AU2012203055B2 - Fusion proteins - Google Patents
Fusion proteins Download PDFInfo
- Publication number
- AU2012203055B2 AU2012203055B2 AU2012203055A AU2012203055A AU2012203055B2 AU 2012203055 B2 AU2012203055 B2 AU 2012203055B2 AU 2012203055 A AU2012203055 A AU 2012203055A AU 2012203055 A AU2012203055 A AU 2012203055A AU 2012203055 B2 AU2012203055 B2 AU 2012203055B2
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- AU
- Australia
- Prior art keywords
- protease
- fusion protein
- seq
- fusion
- protein according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a Targeting Moiety that is 5 capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non cytotoxic protease or fragment thereof and the Targeting Moiety; and a translocation L0 domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described.
Description
P100/0oI Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Fusion proteins The following statement is a full description of this invention, including the best method of performing it known to us: l A Fusion Proteins This inventioW r6[ates to non-cytotoxic fusion proteins, and to the therapeutic 5 application thereof as analgesic molecules. Toxins may be generally divided into two groups according to the type of effect that they have on a target cell. In more detail, the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules. This 10 group of toxins is exemplified inter alia by plant toxins such as ricin, and abrin, and by bacterial toxins such as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxins have attracted much interest in the design of "magic bullets" (e.g. immunoconjugates, which comprise a cytotoxic toxin component and an antibody that binds to a specific marker on a target cell) for the treatment of cellular 15 disorders and conditions such as cancer. Cytotoxic toxins typically kill their target cells by inhibiting the cellular process of protein synthesis. The second group of toxins, which are known as non-cytotoxic toxins, do not (as their name confirms) kill their natural target cells. Non-cytotoxic toxins have 20 attracted much less commercial interest than have their cytotoxic counterparts, and exert their effects on a target cell by inhibiting cellular processes other than protein synthesis. Non-cytotoxic toxins are produced by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria sp. 25 Clostridial neurotoxins are proteins that typically have a molecular mass of the order of 150 kDa. They are produced by various species of bacteria, especially of the genus Clostridium, most importantly C. tetani and several strains of C. botuiinum, C. butyricum and C. argentinense. There are at present eight different classes of the clostridial neurotoxin, namely: tetanus toxin, and botulinum 30 neurotoxin in its serotypes A, B, C1, D, E, F and G, and they all share similar structures and modes of action.
Clostridial neurotoxins represent a major group of non-cytotoxic toxin molecules, and are synthesised by the host bacterium as single polypeptides that are modified post-translationally by a proteolytic cleavage event to form two 5 polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. 10 L-chains possess a protease function (zinc-dependent endopeptidase activity) and exhibit a high substrate specificity for vesicle and/or plasma membrane associated proteins involved in the exocytic process. L-chains from different clostridial species or serotypes may hydrolyse different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25. 15 These substrates are important components of the neurosecretory machinery. Neisseria sp., most importantly from the species N. gonorrhoeae, produce functionally. similar non-cytotoxic proteases. An example of such a protease is IgA protease (see W099/58571). 20 It has been well documented in the art that toxin molecules may be re-targeted to a cell that is not the toxin's natural target cell. When so re-targeted, the modified toxin is capable of binding to a desired target cell and, following subsequent translocation into the cytosol, is capable of exerting its effect on the target cell. 25 Said re-targeting is achieved by replacing the natural Targeting Moiety (TM) of the toxin with a different TM. In this regard, the TM is selected so that it will bind to a desired target cell, and allow subsequent passage of the modified toxin into an endosome within the target cell. The modified toxin also comprises a translocation domain to enable entry of the non-cytotoxic protease into the cell 30 cytosol. The translocation domain can be the natural translocation domain of the toxin or it can be a different translocation domain obtained from a microbial protein with translocation activity.
3 For example, W094/21300 describes modified clostridial neurotoxin molecules that are capable of regulating Integral Membrane Protein (IMP) density present at the cell surface of the target cell. The modified neurotoxin molecules are thus 5 capable of controlling cell activity (e.g. glucose uptake) of the target cell. W096/33273 and W099/17806 describe modified clostridial neurotoxin molecules that target peripheral sensory afferents. The modified neurotoxin molecules are thus capable of demonstrating an analgesic effect. WOOO/10598 describes the preparation of modified clostridial neurotoxin molecules that target 10 mucus hypersecreting cells (or neuronal cells controlling said mucus hypersecreting cells), which modified neurotoxins are capable of inhibiting hypersecretion from said cells. WO01/21213 describes modified clostridial neurotoxin molecules that target a wide range of different types of non-neuronal target cells. The modified molecules are thus capable of preventing secretion 15 from the target cells. Additional publications in the technical field of re-targeted toxin molecules include: WOOO/62814; WOOO/04926; US5,773,586; W093/15766; WOOO/61192; and W099/58571. The above-mentioned TM replacement may be effected by conventional chemical 20 conjugation techniques, which are well known to a skilled person. In this regard, reference is made to Hermanson, G.T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S.S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press. 25 Chemical conjugation is, however, often imprecise. For example, following conjugation, a TM may become joined to the remainder of the conjugate at more than one attachment site. Chemical conjugation is also difficult to control. For example, a TM may become 30 joined to the remainder of the modified toxin at an attachment site on the protease component and/ or on the translocation component. This is problematic 4 when attachment to only one of said components (preferably at a single site) is desired for therapeutic efficacy. Thus chemical conjugation results in a mixed population of modified toxin molecules, which is undesirable. 5 As an alternative to chemical conjugation, TM replacement may be effected by recombinant preparation of a single polypeptide fusion protein (see W098/07864). This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (i.e. holotoxin) is prepared, and results in a fusion protein having the following structural arrangement: 10 NH 2 - [protease component] - [translocation component] - [TM] - COOH According to W098/07864, the TM is placed towards the C-terminal end of the fusion protein. The fusion protein is then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component. A di-chain protein is thus produced, comprising the protease component as a single polypeptide 15 chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM. Whilst the W098/07864 methodology follows (in terms of structural arrangement of the fusion protein) the natural expression system of clostridial holotoxin, the present inventors have found that this system may result in the production of certain fusion proteins that have a substantially-reduced binding 20 ability for the intended target cell. There is therefore a need for an alternative or improved system for constructing a non cytotoxic fusion protein. Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common 25 general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not 30 intended to exclude other additives, components, integers or steps.
5 The present invention addresses one or more of the above-mentioned problems by providing a single chain, polypeptide fusion protein, comprising: a. a non-cytotoxic protease, or a fragment thereof, which protease or protease .fragment is capable of cleaving a protein- of-the- exocytic fusion- apparatus in a nociceptive sensory afferent; b. a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; c. a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and d. a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent. The present invention provides a single chain, polypeptide fusion protein, comprising: a) a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; b) a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; c) a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non cytotoxic protease or fragment thereof and the Targeting Moiety; 5A d) a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the Ondosomal membrane and into the cytosol of the nociceptive sensory afferent; wherein the Targeting Moiety is located between the protease cleavage site and the translocation domain, and wherein the protease (or fragment thereof) and the translocation domain are distanced apart from one another by a maximum of 100 amino acid residues. The W098/07864 system works well for the preparation of conjugates having a TM that requires a C-terminal domain for interaction with a Binding Site on a target cell. In this regard, W098/07864 provides fusion proteins having a C terminal domain that is "free" to interact with a Binding Site on a target cell. The present inventors have found that this structural arrangement is not suitable for all TMs. One such category of TM is a group of TMs that binds to nociceptive sensory afferents. In more detail, the present inventors have found that the WO 98/07864 fusion protein system is not optimal for TMs requiring a N-terminal domain for interaction with a binding site on a nociceptive sensory afferent. This problem is particularly acute with TMs that require a specific N-terminus amino acid residue or a specific sequence of amino acid residues including the N terminus amino acid residue for interaction with a binding site on a nociceptive sensory afferent.
6 In contrast to W098/07864, the present invention provides a system for preparing non-cytotoxic conjugates, wherein the TM component of the conjugate includes the relevant binding domain in an intra domain or an amino acid sequence located--towards -the--middle (ie: of the -linear-peptide sequence) of the TM, or 5 preferably located towards the N-terminus of the TM, or more preferably at or near to the N-terminus. The N-terminal domain is capable of binding to a Binding Site on a nociceptive sensory afferent, and the TM preferably has a requirement for a specific and defined sequence of amino acid residue(s) to be free at its N terminus. 10 The non-cytotoxic protease component of the present invention is a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25, of the exocytic fusion apparatus in a 15 -nociceptive sensory afferent. These substrates-are important components of the neurosecretory machinery. The non-cytotoxic protease component of the present invention is preferably a neisserial IgA protease or a fragment thereof or a clostridial neurotoxin L-chain or a fragment thereof. A particularly preferred non cytotoxic protease component is a botulinum neurotoxin (BoNT) L-chain or a 20 fragment thereof. The translocation component of the present invention enables translocation of the non-cytotoxic protease (or fragment thereof) into the target cell such that functional expression of protease activity occurs within the cytosol of the target 25 cell. The translocation component is preferably capable of forming ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore formation within the endosomal membrane. The translocation component may be obtained from a microbial protein source, in particular from a bacterial or viral 30 protein source. Hence, in one embodiment, the translocation component is a translocating domain of an enzyme, such as a bacterial toxin or viral protein. The translocation component of the present invention is preferably a clostridial 7 neurotoxin H-chain or a fragment thereof. Most preferably it is the HN domain (or a functional component thereof), wherein HN means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or-the domain-corresponding-to that fragment in the intact H 5 chain. The TM component of the present invention is responsible for binding the conjugate of the present invention to a Binding Site on a target cell. Thus, the TM component is simply a ligand through which a conjugate of the present 10 invention binds to a selected target cell. In the context of the present invention, the target cell is a nociceptive sensory afferent, preferably a primary nociceptive afferent (e.g. an A-fibre such as an AS fibre or a C-fibre). Thus, the conjugates of the present invention are capable of 15 inhibiting neurotransmitter or~ neuromodulator [e.g. glutamate, substance P, calcitonin-gene related peptide (CGRP), and/ or neuropeptide Y] release from discrete populations of nociceptive sensory afferent neurons. In use, the conjugates reduce or prevent the transmission of sensory afferent signals (e.g. neurotransmitters or neuromodulators) from peripheral to central pain fibres, and 20 therefore have application as therapeutic molecules for the treatment of pain, in particular chronic pain. It is routine to confirm that a TM binds to a nociceptive sensory afferent. For example, a simple radioactive displacement experiment may be employed in 25 which tissue or cells representative of the nociceptive sensory afferent (for example DRGs) are exposed to labelled (e.g. tritiated) ligand in the presence of an excess of unlabelled ligand. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the ligand binds to the nociceptive sensory afferent target cell. Optionally, 30 the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of ligand binding. Examples of this type of 8 experiment can be found in Hulme, E.C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, In Receptor biochemistry, A Practical Approach, Ed. E.C. Hulme, Oxford University Press. 5 The fusion proteins of the present invention generally demonstrate a reduced binding affinity (in the region of up to 100-fold) for nociceptive sensory afferent target cells when compared with the corresponding 'free' TM. However, despite this observation, the fusion proteins of the present invention surprisingly demonstrate good efficacy. This can be attributed to two principal features. First, 10 the non-cytotoxic protease component is catalytic - thus, the therapeutic effect of a few such molecules is rapidly amplified. Secondly, the receptors present on the nociceptive sensory afferents need only act as a gateway for entry of the therapeutic, and need not necessarily be stimulated to a level required in order to achieve a ligand-receptor mediated pharmacological response. Accordingly, the 15 -fusion proteins of the present invention may be administered at a dosage that is much lower that would be employed for other types of analgesic molecules such as NSAIDS, morphine, and gabapentin. The latter molecules are typically administered at high microgram to milligram (even up to hundreds of milligram) quantities, whereas the fusion proteins of the present invention may be 20 administered at much lower dosages, typically at least 10-fold lower, and more typically at 100-fold lower. The TM preferably comprises a maximum of 50 amino acid residues, more preferably a maximum of 40 amino acid residues, particularly preferably a 25 maximum of 30 amino acid residues, and most preferably a maximum of 20 amino acid residues. Opioids represent a preferred group of TMs of the present invention. Within this family of peptides is included enkephalins (met and leu), endomorphins 1 and 2, 30 p-endorphin and orphi Opioid peptides are frequently used in the clinic to modify the activity to nociceptors, and other cells involved in the pain response. As exemplified by the three-step World Health Organisation Analgesic Ladder, 9 opioids have entry points into the pharmacological treatment of chronic cancer and non-cancer pain at all three stages, underlining their importance to the treatment of pain. Reference to opioids embraces fragments, variants and derivatives -thereof,-which - retain -the- ability -to bind to- nociceptive -sensory 5 afferents. The TM of the invention can also be a molecule that acts as an "agonist" at one or more of the receptors present on a nociceptive sensory afferent, more particularly on a primary nociceptive afferent. Conventionally, an agonist has 10 been considered any molecule that can either increase or decrease activities within a cell, namely any molecule that simply causes an alteration of cell activity. For example, the conventional meaning of an agonist would include a chemical substance capable of combining with a receptor on a cell and initiating a reaction or activity, or a drug that induces an active response by activating receptors, 15 whether the response is- an increase or decrease in cellular activity. However, for the purposes of this invention, an agonist is more specifically defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, which process is susceptible to inhibition by a protease (or 20 fragment thereof) capable of cleaving a protein of the exocytic fusion apparatus in said target cell. Accordingly, the particular agonist definition of the present invention would exclude many molecules that would be conventionally considered as agonists. 25 For example, nerve growth factor (NGF) is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic fusion. In addition, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by the protease activity of a non 30 cytotoxic toxin molecule.
