted for VAMP-2 (BoNT / F1 and BoNT / FA) or SNAP-25 (BoNT / A1). The percentage of SNARE cleavage was determined by densitometry analysis of the ratio of full-length to cleaved protein.

[0338] The results are as follows Figure 5 As shown. VAMP-2 cleaved by recombinant BoNT / FA, potency pEC50 = 12.75 ± 0.14, n = 4. VAMP-2 cleaved by native BoNT / F1, potency pEC50 = 10.77 ± 0.12, n = 3. SNAP-25 cleaved by native BoNT / A1, potency pEC50 = 12.38 ± 0.14, n = 3.

[0339] Example 3: Detection of VAMP proteolytic cleavage of BoNT / B in rat cortical neurons

[0340] A-Method

[0341] 1. Antibody Generation

[0342] Abcam generated monoclonal antibodies using rabbits immunized with the peptide Pep4: FETSAAKLKRKYWWK (SEQ ID NO: 49).

[0343] BoNT / B cleavage-specific anti-VAMP2 antibody (Kegel et al., Toxicology in Vitro; 2007, 21: p1641-1649) was used for comparative studies.

[0344] 2. Rat Cortical Neuron Cell Culture

[0345] Rat cortical neurons were prepared from E17-E18 CD rat embryos. Dissected cortical tissue was collected into ice-cold Hank's balanced salt solution (HBSS) w / o Ca2+ or Mg2+ and then dissociated in papain solution at 37°C for 40 minutes according to the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded at a density of 20,000 cells / well on poly-L-ornithine (PLO)-coated 96-well plates 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. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. At DIV (days in vitro) 4, another 125 μl of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added. The cells were maintained by replacing half of the medium twice a week. At DIV11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the culture medium to prevent the proliferation of non-neuronal cells.

[0346] 3. BoNT treatment

[0347] Rat cortical neurons were cultured in T25 flasks and treated with 1 nM and 10 pM BoNT / B (provided by List Biological Laboratories, Inc.) (SEQ ID NO: 2) at 37°C for 24 hours. The culture 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.

[0348] 4. SDS-PAGE and Western Blotting

[0349] Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysate was added to a 12% Bis-Tris gel per lane and run in MES buffer at 200 μV for 50 minutes. Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using a low MW program. The membranes were blocked with 5% low-fat milk / PBS-Tween for 1 hour at room temperature and then incubated with custom anti-Pep1, anti-Pep2, anti-Pep3, or anti-Pep4 antibodies or with BoNT-B cleavage-specific antibodies overnight at 4°C. The membranes were washed three times in PBS-Tween and incubated with anti-rabbit-HRP secondary antibody for 1 hour at room temperature. The membranes were washed 3×5 minutes in PBS-Tween and then developed with SuperSignal West Femto chemiluminescent substrate and visualized using a Syngene PXi system.

[0350] B-Results

[0351] Based on the results obtained in Examples 1 and 2 above, which suggested that the location of the epitope is key to detecting cleaved VAMP in vitro, new monoclonal antibodies were generated against an epitope located near the BoNT / B cleavage site on the C-terminal side.

[0352] This antibody was tested in the same rat cortex assay after BoNT / B and BoNT / F treatment and compared with anti-Pep1, anti-Pep2, anti-Pep3, and BoNT / B cleavage-specific antibodies ( Figure 6 The epitope regions of all compared antibodies were located on the N-terminal side of the BoNT / B cleavage site. Figure 6 The location of the novel antibody epitope targeting Pep4 enabled detection of full-length VAMP2, as well as cleavage products for both BoNT / B and BoNT / F treatments. In contrast, anti-Pep2 and anti-Pep3 antibodies detected only BoNT / F cleavage products, but not BoNT / B cleavage products. Anti-Pep1 antibodies did not detect any expected cleavage products. BoNT / B cleavage-specific antibodies also failed to detect any BoNT / B cleavage products in these cell lysates.

[0353] In summary, the present data show that an important consideration for the detection of cleaved VAMP is the location of the antibody epitope. Only antibodies raised against an epitope located on the membrane-bound VAMP fragment after cleavage are able to detect this fragment. By localizing the monoclonal antibody epitope to the C-terminus of VAMP, it was hypothesized that this region should be present in the VAMP fragments produced by neurotoxin serotypes B, D, and F that cleave VAMP. This turned out to be the case, enabling the generation of a single antibody (anti-Pep4 Mab) that provided positive results for neurons treated with BoNT / B and BoNT / F. Furthermore, since TeNT and BoNT / B share the same cleavage site, and the BoNT / D cleavage site is closely adjacent to the BoNT / F cleavage site, this antibody is also expected to be suitable for TeNT and BoNT / D cleavage.

[0354] Another 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 powerful tool for normalization without the need for additional housekeeping protein blotting. This provides a very useful and straightforward signal-gain western blot analysis for quantifying BoNT potency in cell models.

