Capture antibodies

Abstract

The present invention provides an antibody or antigen binding fragment that binds to ALQAGASQ (SEQ ID NO: 59), wherein the ALQAGASQ (SEQ ID NO: 59) is located at the C- terminus of a polypeptide. The present invention also provides corresponding nucleic acid compositions, vector systems, host cells, conjugates, kits, uses and methods.

Description

[0001] Capture Antibodies

[0002] The present invention provides an antibody or antigen binding fragment that binds to ALQAGASQ (SEQ ID NO: 59), wherein the ALQAGASQ (SEQ ID NO: 59) is located at the C- terminus of a polypeptide. The present invention also provides corresponding nucleic acid compositions, vector systems, host cells, conjugates, kits, uses and methods.

[0003] Background

[0004] The closely related Clostridial neurotoxin family includes tetanus neurotoxin (TeNT) and seven distinct botulinum neurotoxins (BoNTs; types A to G), which cause the diseases tetanus and botulism respectively. This invention focuses on BoNT type B (BoNT / B) and tetanus, both of which have been shown to cause disease in humans. BoNT / B is associated with foodborne illness while tetanus is most commonly caused by cuts or punctures with contaminated objects.

[0005] Each of the eight Clostridial neurotoxins is synthesised as a -150 kDa single chain protein with three structurally independent domains: a SNARE peptidase domain, a translocation domain, and a neuronal binding domain [1].The single chain protein is subsequently cleaved by Clostridial or host proteases to generate an N-terminal -50 kDa enzymatic Light chain (Lc; comprising the SNARE peptidase domain) and a -100 kDa Heavy chain (He; comprising the translocation domain and the neuronal binding domain). Both chains remain attached via a single disulphide bond, a peptide loop and further non-covalent interactions.

[0006] The three protein domains of a Clostridial neurotoxin perform distinct roles in toxin delivery and activity within host cells. The neuronal binding domain is required to specifically bind to target host cells (neurons); the translocation domain facilitates endocytosis of the toxin into the cytosol of the host cell; and the peptidase domain catalyses the proteolysis of one of three soluble / V-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs) in the cell, namely VAMP / synaptobrevin, a synaptosome-associated protein of 25 kDa (SNAP25), or syntaxin. As these substrate proteins are essential components of the host vesicular membrane fusion apparatus, cleavage of any one of these proteins blocks neurotransmitter release from the host cell. Specifically, BoNT B, and TeNT cleave VAMP on synaptic vesicles. However, due to differences in target host cells between the two toxins, they have different effects on their host. BoNT / B cleaves VAMP in motor neurons, where the blockage of neurotransmitter release results in flaccid paralysis, as the muscles are unable to contract. Conversely, TeNT does not cleave VAMP at the motor neurons, but is instead transported through the neurons by retrograde transport until it reaches the Central Nervous System (CNS), which is responsible for preventing muscle contraction. The resulting cleavage of VAMP at the CNS by TeNT causes the host to be unable to stop contracting their muscles, resulting in the spastic paralysis characteristic of tetanus infections.

[0007] Despite its toxic properties, the flaccid paralysis caused by BoNT / B can be used to treat neuromuscular spasms and other medical applications such as hyperhidrosis, bladder dysfunction and spasmodic dysphonia. As a result, BoNT / B is produced and sold in the US and in the EU, where each individual batch undergoes rigorous testing to ensure that the active dosage of toxin is correct before it is used for human treatment. Highly sensitive testing is also required for TeNT activity, as the vaccine for tetanus is made by growing and then attenuating TeNT into a non-harmful tetanus toxoid, so it is important to ensure that no active TeNT remains in the vaccine.

[0008] However, the current “gold standard” testing method used by commercial producers of BoNT / B and TeNT, the Lethal Dose 50 (LD50) assay, has a number of serious disadvantages. The LD50 is determined by injecting a range of dilutions of the Toxin or Toxoid samples into groups of animals, such as mice or Guinea pigs, and calculating the dose that is required to kill 50% of a group. This test is imprecise, and requires a large number of animals to be injected, which has a large financial cost and causes suffering to many animals in the process.

[0009] Due to these issues, other methods for testing BoNT / B and TeNT activity have been developed, including cell based assays for BoNT / B and TeNT [2], and a Binding ANd CLeavage (BINACLE) assay for TeNT [3], However, many of these assays contain a step that relies on the ability to specifically capture the cleaved end of the VAMP peptide, without also binding non-cleaved VAMP protein. Some of these tests, such as the BINACLE assay, are currently using stocks of a polyclonal antibody against the cytosolic cleaved-end of the VAMP peptide, but polyclonal antibodies are produced in finite supply, with variability between each batch. Therefore, new antibodies will need to be continually developed and tested if these tests continue to use polyclonal antibodies for the cleavage-specific capture of VAMP1-3.

[0010] Brief summary of the disclosure

[0011] The present invention is based on the generation and identification of a series of recombinant rabbit monoclonal antibodies that selectively bind a neopeptide which is generated from any one of VAMPs 1-3 when they are cleaved by either BoNT / B or tetanus toxin (TeNT). The antibodies of the presently claimed invention were generated by immunising rabbits with a synthetic peptide, which mimics the 8 amino acids at the C-terminus of BoNT / B or TeNT cleaved VAMP. These antibodies were isolated by generating hybridomas from the splenic B- cells of the immunised rabbits. The resultant hybridomas were then screened for their selective binding to cleaved VAMP, and the most promising clones sequenced, and further characterised in a series of in-vitro and cell-based experiments (Figure 1A). The antibodies described herein selectively bind to the neoepitope of VAMPs 1-3 which is produced when they are cleaved by BoNT / B or tetanus toxin as determined by ELISA based assays (either using peptides, recombinant NanoLuciferase tagged VAMPs 1-3 or a cell derived NanoLuciferase VAMP2 reporter) and / or immunoblotting from extracts prepared from cell lines expressing NanoLuciferase tagged VAMP2. Based on the antibody light and heavy chain variable domain sequences, three main groups of antibodies were isolated which either bind to the final glutamine or penultimate serine at the C-terminus of the epitope as determined by ELISA and / or immunoblotting. Advantageously, the antibodies described herein can be used to quantify the biological activity of BoNT / B or TeNT as they are capable of selectively detecting the neopeptide which is generated when VAMPs 1-3 are proteolytically cleaved by these toxins. Accordingly, these antibodies can be used to quantify the levels of the neopeptide in a range of in vitro and in vivo assays such as a one step-ELISA, sandwich- ELISA, immunoblotting and Luminex bead assays.

[0012] Advantageously, the invention provides an antibody or antigen binding fragment, comprising:

[0013] (i) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6;

[0014] (ii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20; (iii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34; or

[0015] (iv) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0016] Suitably:

[0017] (i) a) the heavy chain variable region (VH) of (i) may comprise the amino acid sequence of SEQ ID NO: 7; and b) the light chain variable region (VL) of (i) may comprise the amino acid sequence of SEQ ID NO: 8;

[0018] (ii) a) the heavy chain variable region (VH) of (ii) may comprise the amino acid sequence of SEQ ID NO: 21 ; and b) the light chain variable region (VL) of (ii) may comprise the amino acid sequence of SEQ ID NO: 22;

[0019] (iii) a) the heavy chain variable region (VH) of (iii) may comprise the amino acid sequence of SEQ ID NO: 35; and b) the light chain variable region (VL) of (iii) may comprise the amino acid sequence of SEQ ID NO: 36; or

[0020] (iv) a) the heavy chain variable region (VH) of (iv) may comprise the amino acid sequence of SEQ ID NO: 49; and b) the light chain variable region (VL) of (iv) may comprise the amino acid sequence of SEQ ID NO: 50.

[0021] Suitably, the antibody or antigen binding fragment may bind to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment. Suitably, the VAMP fragment may be a VAMP2, VAMP1 , or VAMP3 fragment.

[0022] Suitably, the VAMP fragment may be a VAMP2 fragment.

[0023] Suitably, the VAMP2 fragment may comprise SEQ ID NO: 79.

[0024] Suitably, the antibody may be a rabbit antibody or antigen binding fragment.

[0025] A nucleic acid composition encoding an antibody or antigen binding fragment described herein is also provided.

[0026] An expression vector system comprising a nucleic acid described herein is also provided.

[0027] Further provided is a host cell comprising a nucleic acid described herein or an expression vector described herein.

[0028] Further provided is a conjugate comprising an antibody or antigen binding fragment described herein conjugated to or recombinantly fused to a diagnostic agent, detectable agent, or therapeutic agent.

[0029] A kit for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, the kit comprising an antibody or antigen binding fragment described herein, a nucleic acid described herein, an expression vector described herein, a host cell described herein and / or a conjugate described herein, optionally wherein the polypeptide comprises or consists of a VAMP fragment wherein the ALQAGASQ is part of the VAMP fragment, is provided.

[0030] Suitably, the kit may comprise an antibody or antigen binding fragment described herein.

[0031] Suitably, the kit may further comprises a nucleic acid encoding:

[0032] (i) a VAMP polypeptide; or

[0033] (ii) a VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C- terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity. Suitably, the nucleic acid encoding:

[0034] (i) the VAMP polypeptide; or

[0035] (ii) the VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C- terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity; may be part of an expression vector.

[0036] Suitably, the nucleic acid encoding (i) or (ii) may be within a host cell.

[0037] Suitably, the host cell may be selected from the group consisting of a SiMa neuroblastoma cell, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, immortalised neuron, a BE(2)C cell and primary neuron.

[0038] Suitably, the kit may be for detecting a VAMP fragment, wherein ALQAGASQ is located at the C-terminus of the VAMP fragment.

[0039] Suitably, the kit may be for detecting tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0040] Use of an antibody or antigen binding fragment described herein, a nucleic acid described herein, an expression vector described herein, a host cell described herein, a conjugate described herein and / or a kit described herein for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment is provided.

[0041] Suitably, the antibody or antigen binding fragment, nucleic acid, expression vector, host cell, conjugate and / or kit may be for detecting:

[0042] (a) ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment; and / or

[0043] (b) tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0044] Suitably, the neurotoxin activity may be detected in a test sample comprising:

[0045] (a) a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof;

[0046] (b) a tetanus toxoid, or a botulinum toxoid or a combination thereof; and / or (c) tetanus neurotoxin, botulinum neurotoxin type B, or a combination thereof.

[0047] Also provided herein is a method of binding a polypeptide comprising ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of the polypeptide, the method comprising contacting the polypeptide with an antibody or antigen binding fragment described herein, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment.

[0048] Suitably, the method may be for detecting:

[0049] (a) ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment; and / or

[0050] (b) tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0051] A method of detecting neurotoxin activity in a test sample is provided herein, the method comprising:

[0052] (a) providing a host cell that comprises a nucleic acid encoding:

[0053] (i) a VAMP polypeptide; or

[0054] (ii) a VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1, VAMP2 or VAMP3 activity;

[0055] (b) culturing the host cell under conditions that allow for expression of the polypeptide;

[0056] (c) culturing the host cell of (b) in the presence of the test sample under conditions that allow for neurotoxin-induced cleavage of the polypeptide; and

[0057] (d) using an antibody or antigen binding fragment described herein to determine the level of neurotoxin-induced cleavage of the polypeptide, wherein detection of neurotoxin-induced cleavage of the polypeptide is indicative of neurotoxin activity; wherein the neurotoxin activity is tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0058] Suitably, step (d) may comprise preparing a cell lysate from the host cell of step (c) and contacting the cell lysate with the antibody or antigen binding fragment to determine the level of neurotoxin-induced cleavage of the polypeptide.

[0059] Suitably, the neurotoxin-induced cleavage of the polypeptide may be detected by ELISA, immunoblot, or live cell imaging. Suitably, the test sample may comprise:

[0060] (a) a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof;

[0061] (b) a tetanus toxoid, or a botulinum toxoid, or a combination thereof; and / or

[0062] (c) tetanus neurotoxin, botulinum neurotoxin type B, or a combination thereof.

[0063] Suitably, the antibody or antigen binding fragment may be:

[0064] (a) selected from the group consisting of: Fab, Fab’, F(ab’)2, Fv, scFv, di-scFv, bis-scFv, tri- scFv, scFv-Fc, an antibody domain, sdAb, diabody, triabody, tetrabody, minibody, a nanobody, “third generation” (3G) fragment, and full length antibody; and / or

[0065] (b) selected from lgG1 , lgG2, lgG3 and lgG4; and / or

[0066] (c) monospecific or bispecific.

[0067] Suitably, the antibody may be a monoclonal antibody.

[0068] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0069] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0070] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0071] Various aspects of the invention are described in further detail below.

[0072] Brief description of the Figures

[0073] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows antibodies can be generated which selectively bind to a neoepitope that is formed when VAMP2 is cleaved by Botulinum B and Tetanus toxin. A Amino acid alignment showing the sequences and similarities of VAMP1-3 proteins in the regions where BoNT and Tetanus toxins cleave (arrowheads show the position where the indicated toxins cleave). B Schematic showing the immunisation process used to develop antibodies against the peptide epitope formed when BoNT / B and Tetanus cleave VAMP1-3. C To determine if cleavage selective antibodies had been generated, sera was taken from the rabbits after the fourth immunisation and used to probe extracts from cells expressing the NanoLuc VAMP2 reporter treated with either BoNT / D or BoNT / B. As expected no signal was observed in the samples treated with BoNT / D. D Bar chart showing the performance of the 7 most promising cleavage- selective antibodies. The ELISA plates were coated with either the peptide used for immunisation (ALQAGASQ, SEQ ID NO: 59) or a peptide which was 2 amino acids longer (ALQAGASQFE, SEQ ID NO: 74) (2pg / ml, 100pl / well in Phosphate Buffered Saline, pH 7.4). The hybridoma supernatant was the diluted 1 / 2000 and the amount of bound antibody detected using Anti-Rabbit IgG Fc Monoclonal Secondary Antibody conjugated to HRP. Coating Antigens: A: ALQAGASQ (SEQ ID NO: 59)(Lot: U3823EC120-1)B: ALQAGASQFE (SEQ ID NO: 74) (Lot: U3823EC120-2) Coating Concentration: 2pg / ml, 100pl / wellCoating Buffer: Phosphate Buffered Saline, pH 7.4Secondary Antibody: Anti-Rabbit IgG Fc Monoclonal Secondary Antibody (Min X Hu, Ms, Rt, Sh, Bv, Gt, Camel) (HRP conjugate) (GenScript, Cat. No.A01856). The hybridoma supernatant was diluted 1 / 2000.

[0074] Figure 2 shows that cleavage selective antibodies can be used to detect the action of Botulinum B using an One Step ELISA. A Bar chart showing results of a luciferase-based ELISA assay where 50 pL of 0.5 pg / mL of the cleavage specific antibodies were captured on protein A plates, then incubated with 1.2 pg / mL solutions of purified Nanoluc-tagged VAMP2 (VAMP) or purified Nanoluc-tagged VAMP2 that had been treated with BoNT / B (Cleaved VAMP) for 90 minutes to allow the cleavage-selective antibodies to bind it. Nanoluc activity was measured using NanoGio luciferase assay system. B Graph showing ELISA assay of a titration of the 2 most promising cleavage specific antibodies tested against 0.3 pg / mL solutions of uncleaved and BoNT / B-cleaved Nanoluc-VAMP2 proteins. C Graph showing ELISA assay of a titration of uncleaved and BoNT / B-cleaved Nanoluc-VAMP2 proteins against the optimal concentrations of the 2 best cleavage selective antibodies (5 ug / mL 2F7 and 10 ug / mL 131G9).

[0075] Figure 3 shows that the glutamine and serine residues of the epitope are critical for antibody binding. A T able showing the amino acid sequence of the binding region of the wild-type and mutant Nanonluc-cleaved-VAMP recombinant proteins. B Bar chart showing ELISA assay of Uncleaved, Cleaved and mutated Nanoluc-tagged VAMPs using 1 ug / mL of the cleavage specific antibodies (n=6). C Immunoblots of the wild-type and mutant Nanonluc-cleaved- VAMP recombinant proteins blotted with the 4 best cleavage selective antibodies. D Bar chart showing normalized quantification of signal from the immunoblots of wild-type and mutant Nanoluc-cleaved-VAMP proteins blotted with the 4 best cleavage specific antibodies. (n=3)

[0076] Figure 4 shows that the cleavage selective antibodies can be used to effectively detect the action of Botulinum B or Tetanus toxin in cells using a range of approaches. A Immunoblot of untreated and BoNT / B treated cells blotted with the cleavage-specific antibodies. B Antibodies could detect cleaved VAMP2 in cell-based assay. Bar chart showing results of ELISA. NanoLuc VAMP2 LAN5 cells were treated either with TeNT or BoNT / B at 100 pM concentration. Final concentration of cleavage specific antibodies is 1 ug / mL. NTC: nontreated control.