10 The agonist properties of a TM that binds to a receptor on a nociceptive afferent can be confirmed using the methods described in Example 10. In a preferred embodiment of the invention, the target for the TM is the ORL 1 5 receptor. This receptor is a member of the G-protein-coupled class of receptors, and has a seven transmembrane domain structure. The properties of the ORL 1 receptor are discussed in detail in Mogil & Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages 381-415. 10 In one embodiment, the TM is a molecule that binds (preferably that specifically binds) to the ORL 1 receptor. More preferably, the TM is an "agonist" of the ORL 1 receptor. The term "agonist" in this context is defined as above. The agonist properties of a TM that binds to an ORL 1 receptor can be confirmed 15 using the methods described in Example 10. - These methods are based on previous experiments [see Inoue et al. 1998 [Proc. Nati. Acad. Sci., 95, 10949 10953]), which confirm that the natural agonist of the ORL 1 receptor, nociceptin, causes the induction of substance P release from nociceptive primary afferent neurons. This is supported by the fact that: 20 > the nociceptin-induced responses are abolished by specific NK1 receptor (the substance P receptor) antagonists; and > pre-treatment of the cells with capsaicin (which depletes substance 25 P from small diameter primary afferent neurons) attenuates the nociceptin-induced responses. Similarly, Inoue et al. confirm that an intraplantar injection of botulinum neurotoxin type A abolishes the nociceptin-induced responses. Since it is known that BoNT 30 inhibits the release of substance P from primary afferent neurons (Welch et al., 2000, Toxicon, 38, 245-258), this confirms the link between nociceptin-ORL1 interaction and subsequent release of substance P.
11 Thus, a TM can be said to have agonist activity at the ORL 1 receptor if the TM causes an induction in the release of substance P from a nociceptive sensory --afferent neuron -(see-Example 10). 5 In a particularly preferred embodiment of the invention, the TM is nociceptin - the natural ligand for the ORL 1 receptor. Nociceptin targets the ORL 1 receptor with high affinity. Examples of other preferred TMs include: Code Sequence Ref. SEQ ID No. Nociceptin 1-17 FGGFTGARKSARKLANQ [1] 37,38 Nociceptin 1-11 FGGFTGARKSA [1] 39,40 Nociceptin-[Y1O]1-11 FGGFTGARKYA [1] 41,42 Nociceptin [Y11]1-11 FGGFTGARKSY [1] 43,44 Nociceptin [Y14]1-17 FGGFTGARKSARKYANQ [1] 45,46 Nociceptin 1-13 FGGFTGARKSARK [2] 47,48 Nociceptin [R14K15] 1- FGGFTGARKSARKRKNQ [3,4] 49,50 17 (also known in this specification as "variant" nociceptin) Peptide agonist Peptide agonists from [5] combinatorial library approach 10 [1] Mogil & Pasternak, 2001, Pharmacol. Rev., 53, 381-415 12 [21 Maile et al., 2003, Neurosci. Lett., 350, 190-192 [31 Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63 [41 Okada et al., 2000, Biochem. Biophys. Res. Commun., 278, 493-498 [5}-Dooley-et a/:, 1997, J Pharmacol Exp -Ther. 283(2), 735-41. 5 The above-identified "variant" TM demonstrates particularly good binding affinity (when compared with natural nociceptin) for nociceptive sensory afferents. This is surprising as the amino acid modifications occur at a position away from the N terminus of the TM. Moreover, the modifications are almost at the C-terminus of 10 the TM, which in turn is attached to a large polypeptide sequence (i.e. the translocation domain). Generally speaking, a TM-containing fusion protein will demonstrate an approximate 100-fold reduction in binding ability vis-b-vis the TM per se. The above-mentioned "variant" TM per se demonstrates an approximate 3- to 10-fold increase in binding ability for a nociceptive sensory afferent (e.g. via 15 the ORL1 receptor) vis-b-vis natural nociceptin. Thus, a "variant" TM-containing fusion might be expected to demonstrate an approximate 10-fold reduction in binding ability for a nociceptive sensory afferent (e.g. via the ORL1 receptor) vis &-vis 'free' nociceptin. However, the present inventors have demonstrated that such "variant" TM-containing fusion proteins demonstrate a binding ability that 20 (most surprisingly) closely mirrors that of 'free' nociceptin - see Figure 14. In the context of the present invention, the term opioid or an agonist of the ORL 1 receptor (such as nociceptin, or any one of the peptides listed in the table above) embraces molecules having at least 70%, preferably at least 80%, more 25 preferably at least 90%, and most preferably at least 95% homology with said opioid or agonist. The agonist homologues retain the agonist properties of nociceptin at the ORL 1 receptor, which may be tested using the methods provided in Example 10. Similarly, an opioid homologue substantially retains the binding function of the opioid with which it shows high homology. 30 13 The invention also encompasses fragments, variants, and derivatives of any one of the TMs described above. These fragments, variants, and derivatives substantially retain the properties that are ascribed to said TMs. 5 In addition to the above-mentioned opioid and non-opioid classes of TMs, a variety of other polypeptides are suitable for targeting the conjugates of the present invention to nociceptive sensory afferents (e.g. to nociceptors). In this regard, particular reference is made to galanin and derivatives of galanin. Galanin receptors are found pre- and post-synaptically in DRGs (Liu & Hokfelt, 10 (2002), Trends Pharm. Sci., 23(10), 468-74), and are enhanced in expression during neuropathic pain states. Proteinase-activated receptors (PARs) are also a preferred group of TMs of the present invention, most particularly PAR-2. It is known that agonists of PAR-2 induce/ elicit acute inflammation, in part via a neurogenic mechanism. PAR2 is expressed by primary spinal afferent neurons, 15 and PAR2 agonists stimulate release of substance P (SP) and calcitonin gene related peptide (CGRP) in peripheral tissues A particularly preferred set of TMs of the present invention includes: Ligand Reference Nociceptin Guerrini, et al., (1997) J. Med. Chem., 40, pp. 1789-1793 P-endorphin Blanc, et al, (1983) J. Biol. Chem., 258(13), pp. 8277-8284 Endomorphin-1; Zadina, etal., (1997). Nature, 386, pp. Endomorphin-2 499-502 Dynorphin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Met-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds.
14 Ligand Reference Leu-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Xu et al., (2000) Neuropeptides, 34 (3&4), 137-147 PAR-2 peptide Vergnolle et al., (2001) Nat. Med., 7(7), 821-826 The protease cleavage site of the present invention allows cleavage (preferably controlled cleavage) of the fusion protein at a position between the non-cytotoxic protease component and the TM component. It is this cleavage reaction that 5 converts the fusion protein from a single chain polypeptide into a disulphide linked, di-chain polypeptide. According to a preferred embodiment of the present invention, the TM binds via a domain or amino acid sequence that is located away from the C-terminus of the 10 TM. For example, the relevant binding domain may include an intra domain or an amino acid sequence located towards the middle (i.e. of the linear peptide sequence) of the TM. Preferably, the relevant binding domain is located towards the N-terminus of the TM, more preferably at or near to the N-terminus. 15 In one embodiment, the single chain polypeptide fusion may include more than one proteolytic cleavage site. However, where two or more such sites exist, they are different, thereby substantially preventing the occurrence of multiple cleavage events in the presence of a single protease. In another embodiment, it is preferred that the single chain polypeptide fusion has a single protease cleavage 20 site. The protease cleavage sequence(s) may be introduced (and/ or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage 15 sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed, the following are preferred: 5 Enterokinase (DDDDKI) Factor Xa (IEGRI / IDGRJ,) TEV(Tobacco Etch virus) (ENLYFQI.G) Thrombin (LVPRI.GS) 10 PreScission (LEVLFQIGP). Also embraced by the term protease cleavage site is an intein, which is a self cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present. 15 In use, the protease cleavage site is cleaved and the N-terminal region (preferably the N-terminus) of the TM becomes exposed. The resulting polypeptide has a TM with an N-terminal domain or an intra domain that is substantially free from the remainder of the conjugate. This arrangement 20 ensures that the N-terminal component (or intra domain) of the TM may interact directly with a Binding Site on a target cell. In a preferred embodiment, the TM and the protease cleavage site are distanced apart in the fusion protein by at most 10 amino acid residues, more preferably by 25 at most 5 amino acid residues, and most preferably by zero amino acid residues. Thus, following cleavage of the protease cleavage site, a conjugate is provided with a TM that has an N-terminal domain that is substantially free from the remainder of the conjugate. This arrangement ensures that the N-terminal component of the Targeting Moiety may interact directly with a Binding Site on a 30 target cell.
16 One advantage associated with the above-mentioned activation step is that the TM only becomes susceptible to N-terminal degradation once proteolytic cleavage of the fusion protein has occurred. In addition, the selection of a specific protease cleavage site permits selective activation of the polypeptide 5 fusion into a di-chain conformation. Construction of the single-chain polypeptide fusion of the present invention places the protease cleavage site between the TM and the non-cytotoxic protease component. 10 It is preferred that, in the single-chain fusion, the TM is located between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (i.e. as occurs with native clostridial holotoxin), though in the case of the present invention the order of the two 15 components is reversed vis-b-vis native holotoxin. A further advantage with this arrangement is that the TM is located in an exposed loop region of the fusion protein, which has minimal structural effects on the conformation of the fusion protein. In this regard, said loop is variously referred to as the linker, the activation loop, the inter-domain linker, or just the surface exposed loop (Schiavo 20 et al 2000, Phys. Rev., 80, 717-766; Turton et al., 2002, Trends Biochem. Sci., 27, 552-558). In one embodiment, in the single chain polypeptide, the non-cytotoxic protease component and the translocation component are linked together by a disulphide 25 bond. Thus, following cleavage of the protease cleavage site, the polypeptide assumes a di-chain conformation, wherein the protease and translocation components remain linked together by the disulphide bond. To this end, it is preferred that the protease and translocation components are distanced apart from one another in the single chain fusion protein by a maximum of 100 amino 30 acid residues, more preferably a maximum of 80 amino acid residues, particularly preferably by a maximum of 60 amino acid residues, and most preferably by a maximum of 50 amino acid residues.