[0355] The present data also show differences in VAMP detection between cell-free recombinant protein assays and whole-cell models. It is this inability to detect cellular cleaved VAMP that forms the basis for the hypothesis that VAMP degradation occurs very rapidly in cells (Foran et al., "Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E and F compared with the long-lasting type A". J. Biol Chem 278(2) pp1363–1371 2003). In contrast to the antibodies of the present invention, most commercially available VAMP antibodies are raised against epitopes within the N-terminal region of the protein, and therefore the N-terminal VAMP fragment was the focus of those earlier studies. Although the present invention shows that the smaller C-terminal VAMP fragment is not degraded in cells, the larger N-terminal fragment was not detected either. Interestingly, however, the cell-free results show that not all commercial antibodies are able to detect the expected N-terminal fragment even when it is present in a cell-free system lacking any proteases. From the present data, it can be concluded that the VAMP degradation hypothesis is most certainly only relevant to the N-terminal fragment, and that the C-terminal VAMP fragment is not degraded and remains associated with the vesicle membrane.

[0356] Sequence information

[0357] SEQ ID NO: 1-BoNT / A1-UniProtKB accession number P10845 (Clostridium botulinum)

[0358]

[0359] SEQ ID NO: 2 - BoNT / B1 - UniProtKB Accession No. P10844 (Clostridium botulinum)

[0360]

[0361] SEQ ID NO:3-BoNT / C1-UniProtKB accession number P18640 (Clostridium botulinum)

[0362]

[0363] SEQ ID NO: 4-BoNT / D-UniProtKB accession number P19321 (Clostridium botulinum)

[0364]

[0365] SEQ ID NO: 5 - BoNT / E - Accession No. WP_003372387 (Clostridium botulinum)

[0366]

[0367] SEQ ID NO:6 - BoNT / F-UniProtKB Accession No. YP_001390123 (Botulinum toxin)

[0368]

[0369] SEQ ID NO:7-BoNT / G-UniProtKB accession number WP_039635782 (Clostridium botulinum)

[0370]

[0371] SEQ ID NO:8-TeNT-UniProtKB accession number P04958 (Clostridium tetani)

[0372]

[0373] ·SEQ ID NO:9 - VAMP1_Rat (Q63666) MSAPAQPPAEGTEGAAPGGGPPGPPPNTTSNRRLQQTQAQVEEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYIFT

[0374] ·SEQ ID NO:10 - VAMP1_Human (P23763)

[0375] MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT

[0376] ·SEQ ID NO:11 - VAMP2_Rat (P63045)

[0377] MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

[0378] ·SEQ ID NO:12 - VAMP2_Human (P63027)

[0379] MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

[0380] ·SEQ ID NO:13 - VAMP3_Rat (P63025)

[0381] MSTGVPSGSSAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGISVLVIIVIIIIVWCVS

[0382] SEQ ID NO: 14 - VAMP3_human (Q15836)

[0383] MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

[0384] SEQ ID NO: 15—VAMP epitope

[0385] KLSELDDRADALQ

[0386] SEQ ID NO: 16—VAMP epitope

[0387] QKLSELDDRADALQ

[0388] SEQ ID NO: 17—VAMP epitope KLSELDDRAD

[0389] SEQ ID NO: 18—VAMP epitope KLSELDDRADALQAGAS

[0390] SEQ ID NO: 19—VAMP epitope LSELDDRADALQ

[0391] SEQ ID NO:20—VAMP epitope LSELDDRADA

[0392] SEQ ID NO:21—VAMP epitope LSELDDRADALQAGAS

[0393] SEQ ID NO: 22—VAMP epitope FETSAAKLKRKYW

[0394] SEQ ID NO: 23—VAMP epitope FESSAAKLKRKYW

[0395] SEQ ID NO: 24—VAMP epitope QFETSAAKLKRKYW

[0396] SEQ ID NO: 25—VAMP epitope FETSAAKLKR

[0397] SEQ ID NO: 26—VAMP epitope

[0398] FETSAAKLKRKYWWKN

[0399] SEQ ID NO: 27—VAMP epitope

[0400] AKLKRKYWWKN

[0401] SEQ ID NO: 28—VAMP epitope

[0402] AAKLKRKYWWKN

[0403] SEQ ID NO: 29—VAMP epitope

[0404] AKLKRKYWWKNCKM

[0405] SEQ ID NO:30—VAMP epitope

[0406] AKLKRKYWWKNLKM

[0407] SEQ ID NO:31 – VAMP epitope

[0408] DQKLSELDDRADALQ

[0409] SEQ ID NO:32—VAMP epitope

[0410] ERDQKLSELDDRA

[0411] SEQ ID NO: 33—VAMP epitope

[0412] LERDQKLSELDDRA

[0413] SEQ ID NO:34—VAMP epitope

[0414] VLERDQKLSELDDRA

[0415] SEQ ID NO:35-LH N D

[0416] MGSMTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEAHHHHHHHHHH

[0417] ·SEQ ID NO:36-MBP-LF

[0418] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPVAINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDSIPDKGLVEKIVKFAVDKLAAALEHHHHHH