[0077] Figure 5 shows the sequence alignment of the heavy and light chain variable domains of the cleavage selective antibodies. The cDNA encoding the heavy and light chain variable domains for each antibody molecule was isolated from the specific hybridoma using RT-PCR, cloned and sequenced. The predicted protein sequences were aligned using Clustal Omega and the positions of the complementarity determining regions (CDRs) predicted using homology and Kabat based alignments. The sequence homology was also used to create simple phylograms for the heavy and light chains.

[0078] Figure 6 shows that antibodies can detect cleavage of VAMPs 1 and 3. Bar chart showing ELISA assay of uncleaved and BoNT / B-cleaved, Nanoluc-tagged VAMP1-3 proteins using 1 ug / mL of the 4 best cleavage selective antibodies. Results for each graph were normalized to the signal from cleaved Nanoluc-VAMP. A Nanoluc-VAMP2 results. B Nanoluc-VAMP1 results. C Nanoluc-VAMP3 results.

[0079] Figure 7 shows that currently available mouse antibodies B1148 and B226 can detect cleavage of VAMP2 in western blot, but not in nanoluc-based ELISA assay. A Immunoblot of BoNT / D and BoNT / B treated cells blotted with the cleavage-specific mouse antibodies B1148 and B226. B-C Bar charts showing results of ELISA using protein A (B) or Protein G (C) coated plates. NanoLuc VAMP2 LAN5 cells were treated with BoNT / B at 100 pM concentration. Final concentration of cleavage specific antibodies is 1 ug / mL. Cq8: Positive control, polyclonal, cleavage-specific VAMP2 rabbit antibody [4], NTC: non-treated control.

[0080] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0081] Various aspects of the invention are described in further detail below.

[0082] Detailed Description

[0083] As discussed above, the present invention advantageously provides a series of antibodies that selectively bind a neopeptide which is generated from any one of VAMPs 1-3 when they are cleaved by either BoNT / B or tetanus toxin (TeNT). Advantageously, the antibodies described herein can be used to quantify the biological activity of BoNT / B or TeNT as they are capable of selectively detecting the neopeptide which is generated when any of VAMPs 1-3 are proteolytically cleaved by these toxins. Accordingly, these antibodies can be used to quantify the levels of the neopeptide in a range of in vitro and in vivo assays.

[0084] Antibodies and antigen binding fragments

[0085] A rabbit antibody or antigen binding fragment that binds to ALQAGASQ (SEQ ID NO: 59) is provided herein, wherein the ALQAGASQ sequence is located at the C-terminus of a polypeptide. The polypeptide may comprise or consist of the ALQAGASQ sequence, provided that this sequence is located at the C-terminus of the polypeptide.

[0086] The ALQAGASQ sequence in the polypeptide may be part of a VAMP fragment. In other words, the polypeptide may comprise a VAMP fragment located at its C-terminus, wherein the VAMP fragment has the ALQAGASQ sequence at its C-terminus. The polypeptide may alternatively consist of a VAMP fragment, wherein the VAMP fragment has the ALQAGASQ sequence at its C-terminus.

[0087] The polypeptide may be one of the products that is generated when BoNT / B and / or TeNT cleave a VAMP polypeptide or a VAMP reporter polypeptide. Suitable VAMP polypeptides and suitable VAMP reporter polypeptides are described in more detail below.

[0088] Polypeptides having the ALQAGASQ sequence at their C-terminus are referred to as “C- terminus ALQAGASQ polypeptides” herein. The C-terminus ALQAGASQ polypeptide may comprise or consist of a VAMP fragment as described elsewhere herein.

[0089] VAMPs cover a family of proteins which are characterised by a C-terminal integral membrane domain. The N-terminus (aa 1-90 or more depending on isoform and species) faces the cytosol and comprises a SNARE motif for the interaction with SNAP-25 and syntaxin. All VAMPs are engaged in membrane fusion processes. VAMPs interact by their N-terminal chain with syntaxin and SNAP-25 family members to form the fusion core or SNARE complex, as a prerequisite for exocytosis. The fundamental role of VAMP for vesicle fusion has been shown when analysing VAMP knockout animal models.

[0090] VAMP1 and VAMP2 are known as synaptobrevins and are expressed in brain, spinal cord and in peripheral neurons. They are constituents of the synaptic vesicles, where they participate in neurotransmitter release. VAMP3 (also known as cellubrevin) is ubiquitously expressed and participates in regulated and constitutive exocytosis as a constituent of secretory granules and secretory vesicles [Nature Reviews Molecular Cell Biology 2, 98-106 (February 2001) ‘SNARE-mediated membrane fusion’ Y. A. Chen & R. H. Scheller],

[0091] VAMP1 , VAMP2 and VAMP3 are all known substrates for tetanus neurotoxin (TeNT), botulinum neurotoxin type B (BoNT / B), botulinum neurotoxin type D (BoNT / D), botulinum neurotoxin type F (BoNT / F), and botulinum neurotoxin type G (BoNT / G). The neurotoxins cleave the VAMP1 , VAMP2 and VAMP3 substrates at conserved cleavage site sequences, as shown in table 1 below for BoNT / B and TeNT.

[0092] Table 1

[0093] The antibodies and antigen binding fragments provided herein may be used to detect BoNT / B- and / or TeNT- cleaved human or non-human VAMP since the BoNT / B- and / or TeNT- cleavage sites in VAMP proteins are very highly conserved between species. Accordingly, in some examples, reference to VAMP (e.g. a VAMP polypeptide, a VAMP fragment, or a VAMP reporter polypeptide) herein may refer to human or non-human VAMP. For instance, the VAMP polypeptide or VAMP fragment may be mammalian. For instance, the VAMP polypeptide or VAMP fragment may be human, mouse, rat, guinea pig, horse or sheep. For instance, the VAMP polypeptide or VAMP fragment may be human. In some examples, the VAMP molecule (e.g. a VAMP polypeptide, a VAMP fragment, or a VAMP reporter polypeptide) is a human VAMP (e.g. a human VAMP polypeptide, VAMP fragment, or VAMP reporter polypeptide).

[0094] The antibodies and antigen binding fragments provided herein can detect BoNT / B- and / or TeNT- cleaved VAMP1 , VAMP2 and VAMP3. When cleaved by BoNT / B and / or TeNT, a VAMP fragment is generated, which comprises the ALQAGASQ sequence at its C-terminus.

[0095] The antibodies and antigen binding fragments provided herein can also detect BoNT / B- and / or TeNT-cleaved VAMP1 , VAMP2 and VAMP3 reporter polypeptides. Such reporter polypeptides include VAMP1 , VAMP2 and / or VAMP3 sequences that are recognised and cleaved by BoNT / B and / or TeNT. They comprise the core cleavage site of a VAMP protein (e.g. the GASQFESS or GASQFETS sequence shown in Table 1). When cleaved by BoNT / B and / or TeNT, a cleaved polypeptide is generated from the VAMP reporter polypeptide, where the cleaved polypeptide comprises the ALQAGASQ sequence located at its C-terminus. Examples of suitable VAMP reporter molecules are provided below.

[0096] In one example, a reporter polypeptide capable of being cleaved by BoNT / B may comprise amino acids 60-87 of VAMP2 (i.e. the sequence of LSELDDRADALQAGASQ*FETSAAKLKRK (SEQ ID NO: 75)), where the star identifies the cleavage site. In another example, a reporter polypeptide capable of being cleaved by TeNT may comprise amino acids 40-87 of VAMP2 (i.e. the sequence of DEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQ*FETSAAKLKRK (SEQ ID NO: 76)). Again, the star identifies the cleavage site. The above sequences include VAMP1 , VAMP2 and / or VAMP3 sequences that are recognised and cleaved by BoNT / B and / or TeNT.

[0097] A “VAMP fragment” refers to a portion of a VAMP protein (a VAMP protein is also referred to as a VAMP polypeptide herein). In other words, a VAMP fragment is a portion of the VAMP protein amino acid sequence. For example, a VAMP fragment, as defined herein, may be generated upon tetanus neurotoxin (TeNT) or botulinum neurotoxin type B (BoNT / B) mediated cleavage of a VAMP protein (e.g. a VAMP1-3 protein). A VAMP fragment, as defined herein, may also be generated upon tetanus neurotoxin (TeNT) or botulinum neurotoxin type B (BoNT / B) mediated cleavage of a VAMP reporter polypeptide. A VAMP fragment does not need to be capable of being cleaved by BoNT / B or TeNT nor have VAMP activity.

[0098] As discussed above, BoNT / B and TeNT cleave VAMP1 , VAMP2 and VAMP3 at conserved cleavage site sequences. Advantageously, the antibodies described herein can be used to quantify the biological activity of BoNT / B or TeNT as they are capable of selectively detecting a neopeptide (SEQ ID NO: 59) generated when VAMPs 1-3 are proteolytically cleaved by BoNT / B or TeNT.

[0099] As stated above, the ALQAGASQ sequence in the polypeptide described herein may be part of a VAMP fragment. In other words, the polypeptide may comprise a VAMP fragment located at its C-terminus, wherein the VAMP fragment has the ALQAGASQ sequence at its C- terminus. In this context, the polypeptide may include additional amino acids (e.g. further to the VAMP fragment having the ALQAGASQ sequence at its C-terminus). In the context of the invention, such additional amino acids may be N-terminal to VAMP fragment. The polypeptide may alternatively consist of a VAMP fragment, wherein the VAMP fragment has the ALQAGASQ sequence at its C-terminus.

[0100] A VAMP fragment as described herein may comprise or consist of the amino acid sequence of SEQ ID NO: 59. In the context of the invention, the amino acid sequence of SEQ ID NO: 59 may be located at the C-terminus of the VAMP fragment. In some examples, the VAMP fragment may include additional amino acids (e.g. further to SEQ ID NO: 59). In the context of the invention, such additional amino acids may be N-terminal to the sequence of SEQ ID NO:59 (such that the sequence of SEQ ID NO:59 is at the C-terminus of the VAMP fragment).

[0101] The VAMP fragment may be any suitable length, provided that it is shorter than the corresponding full length VAMP protein (e.g. a native VAMP protein). Typically, a VAMP fragment as described herein will be at least 8 amino acids long (because it will have the sequence of SEQ ID NO:59). A person of skill in the art would readily be able to identify a VAMP fragment by comparing the amino acid sequence of a fragment with a full length VAMP amino acid sequence (e.g. a native VAMP amino acid sequence) to determine if the fragment corresponds to a portion of the full length VAMP sequence.

[0102] In one example, the VAMP fragment is a VAMP2, VAMP1 or VAMP3 fragment.

[0103] In one example, the VAMP fragment is a VAMP1 fragment (i.e. a portion of a full length VAMP1). The VAMP1 fragment may comprise the sequence of SEQ ID NO: 59 at its C- terminus. A person of skill in the art would readily be able to identify a VAMP1 fragment using routine experiments known in the art. For instance, a skilled person would readily be able to identify a VAMP1 fragment by comparing the amino acid sequence of the fragment with a full length VAMP1 amino acid sequence to determine if the fragment corresponded to a portion of the VAMPI sequence. In one example, the VAMP1 fragment comprises or consists of the sequence of SEQ ID NO: 77.

[0104] In one example, the VAMP fragment is a VAMP3 fragment. The VAMP3 fragment may comprise the sequence of SEQ ID NO: 59 at its C-terminus. A person of skill in the art would readily be able to identify a VAMP3 fragment using routine experiments known in the art. For instance, a skilled person would readily be able to identify a VAMP3 fragment by comparing the amino acid sequence of the fragment with a full length VAMP3 amino acid sequence to determine if the fragment corresponded to a portion of the VAMP3 sequence.

[0105] In one example, the VAMP3 fragment comprises the sequence of SEQ ID NO: 78.

[0106] In one example, the VAMP fragment is a VAMP2 fragment. The VAMP1 fragment may comprise the sequence of SEQ ID NO: 59 at its C-terminus. A person of skill in the art would readily be able to identify a VAMP2 fragment using routine experiments known in the art. For instance, a skilled person would readily be able to identify a VAMP2 fragment by comparing the amino acid sequence of the fragment with a full length VAMP2 amino acid sequence to determine if the fragment corresponded to a portion of the VAMP2 sequence.

[0107] In one example, the VAMP2 fragment comprises the sequence of SEQ ID NO: 79.

[0108] The N-terminus of a protein (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2). By convention, peptide sequences are written N-terminus to C-terminus (from left to right). The C-terminus (also known as the carboxyl-term in us, carboxyterminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0109] The rabbit antibody or antigen binding fragment provided herein advantageously binds to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide. In other words, the rabbit antibody or antigen binding fragment provided herein binds ALQAGASQ, wherein the ALQAGASQ may be located at the end of the polypeptide terminated by an amino acid with a free carboxyl group (e.g. wherein the last amino acid of SEQ ID NO: 59 is the last amino acid of the polypeptide). As discussed above, the inventors have advantageously identified antibodies that selectively bind a neopeptide generated from VAMPs 1-3 when they are cleaved by either BoNT / B or tetanus toxin (TeNT). Advantageously, the antibodies described herein can be used to quantify the biological activity of BoNT / B or TeNT as they are capable of selectively detecting the neopeptide which is generated when VAMPs 1-3 are proteolytically cleaved by these toxins. Each of these antibodies is discussed in more detail below.

[0110] (i) antibody clone 131G9

[0111] As provided elsewhere herein, the inventors identified antibody clone 131G9, which binds to ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). The sequences provided herein that correspond to antibody clone 131G9 are SEQ ID NOs: 1-14, see also table 6.

[0112] Accordingly, in one example, an antibody or antigen binding fragment is provided herein comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6.

[0113] In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 7 (or a functional variant thereof); and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 8 (or a functional variant thereof). When part of an antibody or antigen binding fragment described herein, functional VH and VL variants retain the ability to bind ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7 or 8. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 7 or 8, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein. Non-functional variants are amino acid sequence variants of SEQ ID NO: 7 or 8 that do not selectively bind to ALQAGASQ. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:7 or 8, or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

[0114] In one example, the heavy chain variable region (VH) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7 whilst retaining the ability to selectively bind to ALQAGASQ, and / or the light chain variable region (VL) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 8 whilst retaining the ability to selectively bind to ALQAGASQ. In other words, a functional heavy chain variable region (VH) and a light chain variable region (VL) with one or several amino acid substitutions compared to the sequence of SEQ ID NO:7 or 8 are also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:7 or 8 may all be in regions of the variable domains that do not form CDRs (i.e. the variant heavy chain variable region (VH) may have the CDRs of SEQ ID NO: 1 , SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or the variant light chain variable region (VL) may have the CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and / or SEQ ID NO: 6, and still have 25% (or less) sequence variability compared to SEQ ID NO: 7 or 8 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 7 and / or SEQ ID NO: 8 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 7 or SEQ ID NO: 8).

[0115] As an example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 7, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6.

[0116] As another example, the heavy chain variable region (VH) may comprise an amino acid sequence having the sequence of SEQ ID NO: 7, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or the light chain variable region (VL) may comprise an amino acid sequence having the sequence of SEQ ID NO: 8, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6.

[0117] In one example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8. In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 8. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6. In examples where the heavy chain variable region (VH) has the amino acid sequence of SEQ ID NO:7, the heavy chain variable region (VH) may be encoded by the nucleic acid sequence of SEQ ID NO: 9, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0118] In examples where the light chain variable region (VL) has the amino acid sequence of SEQ ID NO:8, the light chain variable region (VL) may be encoded by the nucleic acid sequence of SEQ ID NO: 10, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0119] In one example, the antibody or antigen binding fragment may comprise a heavy chain variable region (VH) with the nucleic acid sequence of SEQ ID NO: 9 and / or a light chain variable region (VL) with the nucleic acid sequence of SEQ ID NO: 10.

[0120] The phrase “genetically degenerate sequence thereof” is used interchangeably with “derivative thereof’ herein.

[0121] Nucleic acids provided by the invention are discussed elsewhere herein in more detail.

[0122] For the avoidance of doubt, the antibody or antigen binding fragment may further comprise constant domains. The invention is not limited to specific constant domains, and encompasses any appropriate constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.

[0123] An example of a specific heavy chain amino acid sequence that includes a heavy chain variable region (VH) described herein with an appropriate constant domain is shown in SEQ ID NO: 11 . An example of a specific light chain amino acid sequence that includes a light chain variable region (VL) described herein with an appropriate constant domain is shown in SEQ ID NO: 12. Appropriate functional variants of SEQ ID NO: 11 and SEQ ID NO: 12 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, wherein the variant amino acid sequences retain the ability to selectively bind to ALQAGASQ when part of an antibody or antigen binding fragment described herein). In other words, a functional heavy chain and a functional light chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 respectively is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:11 and / or SEQ ID NO: 12 may all be in regions of the heavy chain or light chain that do not form CDRs (i.e. the variant heavy chain may have the CDRs of SEQ ID NO: 1 , SEQ ID NO: 2 and / or SEQ ID NO: 3, and / or the variant light chain may have the CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and / or SEQ ID NO: 6, and still have 25% (or less) sequence variability compared to SEQ ID NO: 11 or 12 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 11 and / orSEQ ID NO: 12 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 11 or SEQ ID NO: 12).