17 In one embodiment, the non-cytotoxic protease component forms a disulphide bond with the translocation component of the fusion protein. For example, the --amino -acid residue -of-the-protease component that forms the disulphide bond is 5 located within the last 20, preferably within the last 10 C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the translocation component that forms the second part of the disulphide bond may be located within the first 20, preferably within the first 10 N-terminal amino acid residues of the translocation component. 10 Alternatively, in the single chain polypeptide, the non-cytotoxic protease component and the TM may be linked together by a disulphide bond. In this regard, the amino acid residue of the TM that forms the disulphide bond is preferably located away from the N-terminus of the TM, more preferably towards 15 to C-terminus of the TM. In one embodiment, the non-cytotoxic protease component forms a disulphide bond with the TM component of the fusion protein. In this regard, the amino acid residue of the protease component that forms the disulphide bond is preferably 20 located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the TM component that forms the second part of the disulphide bond is preferably located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the TM. 25 The above disulphide bond arrangements have the advantage that the protease and translocation components are arranged in a manner similar to that for native clostridial neurotoxin. By way of comparison, referring to the primary amino acid sequence for native clostridial neurotoxin, the respective cysteine amino acid 30 residues are distanced apart by between 8 and 27 amino acid residues - taken from Popoff, MR & Marvaud, J-C, 1999, Structural & genomic features of I8 clostridial neurotoxins, Chapter 9, in The Comprehensive Sourcebook of Bacterial Protein Toxins. Ed. Alouf & Freer: Serotype .. Sequence.-- - 'Native' length between C-C BoNT/A1 CVRGIITSKTKS----LDKGYNKALNDLC 23 BoNT/A2 CVRGIIPFKTKS--LDEGYNKALNDLC 23 BoNT/B CKSVKAPG------------C 8 BoNT/C CHKAIDGRS--------LYNKTLDC 15 BoNT/D CLRLTK-----------NSRDDSTC 12 BoNT/E CKN-IVSVK-----GIRK---SIC 13 BoNT/F CKS-VIPRK--------GTKAPP-RLC 15 BoNT/G CKPVMYKNT ----- GKSE---QC 13 TeNT CKKIIPPTNIRENLYNRTASLTDLGGELC 27 5 'Information from proteolytic strains only The fusion protein may comprise one or more purification tags, which are located N-terminal to the protease component and/ or C-terminal to the translocation component. 10 Whilst any purification tag may be employed, the following are preferred: His-tag (e.g. 6 x histidine), preferably as a C-terminal and/ or N-terminal tag MBP-tag (maltose binding protein), preferably as an N-terminal tag 15 GST-tag (glutathione-S-transferase), preferably as an N-terminal tag His-MBP-tag, preferably as an N-terminal tag GST-MBP-tag, preferably as an N-terminal tag Thioredoxin-tag, preferably as an N-terminal tag CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag. 20 19 According to a further embodiment of the present invention, one or more peptide spacer molecules may be included in the fusion protein. For example, a peptide spacer may be employed between a purification tag and the rest of the fusion -protein -molecule (e.g. -between -an N-terminal -purification- tag -and a protease 5 component of the present invention; and/ or between a C-terminal purification tag and a translocation component of the present invention). A peptide spacer may be also employed between the TM and translocation components of the present invention. 10 A variety of different spacer molecules may be employed in any of the fusion proteins of the present invention. Examples of such spacer molecules include those illustrated in Figures 28 and 29. Particular mention here is made to GS15, GS20, GS25, and Hx27 - see Figures 28 and 29. 15 The present inventors have unexpectedly found that the fusion proteins (eg. CPNv/A) of the present invention may demonstrate an improved binding activity for nociceptive sensory afferents when the size of the spacer is selected so that (in use) the C-terminus of the TM and the N-terminus of the translocation component are separated from one another by 40-105 angstroms, preferably by 20 50-100 angstroms, and more preferably by 50-90 angstroms. In another embodiment, the preferred spacers have an amino acid sequence of 11-29 amino acid residues, preferably 15-27 amino acid residues, and more preferably 20-27 amino acid residues. Suitable spacers may be routinely identified and obtained according to Crasto, C.J. and Feng, J.A. (2000) May, 13(5), pp. 309-312 - see 25 also http://www.fccc./edu/research/labs/fenlglimker.html. In accordance with a second aspect of the present invention, there is provided a DNA sequence that encodes the above-mentioned single chain polypeptide. In a preferred aspect of the present invention, the DNA sequence is prepared as part 30 of a DNA vector, wherein the vector comprises a promoter and terminator. In a preferred embodiment, the vector has a promoter selected from: 20 Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) -Ar-aBAD-- -- - - L-arabinose 0.2% (0:002-0.4%)- 5 T7-lac operator IPTG 0.2 mM (0.05-2.0mM) The DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques. 10 The above-mentioned DNA sequence information is optionally modified for codon-biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed. The DNA backbone is preferably screened for any inherent nucleic acid 15 sequence, which when transcribed and translated would produce an amino acid sequence corresponding to the protease cleave site encoded by the second peptide-coding sequence. This screening may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). 20 According to a further embodiment of the present invention, there is provided a method of preparing a non-cytotoxic agent, comprising: a. contacting a single-chain polypeptide fusion protein of the invention with a protease capable of cleaving the protease cleavage site; 25 b. cleaving the protease cleavage site, and thereby forming a di-chain fusion protein. This aspect provides a di-chain polypeptide, which generally mimics the structure of clostridial holotoxin. In more detail, the resulting di-chain polypeptide typically 30 has a structure wherein: a. the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is 21 capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; b. the second chain comprises the TM and the translocation domain- that- is- capable- of-translocating the -protease or 5 protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; and the first and second chains are disulphide linked together. 10 According to a further aspect of the present invention, there is provided use of a single chain or di-chain polypeptide of the invention, for the manufacture of a medicament for treating, preventing or ameliorating pain. According to a related aspect, there is provided a method of treating, preventing 15 or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a single chain or di-chain polypeptide of the invention. The present invention addresses a wide range of pain conditions, in particular 20 chronic pain conditions. Preferred conditions include cancerous and non cancerous pain, inflammatory pain and neuropathic pain. The opioid-fusions of the present application are particularly suited to addressing inflammatory pain, though may be less suited to addressing neuropathic pain. The galanin-fusions are more suited to addressing neuropathic pain. 25 In use, the polypeptides of the present invention are typically employed in the form of a pharmaceutical composition in association with a pharmaceutical carrier, diluent and/or excipient, although the exact form of the composition may be tailored to the mode of administration. Administration is preferably to a 30 mammal, more preferably to a human.
22 The polypeptides may, for example, be employed in the form of a sterile solution for intra-articular administration or intra-cranial administration. Spinal injection (e.g. epidural or intrathecal) is preferred. 5 The dosage ranges for administration of the polypeptides of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the components, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and 10 the judgement of the attending physician. Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably 0.0001 0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004 0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30mg, but 15 typically will be in the region of 0.01 to I mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly. A particularly preferred dosing regimen is based on 2.5 ng of fusion protein (e.g. CPNv/A) as the 1X dose. In this regard, preferred dosages are in the range 1X 20 1OOX (i.e. 2.5-250 ng). This dosage range is significantly lower (i.e. at least 10 fold, typically 100-fold lower) than would be employed with other types of analgesic molecules such as NSAIDS, morphine, and gabapentin. Moreover, the above-mentioned difference is considerably magnified when the same comparison is made on a molar basis - this is because the fusion proteins of the 25 present invention have a considerably greater Mw than do conventional 'small' molecule therapeutics. Wide variations in the required dosage, however, are to be expected depending on the precise nature of the components, and the differing efficiencies of various 30 routes of administration.
23 Variations in these dosage levels can be adjusted using standard empirical routines for optimisation, as is well understood in the art. Compositions- suitable -for injection may be-in-the-form of solutions, suspensions 5 or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use. Fluid unit dosage forms are typically prepared utilising a pyrogen-free sterile vehicle. The active ingredients, depending on the vehicle and concentration 10 used, can be either dissolved or suspended in the vehicle. In preparing administrable solutions, the polypeptides can be dissolved in a vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques 15 before-filling into suitable sterile vials-or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative 20 or bactericidal, suspending or emulsifying agents may be dissolved in the vehicle. Dry powders which are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilised drug substance and other ingredients into a sterile container using aseptic technique in a sterile area. 25 Alternatively the polypeptides and other ingredients may be dissolved in an aqueous vehicle, the solution is sterilized by filtration and distributed into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically. 30 Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile 24 components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation. Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the 5 composition/s to facilitate uniform distribution of the components. In one embodiment the present invention provides a single chain, polypeptide fusion protein, comprising: a) a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of 10 a nociceptive sensory afferent; b) a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; 15 c) a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non cytotoxic protease or fragment thereof and the Targeting Moiety; and d) a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal 20 membrane and into the cytosol of the nociceptive sensory afferent; wherein the Targeting Moiety is located between the protease cleavage site and the translocation domain. In another embodiment the present invention provides a nucleic acid sequence encoding a polypeptide fusion protein according to the invention. 25 in another embodiment the present invention provides a DNA vector, which comprises a promoter, a nucleic acid sequence according to the invention, wherein said DNA sequence is located downstream of the promoter, and a terminator is located downstream of the DNA construct. In another embodiment the present invention provides a method for preparing a single 30 chain polypeptide fusion protein according to the invention, comprising expressing a 24A nucleic acid sequence according to the invention, or a DNA vector according to the invention, in a host cell. In another embodiment the invention provides a non-cytotoxic polypeptide, obtainable by a method of the invention, wherein the polypeptide is a di-chain polypeptide, and wherein: 5 a) the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; b) the second chain comprises the TM and the translocation domain that is capable of translocating the protease or protease fragment from within an 10 endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; and the first and second chains are disulphide linked together. In a further embodiment, there is provided a method-of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective 15 amount of a fusion protein according to the invention or a polypeptide according to the invention. Definitions Section Targeting Moiety (TM) means any chemical structure associated with an agent that functionally interacts with a Binding Site to cause a physical association between the 20 agent and the surface of a target cell. In the context of the present invention, the target cell is a nociceptive sensory afferent. The term TM embraces any molecule (i.e. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (e.g. endosome formation) - also referred to as receptor-mediated endocytosis. The TM 25 may possess an endosomal membrane translocation function, in which case separate TM 24B and Translocation Domain components need not be present in an agent of the present invention. The TM of the present invention binds (preferably specifically binds) to a nociceptive sensory afferent (e.g. a primary nociceptive afferent). In this regard, specifically binds 5 means that the TM binds to a nociceptive sensory afferent (e.g. a primary nociceptive afferent) with a greater affinity than it binds to other neurons such as non-nociceptive afferents, and/ or to motor neurons (i.e. the natural target for clostridial neurotoxin holotoxin). The term "specifically binding" can also mean that a given TM binds to a given receptor, for example the ORL 1 receptor, with a binding affinity (Ka) of 106 M~' or greater, 10 preferably 10 7
M~
1 or greater, more preferably 108 M~ 1 or greater, and most preferably, 10 9
M-
1 or greater.
25 For the purposes of this invention, an agonist is defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, which process-is susceptible to inhibition by a protease (or fragment thereof) -capable of 5 cleaving a protein of the exocytic fusion apparatus in said target cell. Accordingly, the particular agonist definition of the present invention would exclude many molecules that would be conventionally considered as agonists. 10 For example, nerve growth factor (NGF) is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic fusion. In addition, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by the protease activity of a non 15 cytotoxic toxin-molecule. The term "fragment", when used in relation to a protein, means a peptide having at least thirty-five, preferably at least twenty-five, more preferably at least twenty, and most preferably at least ten amino acid residues of the protein in question. 20 The term "variant", when used in relation to a protein, means a peptide or peptide fragment of the protein that contains one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage. 25 The term "derivative", when used in relation to a protein, means a protein that comprises the protein in question, and a further peptide sequence. The further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the original protein. Two or more peptides (or fragments, or variants) may be joined together to form a derivative. Alternatively, 30 a peptide (or fragment, or variant) may be joined to an unrelated molecule (e.g. a second, unrelated peptide). Derivatives may be chemically synthesized, but will be typically prepared by recombinant nucleic acid methods. Additional 26 components such as lipid, and/or polysaccharide, and/or polyketide components may be included. Throughout this specification, reference to the "ORL 1 receptor" embraces all 5 members of the ORL 1 receptor family. Members of the ORL 1 receptor family typically have a seven transmembrane domain structure and are coupled to G proteins of the Gi and Go families. A method for determining the G-protein stimulating activity of ligands of the ORL1 receptor is given in Example 12. A method for measuring reduction in cellular cAMP levels following ORL 1 activation 10 is given in Example 11. A further characteristic of members of the ORL 1 receptor family is that they are typically able to bind nociceptin (the natural ligand of
ORL
1 ). As an example, all alternative splice variants of the ORL 1 receptor, are members of the ORL1 receptor family. 15 The term non-cytotoxic means that the protease molecule in question does not kill the target cell to which it has been re-targeted. The protease of the present invention embraces all naturally-occurring non cytotoxic proteases that are capable of cleaving one or more proteins of the 20 exocytic fusion apparatus in eukaryotic cells. The protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium or Neisseria (e.g. a clostridial L-chain, or a neisserial IgA 25 protease preferably from N. gonorrhoeae). The present invention also embraces modified non-cytotoxic proteases, which include amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified proteases still demonstrate the above 30 mentioned protease activity.