[0419] · SEQ ID NO:37 (Recombinant VAMP2 - GFP)

[0420] GPLGSSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKLENVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0421] · SEQ ID NO:38 - Recombinant BoNT / FA

[0422]

[0423] SEQ ID NO:39–Pep1

[0424] SNRRLQQTQAQVDEC

[0425] SEQ ID NO:40—Pep3

[0426] CLQAGASQ

[0427] SEQ ID NO:41—BoNT / X Genbank Accession No. BAQ12790 (Clostridium botulinum)

[0428]

[0429] ·SEQ ID NO:42 - VAMP4_Rat (D4A560)

[0430] MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALAAAILLLMIITQIILHLKK

[0431] ·SEQ ID NO:43 - VAMP4_Human (O75379)

[0432] MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

[0433] ·SEQ ID NO:44 - VAMP5_Rat (Q9Z2J5)

[0434] MAGKELERCQRQADQVTEIMLNNFDKVLERDGKLSELQQRSDQLLDMSSAFSKTTKTLAQQKRWENIRCRVYLGLAVAGGLLLILVVLLVIFLPSGEDSSKP

[0435] ·SEQ ID NO:45 - VAMP5_Human (O95183)

[0436] MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN

[0437] ·SEQ ID NO:46 - YKT6_Rat (Q5EGY4)

[0438] MKLYSLSVFYKGEPKAVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSAKGSRASVKEQEYLCHVYVRSDSLAGVVIADSEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYTALDGHLSRYQNPREADPMSKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

[0439] ·SEQ ID NO:47 - YKT6_Human (O15498)

[0440] MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

[0441] ·SEQ ID NO:48 - VAMP Epitope

[0442] ETSAAKLKRKYWWK

[0443] ·SEQ ID NO:49 - VAMP Epitope

[0444] FETSAAKLKRKYWWK

[0445] ·SEQ ID NO:50 - VAMP Epitope

[0446] QFESSAAKLKRKYW

[0447] ·SEQ ID NO:51 - VAMP Epitope

[0448] FESSAAKLKR

[0449] ·SEQ ID NO:52 - VAMP Epitope

[0450] FESSAAKLKRKYWWK

[0451] ·SEQ ID NO:53 - VAMP Epitope ADALQAGASQF

[0452] SEQ ID NO: 54—VAMP epitope ADALQAGASQ

[0453] SEQ ID NO: 55—VAMP epitope RADALQAGASQF

[0454] SEQ ID NO: 56—VAMP epitope ADALQAGASQFE

[0455] SEQ ID NO: 57—VAMP epitope ADALQAGASVF

[0456] SEQ ID NO: 58—VAMP epitope ADALQAGASV

[0457] SEQ ID NO: 59—VAMP epitope ADALQAGASVFE

[0458] SEQ ID NO:60—VAMP epitope RADALQAGASVF

[0459] SEQ ID NO:61 - VAMP epitope RADALQAGAS

[0460] SEQ ID NO:62—VAMP epitope SESLSDNATAF

[0461] SEQ ID NO:63—VAMP epitope SESLSDNATA

[0462] SEQ ID NO:64—VAMP epitope KSESLSDNATAF

[0463] SEQ ID NO:65—VAMP epitope SESLSDNATAFS

[0464] SEQ ID NO:66—VAMP epitope SDQLLDMSSTF

[0465] SEQ ID NO:67—VAMP epitope SDQLLDMSST

[0466] SEQ ID NO:68—VAMP epitope RSDQLLDMSSTF

[0467] SEQ ID NO:69—VAMP epitope SDQLLDMSSTFN

[0468] SEQ ID NO:70—VAMP epitope SDQLLDMSSAF

[0469] SEQ ID NO: 71 - VAMP epitope SDQLLDMSSA

[0470] SEQ ID NO: 72—VAMP epitope RSDQLLDMSSAF

[0471] SEQ ID NO:73—VAMP epitope SDQLLDMSSAFS

[0472] SEQ ID NO: 74—VAMP epitope RSDQLLDMSS

[0473] SEQ ID NO:75—VAMP epitope SEVLGTQSKAF

[0474] SEQ ID NO:76—VAMP epitope SEVLGTQSKA

[0475] SEQ ID NO:77—VAMP epitope KSEVLGTQSKAF

[0476] • SEQ ID NO:78—VAMP epitope SEVLGTQSKAFY.