[0124] As an example, the heavy chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 11 , wherein the heavy chain variable region (VH) of said heavy chain comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or the light chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 12, wherein the light chain variable region (VL) of said light chain comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6.

[0125] In examples where the heavy chain has the amino acid sequence of SEQ ID NO: 11 , the heavy chain may be encoded by the nucleic acid sequence of SEQ ID NO: 13, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0126] In examples where the light chain has the amino acid sequence of SEQ ID NO:12, the light chain may be encoded by the nucleic acid sequence of SEQ ID NO: 14, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). In one example, the antibody or antigen binding fragment may comprise a heavy chain with the nucleic acid sequence of SEQ ID NO: 13 and / or a light chain with the nucleic acid sequence of SEQ ID NO: 14.

[0127] In one example, the heavy chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 ; and / or the light chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12. In one example, the antibody or antibody fragment may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 ; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 12. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6.

[0128] In one example, the antibody may be a rabbit antibody or antigen binding fragment. It may be monoclonal antibody (e.g. a monoclonal rabbit antibody) or antigen binding fragment.

[0129] As provided elsewhere herein, the inventors identified antibody clone 114D9, which binds to ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). The sequences provided herein that correspond to antibody clone 114D9 are SEQ ID NOs: 15 -28, see also table 7.

[0130] In one example, the antibody or antigen binding fragment comprises: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20.

[0131] In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 21 (or a functional variant thereof); and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 22 (or a functional variant thereof). When part of an antibody or antigen binding fragment described herein, functional VH and VL variants retain the ability to bind ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 21 or 22. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 21 or 22, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.

[0132] Non-functional variants are amino acid sequence variants of SEQ ID NO: 21 or 22 that do not selectively bind to ALQAGASQ. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:21 or 22, or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

[0133] In one example, the heavy chain variable region (VH) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21 whilst retaining the ability to selectively bind to ALQAGASQ, and / or the light chain variable region (VL) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 22 whilst retaining the ability to selectively bind to ALQAGASQ. In other words, a functional heavy chain variable region (VH) and a light chain variable region (VL) with one or several amino acid substitutions compared to the sequence of SEQ ID NO:21 or 22 are also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:21 or 22 may all be in regions of the variable domains that do not form CDRs (i.e. the variant heavy chain variable region (VH) may have the CDRs of SEQ ID NO: 15, SEQ ID NO: 16 and / or SEQ ID NO: 17 and / or the variant light chain variable region (VL) may have the CDRs of SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20, and still have 25% (or less) sequence variability compared to SEQ ID NO: 21 or 22 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 21 and / or SEQ ID NO: 22 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 21 or SEQ ID NO: 22).

[0134] As an example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 21 , wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 22, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20.

[0135] As another example, the heavy chain variable region (VH) may comprise an amino acid sequence having the sequence of SEQ ID NO: 21 , with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or the light chain variable region (VL) may comprise an amino acid sequence having the sequence of SEQ ID NO: 22, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20. In one example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21 ; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 22. In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 21 ; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 22. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20.

[0136] In examples where the heavy chain variable region (VH) has the amino acid sequence of SEQ ID NO:21 , the heavy chain variable region (VH) may be encoded by the nucleic acid sequence of SEQ ID NO: 23, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0137] In examples where the light chain variable region (VL) has the amino acid sequence of SEQ ID NO:22, the light chain variable region (VL) may be encoded by the nucleic acid sequence of SEQ ID NO: 24, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0138] In one example, the antibody or antigen binding fragment may comprise a heavy chain variable region (VH) with the nucleic acid sequence of SEQ ID NO: 23 and / or a light chain variable region (VL) with the nucleic acid sequence of SEQ ID NO: 24.

[0139] Nucleic acids provided by the invention are discussed elsewhere herein in more detail.

[0140] For the avoidance of doubt, the antibody or antigen binding fragment may further comprise constant domains. The invention is not limited to specific constant domains, and encompasses any appropriate constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities. An example of a specific heavy chain amino acid sequence that includes a heavy chain variable region (VH) described herein with an appropriate constant domain is shown in SEQ ID NO: 25. An example of a specific light chain amino acid sequence that includes a light chain variable region (VL) described herein with an appropriate constant domain is shown in SEQ ID NO: 26. Appropriate functional variants of SEQ ID NO:25 and SEQ ID NO: 26 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26, wherein the variant amino acid sequences retain the ability to selectively bind to ALQAGASQ when part of an antibody or antigen binding fragment described herein). In other words, a functional heavy chain and a functional light chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:25 or SEQ ID NO: 26 respectively is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:25 and / or SEQ ID NO: 26 may all be in regions of the heavy chain or light chain that do not form CDRs (i.e. the variant heavy chain may have the CDRs of SEQ ID NO: 15, SEQ ID NO: 16 and / or SEQ ID NO: 17, and / or the variant light chain may have the CDRs of SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20, and still have 25% (or less) sequence variability compared to SEQ ID NO: 25 or 26 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 25 and / or SEQ I D NO: 26 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 25 or SEQ ID NO: 26).

[0141] As an example, the heavy chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 25, wherein the heavy chain variable region (VH) of said heavy chain comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or the light chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 26, wherein the light chain variable region (VL) of said light chain comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20.

[0142] In examples where the heavy chain has the amino acid sequence of SEQ ID NO: 25, the heavy chain may be encoded by the nucleic acid sequence of SEQ ID NO: 27, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0143] In examples where the light chain has the amino acid sequence of SEQ ID NO:26, the light chain may be encoded by the nucleic acid sequence of SEQ ID NO: 28, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0144] In one example, the antibody or antigen binding fragment may comprise a heavy chain with the nucleic acid sequence of SEQ ID NO: 27 and / or a light chain with the nucleic acid sequence of SEQ ID NO: 28.

[0145] In one example, the heavy chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 25; and / or the light chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 26. In one example, the antibody or antibody fragment may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 26. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20.

[0146] In one example, the antibody may be a rabbit antibody or antigen binding fragment. It may be monoclonal antibody (e.g. a monoclonal rabbit antibody) or antigen binding fragment.

[0147] 011 As provided elsewhere herein, the inventors identified antibody clone 2F7, which binds to ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). The sequences provided herein that correspond to antibody clone 2F7 are SEQ ID NOs: 29 -42, see also table 8.

[0148] In one example, the antibody or antigen binding fragment comprises: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34.

[0149] In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 35 (or a functional variant thereof); and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 36 (or a functional variant thereof). When part of an antibody or antigen binding fragment described herein, functional VH and VL variants retain the ability to bind ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 35 or 36. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 35 or 36, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.

[0150] Non-functional variants are amino acid sequence variants of SEQ ID NO: 35 or 36 that do not selectively bind to ALQAGASQ. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:35 or 36, or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art. In one example, the heavy chain variable region (VH) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 35 whilst retaining the ability to selectively bind to ALQAGASQ, and / or the light chain variable region (VL) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 36 whilst retaining the ability to selectively bind to ALQAGASQ. In other words, a functional heavy chain variable region (VH) and a light chain variable region (VL) with one or several amino acid substitutions compared to the sequence of SEQ ID NO:35 or 36 are also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:35 or 36 may all be in regions of the variable domains that do not form CDRs (i.e. the variant heavy chain variable region (VH) may have the CDRs of SEQ ID NO: 29, SEQ ID NO: 30 and / or SEQ ID NO: 31 and / or the variant light chain variable region (VL) may have the CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and / or SEQ ID NO: 34, and still have 25% (or less) sequence variability compared to SEQ ID NO: 35 or 36 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 35 and / or SEQ ID NO: 36 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 35 or SEQ ID NO: 36).

[0151] As an example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 35, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 36, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34. As another example, the heavy chain variable region (VH) may comprise an amino acid sequence having the sequence of SEQ ID NO: 35, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or the light chain variable region (VL) may comprise an amino acid sequence having the sequence of SEQ ID NO: 36, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34.

[0152] In one example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 35; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 36. In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 35; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 36. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34.

[0153] In examples where the heavy chain variable region (VH) has the amino acid sequence of SEQ ID NO:35, the heavy chain variable region (VH) may be encoded by the nucleic acid sequence of SEQ ID NO: 37, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0154] In examples where the light chain variable region (VL) has the amino acid sequence of SEQ ID NO:36, the light chain variable region (VL) may be encoded by the nucleic acid sequence of SEQ ID NO: 38, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0155] In one example, the antibody or antigen binding fragment may comprise a heavy chain variable region (VH) with the nucleic acid sequence of SEQ ID NO: 37 and / or a light chain variable region (VL) with the nucleic acid sequence of SEQ ID NO: 38.

[0156] Nucleic acids provided by the invention are discussed elsewhere herein in more detail.

[0157] For the avoidance of doubt, the antibody or antigen binding fragment may further comprise constant domains. The invention is not limited to specific constant domains, and encompasses any appropriate constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.

[0158] An example of a specific heavy chain amino acid sequence that includes a heavy chain variable region (VH) described herein with an appropriate constant domain is shown in SEQ ID NO: 39. An example of a specific light chain amino acid sequence that includes a light chain variable region (VL) described herein with an appropriate constant domain is shown in SEQ ID NO: 40. Appropriate functional variants of SEQ ID NO:39 and SEQ ID NO: 40 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, wherein the variant amino acid sequences retain the ability to selectively bind to ALQAGASQ when part of an antibody or antigen binding fragment described herein). In other words, a functional heavy chain and a functional light chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:39 or SEQ ID NO: 40 respectively is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:39 and / or SEQ ID NO: 40 may all be in regions of the heavy chain or light chain that do not form CDRs (i.e. the variant heavy chain may have the CDRs of SEQ ID NO: 29, SEQ ID NO: 30 and / or SEQ ID NO: 31 , and / or the variant light chain may have the CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and / or SEQ ID NO: 34, and still have 25% (or less) sequence variability compared to SEQ ID NO: 39 or 40 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 39 and / or SEQ ID NO: 40 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 39 or SEQ ID NO: 40). As an example, the heavy chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 39, wherein the heavy chain variable region (VH) of said heavy chain comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or the light chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 40, wherein the light chain variable region (VL) of said light chain comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34.

[0159] In examples where the heavy chain has the amino acid sequence of SEQ ID NO: 39, the heavy chain may be encoded by the nucleic acid sequence of SEQ ID NO: 40, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0160] In examples where the light chain has the amino acid sequence of SEQ ID NQ:40, the light chain may be encoded by the nucleic acid sequence of SEQ ID NO: 42, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0161] In one example, the antibody or antigen binding fragment may comprise a heavy chain with the nucleic acid sequence of SEQ ID NO: 41 and / or a light chain with the nucleic acid sequence of SEQ ID NO: 42.

[0162] In one example, the heavy chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 39; and / or the light chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 40. In one example, the antibody or antibody fragment may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 39; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 40. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 40.

[0163] In one example, the antibody may be a rabbit antibody or antigen binding fragment. It may be monoclonal antibody (e.g. a monoclonal rabbit antibody) or antigen binding fragment.

[0164] [iv

[0165] As provided elsewhere herein, the inventors identified antibody clone 30G7, which binds to ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). The sequences provided herein that correspond to antibody clone 30G7 are SEQ ID NOs: 43-56, see also table 9.

[0166] In one example, the antibody or antigen binding fragment comprises:

[0167] (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0168] In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 49 (or a functional variant thereof); and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 50 (or a functional variant thereof). When part of an antibody or antigen binding fragment described herein, functional VH and VL variants retain the ability to bind ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 49 or 50. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 49 or 50, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.

[0169] Non-functional variants are amino acid sequence variants of SEQ ID NO: 49 or 50 that do not selectively bind to ALQAGASQ. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 49 or 50, or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

[0170] In one example, the heavy chain variable region (VH) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 49 whilst retaining the ability to selectively bind to ALQAGASQ, and / or the light chain variable region (VL) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 50 whilst retaining the ability to selectively bind to ALQAGASQ. In other words, a functional heavy chain variable region (VH) and a light chain variable region (VL) with one or several amino acid substitutions compared to the sequence of SEQ ID NO:49 or 50 are also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:49 or 50 may all be in regions of the variable domains that do not form CDRs (i.e. the variant heavy chain variable region (VH) may have the CDRs of SEQ ID NO: 43, SEQ ID NO: 44 and / or SEQ ID NO: 45 and / or the variant light chain variable region (VL) may have the CDRs of SEQ ID NO: 46, SEQ ID NO: 47 and / or SEQ ID NO: 48, and still have 25% (or less) sequence variability compared to SEQ ID NO: 49 or 50 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 49 and / or SEQ ID NO: 50 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 49 or SEQ ID NO: 50).

[0171] As an example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 49, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 50; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 50, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0172] As another example, the heavy chain variable region (VH) may comprise an amino acid sequence having the sequence of SEQ ID NO: 49, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or the light chain variable region (VL) may comprise an amino acid sequence having the sequence of SEQ ID NO: 50, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions, wherein the light chain variable region (VL) comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0173] In one example, the heavy chain variable region (VH) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 49; and / or the light chain variable region (VL) may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 50. In one example, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 49; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 50. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0174] In examples where the heavy chain variable region (VH) has the amino acid sequence of SEQ ID NO:49, the heavy chain variable region (VH) may be encoded by the nucleic acid sequence of SEQ ID NO: 50, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0175] In examples where the light chain variable region (VL) has the amino acid sequence of SEQ ID NQ:50, the light chain variable region (VL) may be encoded by the nucleic acid sequence of SEQ ID NO: 52, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0176] In one example, the antibody or antigen binding fragment may comprise a heavy chain variable region (VH) with the nucleic acid sequence of SEQ ID NO: 51 and / or a light chain variable region (VL) with the nucleic acid sequence of SEQ ID NO: 52.

[0177] Nucleic acids provided by the invention are discussed elsewhere herein in more detail.

[0178] For the avoidance of doubt, the antibody or antigen binding fragment may further comprise constant domains. The invention is not limited to specific constant domains, and encompasses any appropriate constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.

[0179] An example of a specific heavy chain amino acid sequence that includes a heavy chain variable region (VH) described herein with an appropriate constant domain is shown in SEQ ID NO: 53. An example of a specific light chain amino acid sequence that includes a light chain variable region (VL) described herein with an appropriate constant domain is shown in SEQ ID NO: 54. Appropriate functional variants of SEQ ID NO:53 and SEQ ID NO: 54 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 54, wherein the variant amino acid sequences retain the ability to selectively bind to ALQAGASQ when part of an antibody or antigen binding fragment described herein). In other words, a functional heavy chain and a functional light chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:53 or SEQ ID NO: 54 respectively is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO: 53 and / or SEQ ID NO: 54 may all be in regions of the heavy chain or light chain that do not form CDRs (i.e. the variant heavy chain may have the CDRs of SEQ ID NO: 43, SEQ ID NO: 44 and / or SEQ ID NO: 45, and / or the variant light chain may have the CDRs of SEQ ID NO: 46, SEQ ID NO: 47 and / or SEQ ID NO: 48, and still have 25% (or less) sequence variability compared to SEQ ID NO: 53 or 54 respectively). In other words, the sequence of the CDRs of SEQ ID NO: 53 and / or SEQ ID NO: 54 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 53 or SEQ ID NO: 54).

[0180] As an example, the heavy chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 53, wherein the heavy chain variable region (VH) of said heavy chain comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or the light chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 54, wherein the light chain variable region (VL) of said light chain comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0181] In examples where the heavy chain has the amino acid sequence of SEQ ID NO: 53, the heavy chain may be encoded by the nucleic acid sequence of SEQ ID NO: 54, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0182] In examples where the light chain has the amino acid sequence of SEQ ID NO:54, the light chain may be encoded by the nucleic acid sequence of SEQ ID NO: 56, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). In one example, the antibody or antigen binding fragment may comprise a heavy chain with the nucleic acid sequence of SEQ ID NO: 55 and / or a light chain with the nucleic acid sequence of SEQ ID NO: 56.

[0183] In one example, the heavy chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 53; and / or the light chain may comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 54. In one example, the antibody or antibody fragment may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 53; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 54. In these examples, the heavy chain variable region (VH) may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or the light chain variable region (VL) may comprise a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0184] In one example, the antibody may be a rabbit antibody or antigen binding fragment. It may be monoclonal antibody (e.g. a monoclonal rabbit antibody) or antigen binding fragment.

[0185] Nucleic acid compositions

[0186] A nucleic acid composition encoding an antibody or antigen binding fragment described herein is also provided.

[0187] The nucleic acid composition provided herein comprises one or more nucleic acid sequences that encode an antibody or antigen binding fragment described herein.

[0188] For instance, the nucleic acid composition may comprise (a) a nucleic acid sequence that encodes part of an antibody or antigen binding fragment (e.g. a heavy chain variable region (VH) with the specified features described herein); and (b) a nucleic acid sequence that encodes another part of an antibody or antigen binding fragment (e.g. a light chain variable region (VL) with the specified features described herein). The encoded components may form an antibody or antigen specific binding protein that selectively binds to ALQAGASQ, for instance wherein ALQAGASQ is located at the C-terminus of a polypeptide (e.g. wherein the ALQAGASQ is part of a VAMP fragment that is present within the polypeptide, such as a VAMP1 , a VAMP 2 or a VAMP3 fragment).