27 The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevinNAMP, or syntaxin). 5 Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins. 10 Exemplary neurotoxins are produced by clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum. The above-mentioned abbreviations are used throughout the present specification. For example, the 15 nomenclature BoNT/A denotes the source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes. The term L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of 20 proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis. A Translocation Domain is a molecule that enables translocation of a protease (or fragment thereof) into a target cell such that a functional expression of protease 25 activity occurs within the cytosol of the target cell. Whether any molecule (e.g. a protein or peptide) possesses the requisite translocation function of the present invention may be confirmed by any one of a number of conventional assays. For example, Shone C. (1987) describes an in vitro assay employing liposomes, 30 which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K* and/or 28 labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-1801. A-further example is-provided by Blaustein R. (1987), which describes a simple in 5 vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120]. 10 Additional methodology to enable assessment of membrane fusion and thus identification of Translocation Domains suitable for use in the present invention are provided by Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993. 15 The Translocation Domain is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore formation within the endosomal membrane. 20 The Translocation Domain may be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Hence, in one embodiment, the Translocation Domain is a translocating domain of an enzyme, such as a bacterial toxin or viral protein. 25 It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be employed in the present invention. 30 The Translocation Domain may be of a clostridial origin, namely the HN domain (or a functional component thereof). HN means a portion or fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal 29 half of the H-chain, or the domain corresponding to that fragment in the intact H chain. It is preferred that the H-chain substantially lacks the natural binding function of the Hc component of the H-chain. In this regard, the He function may be removed by deletion of the Hc amino acid sequence (either at the DNA 5 synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the H 0 function may be inactivated by chemical or biological treatment. Thus, the H-chain is preferably incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds. 10 In one embodiment, the translocation domain is a HN domain (or a fragment thereof) of a clostridial neurotoxin. Examples of suitable clostridial Translocation Domains include: Botulinum type A neurotoxin - amino acid residues (449-871) 15 Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) 20 Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879) For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al (1997) in The Clostridia: 25 Molecular Biology and Pathogenesis, Academic press. The term HN embraces naturally-occurring neurotoxin HN portions, and modified HN portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified HN portions still 30 demonstrate the above-mentioned translocation function.
30 Alternatively, the Translocation Domain may be of a non-clostridial origin (see Table 4). Examples of non-clostridial Translocation Domain origins include, but not be restricted to, the translocation domain of diphtheria toxin [O=Keefe et al., Proc. Natl.-Acad. Sci. USA (499-2)- 89,-6202-6206; Silverman et al., J. Biol. Chem. 5 (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the translocation domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-35591, the translocation domains of anthrax toxin [Blanke et al. Proc. Nati. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides of translocating function [Plank et al. J. Biol. 10 Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp.7934 7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp.1986 1992]. The Translocation Domain may mirror the Translocation Domain present in a naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain. 15 Particular examples of viral Translocation Domains suitable for use in the present invention include certain translocating domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e. membrane fusion and vesiculation) function of a 20 number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus haemagglutinin. Other virally expressed membrane fusion proteins known to have the desired translocating activity are a translocating domain of a fusogenic peptide of Semliki Forest Virus (SFV), a translocating domain of vesicular stomatitis virus (VSV) glycoprotein G, a translocating domain 25 of SER virus F protein and a translocating domain of Foamy virus envelope glycoprotein. Virally encoded Aspike proteins have particular application in the context of the present invention, for example, the El protein of SFV and the G protein of the G protein of VSV. 30 Use of the Translocation Domains listed in Table (below) includes use of sequence variants thereof. A variant may comprise one or more conservative nucleic acid substitutions and/ or nucleic acid deletions or insertions, with the 31 proviso that the variant possesses the requisite translocating function. A variant may also comprise one or more amino acid substitutions and/ or amino acid deletions or insertions, so long as the variant possesses the requisite translocating function. 5 Translocation Amino acid References domain source residues Diphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain 11 of 405-613 Prior et al., 1992, Biochemistry pseudomonas 31, 3555-3559 exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus GLFGAIAGFIENGWE Plank et al., 1994, J. Biol. Chem. haemagglutinin GMIDGWYG, and 269, 12918-12924 Variants thereof Wagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 Semliki Forest virus Translocation domain Kielian et al., 1996, J Cell Biol. fusogenic protein 134(4), 863-872 Vesicular Stomatitis 118-139 Yao et al., 2003, Virology 310(2), virus glycoprotein G 319-332 SER virus F protein Translocation domain Seth et al., 2003, J Virol 77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al., 2003, J envelope Virol. 77(8), 4722-4730 glycoprotein 32 Figures -Figure-I - Purification-of-a-LC/A-nociceptin-HN/A-fusion protein 5 Figure 2 Purification of a nociceptin-LC/A-HN/A fusion protein Figure 3 Purification of a LC/C-nociceptin-HN/C fusion protein Figure 4 Purification of a LC/A-met enkephalin-HN/A fusion protein Figure 5 Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a nociceptin-LC/A-HN/A fusion protein 10 Figure 6 In vitro catalytic activity of a LC/A-nociceptin-HN/A fusion protein Figure 7 Purification of a LC/A-nociceptin variant-HN/A fusion protein Figure 8 Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a LC/A-nociceptin variant-HN/A fusion protein Figure 9 Expressed / purified LC/A-nociceptin-HN/A fusion protein family with 15 variable spacer length product(s) Figure 10 Inhibition of SP release and cleavage of SNAP-25 by CPN-A Figure 11 Inhibition of SP release and cleavage of SNAP-25 over extended time periods after exposure of DRG to CPN-A Figure 12 Cleavage of SNAP-25 by CPNv-A 20 Figure 13 Cleavage of SNAP-25 over extended time periods after exposure of DRG to CPNv-A Figure 14 CPNv-A fusion-mediated displacement of [3H]-nociceptin binding Figure 15 Expressed / purified CPNv(Ek)-A product Figure 16 Cleavage of SNAP-25 by CPNv(Ek)-A 25 Figure 17 Expressed / purified CPNv-C product Figure 18 Cleavage of syntaxin by CPNv-C Figure 19 CPN-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model Figure 20 CPN-A efficacy in the Streptozotocin (STZ)-induced Peripheral 30 Diabetic Neuropathy (Neuropathic Pain) model Figure 21 CPNv-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model 33 Figure 22 Expressed / purified LC/A-CPLE-HN/A product Figure 23 Expressed / purified LC/A-CPBE-HN/A product Figure 24 Expressed / purified CPOP-A product -Figure-25 Expressed / purified CPOPv-A product 5 Figure 26 In vitro SNAP-25 cleavage in a DRG cell model Figure 27 Expressed / purified CPNv-A-FXa-HT (removable his-tag) Figure 28 In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by ligand competition assay Figure 29 In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with 10 variable spacer length, as assessed by in vitro SNAP-25 cleavage The Figures are now described in more detail. Figure 1 - Purification of a LC/A-nociceptin-HN/A fusion protein 15 Using the methodology outlined in Example 9, a LC/A-nociceptin-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to 20 activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in 25 the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 2 - Purification of a nociceptin-LC/A-HN/A fusion protein 30 Using the methodology outlined in Example 9, a nociceptin-LC/A-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column.
34 Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure-were -assessed by SDS-PAGE (Panel A) and 5 Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. 10 Figure 3 - Purification of a LC/C-nociceptin-HNIC fusion protein Using the methodology outlined in Example 9, an LC/C-nociceptin-HN/C fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. 15 Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were 20 used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 4 - Purification of a LCIA-met enkephalin-HN/A fusion protein 25 Using the methodology outlined in Example 9, an LC/A-met enkephalin-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor 30 Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final purified 35 material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 5 - Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion 5 protein and a nociceptin-LC/A-HN/A fusion protein The ability of nociceptin fusions to bind to the ORL 1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM 10 [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin-HN/A fusion is far superior to the nociceptin-LC/A-HN/A fusion at interacting with the
ORL
1 receptor. 15 Figure 6 - In vitro catalytic activity of a LC/A-nociceptin-HN/A fusion protein The in vitro endopeptidase activity of the purified LC/A-nociceptin-HN/A fusion protein was determined essentially as described in Chaddock et al 2002, Prot. 20 Express Purif. 25, 219-228. Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed to varying concentrations of fusion protein for 1 hour at 37 0 C. Following a series of washes, the amount of cleaved SNAP-25 peptide was quantified by reactivity with a specific antisera. 25 Figure 7 - Purification of a LC/A-nociceptin variant-HN/A fusion protein Using the methodology outlined in Example 9, an LC/A-nociceptin variant-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture 30 column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples 36 from the purification procedure were assessed by SDS-PAGE. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. 5 Figure 8 - Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a LC/A-nociceptin variant-HN/A fusion protein The ability of nociceptin fusions to bind to the ORL1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) 10 were exposed to varying concentrations of test material in the presence of 1nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant HN/A fusion (CPNv-LHA) is superior to the LC/A-nociceptin variant-HN/A fusion 15 -(CPN-LHA) at interacting with the ORL 1 receptor. Figure 9 - Expressed / purified LC/A-nociceptin-HN/A fusion protein family with variable spacer length product(s) 20 Using the methodology outlined in Example 9, variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are purified from E. coli cell paste. Samples from the purification of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A were assessed by SDS-PAGE prior to staining with Coomassie Blue. The 25 electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Top panel: M = benchmark molecular mass markers: S = total E. coli protein soluble fraction; FT = proteins that did not bind to the Ni 2 +-charged Sepharose column; FUSION = fusion protein eluted by the addition of imidazole. Bottom panel: Lane 1 = benchmark 30 molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 --charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; 37 Lane 5 = purified final material post activation with Factor Xa (5 pl); Lane 6 = purified final material post activation with Factor Xa (10 pl); Lane 7 = purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material -post-activation-with-Factor -X-a-+-DTT-(-5-pl-);--L-ane-9-=-purified-final-material post 5 activation with Factor Xa + DTT (10 pl); Lane 10 = purified final material post activation with Factor Xa + DTT (20 pl). Figure 10 - Inhibition of SP release and cleavage of SNAP-25 by CPN-A 10 Briefly, primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A for 24 hours. Cellular proteins were separated by SDS PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and plotted against fusion concentration 15 (dashed line). Material was also recovered for an analysis of substance P content using a specific EIA kit. Inhibition of substance P release is illustrated by the solid line. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 6.30±2.48 nM. 20 Figure 11 - Inhibition of SP release and cleavage of SNAP-25 over extended time periods after exposure of DRG to CPN-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) was used 25 as a control. After this initial exposure, extracellular material was removed by washing, and the cells incubated at 37 0 C for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis 30 and illustrated by the dotted lines. Material was also recovered for an analysis of substance P content using a specific EIA kit. Inhibition of substance P release is illustrated by the solid lines.
38 Figure 12 - Cleavage of SNAP-25 by CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying 5 concentrations of CPNv-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM. 10 Figure 13 - Cleavage of SNAP-25 over extended time periods after exposure of DRG to CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying 15 concentrations of CPNv-A for 24 hours. CPN-A was used as a control. After this initial exposure, extracellular material was removed by washing, and the cells incubated at 37"C for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of 20 cleaved SNAP-25 was calculated by densitometric analysis. Figure 14 - CPNv-A fusion-mediated displacement of [31-]-nociceptin binding 25 The ability of nociceptin fusions to bind to the ORL 1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H1-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of 30 unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant HN/A fusion (labelled as CPNv-LHnA) is superior to the LC/A-nociceptin-HN/A fusion (labelled as CPN-LHnA) at interacting with the ORL1 receptor.