Claims

1. An antigenic polypeptide comprising a VAMP epitope, wherein the antigenic polypeptide consists of 10 to 65 amino acid residues, wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to a VAMP sequence comprising at least 8 amino acid residues, wherein the at least 8 amino acid residues are directly C-terminal to the clostridial neurotoxin cleavage site in the VAMP.

2. The antigenic polypeptide according to claim 1, wherein the polypeptide consists of 10 to 17 amino acid residues, preferably 10 to 16 amino acid residues, more preferably 10 to 15 amino acid residues.

3. The antigenic polypeptide according to claim 1 or 2, wherein the VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and / or YKT6.

4. The antigenic polypeptide according to any one of claims 1 to 3, wherein the VAMP epitope comprises or consists of an amino acid sequence that is at least 90% identical to a VAMP sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 34, and SEQ ID NO: 48 to SEQ ID NO:

78.

5. The antigenic polypeptide according to any one of claims 1 to 4, wherein the VAMP epitope is a VAMP1, VAMP2 and / or VAMP3 epitope and is selected from: - a BoNT / F or BoNT / DVAMP epitope comprising or consisting of KLSELDDRADALQ (SEQ ID NO: 15); - a BoNT / F5 or BoNT / FA VAMP epitope comprising or consisting of ERDQKLSELDDRA (SEQ ID NO: 32); A BoNT / B or TeNT VAMP epitope comprising or consisting of FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49); - a BoNT / G VAMP epitope comprising or consisting of AKLKRKYWWKN (SEQ ID NO: 27); and - A BoNT / X VAMP epitope comprising or consisting of ADALQAGASQF (SEQ ID NO: 53).

6. The antigenic polypeptide according to any one of claims 1 to 4, wherein the VAMP epitope is a VAMP4, VAMP5 and / or YKT6 epitope and is selected from: - A BNT / X VAMP epitope comprising or consisting of SESLSDNATAF (SEQ ID NO: 62), SDQLLDMSSTF (SEQ ID NO: 66) or SEVLGTQSKAF (SEQ ID NO: 75).

7. A polypeptide comprising the antigenic polypeptide of any one of the preceding claims, wherein the polypeptide does not comprise a region of greater than 17, preferably 16, more preferably 15 consecutive amino acids having 100% sequence identity with the naturally occurring VAMP amino acid sequence.

8. An antigenic protein comprising the polypeptide according to any one of claims 1 to 6, or the polypeptide according to claim 7, wherein the antigenic protein is covalently linked to a carrier.

9. Use of an antigenic polypeptide according to any one of claims 1 to 6, or a polypeptide according to claim 7, or an antigenic protein according to claim 8 for generating antibodies against a C-terminal VAMP cleavage product.

10. An antibody which binds to the antigenic polypeptide according to any one of claims 1 to 6, or the polypeptide according to claim 7, or the antigenic protein according to claim 8.

11. The antibody according to claim 10, wherein the antibody is a polyclonal antibody.

12. The antibody according to claim 10, wherein the antibody is a monoclonal antibody.

13. The antibody according to any one of claims 10 to 12, wherein the K between the antibody and the epitope is D Less than 10 - 7 M.

14. Use of an antibody according to any one of claims 10 to 13 in a signal gain cellular assay for VAMP cleavage by a clostridial neurotoxin that cleaves VAMP.

15. A method for determining cleavage of VAMP by a clostridial neurotoxin that cleaves VAMP in a cell, comprising: a. contacting the cells with the Clostridial neurotoxin under conditions suitable for the activity of the Clostridial neurotoxin; b. after cleavage of VAMP by a clostridial neurotoxin that cleaves VAMP, contacting the cytoplasmic contents of the cell with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to any one of claims 10 to 13; and c. Detecting the binding of the first detection antibody to the C-terminal VAMP cleavage product by appropriate means.

16. The method according to claim 15, further comprising d) quantifying the amount of the C-terminal VAMP cleavage product bound to the first detection antibody by suitable means.

17. A method for determining immunoresistance to a clostridial neurotoxin that cleaves VAMP in a subject, comprising: a. adding a Clostridial neurotoxin that cleaves VAMP to a test sample obtained from a subject; b. contacting the cells with the test sample of step a) under conditions suitable for the activity of the Clostridial neurotoxin; c. after cleavage of VAMP by a clostridial neurotoxin that cleaves VAMP, contacting the cytoplasmic contents of the cell with a first detection antibody directed against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to any one of claims 10 to 13; d. detecting the binding of the first detection antibody to the C-terminal VAMP cleavage product by appropriate means; e. quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody; f. Repeat steps a) to e) with a negative control sample instead of the test sample; and 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 detected in step (e) relative to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of neutralizing antibodies to the clostridial neurotoxin that cleaves VAMP.

18. A kit comprising cells susceptible to neurotoxin intoxication by cleaved VAMP; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is the antibody according to any one of claims 10-13.

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