[0189] The nucleic acid sequences of (a) and (b) above may be distinct nucleic acid sequences within the nucleic acid composition. The components of the antibody or antigen binding fragment may therefore be encoded by two (or more) nucleic acid sequences (with distinct nucleotide sequences) which, together, encode all of the components of the antibody or antigen binding fragment. In other words, some of the components may be encoded by one nucleic acid sequence in the nucleic acid composition, and others may be encoded by another (distinct) nucleic acid sequence within the nucleic acid composition.

[0190] Alternatively, the nucleic acid sequences of (a) and (b) may be part of a single nucleic acid sequence. The components of the antibody or antigen binding fragment may therefore all be encoded by a single nucleic acid sequence (for example with a single open reading frame, or with multiple (e.g. 2 or more, three or more etc.) open reading frames).

[0191] The nucleic acid sequences described herein (e.g. the nucleic acid sequences which form part of a nucleic acid composition described herein) may be codon optimized for expression in a host cell, for example they may be codon optimized for expression in a human cell. Codon optimization is a well-known method in the art for maximizing expression of a nucleic acid sequence in a particular host cell.

[0192] In the context of an antibody or antigen binding fragment described herein, a nucleic acid sequence or a nucleic acid composition encoding an antibody or antigen binding fragment may be codon optimized for expression in a host cell such as a CHO cell, a HEK cell or modified version or a derivative thereof. Several suitable cells for expressing antibodies or antigen binding fragments are known in the art and may be selected by a person of skill in the art.

[0193] The nucleic acid sequences described herein encoding a polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors. The nucleotide sequences described herein may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R et al (Science (1988) 239, pp 487-491).

[0194] The nucleotide sequences described herein may be a mix of genomic and exogenous origin in accordance with standard techniques. For example, the nucleotide sequence may be generated using gene editing techniques such as CRISPR / Cas9.

[0195] The term “nucleic acid molecule” or "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). A “nucleic acid composition” as described herein comprises one or more nucleic acid molecules. The nucleotide sequence(s) may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or antisense strand. The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g. mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.

[0196] The nucleic acid molecules described herein can be single-stranded or double-stranded, but preferably are double-stranded DNA, more preferably cDNA for the coding sequence. In a preferred embodiment, the nucleotide sequence per se encoding a polypeptide having the specific properties as defined herein does not cover the native nucleotide sequence in its natural environment when it is linked to its naturally associated sequence(s) that is / are also in its / their natural environment. For instance, the nucleic acid sequence or nucleic acid composition may be isolated. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence" or “non-naturally occurring sequence”. In this regard, the term "native nucleotide sequence" or “naturally occurring sequence” means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. Thus, the polypeptide of the present invention can be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.

[0197] In some examples, the polypeptide is not a native polypeptide. In this regard, the term "native polypeptide" or “naturally occurring polypeptide” means an entire polypeptide that is in its native environment and when it has been expressed by its native nucleotide sequence. Typically, the nucleotide sequence encoding polypeptides having the specific properties as defined herein is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

[0198] As used herein, the term “recombinant” refers to a biomolecule, for example a gene or a protein that (1) has been removed from its naturally occurring (native) environment, (2) is not associated with all or a portion of a nucleic acid molecule or protein as it is found in nature, (3) is operatively linked to a polynucleotide or polypeptide which it is not linked to in nature, or (4) does not occur in nature.

[0199] As discussed above, a nucleic acid composition encoding an antibody or antigen binding fragment described herein is provided.

[0200] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 9; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 10. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 8.

[0201] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 13; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 14. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 8. Further, the heavy chain may comprise the amino acid sequence of SEQ ID NO: 11 ; and / or the light chain may comprise the amino acid sequence of SEQ ID NO: 12.

[0202] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 23; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 24. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 21 ; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 22.

[0203] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 27; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 28. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 21 ; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 22. Further, the heavy chain may comprise the amino acid sequence of SEQ ID NO: 25; and / or the light chain may comprise the amino acid sequence of SEQ ID NO: 26.

[0204] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 37; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 38. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 35; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 36.

[0205] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 41 ; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 42. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO:35; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 36. Further, the heavy chain may comprise the amino acid sequence of SEQ ID NO: 39; and / or the light chain may comprise the amino acid sequence of SEQ ID NO: 40.

[0206] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 51 ; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 52. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO: 49; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 50.

[0207] In one example, the nucleic acid composition comprises a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 55; and / or a nucleotide sequence having at least 80% sequence identity to, comprising or consisting of SEQ ID NO: 56. In these examples, the encoded antibody or antigen binding fragment may comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and / or a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48. Further, the heavy chain variable region (VH) may comprise the amino acid sequence of SEQ ID NO:49; and / or the light chain variable region (VL) may comprise the amino acid sequence of SEQ ID NO: 50. Further, the heavy chain may comprise the amino acid sequence of SEQ ID NO: 53; and / or the light chain may comprise the amino acid sequence of SEQ ID NO: 54.

[0208] Nucleic acid sequences relevant in the context of the antibodies or antigen binding fragments provided herein are also described elsewhere herein. Further, nucleic acid sequences relevant in the context of the kits and methods described herein are discussed elsewhere herein.

[0209] The nucleic acid compositions or nucleic acid sequence(s) described herein may be present within any appropriate host cell (thereby generating a genetically modified cell).

[0210] Vector systems

[0211] A vector system which includes a nucleic acid composition encoding an antibody or antigen binding fragment as described herein is also provided.

[0212] The vector system may have one or more vectors. As discussed previously, the antibody or antigen binding fragments that are encoded by the nucleic acid composition may be encoded by one or more nucleic acid sequences in the nucleic acid composition. In examples where all of the antibody or antigen binding fragment components are encoded by a single nucleic acid sequence, the nucleic acid sequence may be present within a single vector (and thus the vector system described herein may comprise of one vector only). In examples where the antibody or antigen binding fragment components are encoded by two or more nucleic acid sequences (wherein the plurality of nucleic acid sequences, together, encode all of the components of the antibody or antigen binding fragment) these two or more nucleic acid sequences may be present within one vector (e.g. in different open reading frames of the vector), or may be distributed over two or more vectors. In this example, the vector system will comprise a plurality of distinct vectors (i.e. vectors with different nucleotide sequences).

[0213] Accordingly, in one example, a vector system is provided, comprising a nucleic acid composition described herein.

[0214] As used herein, the term “vector” or “construct” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been operably linked. The terms “vector” and “construct” are used interchangeably herein. The vector can be capable of autonomous replication or it can integrate into a host DNA. The vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers. The vector can be a nucleic acid molecule in the form of a plasmid, a bacteriophage or a cosmid. Preferably the vector is suitable for expression in a cell (i.e. the vector is an “expression vector”).

[0215] In the context of a vector system which includes a nucleic acid composition encoding an antibody or antigen binding fragment as described herein, the vector system is suitable for expression in a CHO cell, a HEK cell or a modified version or a derivative thereof for instance.

[0216] Preferably the (expression) vector or the vector system provided herein is capable of propagation in a host cell and is stably transmitted to future generations.

[0217] "Operably linked" as used herein, refers to a single or a combination of the below-described control elements together with a coding sequence in a functional relationship with one another, for example, in a linked relationship so as to direct expression of the coding sequence.

[0218] "Regulatory sequences" as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, Shine Dalgarno sequences, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites, RNA transporting signals or sequences important for UV-light mediated gene response. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and / or inducible sequences.

[0219] "Promoter", as used herein, refers to the nucleotide sequences in DNA or RNA to which RNA polymerase binds to begin transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. Preferably the promoter is selected from SV40, CMV, Actin, EF1 alpha, UB, RCV, PGK, CAG, MMLV-LTR or CMV-LTR hybrid promoters.

[0220] “Transcriptional terminator” as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.

[0221] “Translational control element”, as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are T7 or T3 ribosome binding sites.

[0222] "Restriction enzyme recognition site" as used herein, refers to a motif on the DNA recognized by a restriction enzyme.

[0223] "Selectable marker" as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene. Generally this may be a protein that confers resistance to an antibiotic such as ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. Further examples of antibiotics are Penicillins; Ampicillin HCI, Ampicillin Na, Amoxycillin Na, Carbenicillin sodium, Penicillin G, Cephalosporins, Cefotaxim Na, Cefalexin HCI, Vancomycin, Cycloserine. Other examples include Bacteriostatic Inhibitors such as: Chloramphenicol, Erythromycin, Lincomycin, Tetracyclin, Spectinomycin sulfate, Clindamycin HCI, Chlortetracycline HCI.

[0224] The design of the expression vector or vector system depends on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including an antibody or antigen binding fragment, encoded by nucleic acids (e.g. nucleic acid compositions) as described herein.

[0225] Preferably the vector or vector system comprises those genetic elements which are necessary for expression of the polypeptides described herein by a host cell (e.g. an antibody or antigen binding fragment described herein). The elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.

[0226] Expression vectors of the invention can be standard expression vectors such as pCDNA3, pIRES-NEO or retroviral vectors such as pQCXIP, pQCXIN, pQCXIG, pLXlN, pBMN, pBABE- hygro, and pBABE-puro.

[0227] The terms “expression vector”, “expression construct”, “construct” and “vector” are used interchangeably herein.

[0228] Preferably, the expression vector or vector system is a high-copy-number expression vector; alternatively, the expression vector is a low -copy-number expression vector. A person of skill in the art will be aware of the molecular techniques available for the preparation of expression vectors, and thus vector systems.

[0229] The nucleic acid molecule or nucleic acid composition for incorporation into an expression vector or a vector system of the invention, as described above, can be prepared by synthesizing nucleic acid molecules using mutually priming oligonucleotides and the nucleic acid sequences described herein.

[0230] A number of molecular techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0231] In an alternative embodiment, synthetic linkers containing one or more restriction sites are used to operably link the nucleic acid molecule to the expression vector. In one embodiment, the nucleic acid molecule is generated by restriction endonuclease digestion. Preferably, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3'-single-stranded termini with their 3'-5'- exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a nucleic acid molecule carrying polymeric linker sequences at its ends. These nucleic acid molecules are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the nucleic acid molecule.

[0232] Alternatively, a vector comprising ligation-independent cloning (LIC) sites can be employed. The required PCR amplified nucleic acid molecule can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, Nucl. Acid. Res. 18, 6069-6074, (1990), Haun, et al, Biotechniques 13, 515-518 (1992).

[0233] In order to isolate and / or modify the nucleic acid molecule or nucleic acid composition of interest for insertion into the chosen plasmid, it is preferable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.

[0234] In a preferred embodiment a nucleic acid molecule or nucleic acid composition for incorporation into an expression vector or vector system of the invention, is prepared by the use of the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491 , using appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In a preferred embodiment the amplification primers contain restriction endonuclease recognition sites which allow the amplified sequence product to be cloned into an appropriate vector.

[0235] Preferably, the nucleic acid molecule or nucleic acid composition is obtained by PCR and introduced into an expression vector or vector system using restriction endonuclease digestion and ligation, a technique which is well known in the art. More preferably, the nucleic acid molecule or nucleic acid composition is introduced into an expression vector such as pCDNA3, pIRES-NEO or a retroviral vector such as pQCXIP, pQCXIN, pQCXIG, pLXlN, pBMN, pBABE- hygro, or pBABE-puro.

[0236] The expression vectors or vector systems of the invention can contain a single copy of the nucleic acid molecule described previously, or multiple copies of the nucleic acid molecule described previously.

[0237] The nucleic acid compositions and / or vector systems described herein may be present within any appropriate host cell (thereby generating a genetically modified cell).

[0238] Host cells

[0239] Host cells are also provided herein.

[0240] A host cell comprising a nucleic acid composition encoding an antibody or antigen binding fragment described herein, or a vector system comprising a nucleic acid composition encoding an antibody or antigen binding fragment as described herein, is also provided.

[0241] The term "host cell" in relation to the present invention includes any cell that comprises a nucleotide sequence, nucleic acid composition, vector or vector system according to the present invention. Preferably the nucleotide sequence or nucleic acid composition is incorporated in the genome of the cell. Accordingly, the host cell may comprise a nucleic acid or a nucleic acid composition encoding an antibody or antigen binding fragment described herein, or a vector or vector system comprising a nucleic acid composition encoding an antibody or antigen binding fragment as described herein.

[0242] The terms “host cell”, “genetically modified cell” and “recombinant host cell” are used interchangeably. These terms do not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment, but refer to a genetically altered (e.g. transformed or transfected) cell. The terms refer to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0243] The genetically modified cell may be a eukaryotic cell or a prokaryotic cell. A person of skill in the art would readily be able to identify suitable cells.

[0244] In the context of a host cell comprising a nucleic acid composition encoding an antibody or antigen binding fragment described herein, or a vector system comprising a nucleic acid composition encoding an antibody or antigen binding fragment as described herein, the genetically modified cell may be a CHO cell, a HEK cell or a modified version or a derivative thereof.

[0245] The host cell genome may be altered to generate the nucleic acid sequence or nucleic acid composition of the invention, using any suitable means e.g. gene editing techniques that introduce a nucleic acid sequence encoding an antibody or antigen binding fragment. Alternatively or additionally, host cells may be transformed, infected or transfected with an expression vector or vector system of the invention using standard techniques known in the art.

[0246] A host cell transformed, infected or transfected with a nucleic acid , a nucleic acid composition or a vector, or vector system described herein can be used to produce (i.e. express) a polypeptide as provided herein (e.g. an antibody or antigen binding fragment described herein). The nucleic acid molecule, nucleic acid composition, vector and / or vector system of the present invention can be introduced into cells by any convention method known in the art, for instance by conventional transformation, transfection or transduction techniques. “Transformation”, “transfection” and “transduction” refer to techniques for introducing foreign nucleic acids into a cell. The specific method used typically depends on both the type of vector and the cell. Said techniques encompass methods such as electroporation, microinjection, gene gun delivery, transduction with retroviral, lentiviral or adeno-associated vectors, lipofection, superfection etc, and also include, but are not limited to calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, chemoporation or electroporation. Appropriate methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known in the art.

[0247] Techniques known in the art for the transformation, transfection or transduction of cells are disclosed in for example, Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637-646.

[0248] Successfully transformed, transfected or transduced cells (or genetically modified cells), that is, those cells containing the nucleic acid molecule, nucleic acid composition, vector or vector system of the present invention, can be identified by techniques that are well known in the art. For example, the nucleic acid molecule, nucleic acid composition, vector or vector system may be introduced using CRISPR technology. Insertion of the nucleic acid sequences at a specific locus by engineering with CRISPR / Cas9 and homologous directed repair (HDR) or non- homologous end joining (NHEJ) is therefore encompassed. Other conventional methods such as transfection, transduction or transformation of the cell may also be used. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

[0249] In a preferred embodiment the invention comprises a culture of transformed cells. Preferably the culture is clonally homogeneous.

[0250] The cell can contain a single copy of an expression vector or vector system described previously, or alternatively, multiple copies of an expression vector or vector system.

[0251] Conjugates A conjugate comprising an antibody or antigen binding fragment described herein conjugated to or recombinantly fused to a diagnostic agent, detectable agent, or therapeutic agent is provided herein.

[0252] In one example, the conjugate comprises a linker, for example between the antibody or antigen binding fragment described herein and the diagnostic agent, detectable agent, or therapeutic agent of the conjugate. Linkers can for example improve folding and stability of the conjugate, expression of the conjugate, or bioactivity of the conjugate.

[0253] An antibody or antigen binding fragment according to the invention can be produced recombinantly. Such a recombinant polypeptide can be recombinantly fused to a diagnostic agent, detectable agent, or therapeutic agent, by for example introducing a nucleotide sequence encoding a diagnostic agent, detectable agent, or therapeutic agent, behind, in front or in the nucleotide sequence encoding the antibody or antigen binding fragment. A person skilled in the art understands that such an introduction would retain the intended purpose and effect of the antibody or antigen binding fragment described herein.

[0254] A nucleotide sequence encoding a diagnostic agent, detectable agent, or therapeutic agent may comprise a linker sequence encoding a linker, thereby recombinantly fusing the antibody or antigen binding fragment according to the invention to the agent via the linker. A linker may be an amino acid sequence, a peptide, or a polypeptide and may be linear or circular. Various linkers are known in the art and can be used for the present invention.