39 Figure 15 - Expressed / purified CPNv(Ek)-A product -Proteins-were subjected to SDS-PAGE prior-to staining-with Coomassie Blue. 5 The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv(Ek)-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 -charged Sepharose; Lane 4 = purified final material post activation with enterokinase (5 pl); Lane 5 = purified 10 final material post activation with enterokinase (10 pl); Lane 6 = purified final material post activation with enterokinase (20 pl); Lane 7 purified final material post activation with enterokinase + DTT (5 pl); Lane 8 = purified final material post activation with enterokinase + DTT (10 p1); Lane 9 = purified final material post activation with enterokinase + DTT (20 pl). 15 Figure 16 - Cleavage of SNAP-25 by CPNv(Ek)-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv(Ek)-A for 24 hours. Cellular proteins were separated by 20 SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. CPNv-A as prepared in Example 9 was used for comparison purposes. The percentage cleavage of SNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled as Xa activated) are 25 illustrated. Figure 17 - Expressed / purified CPNv-C product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. 30 The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-C. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = 40 purified material following initial capture on Ni 2 *-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 *-charged Sepharose; Lane 5 = purified material following second capture on Ni 2 *-charged Sepharose; -Lane 6 =-final purified material; Lane 7 = final purified material + DTT; Lane 8 = 5 benchmark molecular mass markers. Figure 18 - Cleavage of syntaxin by CPNv-C Primary cultures of dorsal root ganglia (DRG) were exposed to varying 10 concentrations of CPNv-C for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-syntaxin to facilitate an assessment of syntaxin cleavage. The percentage of cleaved syntaxin was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal syntaxin cleavage is estimated to be 3.13±1.96 nM. 15 Figure 19 - CPN-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit capsaicin-induced 20 mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat); after subcutaneous intraplantar treatment with CPN/A but before capsaicin (Pre-CAP); and following 25 capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 pL of a 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline. 30 41 Figure 20 - CPN-A efficacy in the Streptozotocin (STZ)-Induced Peripheral Diabetic Neuropathy (Neuropathic Pain) model -Male-Sprague-Dawley rats (250-300 g)-are-treated with 65-mg/kg STZ in citrate 5 buffer (I.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in response to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomized to 10 either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a test group (CPN/A). Test materials (20-25 pl) are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured at 1 day post-treatment and periodically thereafter over a 2 week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) is injected daily, 15 2 hours prior to the start of PWT testing. Figure 21 - CPNv-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model 20 The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin-induced mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat), after 25 subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre CAP), and following capsaicin challenge post-injection of CPNv/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 pL of a 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline. These data are expressed as a normalized paw withdrawal frequency differential, 30 in which the difference between the peak response (post-capsaicin) and the baseline response (pre-capsaicin) is expressed as a percentage. With this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower 42 dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A. -Figure 22---Expressed /[purified LC/A-CPLE-HN/A product 5 Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPLE-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. co/i protein soluble fraction; Lane 3 = 10 purified material following initial capture on Ni'--charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified material following second capture on Ni*-charged Sepharose; Lane 6 = final purified material; Lane 7 = final purified material + DTT. 15 Figure 23 - Expressed / purified LC/A-CPBE-HN/A product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Lane 1 = total E. coli 20 protein soluble fraction; Lane 2 = purified material following initial capture on Ni2+. charged Sepharose; Lane 3 = Factor Xa treated material prior to final capture on Ni 2 -charged Sepharose; Lane 4 = purified final material post activation with Factor Xa (5 pl); Lane 5 = purified final material post activation with Factor Xa (10 pl); Lane 6 = purified final material post activation with Factor Xa (20 pl); Lane 7 = 25 purified final material post activation with Factor Xa + DTT (5 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (20 pl); Lane 10 = benchmark molecular mass markers. 30 43 Figure 24 - Expressed / purified CPOP-A product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. -The-electrophoresis profile- indicates purification of a-disulphide-bonded di-chain 5 species of the expected molecular mass of CPOP-A. Lane 1 = benchmark molecular mass markers; Lane 2 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 3 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 4 = purified material following second capture on Niz+-charged Sepharose; Lane 5 = purified final material post 10 activation with Factor Xa (5 pl); Lane 6 = purified final material post activation with Factor Xa (10 pl); Lane 7 = purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 10 = purified final material post activation with Factor Xa + DTT (20 pl). 15 Figure 25 - Expressed / purified CPOPv-A product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain 20 species of the expected molecular mass of CPOPv-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified final material post activation with Factor Xa (5 pl); Lane 6 = 25 purified final material post activation with Factor Xa (10 pl); Lane 7 = purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 10 purified final material post activation with Factor Xa + DTT (20 pl). 30 44 Figure 26 - In vitro SNAP-25 cleavage in a DRG cell model Primary cultures of dorsal root ganglia (DRG) were exposed to varying --concentrations- of-GPOPv-A-for-24- hours- - Cellular proteins were separated by 5 SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. Figure 27 - Expressed / purified CPNv-A-FXa-HT (removable his-tag) 10 Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-A-FXa-HT. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble 15 ---fraction; Lane 3 = Factor-Xa -treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 4 = purified final material post activation with Factor Xa; Lane 5 = purified final material post activation with Factor Xa + DTT. Figure 28 - In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with 20 variable spacer length, as assessed by ligand competition assay The ability of LC/A-nociceptin-HN/A fusions of variable spacer length to bind to the ORL, receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying 25 concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). The upper panel illustrates the displacement characteristics of the GSO, GS20, GS30 and Hx27 spacers, whilst the lower 30 panel illustrates the displacement achieved by the GS10, GS15 and GS25 spaced fusion proteins. It is concluded that the GSO and GS30 spacers are 45 ineffective, and the GS10 is poorly effective, at displacing nociceptin from the ORL1 receptor. Figure-29- --in-vitro- efficacy of LC/A-nociceptin-HN/A fusion proteins with 5 variable spacer length, as assessed by in vitro SNAP-25 cleavage Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A (of variable spacer length) for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti 10 SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The poorly effective binding characteristics of the GS10 spaced fusion protein (see Figure 28) are reflected in the higher concentrations of fusion required to achieve cleavage of intracellular SNAP-25. GSO and GS30 spaced fusion proteins were completely 15 ineffective (date not shown). GS15, 20 and 25 spaced fusion proteins were similarly effective. SEQ ID NOs 20 SEQ ID1 DNA sequence of the LC/A SEQ ID2 DNA sequence of the HN/A SEQ ID3 DNA sequence of the LC/B SEQ ID4 DNA sequence of the HN/B SEQ ID5 DNA sequence of the LC/C 25 SEQ ID6 DNA sequence of the HN/C SEQ ID7 DNA sequence of the CPN-A linker SEQ ID8 DNA sequence of the A linker SEQ ID9 DNA sequence of the N-terminal presentation nociceptin insert SEQ ID10 DNA sequence of the CPN-C linker 30 SEQ ID11 DNA sequence of the CPBE-A linker SEQ ID12 DNA sequence of the CPNvar-A linker SEQ ID13 DNA sequence of the LC/A-CPN-HN/A fusion 46 SEQ ID14 Protein sequence of the LC/A-CPN-HN/A fusion SEQ ID15 DNA sequence of the N-LC/A-HN/A fusion SEQ ID16 Protein sequence of the N-LC/A-HN/A fusion -SEQ-ID-17 DNA sequence of the LC/C-CPN-HN/C fusion 5 SEQ ID18 Protein sequence of the LC/C-CPN-HN/C fusion SEQ ID19 DNA sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID20 Protein sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID21 DNA sequence of the LC/A-CPME-HN/A fusion SEQ ID22 Protein sequence of the LC/A-CPME-HN/A fusion 10 SEQ ID23 DNA sequence of the LC/A-CPBE-HN/A fusion SEQ ID24 Protein sequence of the LC/A-CPBE-HN/A fusion SEQ ID25 DNA sequence of the LC/A-CPNv-HN/A fusion SEQ ID26 Protein sequence of the LC/A-CPNv-HN/A fusion SEQ ID27 DNA sequence of the LC/A-CPN[1-11]-HN/A fusion 15 -SEQ ID28 Protein sequence of the LC/A-CPN[1 -11]-HN/A fusion SEQ ID29 DNA sequence of the LC/A-CPN[[Y101 -11I-HN/A fusion SEQ ID30 Protein sequence of the LC/A-CPN[[Y10]1-1 1]-HN/A fusion SEQ ID31 DNA sequence of the LC/A-CPN[[Yi 1]1-1 1]-HN/A fusion SEQ ID32 Protein sequence of the LC/A-CPN[[Y 11]1-1 1]-HN/A fusion 20 SEQ ID33 DNA sequence of the LC/A-CPN{{Y14]1-17]-HN/A fusion SEQ ID34 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion SEQ ID35 DNA sequence of the LC/A-CPN[ -1 3]-HN/A fusion SEQ ID36 Protein sequence of the LC/A- CPN[1-1 3]-HN/A fusion SEQ ID37 DNA sequence of CPN[1-17] 25 SEQ ID38 Protein Sequence of CPN[1 -17] SEQ 1039 DNA sequence of CPN[1-1 1] SEQ ID40 Protein sequence of CPN[1-11] SEQ ID41 DNA sequence of CPN[[Y10]1-11] SEQ ID42 Protein sequence of CPN[[Y10]1-1 111 30 SEQ ID43 DNA sequence of CPN[[Y1111-11] SEQ ID44 Protein sequence of CPN[[Y11]1-11I SEQ ID45 DNA sequence of CPN[[Y14]1-17] 47 SEQ ID46 Protein sequence of CPN[[Y14)1-17] SEQ ID47 DNA sequence of CPN[1-13] SEQ ID48 Protein sequence of CPN[1-13] -- SEQ ID49 - DNA-sequence -of -CPNv-(-alsc-known-as-N[[R14K-15]1--1-7) 5 SEQ ID50 Protein sequence of CPNv (also known as N[[R14K15]1-17]) SEQ ID51 DNA sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID52 Protein sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID53 DNA sequence of the CPN-A GS1 0 linker SEQ ID54 DNA sequence of the CPN-A GS1 5 linker 10 SEQ ID55 DNA sequence of the CPN-A GS25 linker SEQ ID56 DNA sequence of the CPN-A GS30 linker SEQ ID57 DNA sequence of the CPN-A HX27 linker SEQ ID58 DNA sequence of the LC/A-CPN(GS15)-HN/A fusion SEQ ID59 Protein sequence of the LC/A-CPN(GS15)-HN/A fusion 15 SEQ ID60 DNA sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID61 Protein sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID62 DNA sequence of the CPNvar-A Enterokinase activatable linker SEQ ID63 DNA sequence of the LC/A-CPNv(Ek)-HN/A fusion SEQ ID64 Protein sequence of the LC/A-CPNv(Ek)-HN/A fusion 20 SEQ ID65 DNA sequence of the CPNvar-A linker SEQ ID66 DNA sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID67 Protein sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID68 DNA sequence of the LC/A-CPLE-HN/A fusion SEQ ID69 Protein sequence of the LC/A-CPLE-HN/A fusion 25 SEQ ID70 DNA sequence of the LC/A-CPOP-HN/A fusion SEQ ID71 Protein sequence of the LC/A-CPOP-HN/A fusion SEQ ID72 DNA sequence of the LC/A-CPOPv-HN/A fusion SEQ ID73 Protein sequence of the LC/A-CPOPv-HN/A fusion SEQ ID74 DNA sequence of the IgA protease 30 SEQ ID75 DNA sequence of the IgA-CPNv-HN/A fusion SEQ ID76 Protein sequence of the IgA-CPNv-HN/A fusion SEQ ID77 DNA sequence of the FXa-HT 48 SEQ 1078 DNA sequence of the CPNv-A-FXa-HT SEQ ID79 Protein sequence of the CPNv-A-FXa-HT fusion SEQ ID80 DNA sequence of the DT translocation domain SEQ ID81 DNA sequence of the CPLE-DT-A 5 SEQ ID82 Protein sequence of the CPLE-DT-A fusion SEQ ID83 DNA sequence of the TeNT LC SEQ ID84 DNA sequence of the CPNv-TENT LC SEQ ID85 Protein sequence of the CPNV-TeNT LC fusion SEQ ID86 DNA sequence of the CPNvar-C linker 10 SEQ ID87 DNA sequence of the LC/C-CPNv-HN/C fusion (act. C) SEQ ID88 Protein sequence of the LC/C-CPNv-HN/C fusion (act. C) Examples 15 Example i - Preparation of a LC/A and HN/A backbone clones The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID1 and SEQ 102), though the 20 procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)]. Preparation of cloning and expression vectors 25 pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHI-Sall 30 Pstl-Hindll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