[0255] As used herein, a “diagnostic agent” is a substance that can be used to aid in the diagnosis or monitoring of a disease. It can be administered in vivo or to a subject and it can be used to determine the location or status of a disease causing process. A” detectable agent” as used herein is a substance that can be used to determine the presence of a desired molecule, such as a polypeptide according to the invention, in a sample, in vivo, or in a subject. Many different diagnostic or detectable agents are known in the art, examples of which are inorganic or organic compounds, radioactive tracers, dyes, proteins, and peptides. Tracers in general and other tracers such as stable isotopes or radioisotopes are also examples of diagnostic or detectable agents. In one example, a diagnostic or detectable agent is selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photoactive agent. Many different labels are known in the art, and a label typically comprises or consists of a detectable substance which is attached to the molecule to be detected. As used herein, a “therapeutic agent” is a substance used to treat, cure, prevent, or ameliorate a disease or to promote well-being. Many different therapeutic agents are known in the art, examples of which are small molecules, oligonucleotides, radionuclides, prodrugs, cytotoxic or cytostatic agents such as chemotherapeutic agents or toxins, photoactive agents, anti- angiogenic agents, enzymes, immunomodulators, cytokines, growth factors, chemokines, hormones, or recombinant proteins.

[0256] An antibody or antigen binding fragment according to the invention can also be conjugated to a diagnostic agent, detectable agent, or therapeutic agent. Various methods and techniques are known in the art to conjugate various agents to polypeptides, which can also be used for producing the polypeptide according to the invention conjugated to a diagnostic agent, detectable agent, or therapeutic agent. Conjugation can be done post-translationally by chemical crosslinking of the polypeptide according to the invention to the agent by using crosslinking reagents, thereby generating a covalent or a non-covalent bond between the polypeptide and the agent. Crosslinking reagents preferably have similar functions as linkers. Various crosslinking reagents and methods are known in the art and can be used for the present invention.

[0257] Kits, Uses and Methods

[0258] A kit for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide is provided, the kit comprising an antibody or antigen binding fragment described herein, a nucleic acid composition encoding an antibody or antigen binding fragment described herein, a vector system comprising a nucleic acid composition encoding an antibody or antigen binding fragment described herein, a host cell comprising said nucleic acid composition or said vector system and / or a conjugate according described herein.

[0259] As would be clear to the skilled person, the kit includes reagents suitable for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide. Appropriate C-terminus ALQAGASQ polypeptides that could be bound by the reagents in the kit are described elsewhere herein and the definitions provided elsewhere herein apply equally here.

[0260] In an example, the kit comprises an antibody or antigen binding fragment. The antibodies or antigen binding fragments provided herein are capable of binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide. They are therefore reagents suitable for binding to ALQAGASQ in the context of the kits provided herein. In particular, the kit may comprise an antibody or antigen binding fragment, comprising:

[0261] (i) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6; (ii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20; (iii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34; or (iv) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

[0262] In an example, the kit further comprises a:

[0263] (i) a VAMP polypeptide; or (ii) a VAMP reporter polypeptide, such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity.

[0264] VAMP polypeptides are well known in the art, e.g. VAMP1 , VAMP2 or VAMP3. See for example SEQ ID NO: 61 , SEQ ID NO:63 and SEQ ID NO:65.

[0265] A “VAMP reporter polypeptide” refers to a polypeptide that includes VAMP1 , VAMP2 and / or VAMP3 sequences that are recognised and cleaved by BoNT / B and / or TeNT. Thus, a VAMP reporter polypeptide is capable of being cleaved by either or both of BoNT / B or TeNT. VAMP reporter polypeptides comprise the core cleavage site of a VAMP protein (e.g. the GASQFESS or GASQFETS sequence shown in Table 1). When cleaved by BoNT / B and / or TeNT, a cleaved polypeptide is generated from the VAMP reporter polypeptide, where the cleaved polypeptide comprises the ALQAGASQ sequence located at its C-terminus. In an example, a VAMP reporter polypeptide may comprise the sequence of SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 66.

[0266] In one example, the VAMP reporter polypeptide comprises amino acids 60-87 of VAMP2 (i.e. the sequence of LSELDDRADALQAGASQ*FETSAAKLKRK (SEQ ID NO: 75)), where the star identifies the cleavage site. In another example, the VAMP reporter polypeptide comprises amino acids 40-87 of VAMP2 (i.e. the sequence of DEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQ*FETSAAKLKRK (SEQ ID NO:76)). Again, the star identifies the cleavage site. The above sequences include VAMP1 , VAMP2 and / or VAMP3 sequences that are recognised and cleaved by BoNT / B and / or TeNT

[0267] An example of a VAMP reporter polypeptide that is particularly relevant in the context of the present invention is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity. This VAMP reporter molecule is capable of being cleaved by BoNT / B and TeNT (and modified versions thereof, including chimeras, that retain neurotoxin activity). Cleavage of the VAMP reporter polypeptide generates two cleavage products; a first fragment that comprises the N-terminal polypeptide domain having luciferase activity and a portion of the polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity (wherein the portion of the polypeptide having VAMP 1 , VAMP2 or VAMP3 activity has the sequence of SEQ ID NO:59 at its C- terminus); and a second fragment comprising the remaining portion of the polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity. The N-terminal polypeptide domain having luciferase activity reduces or prevents intracellular degradation of the first fragment. The VAMP fragment generated as a result of neurotoxin mediated cleavage of said polypeptide can advantageously be detected using an antibody or antigen binding fragment described herein. Such polypeptides are described in detail in WO 2018 / 150177 which is hereby incorporated by reference in it’s entirety. A polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity is also described in more detail below.

[0268] In an example, the kit further comprises a nucleic acid encoding: (i) a VAMP polypeptide; or (ii) a VAMP reporter polypeptide, such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity.

[0269] Nucleic acids, in other words, nucleic acid sequences or nucleic acid molecules are described generally elsewhere herein and the definitions provided elsewhere herein apply equally here.

[0270] In some examples, the nucleic acid encoding (i) the VAMP polypeptide or (ii) the VAMP reporter polypeptide (such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity) is part of an expression vector.

[0271] Expression vectors are described generally elsewhere herein and the definitions provided elsewhere herein apply equally here.

[0272] In some examples, the nucleic acid encoding (i) a VAMP polypeptide or (ii) a VAMP reporter polypeptide (such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity) is within a host cell. Examples of suitable host cells in the context of these particular nucleic acids include but are not limited to a SiMa neuroblastoma cell, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, immortalised neuron, BE(2)-C cell and primary neuron.

[0273] In some examples, the kit is for detecting a polypeptide, wherein ALQAGASQ (SEQ ID NO: 59) is located at the C-terminus of the polypeptide. In other words, the kit may be for detecting a C-terminus ALQAGASQ polypeptide as described elsewhere herein.

[0274] In some examples, the kit is for detecting tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof. In some examples, the kit comprises instructions for use.

[0275] As used herein “neurotoxin activity” refers to the ability of any one of naturally occurring tetanus, or botulinum type B neurotoxin(s) to cleave their substrate (e.g. a VAMP polypeptide or a VAMP reporter polypeptide). Naturally occurring neurotoxins and their respective neurotoxin activity are well known in the art, and are described elsewhere in more detail (see Table 1 and also for example Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013).

[0276] For example, “tetanus neurotoxin activity” refers to the cleavage of VAMP1 (or a VAMP1 reporter polypeptide) at the cleavage site GASQ78FESS (or a VAMP2 reporter polypeptide) at the cleavage site GASQ76FETS, and / or cleavage of VAMP3 (or a VAMP3 reporter polypeptide) at the cleavage site GASQ59FETS. For VAMP reporter sequences, the cleavage site will have the amino acid sequence set out above, but the position within the polypeptide may differ (e.g. in a VAMP2 reporter polypeptide, the cleavage site of GASQFETS may be present, but the Q amino acid may not be at position 76 in the reporter polypeptide).

[0277] For example, “botulinum type B neurotoxin activity” refers to the cleavage of VAMP1 (or a VAMP1 reporter polypeptide) at the cleavage site GASQ78FESS, cleavage of VAMP2 (or a VAMP2 reporter polypeptide) at the cleavage site GASQ76FETS, and / or cleavage of VAMP3 (or a VAMP3 reporter polypeptide) at the cleavage site GASQ59FETS. For VAMP reporter sequences, the cleavage site will have the amino acid sequence set out above, but the position within the polypeptide may differ (e.g. in a VAMP2 reporter polypeptide, the cleavage site of GASQFETS may be present, but the Q amino acid may not be at position 76 in the reporter polypeptide).

[0278] Modified versions (e.g. artificially modified, recombinants, chimeras, toxoids etc) of the tetanus and / or botulinum type B neurotoxin(s) may retain “neurotoxin activity” (i.e. their ability to cleave VAMP1 , VAMP2 and / or VAMP3 in the manner shown in Table 1). The invention can therefore be used to detect whether such modified neurotoxins retain “neurotoxin activity”. Such uses and methods are encompassed by the invention.

[0279] The diversity of botulinum neurotoxins is summarized in, for example, [Research in Microbiology, Volume 166, Issue 4, May 2015, Pages 303-317, Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II, Andrew T. Carter, Michael W. Peck], The invention can thus be used to detect the neurotoxin activity (and / or presence) of the naturally occurring forms of these neurotoxins, as well as the neurotoxin activity of chimeras, or artificially modified forms thereof. Advantageously, the invention therefore provides a means for testing modified forms of these neurotoxins for increased or decreased neurotoxin activity.

[0280] The present invention is based on the surprising generation and identification of a series of antibodies that selectively bind a neopeptide (SEQ ID NO: 59). The neopeptide of SEQ ID NO: 59 may be generated from VAMPs 1-3 when they are cleaved by either BoNT / B or Tetanus toxin (TeNT) (or versions thereof).

[0281] Accordingly, use of an antibody or antigen binding fragment, a nucleic acid composition, a vector system, a host cell, a conjugate and / or a kit described herein for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide is provided. The polypeptide may be any C-terminus ALQAGASQ polypeptide described herein.

[0282] In some examples, the antibody or antigen binding fragment, nucleic acid, expression vector, host cell, conjugate and / or kit may be used for detecting:

[0283] (a) ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide; and / or

[0284] (b) tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0285] In examples where the antibody or antigen binding fragment, nucleic acid, expression vector, host cell, conjugate and / or kit may be used for detecting tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof, the neurotoxin activity may be detected in a test sample.

[0286] As used herein, “a test sample” may be any sample (with a known, unknown or partially known composition) that is to be tested for the presence of neurotoxin activity, wherein the neurotoxin activity is selected from the group consisting of: tetanus neurotoxin activity and botulinum type B neurotoxin activity, or any combination thereof.

[0287] A “test sample” may comprise drug product (e.g. a vaccine for administration to a subject, such as a toxoid, where a toxoid a chemically modified toxin from a pathogenic microorganism (e.g. tetanus or botulinum), which is no longer toxic but is still antigenic and can be used as a vaccine). In such cases, the invention may be used to detect (unwanted) residual neurotoxin activity. A suitable test sample may also comprise a food sample, a clinical sample or an environmental sample (wherein the food sample, clinical sample or environmental sample may have been contaminated with Clostridium, and / or may contain a neurotoxin), or any combination thereof. In such cases, the invention may be used to detect the presence of contamination.

[0288] Suitable test samples also comprise a tetanus neurotoxin, botulinum neurotoxin type B (for example a test sample from a known batch of neurotoxin, optionally manufactured for therapeutic or non-therapeutic use), or any combination thereof. In such cases, the invention may be used to detect or determine the potency of the neurotoxin. For the avoidance of doubt, the neurotoxins in the test sample may be naturally occurring neurotoxins, or may be modified (e.g. artificially modified, including chimeras) versions thereof.

[0289] The test sample may comprise:

[0290] (a) a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof;

[0291] (b) a tetanus toxoid, or a botulinum toxoid or a combination thereof; and / or

[0292] (c) tetanus neurotoxin, botulinum neurotoxin type B, or a combination thereof.

[0293] Any suitable means for detecting neurotoxin activity may be used. Several standard techniques may be used to detect cleavage of the VAMP polypeptides or VAMP reporter polypeptides described herein. Exemplary techniques include, but are not limited to immunoblotting (also known as western blotting), sandwich ELISA assays or live cell imaging. Such methods or routine in the field and the details of how to perform each is well known (see for example [Specificity of botulinum protease for human VAMP family proteins. Yamamoto H, et al. Microbiol Immunol, 2012 Apr. PMID 22289120; Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin. Chen S, et al. J Biol Chem, 2008 Jul 25. PMID 18511417]).

[0294] Detection of neurotoxin activity may be performed in vitro, in the absence of a genetically modified cell. For example, a VAMP polypeptide or VAMP reporter polypeptide as described herein may be contacted with a neurotoxin (or test sample as described below) and the presence of cleaved product may be determined (e.g. by ELISA or western blotting). The development of such detection methods is well within the routine capabilities of a person of skill in the art.

[0295] Detection of neurotoxin activity may alternatively be performed using a host cell, as described elsewhere herein. Advantageously, a host cell provides a means for testing all three stages of neurotoxin action, namely binding to the cell surface, delivery of the neurotoxin peptidase into the cell cytosol and neurotoxin-induced cleavage of its substrate (in this case a polypeptide of the invention). The presence of cleaved product may be determined (e.g. by ELISA or western blotting of a cell lysate) by determining binding of an antibody or antigen binding fragment as described herein to the cleaved product.

[0296] In a preferred method, neurotoxin activity is detected (or determined) using a host cell, wherein the cell is cultured under conditions that allow for expression of a VAMP polypeptide or a VAMP reporter polypeptide, such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide having VAMP1 , VAMP2 or VAMP3 activity. In this example, the host cell may be contacted with a neurotoxin (or test sample) and the presence of cleaved product may be determined (e.g. by ELISA, western blotting or live cell imaging). The development of such methods is also well within the routine capabilities of a person of skill in the art.

[0297] Accordingly, in one aspect of the invention, a method of detecting neurotoxin activity in a test sample is provided, the method comprising:

[0298] (a) providing a host cell that comprises a nucleic acid encoding:

[0299] (i) a VAMP polypeptide; or

[0300] (ii) a VAMP reporter polypeptide, such as a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity;

[0301] (b) culturing the host cell under conditions that allow for expression of the polypeptide;

[0302] (c) culturing the host cell of (b) in the presence of the test sample under conditions that allow for neurotoxin-induced cleavage of the polypeptide; and

[0303] (d) using an antibody or antigen binding fragment according to the invention to determine the level of neurotoxin-induced cleavage of the polypeptide, wherein detection of neurotoxin- induced cleavage of the polypeptide is indicative of neurotoxin activity; wherein the neurotoxin activity is tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0304] In one example, the host cell is a genetically modified host cell host cell that comprises a nucleic acid encoding: (i) the VAMP polypeptide; or (ii) the polypeptide comprising an N- terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity. In other words, in this example, the nucleic acid encoding (i) or (ii) is not endogenous to the host cell. Optimising culture conditions to allow for expression of the polypeptide is well within the routine capabilities of a person of skill in the art. In addition, identifying whether or not the chosen culture conditions allow for polypeptide expression is also routine, and the amount of polypeptide expression may be detected using standard techniques such as ELISA or western blotting.

[0305] Cells are grown or cultured in the manner with which the skilled worker is familiar, depending on the host cell. The culture medium (also called “growth medium”, “medium” or “media” herein) to be used must suitably meet the requirements of the cells in question. Preferably, the culture media is sufficient to support the growth of the host cell. Descriptions of suitable culture media for various cells can be found in the textbook “Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition, R. Ian Freshney, 2010 John Wiley & Sons, Inc.” By way of example only, SiMa neuroblastoma cells are preferably cultured under the following conditions: SiMa cells (from DSMZ cell collection) are grown in RPMI media supplemented with 10% Fetal Bovine Serum. For differentiation, plates were pre-coated with 10 pg / ml laminin. SiMa cells were seeded at a density of 1 x104cells per well in 96-well plates or 2 x104cells per well in 48-well plates and incubated for 72 hours in differentiation medium (RPMI, B27 or GS21 supplement, 1 mM HEPES and 1 % NEAA with 10 pM AT- retinoic acid).

[0306] Cells may be grown in a liquid medium comprising one or more of a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, inorganic salts, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 25°C and 40°C, while gassing in carbon dioxide.

[0307] Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, carbon dioxide, sodium bicarbonate, bicarbonate, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Preferably, the carbon source is carbon dioxide. Alternatively, the carbon source is bicarbonate. The addition of mixtures of a variety of carbon sources may also be advantageous.

[0308] Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

[0309] Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0310] Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.

[0311] Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

[0312] Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

[0313] The culture media used may also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

[0314] The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not.

[0315] An overview of known cultivation methods can be found in the textbook by Chmiel (BioprozeBtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, BrunswickA / Viesbaden, 1994)).

[0316] All media components are sterilized, either by heat (20 min at 1.5 bar and 121°C) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

[0317] The culture temperature will vary depending on the particular experiment and the host cell. The culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40°C, more preferably at from 25 to 37 °C and may be kept constant or may be altered during the experiment. By way of example only, for SiMa neuroblastoma cells, the temperature is preferably at from 25 to 40 °C and more preferably at 37°C.

[0318] The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of vector it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C. The culture is continued until expression of the polypeptide has occurred. This aim is normally achieved within 6 to 96 hours.

[0319] The culture conditions in step (c) are in the presence of the test sample and are such that they allow for neurotoxin-induced cleavage of the polypeptide. In other words, the culture conditions are such that, if a functional neurotoxin were present in the test sample, at least some neurotoxin-induced cleavage of the polypeptide would occur. Optimising culture conditions to allow for neurotoxin-induced cleavage of the polypeptide is well within the routine capabilities of a person of skill in the art. In addition, identifying whether or not the chosen culture conditions allow for neurotoxin-induced cleavage of the polypeptide is also routine, and the amount of neurotoxin-induced cleavage of the polypeptide may be detected using standard techniques such as ELISA or western blotting (as discussed elsewhere). In one embodiment, culturing steps (b) and (c) are carried out simultaneously. In other words, the test sample may be added to the host cell at the start of cell culture, or may be added later, once polypeptide expression has already commenced. Optimising the time point at which the host cell is cultured in the presence of the test sample is well within the routine capabilities of a person of ordinary skill in the art.

[0320] Optionally, the test sample is provided in the culture medium.

[0321] As described elsewhere, the test sample may comprise a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof. Alternatively, the test sample may comprise a tetanus toxoid, or a botulinum toxoid, or a combination thereof.

[0322] The test sample may comprise tetanus neurotoxin, botulinum neurotoxin type B, or any combination thereof. For the avoidance of doubt, neurotoxins in the test sample may be naturally occurring neurotoxins, or may be modified (e.g. artificially modified, including chimeras) versions thereof.

[0323] The methods provided herein comprise step (d), i.e. using an antibody or antigen binding fragment according to the invention to determine the level of neurotoxin-induced cleavage of the polypeptide, wherein detection of neurotoxin-induced cleavage of the polypeptide is indicative of neurotoxin activity; wherein the neurotoxin activity is tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

[0324] In one example, the method comprises the step of contacting the host cell with the antibody or antigen binding fragment (under conditions that allow for binding of the antibody or antigen binding fragment to a neurotoxin-cleaved polypeptide) after step (C) to determine the level of neurotoxin-induced cleavage of the polypeptide.

[0325] In one example, the method comprises the step of preparing a cell lysate from the host cell after step (c) and contacting the cell lysate with the antibody or antigen binding fragment (under conditions that allow for binding of the antibody or antigen binding fragment to a neurotoxin-cleaved polypeptide) to determine the level of neurotoxin-induced cleavage of the polypeptide.

[0326] The level of neurotoxin-induced cleavage of the polypeptide may be determined (or detected) using any of the suitable standard techniques discussed elsewhere herein (e.g. ELISA, western blotting, live cell imaging etc). As used here, “detection” of neurotoxin-induced cleavage encompasses any level or amount of neurotoxin-induced cleavage that is detectable, e.g. visible, quantifiable etc. By way of example, this includes cleavage of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the substrate (polypeptide) at the point of testing (where the point of testing may be, for example, 6 or more hours after the addition of the test sample to the cells. Other examples of suitable time points (e.g. “point of testing” as used above) after the addition of the test sample to the cells include at least 24, 48 hours or 72 hours.

[0327] “Neurotoxin-induced cleavage” refers to the cleavage of a polypeptide, wherein the cleavage is due to the activity of a neurotoxin (i.e. this does not include cleavage of the polypeptide due to other mechanisms such are non-specific polypeptide degradation or cleavage by other nonneurotoxin enzymes). Neurotoxin-induced cleavage may be identified using standard techniques in the art, such as ELISA or the size of the cleavage products (e.g. on a western blot) to confirm that neurotoxin-induced cleavage has occurred. The detection of neurotoxin- induced cleavage of the polypeptide is indicative of neurotoxin activity (e.g. the level or amount of cleavage is proportional to the level or amount of neurotoxin activity in the test sample). In the context of the invention, the terms “neurotoxin-induced cleavage” and “neurotoxin activity” can be used interchangeably.

[0328] In certain embodiments of the invention, it may be advantageous to use a negative control. For example, the level of neurotoxin induced cleavage of the polypeptide may be determined and then compared to the level of cleavage of the polypeptide in a negative control sample or with a predetermined reference level for cleavage of the polypeptide, wherein an increased level of cleavage in the presence of the test sample compared to the control sample or compared to the predetermined reference level identifies the presence of neurotoxin-induced cleavage. Negative controls include culturing the same cell under the same conditions in the absence of a test sample or in the presence of a sample that is known to lack neurotoxin activity e.g. a heat inactivated test sample.

[0329] Additionally, or alternatively, it may be advantageous to use a positive control (for example to ensure that false negatives are not generated when testing for residual neurotoxin activity of toxoids). An example of a positive control may be culturing the same cell under the same conditions in the presence of a test sample known to comprise functional neurotoxin.

[0330] It is advantageous to use an antibody or antigen fragment of the invention as it binds selectively to the neurotoxin cleavage product of interest. Exemplary VAMP reporter polypeptide: A polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity

[0331] As discussed above, a VAMP reporter polypeptide may be particularly useful in the context of the present invention. An example of such a polypeptide is a polypeptide comprising an N- terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity.

[0332] The term “polypeptide domain” refers to a portion of a polypeptide seguence that can evolve, function and exist independently of the rest of the polypeptide chain. Typically, each domain within a polypeptide may form a compact three-dimensional structure and often can be independently stable and folded.

[0333] The terms “N-terminal” and “C-terminal” are used to describe the relative position of e.g. a domain within a polypeptide. Accordingly, a domain that is “N-terminal” is positioned closer (in relative terms) to the N-terminus than to the C-terminus of the polypeptide. Conversely, a domain that is “C-terminal” is positioned (in relative terms) closer to the C-terminus than to the N-terminus of the polypeptide. As used herein, the term “positioned” refers to the location of the e.g. domain within the linear amino acid seguence of the polypeptide.

[0334] The terms “N-terminal” and “C-terminal” can be used to describe the relative position of two or more domains within a polypeptide. In this context, a domain that is “N-terminal” is positioned closer (in relative terms) to the N-terminus of the polypeptide than a domain that is “C-terminal”. Conversely, a domain that is “C-terminal” is positioned closer (in relative terms) to the C-terminus of the polypeptide than a domain that is “N-terminal”.

[0335] A domain that is “N-terminal” may be, but does not have to be, at the N-terminus of the polypeptide (i.e. it may be, but does not have to be, at the start of the polypeptide terminated by an amino acid with a free amine group). In other words, the first amino acid of an N-terminal domain does not need to be (but may be) the first amino acid of the polypeptide. This means that there may be other amino acids, polypeptide domains (e.g. tags such as HA tags) etc between the N-terminus of the polypeptide and the start of the “N-terminal” domain (provided that the domain is positioned closer to the N-terminus than to the C-terminus of the polypeptide; or when used to describe the relative positions of two or more domains, provided that the domain is positioned closer to the N-terminus than a domain that is “C-terminal”). Likewise, a domain that is “C-terminal” may be, but does not have to be, at the C-terminus of the polypeptide (i.e. it may be, but does not have to be, at the end of the polypeptide terminated by any amino acid with a free carboxyl group). In other words, the last amino acid of a C- terminal domain does not need to be (but may be) the last amino acid of the polypeptide. This means that there may be other amino acids, polypeptide domains etc (e.g. tags) between the C-terminus of the polypeptide and the end of the “C-terminal” domain (provided that the domain is positioned closer to the C-terminus than to the N-terminus of the polypeptide; or when used to describe the relative positions of two or more domains, provided that the domain is positioned closer to the C-terminus than a domain that is “N-terminal”).

[0336] Polypeptides comprising an N-terminal polypeptide domain (A) and a C-terminal polypeptide domain (B) are conventionally written as A-B i.e. N-terminal to C-terminal (left to right). By way of example, a polypeptide comprising an N-terminal enzyme luciferase (e.g. NanoLuc or “NIuc”) domain and a C-terminal VAMP2 domain will be conventionally written as Nluc-VAMP2 (or NlucVAMP2).

[0337] By way of example, the polypeptide described in this section may comprise a HA tag N- terminal to the polypeptide domain having luciferase activity. Other suitable tags are well known in the art and may additionally or alternatively be used.

[0338] A linker may also be present between the domain having luciferase activity and the domain having VAMP1 , VAMP2 or VAMP3 activity e.g. a proline glycine linker may be used. Other suitable linkers are well known in the art and may additionally or alternatively be used.

[0339] A polypeptide domain having “luciferase activity” refers to a polypeptide domain that retains the functional activity of a luciferase enzyme i.e. it is capable of producing bioluminescence by oxidising a photon-emitting substrate, such as luciferin and furimazine [ACS Chem Biol. 2012 Nov 16; 7(11): 1848-1857 ‘Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate’ Hall et al]. As used herein, a polypeptide having “luciferase activity” includes any polypeptide from the luciferase class of oxidative enzymes that produce bioluminescence by oxidising luciferin or furimazine (i.e. it includes any functional luciferase). A person of skill in the art is readily aware of how to identify polypeptide domains with luciferase activity, using routine experiments known in the art. A suitable experiment for identifying functional luciferases is summarised in [Beyond D-luciferin: Expanding the Scope of Bioluminescence Imaging in vivo. Spencer T. Adams, Jr., Stephen C. Miller Curr Opin Chem Biol. 2014; 0: 112-120.] In one embodiment, the polypeptide domain having luciferase activity comprises the amino acid sequence shown in SEQ ID NO: 60, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 60. The term “variant” also encompasses homologues.

[0340] Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 60, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO: 60 may therefore be a conservative amino acid sequence variant of SEQ ID NQ:60, wherein the variant has luciferase activity.

[0341] A summary of the critical and non-critical amino acids in luciferase is provided in [Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. Hall MP, llnch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. ACS Chem Biol. 2012, 7(11): 1848-57], Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO: 60. Homologues of SEQ ID NQ:60 can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0342] A polypeptide having luciferase activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 60, or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NQ:60), or portions or fragments thereof.

[0343] The amino acid sequence shown in SEQ ID NO: 60 is that of the luciferase NanoLuc (sold by Promega). The terms “NanoLuc” and “NLuc” are used interchangeably herein. More details on the NanoLuc luciferase can be found in Hall et al., ACS chem Biol 2012, 7, pg 1848 to 1857.

[0344] Alternative examples of suitable luciferases include the firefly luciferase (FLuc; EC 1.13.12.7) from the firefly Photinus pyralis. Firefly luciferase is a euglobulin protein that catalyses the oxygenation of luciferin using ATP and molecular oxygen to yield oxyluciferin, a highly unstable, singlet-excited compound that emits light upon relaxation to its ground state. A variety of other organisms regulate their light production using different luciferases in a variety of light-emitting reactions (e.g the Jack-o-lantern mushroom Omphalotus olearius, several marine creatures such as the sea pansy (Renilla reniformis, with its luciferase Renilla-luciferin 2-monooxygenase; RLuc), and the luciferase of dinoflagellates). Other examples of luciferases include Modified Firefly luciferase (Ultra-GLo; derived from Photuris pennysylvanica), Click beetle luciferase (CBLuc; derived from Pyrophorus plagiophthalamus), Copepod crustacean luciferase (GLuc; derived from Gaussia princeps) and Ostracod crustacean luciferase (CLuc; derived from Cypridina noctiluca). A review of different luciferases commonly used in the art can be found in Thorne et al., Chem Biol. 2010 Jun 25; 17(6); 646-657.

[0345] As used herein, a polypeptide domain “having VAMP1 , VAMP2 or VAMP3 activity” refers to a polypeptide domain that is capable of functioning as a VAMP1 , VAMP2 or VAMP3 protein. VAMPs are discussed elsewhere herein.

[0346] A polypeptide domain having “VAMP1 activity” refers to a polypeptide domain that (i) retains the functional activity of VAMP1 i.e. it resides in vesicles and is capable of SNARE complex formation and (ii) is capable of being cleaved by at least one of, or at least all of tetanus neurotoxin (TeNT), and botulinum neurotoxin type B (BoNT / B).

[0347] In one embodiment, the polypeptide domain having VAMP1 activity comprises the amino acid sequence shown in SEQ ID NO: 61 , or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:61. The term “variant” also encompasses homologues.

[0348] In one embodiment, the polypeptide domain having VAMP1 activity comprises amino acids 40 to 118 of SEQ ID NO:61 , or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:61. The term “variant” also encompasses homologues.

[0349] Amino acids 40 to 118 of SEQ ID NO:61 represent the core VAMP1 amino acid sequence for BoNT interaction and cleavage (amino acids 40 to 118 are also referred to herein as SEQ ID NO:62).

[0350] Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 61 , or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:61 may therefore be a conservative amino acid sequence variant of SEQ ID NO:61 , wherein the variant has VAMP1 activity.

[0351] A summary of the critical and non-critical amino acids in VAMP1 is provided in [Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15781-6. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Fasshauer D, Sutton RB, Brunger AT, Jahn R ]. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO: 61 . Homologues of SEQ ID NO: 61 can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0352] A polypeptide having VAMP1 activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 61 , or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 61), or portions or fragments thereof.

[0353] The amino acid sequence shown in SEQ ID NO:61 is that of naturally occurring human VAMP1. More details on human VAMP1 can be found in [Specificity of botulinum protease for human VAMP family proteins. Yamamoto H, et al. Microbiol Immunol, 2012 Apr. PMID 22289120],

[0354] A polypeptide domain having “VAMP2 activity” refers to a polypeptide domain that (i) retains the functional activity of VAMP2 i.e resides in vesicles and is capable of SNARE complex formation and (ii) is capable of being cleaved by at least one of, or at least all of tetanus neurotoxin (TeNT), and botulinum neurotoxin type B (BoNT / B),

[0355] In one embodiment, the polypeptide domain having VAMP2 activity comprises the amino acid sequence shown in SEQ ID NO: 63, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 63. The term “variant” also encompasses homologues.

[0356] In one embodiment, the polypeptide domain having VAMP2 activity comprises amino acids 38 to 116 of SEQ ID NO: 63, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 63. The term “variant” also encompasses homologues. Amino acids 38 to 116 of SEQ ID NO: 63 represent the core VAMP2 amino acid sequence for BoNT interaction and cleavage (amino acids 38 to 116 are also referred to herein as SEQ ID NO: 64).

[0357] Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 63, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO: 63 may therefore be a conservative amino acid sequence variant of SEQ ID NO: 63, wherein the variant has VAMP2 activity.

[0358] A summary of the critical and non-critical amino acids in vesicle-associated VAMP2 is provided in [Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15781-6. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Fasshauer D, Sutton RB, Brunger AT, Jahn R], and in [Proc Natl Acad Sci U S A. 2006 May 30;103(22):8378-83. Conformation of the synaptobrevin transmembrane domain. Bowen M, Brunger AT.]. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO: 63. Homologues of SEQ ID NO: 63 can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0359] A polypeptide having VAMP2 activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 63, or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 63), or portions or fragments thereof.

[0360] The amino acid sequence shown in SEQ ID NO: 63 is that of naturally occurring human VAMP2. More details on human VAMP2 can be found in [Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin. Chen S, et al. J Biol Chem, 2008 Jul 25. PMID 18511417],

[0361] A polypeptide domain having “VAMP3 activity” refers to a polypeptide domain that (i) retains the functional activity of VAMP3 i.e. it resides in vesicles and is capable of SNARE complex formation and (ii) is capable of being cleaved by at least one of, or at least all of tetanus neurotoxin (TeNT), and botulinum neurotoxin type B (BoNT / B), In one embodiment, the polypeptide domain having VAMP3 activity comprises the amino acid sequence shown in SEQ ID NO: 65, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 65. The term “variant” also encompasses homologues.

[0362] In one embodiment, the polypeptide domain having VAMP3 activity comprises amino acids 21 to 100 of SEQ ID NO: 65, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 65. The term “variant” also encompasses homologues.

[0363] Amino acids 21 to 100 of SEQ ID NO:3 represent the core VAMP3 amino acid sequence for BoNT interaction and cleavage (amino acids 21 to 100 are also referred to herein as SEQ ID NO:66).

[0364] Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 65, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:65 may therefore be a conservative amino acid sequence variant of SEQ ID NO: 65, wherein the variant has VAMP3 activity.

[0365] A summary of the critical and non-critical amino acids in VAMP3 is provided in [Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15781-6. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Fasshauer D, Sutton RB, Brunger AT, Jahn R], Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO:65. Homologues of SEQ ID NO: 65 can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0366] A polypeptide having VAMP3 activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:65, or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:65), or portions or fragments thereof. The amino acid sequence shown in SEQ ID NO:65 is that of naturally occurring human VAMP3. More details on human VAMP3 can be found in [Nature Reviews Molecular Cell Biology 2, 98-106 (February 2001) SNARE-mediated membrane fusion’ Y. A. Chen & R. H. Scheller],

[0367] A person of skill in the art is readily aware of how to identify polypeptide domains with vesicle- associated VAMP1 , VAMP2 or VAMP3 activity, using routine experiments known in the art. Further, a person of skill in the art is also readily aware of how to identify polypeptide domains that are capable of being cleaved by is capable of being cleaved by at least one of, or at least all of tetanus neurotoxin (TeNT), and botulinum neurotoxin type B (BoNT / B), using routine experiments known in the art. Further details can be found in WO 2018 / 150177. The VAMP1 , VAMP 2 and VAMP 3 amino acid sequences, BoNT interactions and cleavage sites are well known in the art (see for example Yamamoto H, Ida T, Tsutsuki H, Mori M, Matsumoto T, Kohda T, Mukamoto M, Goshima N, Kozaki S, lhara H. Microbiol Immunol. 2012 Apr;56(4):245-53). Accordingly, other appropriate polypeptide domains having VAMP1 , VAMP 2 or VAMP 3 activity can also be readily determined by a person of skill in the art. In addition, WO 2018 / 150177 provides ample guidance of appropriate cleavage site sequences.