49 Preparation of protease (e.g. LC/A) insert The LC/A (SEQ ID1) is created by one of two ways: The--DNA sequence -is- designed--by -back- translation of the LC/A amino acid 5 sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_ CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHl/Sall recognition sequences are incorporated at the 5' and 10 3' ends respectively of the sequence, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding 15 sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the 20 LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and Sall restriction enzyme sequences incorporated into 25 the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the 30 pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation 50 temperature of the reaction tube automatically, cycling between approximately 940C (for denaturation), 550C (for oligonucleotide annealing), and 720C (for synthesis). Other reagents required for amplification of a PCR product include a -DNA polymerase (such -as Taq or Pfu polymerase), each of the four nucleotide 5 dNTP building blocks of DNA in equimolar amounts (50-200 pM) and a buffer appropriate for the enzyme optimised for Mg 2 + concentration (0.5-5 mM). The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits 10 commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example, using Quickchange (Stratagene Inc.)]. 15 -Preparation of translocatio-n (e.g. HN) inset The HN/A (SEQ ID2) is created by one of two ways: The DNA sequence is designed by back translation of the HN/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)] 20 using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. A Pstl restriction sequence added to the N-terminus and Xbal stop codon-Hindill to the C-terminus ensuring the correct reading frame is maintained. The DNA sequence is screened (using software such as MapDraw, 25 DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon 30 Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank 51 Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma Genosys) and is provided in the pCR 4 vector. 5 The alternative method is to use PCR amplification from an existing DNA sequence with Pstl and Xbal-stop codon-Hindlli restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not 10 compatible with the cloning system are then removed using site directed mutagenesis [for example using Quickchange (Stratagene Inc.)]. Example 2 - Preparation of a LC/A-nociceptin-HN/A fusion protein (nociceptin is N-terminal of the HN-chain) 15 Preparation of linker-nociceptin-spacer insert The LC-HN tinker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the 20 cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N-terminus of the sequence ALNDL. This sequence information is freely available from available database sources such as GenBank (accession number 25 P10845) or Swissprot (accession locus BXA1 CLOBO). Into this linker a Factor Xa site, nociceptin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction 30 sites are then incorporated into the DNA sequence and can be arranged as BamHI-Sa/1-linker-protease site-nociceptin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-HindIll (SEQ ID7). It is important to ensure the correct reading frame is 52 maintained for the spacer, nociceptin and restriction sequences and that the Xbal sequence is not preceded by the bases, TC, which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation- and .any additional sequences- are removed manually from the 5 remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example, GenBank Release 143, 13 September 2004). This optimised DNA sequence is then 10 commercially synthesized (for example by Entelechon, Geneart or Sigma Genosys) and is provided in the pCR 4 vector. Preparation of the LC/A-nociceptin-HN/A fusion In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID13), the 15 pCR 4 vector encoding the linker (SEQ ID7) is cleaved with BamHI + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ IDI) cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with Pstl + Xbal restriction enzymes and serves as the recipient vector for the insertion and ligation of the HN/A DNA 20 (SEQ ID2) cleaved with Pstl + Xbal. The final construct contains the LC-linker nociceptin-spacer-HN ORF (SEQ ID13) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID14. Example 3 - Preparation of a nociceptin-LC/A-HN/A fusion protein 25 (nociceptin is N-terminal of the LC-chain) The LC/A-HN/A backbone is constructed as described in Example 2 using the synthesised A serotype linker with the addition of a Factor Xa site for activation, arranged as BamH I-Sall-linker-protease site-linker-Pstl-Xbal-stop codon-Hindill 30 (SEQ D8). The LC/A-HN/A backbone and the synthesised N-terminal presentation nociceptin insert (SEQ ID9) are cleaved with BamHI + Hindlll restriction enzymes, gel purified and ligated together to create a nociceptin- 53 spacer-LC-linker-HN. The ORF (SEQ ID15) is then cut out using restriction enzymes Aval + Xbal for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID16. 5 Example 4 - Preparation of a LC/C-nociceptin-HN/C fusion protein Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the C serotype linker arranged as BamHI-Sa/I-linker-protease site-nociceptin-Nhel-spacer-Spel-Pst-XbaI-stop 10 codon-Hindlli (SEQ 1010). The final construct contains the LC-linker-nociceptin spacer-HN ORF (SEQ ID17) for expression as a protein of the sequence illustrated in SEQ ID18. Example 5 - Preparation of a LC/C-nociceptin-HN/C fusion protein with a 15 serotype A activation sequence Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) and HNIC (SEQ ID6) are created and inserted into the A serotype linker arranged as BamHI-Sa/l-linker-protease site-nociceptin-Nhel-spacer-Spel-Pstl-Xbal-stop 20 codon-Hindlli (SEQ ID7). The final construct contains the LC-linker-nociceptin spacer-HN ORF (SEQ ID19) for expression as a protein of the sequence illustrated in SEQ ID20. Example 6 - Preparation of a LC/A-met enkephalin-HN/A fusion protein 25 Due to the small, five-amino acid, size of the met-enkephalin ligand the LC/A-met enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a template. Oligonucleotides are designed encoding the YGGFM 30 met-enkephalin peptide, ensuring standard E.coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID13) 54 either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC-linker-met enkephalin spacer-HN ORF (SEQ ID21) for expression as a protein of the sequence -illustrated in SEQ ID22. 5 Example 7 - Preparation of a LC/A-P endorphin-HN/A fusion protein Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) and HN/A (SEQ ID2) are created and inserted into the A serotype p endorphin linker 10 arranged as BamHI-Sall-linker-protease site-p endorphin-Nhel-spacer-Spel-Psti Xbal-stop codon-Hindlil (SEQ ID1 1). The final construct contains the LC-linker-P endorphin-spacer-HN ORF (SEQ ID23) for expression as a protein of the sequence illustrated in SEQ ID24. 15 Example 8 - Preparation of -a LC/A-nociceptin variant-HN/A fusion protein Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) and HN/A (SEQ 102) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-SaIl-linker-protease site-nociceptin variant-Nhel-spacer 20 Spel-Pst-Xbal-stop codon-Hindill (SEQ ID12). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF (SEQ ID25) for expression as a protein of the sequence illustrated in SEQ ID26. Example 9 - Purification method for LC/A-nociceptin-HN/A fusion protein 25 Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample 30 remains cool. Spin the lysed cells at 18 000 rpm, 40C for 30 minutes. Load the supernatant onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is 55 sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4 0 C overnight and 5 measure the OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100 pg fusion protein and Incubate at 25*C static overnight. Load onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away 10 the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4*C overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20*C. Test purified protein using OD, BCA, purity analysis and SNAP-25 assessments. 15 Example 10 - Confirmation of TM Agonist Activity by measuring release of substance P from neuronal cell cultures Materials 20 Substance P EIA is obtained from R&D Systems, UK. Methods Primary neuronal cultures of eDRG are established as described previously (Duggan et al., 2002). Substance P release from the cultures is assessed by 25 EIA, essentially as described previously (Duggan et al., 2002). The TM of interest is added to the neuronal cultures (established for at least 2 weeks prior to treatment); control cultures are performed in parallel by addition of vehicle in place of TM. Stimulated (100 mM KCl) and basal release, together with total cell lysate content, of substance P are obtained for both control and TM treated 30 cultures. Substance P immunoreactivity is measured using Substance P Enzyme 56 Immunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK) according to manufacturers' instructions. -The amount of Substance P released by the neuronal cells in the presence of the 5 TM of interest is compared to the release obtained in the presence and absence of 100 mM KCI. Stimulation of Substance P release by the TM of interest above the basal release, establishes that the TM of interest is an "agonist ligand" as defined in this specification. If desired the stimulation of Substance P release by the TM of interest can be compared to a standard Substance P release-curve 10 produced using the natural ORL-1 receptor ligand, nociceptin (Tocris). Example 11 - Confirmation of ORLj receptor activation by measuring forskolin-stimulated cAMP production 15 --Confirmation that-a- given TM is acting via-the-ORL1 receptor is- provided by the following test, in which the TMs ability to inhibit forskolin-stimulated cAMP production is assessed. Materials 20 [ 3 H]adenine and [1 4 C]cAMP are obtained from GE Healthcare Methods The test is conducted essentially as described previously by Meunier et al. [Isolation and structure of the endogenous agonist of opioid receptor-like
ORL
1 25 receptor. Nature 377: 532-535, 1995] in intact transfected-CHO cells plated on 24-well plastic plates. To the cells is added [3H]adenine (1.0 pCi) in 0.4 ml of culture medium. The cells remain at 37*C for 2 h to allow the adenine to incorporate into the intracellular 30 ATP pool. After 2 h, the cells are washed once with incubation buffer containing: 130 mM NaCl, 4.8 mM KCI, 1.2 mM KH 2
PO
4 , 1.3 mM CaCl 2 , 1.2 mM MgSO4, 10 57 mM glucose, 1 mg/ml bovine serum albumin and 25 mM HEPES pH 7.4, and replaced with buffer containing forskolin (10 pM) and isobutylmethylxanthine (50 pM) with or without the TM of interest. After 10 min, the medium is aspirated and - replaced-with 0.5 ml, 0.2-M HCI.- -Approximately 1000 cpm of [ 1 4 C]cAMP is added 5 to each well and used as an internal standard. The contents of the wells are then transferred to columns of 0.65 g dry alumina powder. The columns are eluted with 4 ml of 5 mM HCI, 0.5 ml of 0.1 M ammonium acetate, then two additional millilitres of ammonium acetate. The final eluate is collected into scintillation vials and counted for 14C and tritium. Amounts collected are corrected for recovery of 10 [ 14 C]cAMP. TMs that are agonists at the ORL, receptor cause a reduction in the level of cAMP produced in response to forskolin. Example 12 - Confirmation of ORL 1 receptor activation using a GTPyS binding functional assay 15 Confirmation that a given TM is acting via the ORL 1 receptor is also provided by the following test, a GTPyS binding functional assay. Materials 20 [ 3 5 S]GTPyS is obtained from GE Healthcare Wheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare Methods This assay is carried out essentially as described by Traynor and Nahorski 25 [Modulation by p-opioid agonists of guanosine-5 -O-(3-[ 35 S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848-854, 1995]. Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mM 30 ethylenediaminetetraacetic acid, then centrifuged at 500 x g for 10 min. Cells are resuspended in this buffer and homogenized with a Polytron Homogenizer.