[0368] General Definitions

[0369] As used herein, the term "selectively" refers to having a unique effect or influence or reacting in only one way or with only one thing. As used herein, the term "selectively binds," when made in reference to an antibody, refers to the discriminatory binding of the antibody to the indicated target epitope such that the antibody does not substantially cross react with nontarget epitopes. In the context of the present invention, the antibodies or antigen binding fragments provided herein selectively bind ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, the antibodies or antigen binding fragments do not substantially cross react with non-target epitopes (i.e. epitopes other than ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide).

[0370] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0371] In some examples, the antibody or antigen binding fragment is a monoclonal antibody. In a natural antibody molecule, there are two heavy chains and two light chains. Each heavy chain and each light chain has at its N-terminal end a variable domain. Each variable domain is composed of four framework regions (FRs) alternating with three complementarity determining regions (CDRs). The residues in the variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated.

[0372] Accordingly, when referring to CDRs herein, the positions of the complementarity determining regions (CDRs) have been predicted using Kabat based alignments.

[0373] An “antigen binding fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fab, Fab’, F(ab’)2, Fv, scFv, di-scFv, bis-scFv, tri-scFv, scFv-Fc, an antibody domain, sdAb, diabody, triabody, tetrabody, minibody, a nanobody, and “third generation” (3G) fragments. Antigen binding fragment as used herein encompasses an antibody fragment.

[0374] The complete antigen-recognition and antigen-binding site is contained within the variable domains of the antibody, i.e. , the Fv fragment. This fragment includes a dimer of one heavy chain variable domain (VH) and one light chain variable domain (VL) in tight, non-covalent association. Each domain comprises four framework regions (FR), which largely adopting a - sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the p-sheet structure. Each hypervariable region comprises an amino acid sequence corresponding to a complementarity determining region (CDRs). Collectively, it the three-dimensional configuration of the six CDR regions that define an antigen-binding site on the surface of the VH-VL dimmer that confers antigen binding specificity. See e.g., Cyrus Chothia, et al., Conformations of Immunoglobulin Hypervariable Regions, Nature 342(6252): 877-883 (1989); Elvin A. Kabat, et al Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), each of which is incorporated by reference in its entirety. The constant domains of the antibody are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity.

[0375] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs or CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0376] As used herein, a “rabbit antibody or antigen binding fragment” is an antibody or antigen binding fragment which possesses an amino acid sequence which corresponds to that of an antibody or antigen binding fragment produced by a rabbit or a rabbit cell or derived from a non-rabbit source that utilizes rabbit antibody repertoires or other rabbit antibody-encoding sequences. This definition of a rabbit antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In some examples, the rabbit antibody or antigen binding fragment provided herein may be a recombinant antibody or antigen binding fragment.

[0377] In some examples, the antibody or antigen binding fragment described herein is isolated.

[0378] In some examples, the antibody or antigen binding fragment described herein is a monoclonal antibody or antigen binding fragment.

[0379] A “non-essential” or “non-critical” amino acid residue is a residue that can be altered from the wild-type sequence without abolishing or, more preferably, without substantially altering a biological activity, whereas an “essential” amino acid residue results in such a change.

[0380] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.

[0381] As used herein, a “biologically active portion” of protein or a protein portion with “biological activity” includes fragment of protein that participate in an interaction between molecules and non-molecules. Biologically active portions of protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the protein, which include fewer amino acids than the full length protein, and exhibit at least one activity of the encoded protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the protein, e.g., the biologically active portion may retain one of the following activities (as appropriate); luciferase, VAMP1 , VAMP2 or VAMP3 activity.

[0382] Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0383] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0384] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a BLOSLIM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2,

[0385] 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of

[0386] 4, and a frameshift gap penalty of 5.

[0387] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0388] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the N BLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>.

[0389] The polypeptides described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequences provided herein. The terms “sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

[0390] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0391] Aspects of the invention are demonstrated by the following non-limiting examples.

[0392] EXAMPLES

[0393] Example 1 : Generation and selection of the antibodies

[0394] BoNT / B and TeNT both target a specific amino acid sequence, which is conserved across VAMP1-3 proteins (Figure 1A). As such, the C-terminal end of the cytoplasmic protein fragment generated by cleavage of VAMP1-3 by either BoNT / B or TeNT is also conserved, and ends with the 8 amino acids Alanine, Leucine, Glutamine, Alanine, Glycine, Alanine, Serine and Glutamine (ALQAGASQ - SEQ ID NO: 59). This 8-amino acid peptide epitope was synthesised, coupled to KLH and injected into 4 rabbits (Figure 1 B). Sera from these 4 rabbits was collected after the fourth immunisation for characterisation.

[0395] To validate the rabbit sera samples, the inventors used a modified LAN5 cell line which expresses VAMP2 tagged with the Nanoluc (Promega) Luciferase (for detailed methodology see W02018 / 150177). This cell line was used as the cytoplasmic fragment of native VAMP2 is normally degraded by the host cell after cleavage by BoNT / B or TeNT. However, the addition of the Luciferase tag prevents this degradation, allowing the cytoplasmic fragment to be detected by antibodies.

[0396] Following treatment of these modified LAN5 cells with 10 nM BoNT / B, BoNT / D or vehicle for 65 h in 48-well plates, cell culture media was removed before addition of SDS-PAGE loading buffer (56 mM sodium dodecyl sulfate, 0.05 M T ris-HCI, pH 6.8, 1 .6 mM EDTA, 6.25% glycerol, 0.0001 % bromophenol blue, 10 mM MgCh, 26 U / rnL benzonase). Plates were then shaken at 900 rpm for 10 min. Samples were boiled for 3 min at 95°C and then run on 12% Novex SDS- PAGE gels (Invitrogen). Following separation, proteins were transferred onto Immobilin-P membranes, and then incubated for 30 min in blocking solution (5% milk, 0.1% TWEEN 20 in PBS). Rabbit antisera was added at 1 :500 dilution to the blocking solution at 4°C overnight. Membranes were washed three times in 0.1% TWEEN 20 in PBS for 5 min and then incubated for 30 min in the blotting solution containing secondary peroxidase-conjugated donkey antirabbit antibodies (Amersham). Membranes were washed three times for 5 min in 0.1% TWEEN 20 in PBS. Immunoreactive protein bands were visualised using SuperSignal West Dura Extended Duration solution (Thermo Scientific, Cramlington, UK) and a ChemiDoc XRS (BioRad) machine.

[0397] These western immunoblots showed that the antisera from all rabbits bound to cleaved Nanoluc-VAMP protein in the cells treated with BoNT / B, and only 1 set of antisera (rabbit 3) bound to uncleaved Nanoluc-VAMP (Figure 1C). None of the antisera samples bound to Nanoluc-VAMP cleaved by BoNT / D, suggesting that the generated antibodies from rabbits 1 , 2 and 4 would contain antibodies that could selectively bind BoNT / B and Tetanus-cleaved VAMP2 protein. B-cell cloning of the antisera from the rabbits was used to generate a library of monoclonal antibodies, which were screened for selective binding to BoNT / B cleaved VAMP2 using an ELISA assay. The monoclonal antibodies were then expressed and purified under small scale recombinant expression.

[0398] Table 2. Kabat predicted Heavy Chain CDR Sequences

[0399] Table 3. Kabat predicted Light Chain CDR Sequences

[0400] Example 2: Use of the antibodies to detect Botulinum B in an in-vitro ELISA assay

[0401] All recombinant proteins were made as glutathione-S-transferase (GST) C-terminal fusions cleavable by thrombin. The plasmid for expression was the pGEX-KG vector. All proteins were expressed in the BL21-Gold-PLysS-DE3 strain of E. coli (Agilent). GST fusion constructs were purified by glutathione affinity chromatography and eluted from the glutathione beads using thrombin as described previously [5],

[0402] The DNA sequence for the Nanoluc luciferase (GenBank: JQ437370.1) was synthesised upstream of amino acids 1-89 of VAMP2. (UNIPROT: K7ENK9) in the pGEX-KG vector. The Nanoluc-tagged-VAMP2 (1-89) protein was expressed and purified as above, then cleaved in- vitro by addition of purified BoNT / B light chain and translocation domain (amino acids 1-857). Cleavage was verified by running on 12% Novex SDS-PAGE gels (Invitrogen) along with the uncleaved protein.

[0403] An one-step ELISA assay was performed by incubating white Protein-A coated 96-well plates (Thermo Fisher Scientific, UK) with 50 pL of antibody diluted in blocking solution (PBS, 1% w / v BSA) overnight at +4°C with gentle shaking. After antibody incubation, the wells were washed with washing buffer (PBS, 0.05% Tween) 3 times for 5 min on an orbital shaker. 50 pl of recombinant protein diluted in PBS was added to each well and incubated for 90 min at room temperature on a shaker. The samples were removed and the wells washed 3 times. The buffer was then removed and the plate incubated with 50 pl NanoGio solution (2 pl of the substrate mixed with 48 pl of the buffer, Promega) for 5 min in the dark. Luminescence was read on Fluoroskan Ascent FL plate reader (Lab systems).

[0404] For an initial test of the assay and antibodies, the protein A plates were coated with 0.5 pg / mL of antibody and the recombinant protein was diluted to 1.2 pg / mL (Figure 2A). This showed that 5 of the 7 antibodies were capable of detecting the BoNT / B-cleaved Nanoluc-VAMP2 protein in these conditions, with the 131G9 and 2F7 clones being the most promising. In order to optimise the assay, the protein A plates were then coated with varying concentrations of 131G9 and 2F7 antibodies, and tested against 0.3 pg / mL solutions of uncleaved and BoNT / B-cleaved Nanoluc-VAMP2. This showed that the cleaved-specific ELISA signal could be increased by incubating the Protein A plate with greater amounts of the antibody, with the optimal concentrations being 10 pg / mL of 131G9 and 5 pg / mL of 2F7 antibody (Figure 2B).

[0405] These optimal concentrations of 131 G9 and 2F7 antibodies were then tested against a titration of uncleaved and BoNT / B-cleaved Nanoluc-VAMP2 ranging from 0.2 to 430 ng / mL, and both antibodies showed a linear relationship between the concentration of the substrate and the ELISA signal (Figure 2C). For both antibodies, the minimal concentration of protein that could be detected was 5.3 ng / mL, or 265 pg per well.

[0406] Example 3: Use of antibodies to detect cleaved VAMP1 and 3

[0407] In order to determine that the antibodies could detect other cleaved VAMP proteins that have the conserved cleavage site from VAMP2, Nanoluc-tagged versions of VAMP1 (1-91 , UniProt: P23763) and VAMP3 (1-72, UniProt: Q15836) were synthesized, expressed purified and cleaved in the same manner as the Nanoluc-VAMP2 protein in Example 2. Protein A plates were then coated with 1 pg / mL solutions of the four most promising antibodies and tested against 1 pg / mL solutions of uncleaved and cleaved Nanoluc-VAMP1-3.

[0408] Despite having a higher negative signal from the uncleaved Nanoluc-VAMP1 and 3 compared to VAMP2, all of the four antibodies tested (131G9, 2F7, 30G7, 114D9) showed an increase in signal against the cleaved Nanoluc-VAMP1 and 3. In particular, the 2F7 antibody showed a significantly higher signal against the cleaved Nanoluc-VAMP1 compared to the other antibodies (Figure 6).

[0409] Example 4: Glutamine and Serine residues are critical for antibody binding

[0410] In order to determine if the cleavage-selective antibodies were binding as expected to the C- terminal amino acids of the cytosolic cleaved VAMP fragment, a series of Nanoluc-VAMP2 (1- 76) mutants were synthesised with a C-terminal stop codon immediately after the BoNT / B / Tetanus cleavage point (Q76), in order to mimic the effects of cleavage by BoNT / B or TeNT without the need for addition of extra toxin to the proteins.

[0411] These mutations were focused on the Serine and Glutamine residues immediately preceding the cleavage point (S75 and Q76) and a glutamine 5 residues away (Q71) as it was considered most likely that the antibodies would recognise and bind to these amino acids. Mutant 1 has a single mutation (Q76A), as did Mutant 2 (S75A), and Mutant 3 (Q71A). Mutant 4 has a double mutation of the 2 amino acids preceding the cleavage point (S75A, Q76A) and Mutant 5 contained all three mutated residues together (Q71A, S75A, Q76A). These mutations and their sequences compared to the wild-type protein are summarised in figure 3A.

[0412] To characterise the binding of the 4 most promising antibodies (131G9, 2F7, 30G7 and 114D9) to the cleaved VAMP end, an ELISA assay was performed as described in Example 2 using protein A plates coated with 1 pg / mL of antibody and 40 ng / mL of either the Uncleaved version of Nanoluc-VAMP2 (1-89) (SEQ ID NO: 67), Wild-type Nanoluc-VAMP2 (1-76) (SEQ ID NO: 68), (which acts as a mimic of the BoNT / B-cleaved VAMP2 and can be used as a positive control for the antibodies), or Mutants 1-5 (SEQ ID NO: 69-73). As seen before, none of the antibodies detected the uncleaved Nanoluc-VAMP2 (1-89), and all of them detected the wildtype shortened Nanoluc-VAMP2 (1-76). The signal for Mutants 1 , 2, 4 and 5 were either drastically reduced or undetectable across all antibodies, implying that the C-terminal Serine and Glutamine residues are critical for antibody binding. The signal from Mutant 3 was more varied; with 131G9 and 2F7 the signal for Mutant 3 was significantly lower than that of the wild type protein, however for 30G7 and 114D9 there was less difference between Mutant 3 and the wild type protein. This suggests that the Q71 residue may be involved in the binding of some of the cytosolic cleaved-VAMP antibodies, but it is not critical for binding (Figure 2B).

[0413] To further validate the binding patterns of the most promising antibodies, 7.5 ng each of the purified wild-type, shortened Nanoluc-VAMP2 (1-76) and Mutants 1-5 were run on 12% Novex SDS-PAGE gels (Invitrogen). Following separation, proteins were transferred onto Protean nitrocellulose membranes, and then incubated for 30 min in blocking solution (5% milk, 0.1% TWEEN 20 in PBS). 0.5 pg / mL of 131G9, 2F7, 30G7 or 114D9 antibody was added to the blocking solution at 4°C overnight. Membranes were washed three times in 0.1% TWEEN 20 in PBS for 5 min and then incubated for 30 min in the blotting solution containing 1 :5000 dilution of IRDye 680RD Goat Anti-rabbit secondary antibody (LiCor). Membranes were washed three times for 5 min in 0.1 % TWEEN 20 in PBS. Immunofluorescent protein bands were visualised at 700 nm using a LiCor Odyssey Sa imaging system (Figure 3C) and then quantified using the band analysis tools of Image Studio Ver 5.0 software (Figure 3D).

[0414] For 2F7, the results of the western blot were similar to the ELISA, as only the wild-type and Mutant 3 proteins were detected, suggesting that the C-terminal S75 and Q76 residues are needed for this antibody to bind. However, the signal from Mutant 3 was not significantly different from the wild type VAMP2 (1-76), suggesting that the Q71 residue is less involved in binding under the conditions of the western blot than it is in the ELISA. The 30G7 antibody also showed reduced signal against Mutants 1 , 2 and 4, along with no signal for mutant 5. This suggests that the S75 and Q76 residues are involved in binding, but not critical to binding in the western blot conditions as long as the Q71 residue is intact. However the other 2 antibodies, 131G9 and 114D9, had surprisingly different binding patterns in the western blot compared to the ELISA. Both these antibodies were still capable of binding the Mutant 1 protein, with a similar level of signal as they did for the wild-type and Mutant 3 proteins. This would suggest that, in the western blot conditions, only the C-terminal Serine residue is critical for these antibodies to binding cleaved VAMP.

[0415] Example 5: Use of the antibodies to detect Botulinum B and Tetanus in a cell-based assay

[0416] Since the results with pure recombinant protein were promising, the antibodies were then tested against cell cultures treated with the toxin, in order to replicate the conditions of a cellbased BoNT / B or TeNT assay. For the first test, differentiated Nanoluc-VAMP2 expressing LAN5 cells were treated with 1 nM BoNT / B or vehicle control buffer. These cells were lysed and run on 12% SDS-PAGE and western blotted with the 0.5 pg / mL of each of the 7 monoclonal antibodies in the same way as the rabbit sera samples in Example 1. For all 7 antibodies, only the Nanoluc-VAMP2 from cells treated with BoNT / B was detected, showing that the antibodies were capable of selective binding to cleaved VAMP from mammalian cells (Figure 4A).

[0417] As a further example of how these antibodies could be used in a cell-based assay against both BoNT / B and TeNT, an ELISA assay was used to test a lower concentration of both toxins. Differentiated Nanoluc-VAMP2 expressing LAN5 cells were treated with either vehicle buffer, or 100 pM of BoNT / B or TeNT for 65h. After toxin treatment, the LAN5 cells were lysed by incubation with a cell extraction buffer (PBS, 0.5% Triton X-100 and 1X Roche MiniEDTA protease inhibitor) for 15 min on a microplate shaker (600 rpm). The samples were then transferred into pre-chilled microcentrifuge tubes and centrifuged at 14000 rpm for 15 min at 4°C. The supernatant was then collected and diluted in PBS and stored at -80°C until required.

[0418] 50 pL of each sample was then added to wells of a protein A plate coated with 1 ug / mL of the four most promising ELISA antibodies (2F7, 131G9, 30G7, 114D9) in the same way as the One-Step ELISA assay in Example 2. All four of the antibodies showed a significant difference in signal between the untreated and toxin-treated cells, showing that these antibodies would be usable in a cell-based assay for either BoNT / B or TeNT (Figure 4B).

[0419] Example 6: Currently available Mouse-based antibodies were incompatible with ELISA-based cell assay

[0420] In order to determine if available cleavage-specific mouse antibodies could be used, we tested 2 monoclonal, Mouse, cleavage-specific antibodies for BoNT / B-cleaved VAMP2 that were generated by the Robert Koch Institute, B226 and B1148. For the first test, differentiated Nanoluc-VAMP2 expressing LAN5 cells were treated with 1 nM BoNT / B or 1 nM BoNT / D. These cells were lysed and run on 12% SDS-PAGE and western blotted with the 0.5 pg / mL of each of the 2 monoclonal mouse antibodies in the same way as the rabbit sera samples in Example 1. For both antibodies, only the Nanoluc-VAMP2 from cells treated with BoNT / B was detected, showing that the antibodies were capable of selectively binding to cleaved VAMP from mammalian cells in western blot (Figure 7A).

[0421] To determine if these 2 mouse monoclonal antibodies could be used be used in a cell-based assay against both BoNT / B and TeNT, differentiated Nanoluc-VAMP2 expressing LAN5 cells were treated with either vehicle buffer, or 100 pM of BoNT / B for 3 days. After toxin treatment, the LAN5 cells were lysed as in Example 5. 50 pL of each sample was then added to wells of a protein A or protein G plate coated with 1 ug / mL of B226, B1148 and Cq8 (a known polyclonal, rabbit, cleavage-specific VAMP2 antibody used as a standard positive control [4]) in the same way as the One-Step ELISA assay in Example 2. While the rabbit antibody Cq8 was able to detect the cleaved VAMP2 in cells treated with BoNT / B, there was no significant difference in the signal detected from BoNT / B versus untreated cells using the 2 mouse monoclonal antibodies (Figure 7B-C).

[0422] Table 4. Summary of results from the examples using wild-type VAMP sequences. -: No signal, +: Low signal, ++: Medium signal, +++: High signal

[0423] Table 5. Summary of results using mutated VAMP sequences from example 4. -: No signal, +: Low signal, ++: Medium signal, +++: High signal The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0424] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0425] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0426] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0427] Sequences

[0428] In tables 6 to 9 below a “*” denotes a stop codon. In table 6 to 9 below, underlined sequences denote signal peptide sequences.

[0429] Table 6: sequences for antibody 131 G9

[0430] Table 7: sequences for antibody 114D9

[0431] Table 8: sequences for antibody 2F7

[0432] Table 9: sequences for antibody 30G7

[0433] SEQ ID NO: 57 - CALQAGASQ

[0434] SEQ ID NO: 58 - CALQAGASQFE

[0435] SEQ ID NO: 59 - ALQAGASQ

[0436] SEQ ID NO: 60 - NanoLuc Ref: ACS Chem Biol. 2012 Nov 16; 7(11): 1848-1857.

[0437] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

[0438] GLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG

[0439] TLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0440] SEQ ID NO: 61 - Human VAMP1 Ref sequence NM_014231 .4.

[0441] MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEWDIIRVNVDKVLERDQ KLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIWVIVIYFFT

[0442] SEQ ID NO: 62 - Human VAMP1 core sequence

[0443] QVEEWDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYVWVKNCKMMIMLG AICAIIVWIVIYFFT

[0444] SEQ ID NO: 63 - Human VAMP2 Ref sequence NM_014232.2.

[0445] MS ATAATAP PAAPAG EGGPPAPPPNLTSN RRLQQTQAQVDEWDIMRVNVDKVLERDQKLSEL DDRADALQAGASQFETSAAKLKRKYWWKNLKMM I 1 LGVICAI I LI 11 IVYFST

[0446] SEQ ID NO: 64 - Human VAMP2 core sequence

[0447] QVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILG VICAIILIIIIVYFST

[0448] SEQ ID NO: 65 - Human VAMP3 Ref sequence NM_004781 .3

[0449] MSTG PTAATGSNRRLQQTQNQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQFET

[0450] SAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

[0451] SEQ ID NO: 66 - Human VAMP3 core sequence

[0452] QVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYVWVKNCKMWAIGI TVLVIFIIIIIVWVVSS

[0453] SEQ ID NO: 67 - VAMP2 sequence of Nanoluc-VAMP2 (1-89) construct (Uncleaved version)

[0454] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

[0455] SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0456] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0457] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAK

[0458] LKRKYW

[0459] SEQ ID NO: 68 - VAMP2 sequence of Nanoluc-VAMP2 (1-76) construct (Mimic of cleaved version)

[0460] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

[0461] SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0462] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0463] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQ

[0464] SEQ ID NO: 69 - VAMP2 mutated sequence of Mutant 1 : Nanoluc-VAMP2 (1-76) construct with

[0465] Q76A VAMP mutation

[0466] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

[0467] SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0468] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0469] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGASA

[0470] SEQ ID NO: 70 - VAMP2 mutated sequence of Mutant 2: Nanoluc-VAMP2 (1-76) construct with

[0471] S75A VAMP mutation

[0472] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

[0473] SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0474] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGAAQ

[0475] SEQ ID NO: 71- VAMP2 mutated sequence of Mutant 3: Nanoluc-VAMP2 (1-76) construct with Q71A VAMP mutation

[0476] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL

[0477] SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0478] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0479] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALAAGASQ

[0480] SEQ ID NO: 72 - VAMP2 mutated sequence of Mutant 4: Nanoluc-VAMP2 (1-76) construct with S75A and Q76A VAMP mutations

[0481] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0482] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0483] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALQAGAAA

[0484] SEQ ID NO: 73 - VAMP2 mutated sequence of Mutant 5: Nanoluc-VAMP2 (1-76) construct with Q71A, S75A and Q76A VAMP mutations

[0485] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKWYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL

[0486] WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGPGPGPMSATAATAPPAAPAGEGGPP

[0487] APPPNLTSNRRLQQTQAQVDEWDIMRVNVDKVLERDQKLSELDDRADALAAGAAA

[0488] SEQ ID NO: 74 - ALQAGASQFE

[0489] SEQ ID NO: 75 - LSELDDRADALQAGASQFETSAAKLKRK

[0490] SEQ ID NO: 76 - DEWDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRK

[0491] SEQ ID NO: 77

[0492] MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEWDIIRVNVDKVLERDQKLS

[0493] ELDDRADALQAGASQ

[0494] SEQ ID NO: 78

[0495] MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQ

[0496] SEQ ID NO: 79

[0497] MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSEL

[0498] DDRADALQAGASQ

[0499] SEQ ID NO: 80 - GASQFESS

[0500] SEQ ID NO: 81 - GASQFETS

[0501] SEQ ID NO: 82 - ALQAGASA

[0502] SEQ ID NO: 83 - ALQAGAAQ

[0503] SEQ ID NO: 84 - ALAAGASQ

[0504] SEQ ID NO: 85 - ALQAGAAA SEQ ID NO: 86 - ALAAGAAA

[0505] References

[0506] [1] O. Rossetto, M. Pirazzini, and C. Montecucco, “Botulinum neurotoxins: genetic, structural and mechanistic insights,” Nat. Rev. Microbiol., vol. 12, no. 8, pp. 535-549, Aug. 2014, doi: 10.1038 / nrmicro3295.

[0507] [2] A. Rust et al., “A Cell Line for Detection of Botulinum Neurotoxin Type B,” Front. Pharmacol., vol. 8, p. 796, 2017, doi: 10.3389 / fphar.2017.00796.

[0508] [3] H. A. Behrensdorf-Nicol, K. Weisser, and B. Kramer, “‘BINACLE’ assay for in vitro detection of active tetanus neurotoxin in toxoids,” ALTEX, vol. 32, no. 2, pp. 137-142, 2015, doi: 10.14573 / altex.1412181.

[0509] [4] R. Riches-Duit et al., “Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay,” Biologicals, vol.

[0510] 71 , pp. 31-41 , Jun. 2021 , doi: 10.1016 / j.biologicals.2021.04.002.

[0511] [5] F. Darios et al., “SNARE tagging allows stepwise assembly of a multimodular medicinal toxin,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 42, pp. 18197-18201 , Oct. 2010, doi: 10.1073 / pnas.1007125107.

Claims

Claims1 . An antibody or antigen binding fragment, comprising:(i) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6;(ii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 15, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 16, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 17; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 20;(iii) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 29, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 30, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 32, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 33, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 34; or(iv) a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 44, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 45; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ IDNO: 47, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48.

2. The antibody or antibody fragment of claim 1 wherein:(i) a) the heavy chain variable region (VH) of claim 1 (i) comprises the amino acid sequence of SEQ ID NO: 7; and b) the light chain variable region (VL) of claim 1 (i) comprises the amino acid sequence of SEQ ID NO: 8;(ii) a) the heavy chain variable region (VH) of claim 1 (ii) comprises the amino acid sequence of SEQ ID NO: 21 ; and b) the light chain variable region (VL) of claim 1 (ii) comprises the amino acid sequence of SEQ ID NO: 22;(iii) a) the heavy chain variable region (VH) of claim 1 (iii) comprises the amino acid sequence of SEQ ID NO: 35; and b) the light chain variable region (VL) of claim 1 (iii) comprises the amino acid sequence of SEQ ID NO: 36; or(iv) a) the heavy chain variable region (VH) of claim 1 (iv) comprises the amino acid sequence of SEQ ID NO: 49; and b) the light chain variable region (VL) of claim 1 (iv) comprises the amino acid sequence of SEQ ID NO: 50.

3. The antibody or antigen binding fragment of claim 1 or 2, wherein the antibody or antigen binding fragment binds to ALQAGASQ, wherein the ALQAGASQ is located at the C- terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment.

4. The antibody or antigen binding fragment of claim 3, wherein the VAMP fragment is a VAMP2, VAMP1 , or VAMP3 fragment.

5. The antibody or antigen binding fragment of claim 4, wherein the VAMP fragment is a VAMP2 fragment.

6. The antibody or antigen binding fragment of claim 5, wherein the VAMP2 fragment comprises SEQ ID NO: 79.

7. The antibody or antigen binding fragment of any one of claims 1 to 6, wherein the antibody is a rabbit antibody or antigen binding fragment.

8. A nucleic acid composition encoding the antibody or antigen binding fragment of any one of claims 1 to 7.

9. An expression vector system comprising a nucleic acid according to claim 8.

10. A host cell comprising the nucleic acid according to claim 8 or an expression vector according to claim 9.

11. A conjugate comprising an antibody or antigen binding fragment according to any one of claims 1 to 7 conjugated to or recombinantly fused to a diagnostic agent, detectable agent, or therapeutic agent.

12. A kit for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, the kit comprising an antibody or antigen binding fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, an expression vector according to claim 9, a host cell according to claim 10 and / or a conjugate according to claim 11, optionally wherein the polypeptide comprises or consists of a VAMP fragment wherein the ALQAGASQ is part of the VAMP fragment.

13. The kit according to claim 12, wherein the kit comprises an antibody or antigen binding fragment according to any one of claims 1 to 7.

14. The kit according to claim 12 or 13, wherein the kit further comprises a nucleic acid encoding:(i) a VAMP polypeptide; or(ii) a VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C- terminal polypeptide domain having VAMP1, VAMP2 or VAMP3 activity.

15. The kit of claim 14, wherein the nucleic acid encoding:(i) the VAMP polypeptide; or(ii) the VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C- terminal polypeptide domain having VAMP1, VAMP2 or VAMP3 activity; is part of an expression vector.

16. The kit of claim 14 or 15, wherein the nucleic acid encoding (i) or (ii) is within a host cell.

17. The kit of claim 16, wherein the host cell is selected from the group consisting of a SiMa neuroblastoma cell, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, immortalised neuron, a BE(2)C cell and primary neuron.

18. The kit of any one of claims 12 to 17, wherein the kit is for detecting a VAMP fragment, wherein ALQAGASQ is located at the C-terminus of the VAMP fragment.

19. The kit of any one of claims 12 to 18, wherein the kit is for detecting tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

20. Use of an antibody or antigen binding fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, an expression vector according to claim 9, a host cell according to claim 10, a conjugate according to claim 11 and / or a kit according to any one of claims 12 to 19 for binding to ALQAGASQ, wherein the ALQAGASQ is located at the C- terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment.

21. The use according to claim 20, wherein the antibody or antigen binding fragment, nucleic acid, expression vector, host cell, conjugate and / or kit is for detecting:(a) ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment; and / or(b) tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

22. The use according to claim 21 (b), wherein the neurotoxin activity is detected in a test sample comprising:(a) a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof;(b) a tetanus toxoid, or a botulinum toxoid or a combination thereof; and / or(c) tetanus neurotoxin, botulinum neurotoxin type B, or a combination thereof.

23. A method of binding a polypeptide comprising ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of the polypeptide, the method comprising contacting the polypeptide with an antibody or antigen binding fragment according to any one of claims 1 to7, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment.

24. The method according to claim 23, wherein the method is for detecting:(a) ALQAGASQ, wherein the ALQAGASQ is located at the C-terminus of a polypeptide, optionally wherein the polypeptide comprises or consists of a VAMP fragment and the ALQAGASQ is part of the VAMP fragment; and / or(b) tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

25. A method of detecting neurotoxin activity in a test sample, the method comprising:(a) providing a host cell that comprises a nucleic acid encoding:(i) a VAMP polypeptide; or(ii) a VAMP reporter polypeptide, optionally wherein the VAMP reporter polypeptide is a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1 , VAMP2 or VAMP3 activity;(b) culturing the host cell under conditions that allow for expression of the polypeptide;(c) culturing the host cell of (b) in the presence of the test sample under conditions that allow for neurotoxin-induced cleavage of the polypeptide; and(d) using an antibody or antigen binding fragment according to any one of claims 1 to 7 to determine the level of neurotoxin-induced cleavage of the polypeptide, wherein detection of neurotoxin-induced cleavage of the polypeptide is indicative of neurotoxin activity; wherein the neurotoxin activity is tetanus neurotoxin activity, botulinum type B neurotoxin activity, or a combination thereof.

26. The method of claim 25, wherein step (d) comprises preparing a cell lysate from the host cell of step (c) and contacting the cell lysate with the antibody or antigen binding fragment to determine the level of neurotoxin-induced cleavage of the polypeptide.

27. The method of claim 25 or 26, wherein the neurotoxin-induced cleavage of the polypeptide is detected by ELISA, immunoblot, or live cell imaging.

28. The method of any one of claims 25 to 27, wherein the test sample comprises:(a) a drug product, a food sample, a clinical sample, or an environmental sample, or any combination thereof;(b) a tetanus toxoid, or a botulinum toxoid, or a combination thereof; and / or(c) tetanus neurotoxin, botulinum neurotoxin type B, or a combination thereof.

29. The antibody or antigen binding fragment according to any one of claims 1 to 7, the nucleic acid according to claim 8, the expression vector according to claim 9, the host cell according to claim 10, the conjugate according to claim 11 , the kit according to any one of claims 12 to 19, the use according to any one of claims 20 to 22, or the method according to any one of claims 23 to 28, wherein the antibody or antigen binding fragment:(a) is selected from the group consisting of: Fab, Fab’, F(ab’)2, Fv, scFv, di-scFv, bis-scFv, tri- scFv, scFv-Fc, an antibody domain, sdAb, diabody, triabody, tetrabody, minibody, a nanobody, “third generation” (3G) fragment, and full length antibody; and / or(b) is selected from lgG1 , lgG2, lgG3 and lgG4; and / or(c) is monospecific or bispecific.

30. The antibody or antigen binding fragment, nucleic acid, expression vector, host cell, conjugate, kit, use, or method according to any one of the preceding claims, wherein the antibody is a monoclonal antibody.

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