58 The homogenate is centrifuged at 27,000 x g for 15 min, and the pellet resuspended in buffer A, containing: 20 mM HEPES, 10 mM MgCI2, 100 mM NaCl, pH 7.4. The suspension is recentrifuged at 20,000 x g and suspended once-more-in buffer A.- For-the-binding assay, membranes (8-15 pg protein) are 5 incubated with [31S]GTP S (50 pM), GDP (10 pM), with and without the TM of interest, in a total volume of 1.0 ml, for 60 min at 250C. Samples are filtered over glass fibre filters and counted as described for the binding assays. Example 13 - Preparation of a LC/A-nociceptin-HN/A fusion protein 10 (nociceptin is N-terminal of the HN-chain) The linker-nociceptin-spacer insert is prepared as described in Example 2. Preparation of the LC/A-nociceptin-HNIA fusion 15 In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved with BamHl + Sal restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHl + Sall. The resulting plasmid DNA is then cleaved with BamHl + Hindlll restriction enzymes 20 and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHl, Sall, Pstl, and HindIll such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with Pstl + Hindill restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC-linker-nociceptin-spacer-HN ORF 25 (SEQ ID1 3) for expression as a protein of the sequence illustrated in SEQ ID14. Example 14 - Preparation of a nociceptin-LC/A-HN/A fusion protein (nociceptin is N-terminal of the LC-chain) 30 In order to create the nociceptin-spacer-LC/A-HNIA construct, an A serotype linker with the addition of a Factor Xa site for activation, arranged as BamHI-Sall- 59 linker-protease site-linker-Psti-Xbal-stop codon-Hindill (SEQ ID8) is synthesised as described in Example 13. The pCR 4 vector encoding the linker is cleaved with BamHI + Sall restriction enzymes. This cleaved vector then serves as the recipient for insertion and-ligation of the-L-G/A DNA (SEQ IDI) also cleaved with 5 BamHI + Sall. The resulting plasmid DNA is then cleaved with BamHI + HindIll restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing the synthesised N-terminal presentation nociceptin insert (SEQ ID9). This construct is then cleaved with Aval + Hindlll and inserted into an expression vector such as the pMAL plasmid (NEB). The HN/A DNA (SEQ ID2) is 10 then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL-nociceptin-LC/A-linker construct. The final construct contains the nociceptin-spacer-LC/A-H/A ORF (SEQ ID51) for expression as a protein of the sequence illustrated in SEQ ID52. 15 Example 15 - Preparation and purification of an LC/A-nociceptin-HN/A fusion protein family with variable spacer length Using the same strategy as employed in Example 2, a range of DNA linkers were prepared that encoded nociceptin and variable spacer content. Using one of a 20 variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-Sall-linker-protease site-nociceptin-Nhel-spacer-Spel-Pstl 25 Xbal-stop codon-Hindill (SEQ ID53 to SEQ ID57). It is important to ensure the correct reading frame is maintained for the spacer, nociceptin and restriction sequences and that the Xbal sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation and any additional sequences are removed manually 30 from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and 60 codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. 5 The spacers that were created included: Code Protein sequence of the linker SEQ ID of the linker DNA GS10 ALAGGGGSALVLQ 53 GS15 ALAGGGGSGGGGSALVLQ 54 GS25 ALAGGGGSGGGGSGGGGSGGGGSALVLQ 55 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ 56 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 57 Table I By way of example, in order to create the LC/A-CPN(GS15)-HN/A fusion construct 10 (SEQ ID58), the pCR 4 vector encoding the linker (SEQ ID54) is cleaved with BamHl + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ IDI) also cleaved with BamHl + Sall. The resulting plasmid DNA is then cleaved with BamHl + HindIll restriction enzymes and the LC/A-linker fragment inserted into a similarly 15 cleaved vector containing a unique multiple cloning site for BamHl, Sall, Pstl, and HindIll such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A CPN(GS15)-HN/A ORF (SEQ ID58) for expression as a protein of the sequence 20 illustrated in SEQ ID59. As a further example, to create the LC/A-CPN(GS25)-HN/A fusion construct (SEQ ID60), the pCR 4 vector encoding the linker (SEQ ID55) is cleaved with BamHl + Sall restriction enzymes. This cleaved vector then serves as the recipient vector 61 for insertion and ligation of the LC/A DNA (SEQ 101) cleaved with BamHl + Sall. The resulting plasmid DNA is then cleaved with BamHI + Hindlll restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHl, Sall, Psti, and Hindlli such 5 as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with Psti + Hindill restriction enzymes and inserted into the similarly cleaved pMAL-LC/A linker construct. The final construct contains the LC/A-CPN(GS25)-HN/A ORF (SEQ ID60) for expression as a protein of the sequence illustrated in SEQ ID61. 10 Variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are similarly created. Using the purification methodology described in Example 9, fusion protein is purified from E. coli cell paste. Figure 9 illustrates the purified product obtained in the case of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A. 15 - Example 16 - Assessment of in vitro efficacy of an LC/A-nociceptin-HNA fusion Fusion protein prepared according to Examples 2 and 9 was assessed in the 20 eDRG neuronal cell model. Assays for the inhibition of substance P release and cleavage of SNAP-25 have been previously reported (Duggan et al., 2002, J. Bio. Chem., 277, 34846 34852). Briefly, dorsal root ganglia neurons are harvested from 15-day-old fetal 25 Sprague-Dawley rats and dissociated cells plated onto 24-well plates coated with Matrigel at a density of 1 x 106 cells/well. One day post-plating the cells are treated with 10 pM cytosine P-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco's minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and 30 100 ng/ml 2.5S mouse nerve growth factor. Cultures are maintained for 2 weeks at 37 0 C in 95% air/5% C02 before addition of test materials.
62 Release of substance P from eDRG is assessed by enzyme-linked immunosorbent assay. Briefly, eDRG cells are washed twice with low potassium -balanced- salt solution (BSS: -5 mM KCI, 137 mM NaCl, 1.2 mM MgC 2 , 5 mM 5 glucose, 0.44 mM KH 2 PO4, 20 mM HEPES, pH 7.4, 2 mM CaCl2). Basal samples are obtained by incubating each well for 5 min. with 1 ml of low potassium BSS. After removal of this buffer, the cells are stimulated to release by incubation with 1 ml of high potassium buffer (BSS as above with modification to include 100 mM KCI isotonically balanced with NaCl) for 5 min. All samples 10 are removed to tubes on ice prior to assay of substance P. Total cell lysates are prepared by addition of 250 pi of 2 M acetic acid/0.1% trifluoroacetic acid to lyse the cells, centrifugal evaporation, and resuspension in 500 pl of assay buffer. Diluted samples are assessed for substance P content. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits 15 (Cayman Chemical Company or R&D Systems) according to manufacturers' instructions. Substance P is expressed in pg/mi relative to a standard substance P curve run in parallel. SDS-PAGE and Western blot analysis were performed using standard protocols 20 (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycine polyacrylamide gel (Novex) and subsequently transferred to nitrocellulose membrane. The membranes were probed with a monoclonal antibody (SMI-81) that recognises cleaved and intact SNAP-25. Specific binding was visualised using peroxidase conjugated secondary antibodies and a chemiluminescent detection system. 25 Cleavage of SNAP-25 was quantified by scanning densitometry (Molecular Dynamics Personal SI, ImageQuant data analysis software). Percent SNAP-25 cleavage was calculated according to the formula: (Cleaved SNAP 25/(Cleaved+lntact SNAP-25))x100. 30 Following exposure of eDRG neurons to an LC/A-nociceptin-HN/A fusion (termed CPN-A), both inhibition of substance P release and cleavage of SNAP-25 are 63 observed (Figure 10). After 24 h exposure to the fusion, 50% of maximal SNAP 25 cleavage is achieved by a fusion concentration of 6.3±2.5 nM. -The -effect of the- fusion- is-also assessed-at -defined time points following a 16 h 5 exposure of eDRG to CPN-A. Figure 11 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 28 days post exposure. Example 17 - Assessment of in vitro efficacy of an LC/A-nociceptin variant 10 HN/A fusion Fusion protein prepared according to Examples 8 and 9 was assessed in the eDRG neuronal cell mode using the method described in Example 16. 15 Following exposure of eDRG neurons to an LC/A-nociceptin variant-HN/A fusion (termed CPNv-A), both inhibition of substance P release and cleavage of SNAP 25 are observed. After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved by a fusion concentration of 1.4±0.4 nM (Figure 12). 20 The effect of the fusion is also assessed at defined time points following a 16 h exposure of eDRG to CPN-A. Figure 13 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 24 days post exposure. 25 The binding capability of the CPNv-A fusion protein is also assessed in comparison to the CPN-A fusion. Figure 14 illustrates the results of a competition experiment to determine binding efficacy at the ORL-1 receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, thereby confirming that access to the receptor is possible with the ligand in the central presentation format. 30 64 Example 18 - Preparation of an LC/A-nociceptin variant-HN/A fusion protein that is activated by treatment with Enterokinase Following-the methods used in Examples I and 2, the LC/A (SEQ 101) and HN/A 5 (SEQ ID2) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-Sal-linker-enterokinase protease site-nociceptin variant Nhel-spacer-Spel-Psti-Xbal-stop codon-Hindlli (SEQ ID62). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID63) for expression as a protein of the sequence illustrated in SEQ ID64. The fusion 10 protein is termed CPNv(Ek)-A. Figure 15 illustrates the purification of CPNv(Ek) A from E. coli following the methods used in Example 9 but using Enterokinase for activation at 0.00064 pg per 100 pg of fusion protein. Example 19 - Assessment of in vitro efficacy of a LC/A-nociceptin variant 15 HN/A fusion that has been activated by treatment with enterokinase The CPNv(Ek)-A prepared in Example 18 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 16). Figure 16 illustrates the cleavage of SNAP-25 20 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency of cleavage is observed to be similar to that achieved with the Factor Xa-cleaved material, as recorded in Example 17. Example 20 - Preparation of an LC/C-nociceptin variant-HN/C fusion protein 25 with a Factor Xa activation linker derived from serotype A Following the methods used in Example 4, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the A serotype nociceptin variant linker arranged as BamHl-Sal-linker-nociceptin variant-Nhel-spacer-Spel-Pstl-Xbal 30 stop codon-Hindlil (SEQ ID65). The final construct contains the LC-linker nociceptin variant-spacer-HN ORF sequences (SEQ ID66) for expression as a protein of the sequence illustrated in SEQ ID67. The fusion protein is termed 65 CPNv-C (act. A). Figure 17 illustrates the purification of CPNv-C (act. A) from E. coli following the methods used in Example 9. Example 21 - Assessment of in vitro-efficacy of an LC/C-nociceptin variant 5 HN/C fusion protein Following the methods used in Example 9, the CPNv-C (act. A) prepared in Example 20 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 16). After 24 h 10 exposure to the fusion, 50% of maximal syntaxin cleavage is achieved by a fusion concentration of 3.1±2.0 nM. Figure 18 illustrates the cleavage of syntaxin following 24 h exposure of eDRG to CPNv-C (act. A). Example 22 - Assessment of in vivo efficacy of an LC/A-nociceptin-HN/A 15 - fusion The ability of an LC/A-nociceptin- HN/A fusion (CPN/A) to inhibit acute capsaicin induced mechanical allodynia is evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals are evaluated for paw withdrawal 20 frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study, after subcutaneous treatment with CPNIA but before capsaicin, and following capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'). Capsaicin challenge is achieved by injection of 10 pL of a 0.3% solution. Sample dilutions are prepared in 0.5% 25 BSA/saline. Figure 19 illustrates the reversal of mechanical allodynia that is achieved by pre-treatment of the animals with a range of concentrations of LC/A nociceptin-HN/A fusion. The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit streptozotocin 30 (STZ)- induced mechanical (tactile) allodynia in rats is evaluated. STZ-induced mechanical allodynia in rats is achieved by injection of streptozotocin (i.p. or i.v.) which yields destruction of pancreatic -cells leading to loss of insulin production, 66 with concomitant metabolic stress (hyperglycemia and hyperlipidemia). As such, STZ induces Type I diabetes. In addition, STZ treatment leads to progressive development of neuropathy, which serves as a model of chronic pain with hyperalgesia and allodynia that may reflect signs observed in diabetic humans 5 (peripheral diabetic neuropathy). Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ in citrate buffer (I.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in 10 response to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomized to either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a test group (CPN/A). Test materials (20-25 pI) are injected 15 -subcutaneously as -a--single- injection (except -gabapentin) and the PWT is measured at I day post-treatment and periodically thereafter over a 2-week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) is injected daily, 2 hours prior to the start of PWT testing. Figure 20 illustrates the reversal of allodynia achieved by pre-treatment of the animals with 750 ng of CPN/A. Data 20 were obtained over a 2-week period after a single injection of CPN/A Example 23 - Assessment of in vivo efficacy of an LC/A-nociceptin variant HNIA fusion 25 The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin-induced mechanical allodynia is evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals are evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat); after 30 subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre CAP); and following capsaicin challenge post-injection of CPNv/A (average of 67 responses at 15' and 30'; CAP). Capsaicin challenge is achieved by injection of 10 pL of a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline. -Figure-21-illustrates-the-reversal-of-allodynia -that is -achieved -by-pre-treatment of 5 the animals with a range of concentrations of LC/A-nociceptin variant-HN/A fusion in comparison to the reversal achieved with the addition of LC/A-nociceptin-HN/A fusion. These data are expressed as a normalized paw withdrawal frequency differential, in which the difference between the peak response (post-capsaicin) and the baseline response (pre-capsaicin) is expressed as a percentage. With 10 this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A. Example 24 - Preparation of an LC/A-leu enkephalin-HN/A fusion protein 15 Due to the small, five-amino acid, size of the leu-enkephalin ligand the LC/A-leu enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a template. Oligonucleotides are designed encoding the YGGFL 20 leu-enkephalin peptide, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID1 3) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC-linker-leu enkephalin 25 spacer-HN ORF (SEQ ID68) for expression as a protein of the sequence illustrated in SEQ ID69. The fusion protein is termed CPLE-A. Figure 22 illustrates the purification of CPLE-A from E. coli following the methods used in Example 9. 30 68 Example 25 - Expression and purification of an LC/A-beta-endorphin-HNiA fusion protein -Following the methods-used in Example 9, and with the LC/A-beta-endorphin 5 HN/A fusion protein (termed CPBE-A) created in Example 7, the CPBE-A is purified from E. co/i. Figure 23 illustrates the purified protein as analysed by SDS-PAGE. Example 26 - Preparation of an LC/A-nociceptin mutant-HN/A fusion protein 10 Due to the single amino acid modification necessary to mutate the nociceptin sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin mutant-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a 15 template. Oligonucleotides are designed encoding tyrosine at position I of the nociceptin sequence, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID1 3) either side on the nociceptin section. The SDM product is checked by 20 sequencing and the final construct containing the LC/A-nociceptin mutant-spacer HN/A fusion ORF (SEQ ID70) for expression as a protein of the sequence illustrated in SEQ ID71. The fusion protein is termed CPOP-A. Figure 24 illustrates the purification of CPOP-A from E. coli following the methods used in Example 9. 25 Example 27 - Preparation and assessment of an LC/A-nociceptin variant mutant-HN/A fusion protein Due to the single amino acid modification necessary to mutate the nociceptin 30 sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin variant mutant HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin variant-HN/A fusion 69 (SEQ ID25) as a template. Oligonucleotides are designed encoding tyrosine at position 1 of the nociceptin sequence, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by -sequences-complimentary to-the linker-region of the LC/A-nociceptin variant-HN/A 5 fusion (SEQ ID25) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC/A-nociceptin mutant-spacer-HN/A fusion ORF (SEQ ID72) for expression as a protein of the sequence illustrated in SEQ ID73. The fusion protein is termed CPOPv-A. Figure 25 illustrates the purification of CPOPv-A from E. coli following the 10 methods used in Example 9. Using methodology described in Example 16, CPOPv-A is assessed for its ability to cleave SNAP-25 in the eDRG cell model. Figure 26 illustrates that CPOPv-A is able to cleave SNAP-25 in the eDRG model, achieving cleavage of 50% of the 15 maximal SNAP-25 after exposure of the cells to approximately 5.9 nM fusion for 24 h. Example 28 - Preparation of an IgA protease-nociceptin variant-HN/A fusion protein 20 The IgA protease amino acid sequence was obtained from freely available database sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of 25 Neisseria gonorrhoeae IgA protease, Nature, 1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli expression was determined. A BamHI recognition sequence was incorporated at the 5' end and a codon encoding a cysteine amino acid and Sall recognition sequence were incorporated at the 3' end of the IgA 30 DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required for 70 cloning were removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage was assessed Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed -by reference to published codon usage- tables. This 5 optimised DNA sequence (SEQ ID74) containing the IgA open reading frame (ORF) is then commercially synthesized. The IgA (SEQ ID74) is inserted into the LC-linker-nociceptin variant-spacer-HN ORF (SEQ ID25) using BamHI and Sall restriction enzymes to replace the LC 10 with the IgA protease DNA. The final construct contains the IgA-linker-nociceptin variant-spacer-HN ORF (SEQ ID75) for expression as a protein of the sequence illustrated in SEQ ID76. Example 29 - Preparation and assessment of a nociceptin targeted 15 -endopeptidase fusion protein with a removable histidine purification tag. DNA was prepared that encoded a Factor Xa removable his-tag (his6), although it is clear that alternative proteases site such as Enterokinase and alternative purification tags such as longer histidine tags are also possible. Using one of a 20 variety of reverse translation software tools [for example EditSeq best E. co/i reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the Factor Xa removable his-tag region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as Nhel-linker-Spel-Pst\-HN/A-Xbal-LEIEGRSGHHHHHHStop 25 codon-Hindlll (SEQ ID77). The DNA sequence is screened for restriction sequence incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and 30 codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart 71 or Sigma-Genosys) and is provided in the pCR 4 vector. In order to create CPNv-A-FXa-HT (SEQ ID78, removable his-tag construct) the pCR 4 vector encoding the removable his-tag is cleaved with Nhel and HindIll. The Nhel -HindH I -fragment-is then-inserted-into-the- LC/A-CPNv-HN/A vector (SEQ-ID25) that 5 has also been cleaved by Nhel and Hindlll. The final construct contains the LC/A-linker-nociceptin variant-spacer-HN-FXa-Histag-Hindll ORF sequences (SEQ ID78) for expression as a protein of the sequence illustrated in SEQ ID79. Figure 27 illustrates the purification of CPNv-A-FXa-HT from E. coli following the methods used in Example 9. 10 Example 30 - Preparation of a leu-enkephalin targeted endopeptidase fusion protein containing a translocation domain derived from diphtheria toxin The DNA sequence is designed by back translation of the amino acid sequence 15 of the translocation domain of the diphtheria toxin (obtained from freely available database sources such as GenBank (accession number IXDTT) using one of a variety of reverse translation software tools [for example EditSeq best E. co/i reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. Restriction sites are then incorporated into the DNA sequence and can be 20 arranged as Nhel-Linker-Spel-Pstl- diphtheria translocation domain-Xbal-stop codon-HindIll (SEQ ID80). Pstl/Xbal recognition sequences are incorporated at the 5' and 3' ends of the translocation domain respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage 25 sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the 30 %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the diphtheria translocation domain is then 72 commercially synthesized as Nhel-Linker-SpeI-Pstl- diphtheria translocation domain-Xbal-stop codon-Hindill (for example by Entelechon, Geneart or Sigma Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR 4 vector encoding-the-diphtheria-translocation domain is cleaved with Nhel and Xbal. The 5 Nhel - Xbal fragment is then inserted into the LC/A-CPLE-HN/A vector (SEQ ID68) that has also been cleaved by Nhel and Xbal. The final construct contains the LC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF sequences (SEQ ID81) for expression as a protein of the sequence illustrated in SEQ ID82. 10 Example 31 - Preparation of a nociceptin variant targeted endopeptidase ( fusion protein containing a LC domain derived from tetanus toxin. The DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as 15 -GenBank (accession-number--X 044 36 ) using - one- of -a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/Sal recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame (SEQ ID83). The DNA 20 sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is 25 assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by 30 Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHl and Sall. The BamHl - Sall fragment is then inserted into the LC/A-CPNv-HN/A 73 vector (SEQ ID25) that has also been cleaved by BamHl and Sall. The final construct contains the TeNT LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID84) for expression as a protein of the sequence illustrated in -SEQ 1D85: Example 32 - Preparation of an LC/C-nociceptin variant-HN/C fusion protein with a native serotype C linker that is susceptible to Factor Xa cleavage Following the methods used in Example 4, the LC/C (SEQ ID5) and HN/C (SEQ 10 ID6) are created and inserted into the C serotype nociceptin variant linker arranged as BamH I-Sal-linker-nociceptin variant-Nhel-s pacer-Spel-Pst\-Xbal stop codon-Hindill (SEQ ID86). The final construct contains the LC-linker nociceptin variant-spacer-HN ORF sequences (SEQ ID87) for expression as a protein of the sequence illustrated in SEQ ID88. The fusion protein is termed 15 CPNv-C (act. C).
Claims (29)
1. A single chain, polypeptide fusion protein, comprising: a) a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion 5 apparatus of a nociceptive sensory afferent; b) a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; 10 c) a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non cytotoxic protease or fragment thereof and the Targeting Moiety; d) a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal 15 membrane and into the cytosol of the nociceptive sensory afferent; wherein the Targeting Moiety is located between the protease cleavage site and the translocation domain, and wherein the protease or fragment thereof and the translocation domain are distanced apart from one another by a maximum of 100 amino acid residues. 20
2. The fusion protein according to Claim 1, wherein the Targeting Moiety and the protease cleavage site are separated by at most 10 amino acid residues.
3. The fusion protein according to Claim 1, wherein the Targeting Moiety and the protease cleavage site are separated by at most 5 amino acid residues.
4. The fusion protein according to Claim 1, wherein the Targeting Moiety and the 25 protease cleavage site are separated by at most zero amino acid residues.
5. The fusion protein according to any preceding claim, wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease. 75
6. The fusion protein according to any preceding claim, wherein the translocation domain is the HN domain of a clostridial neurotoxin.
7. The fusion protein according to any preceding claim, wherein the Targeting Moiety comprises at most 50 amino acid residues. 5
8. The fusion protein according to any preceding claim, wherein the Targeting Moiety comprises at most 40 amino acid residues.
9. The fusion protein according to any preceding claim, wherein the Targeting Moiety comprises at least 30 amino acid residues.
10. The fusion protein according to any preceding claim, wherein the Targeting 10 Moiety comprises at most 20 amino acid residues.
11. The fusion protein according to any of Claims 1 to 10, wherein the Targeting Moiety: (i) is an opioid; (ii) is an agonist of a receptor present on a nociceptive sensory afferent; or 15 (iii) binds to the ORL, receptor.
12. The fusion protein according to Claim 11, wherein the Targeting Moiety is an agonist of a receptor present on a primary nociceptive sensory afferent.
13. The fusion protein according to Claim 11 or 12, wherein the Targeting Moiety binds specifically to the ORL1 receptor. 20
14. The fusion protein according to any one of Claims 11 to 13, wherein the Targeting Moiety is an agonist of the ORL 1 receptor.
15. The fusion protein according to any one of Claims 11 to 14, wherein the Targeting Moiety has at least 70% homology to SEQ ID No. 38 or a fragment thereof. 76
16. The fusion protein according to any one of Claims 11 to 14, wherein the Targeting Moiety has at least 95% homology to SEQ ID No. 38 or a fragment thereof.
17. The fusion protein according to any one of Claims 11 to 14, wherein the 5 Targeting Moiety is SEQ ID No. 38 or a fragment thereof.
18. The fusion protein according to any one of Claims 11 to 14, wherein the Targeting Moiety is one of SEQ ID Nos: 40, 42, 44, 46, 48 or 50.
19. The fusion protein according to any one of Claims 11 to 14, wherein the Targeting Moiety is nociceptin. 10
20. The fusion protein according to any of Claims 1 to 10, wherein the Targeting Moiety is selected from the group consisting of nociceptin, p-endorphin, endomorphine-1, endomorphine-2, dynorphin, met-enkephalin, leu-enkephalin, galanin, and PAR-2 peptide.
21. The fusion protein according to any preceding claim, wherein the translocation 15 domain is separated from the Targeting Moiety by a peptide spacer molecule.
22. A polypeptide fusion protein according to Claim 1, comprising any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 52, 59, 61, 64, 67, 69, 71, 73, 76, 79, 82, 85, or 88.
23. A nucleic acid sequence encoding the polypeptide fusion protein according to 20 any preceding claim.
24. A nucleic acid sequence encoding the polypeptide fusion protein according to any preceding claim, wherein the nucleic acid molecule comprises any one of SEQ ID NOs: 1-13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 58, 60, 63, 66, 68, 70, 72, 75, 78, 81, 84, or 87; or 25 the complementary DNA strand thereof.
25. A DNA vector, which comprises a promoter, a nucleic acid sequence according to Claim 23 or 24, wherein said DNA sequence is located downstream of the promoter, and a terminator is located downstream of the DNA construct. 77
26. A method for preparing a single-chain polypeptide fusion protein according to any of Claims 1 to 22, comprising expressing a nucleic acid sequence according to Claim 23 or 24, or a DNA vector according to Claim 25, in a host cell.
27. A method of preparing a non-cytotoxic agent, comprising: a) contacting a single-chain polypeptide fusion protein according to any of Claims 1 to 22 with a protease capable of cleaving the protease cleavage site; b) cleaving the protease cleavage site; and thereby forming a di-chain fusion protein.
) 28. A non-cytotoxic polypeptide, obtained by the method of Claim 27, wherein the polypeptide is a di-chain polypeptide, and wherein: a) the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; 5 b) the second chain comprises the TM and the translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; and the first and second chains are disulphide linked together. 0
29. Use of a fusion protein according to any of Claims 1 to 22 or a polypeptide according to Claim 28, for the manufacture of a medicament for treating, preventing or ameliorating pain.
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JP5826635B2 (en) * | 2009-03-13 | 2015-12-02 | アラーガン、インコーポレイテッドAllergan,Incorporated | Immune system-regulated endopeptidase activity assay |
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AU2012200046B2 (en) | 2015-05-28 |
AU2011202219B2 (en) | 2015-05-28 |
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AU2012201491A1 (en) | 2013-10-03 |
AU2012203056A1 (en) | 2012-06-14 |
AU2011202219A1 (en) | 2012-11-29 |
AU2012203055A1 (en) | 2012-06-14 |
AU2012201491B2 (en) | 2015-02-05 |
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