FUSION PROTEINS
A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, which cleaves a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a galanin Targeting Moiety that binds a Binding Site on the nociceptive sensory afferent, which can undergo endocytosis to be incorporated into an endosome; a protease cleavage site where the fusion protein is cleavable by a protease located between the non-cytotoxic protease and the galanin Targeting Moiety; a translocation domain that translocates the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; a first spacer from 4 to 25 amino acids between the non-cytotoxic protease and protease cleavage site; and a second spacer comprising from 4 to 35 residues between the galanin Targeting Moiety and translocation domain. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described.
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The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 39905_Sequence Listing_FINAL—2012-08-24_ST25.txt. The text file is 352 KB; was created on Aug. 24, 2012; and is being submitted via EFS-Web with the filing of the specification.
FIELD OF THE INVENTIONThis invention relates to non-cytotoxic fusion proteins, and to the therapeutic application thereof as analgesic molecules.
BACKGROUNDToxins 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 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 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 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.
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. botulinum, C. butyricum and C. argentinense. There are at present eight different classes of the clostridial neurotoxin, namely: tetanus toxin, and botulinum 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 synthesized by the host bacterium as single polypeptides that are modified post-translationally by a proteolytic cleavage event to form two 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.
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 hydrolyze different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25. 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 WO99/58571).
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. 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 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.
The above-mentioned TM replacement may be effected by conventional chemical 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. Alternatively, recombinant techniques may be employed, such as those described in WO98/07864. All of the above cited references are incorporated by reference herein.
Pain-sensing cells possess a wide range of receptor types. However, not all receptor types are suited (least of all desirable) for receptor-mediated endocytosis. Similarly, binding properties can vary widely between different TMs for the same receptor, and even more so between different TMs and different receptors.
There is therefore a need to develop modified non-cytotoxic fusion proteins that address one or more of the above problems. Of particular interest is the development of an alternative/improved non-cytotoxic fusion protein for use in treating pain.
The present invention seeks to address one or more of the above problems by providing unique fusion proteins.
SUMMARYThe present invention addresses one or more of the above-mentioned problems by providing a single chain, polypeptide fusion protein, comprising:
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- a. a non-cytotoxic protease which protease cleaves a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;
- b. a galanin Targeting Moiety that binds to a Binding Site on the nociceptive sensory afferent, which Binding Site endocytoses 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 and the galanin Targeting Moiety;
- d. a translocation domain that translocates the protease from within an endosome, across the endosomal 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;
- e. a first spacer located between the non-cytotoxic and the protease cleavage site, wherein said first spacer comprises an amino acid sequence of from 4 to 25 amino acid residues;
- f. a second spacer located between the galanin Targeting Moiety and the translocation domain, wherein said second spacer comprises an amino acid sequence of from 4 to 35 amino acid residues.
Using the methodology outlined in Example 3, a LC/A-GS18-galanin-GS20-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 enterokinase to activate the fusion protein and treated with factor Xa to remove the maltose-binding protein (MBP) tag. Activated fusion protein was then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (
Using the methodology outlined in Example 3, an LC/C-galanin-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. Bound proteins were eluted with 100 mM imidazole, treated with enterokinase to activate the fusion protein, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (
Chinese hamster ovary (CHO) cells were transfected so that they express the GALR1 receptor and SNAP-25. Said cells were used to measure cAMP deletion that occurs when the receptor is activated with a galanin ligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). The transfected cells were exposed to varying concentrations of galanin (GA16) fusion proteins having different serotype backbones (i.e., botulinum neurotoxin serotypes A, B, C and D) for 2 hours. cAMP levels were then detected by addition of a detection mix containing a fluorescently labeled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labeled anti-cAMP antibody and incubating at room temperature for 24 hours. Then samples were excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels. The data demonstrate that galanin fusion proteins of the present invention having different serotype backbones activated the GALR1 receptor.
Chinese hamster ovary (CHO) cells were transfected so that they express the GALR1 receptor. Said cells were further transfected to express a SNARE protein (SNAP-25). The transfected cells were exposed to varying concentrations of different galanin fusion proteins 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 data demonstrate that galanin fusion proteins having galanin-16 and galanin-30 ligands cleave SNARE protein. In addition, the data confirm that galanin fusion proteins having GS5, GS10 and GS18 spacers between the non-cytotoxic protease component and the protease cleavage site are functional.
The nociceptive flexion reflex (also known as paw guarding assay) is a rapid withdrawal movement that constitutes a protective mechanism against possible limb damage. It can be quantified by assessment of electromyography (EMG) response in anesthetized rat as a result of low dose capsaicin, electrical stimulation or the capsaicin-sensitized electrical response. Intraplantar pretreatment (24 hour) of fusion proteins of the present invention into 300-380 g male Sprague-Dawley rats. Induction of paw guarding was achieved by 0.006% capsaicin, 10 μl in PBS (7.5% DMSO), injected in 10 seconds. This produced a robust reflex response from biceps feroris muscle. A reduction/inhibition of the nociceptive flexion reflex indicates that the test substance demonstrates an anti-nociceptive effect. The data demonstrated the anti-nociceptive effect of the galanin fusion proteins of the present invention.
The ability of different galanin fusion proteins of the invention to inhibit capsaicin-induced thermal hyperalgesia was evaluated. Intraplantar pretreatment of fusion proteins into Sprague-Dawley rats and 24 hours later 0.3% capsaicin was injected and rats were put on 25° C. glass plate (rats contained in acrylic boxes, on 25° C. glass plate). Light beam (adjustable light Intensity) focused on the hind paw. Sensors detected movement of paw, stopping timer. Paw Withdrawal Latency is defined as the time to remove paw from heat source (Cut-off of 20.48 seconds). A reduction/inhibition of the paw withdrawal latency indicates that the test substance demonstrates an anti-nociceptive effect. No. 1=LHN-GA16; No. 2=LHN-GA30; No. 3=LC-GS5-EN-CPGA16-GS20-HN-HT; No. 4=LC-GS18-EN-CPGA16-GS20-HN-HT; No. 5=BOTOX; No. 6=morphine. The data demonstrated the enhanced anti-nociceptive effect of the galanin fusion proteins of the present invention compared to fusion proteins with a C-terminally presented ligand.
The ability of different galanin fusion proteins of the invention to inhibit capsaicin-induced thermal hyperalgesia was evaluated. Intraplantar pretreatment of fusion proteins into Sprague-Dawley rats and 24 hours later 0.3% capsaicin was injected and rats were put on a 25° C. glass plate (rats were contained in acrylic boxes, on 25° C. glass plate). Light beam (adjustable light Intensity) focused on the hind paw. Sensors detected movement of a paw, stopping a timer. Paw Withdrawal Latency is defined as the time to remove paw from heat source (Cut-off of 20.48 seconds). A reduction/inhibition of the paw withdrawal latency indicated that the test substance demonstrates an anti-nociceptive effect. The data demonstrated the anti-nociceptive effect of the galanin fusion proteins of the present invention having different serotype backbones (i.e., A, B, C and D).
Galanin fusion proteins lacking a first spacer (spacer 1) of the present invention located between the non-cytotoxic protease component and the Targeting Moiety component showed poor activation with protease (
The non-cytotoxic protease component of the present invention is a non-cytotoxic protease, which protease 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 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 clostridial neurotoxin L-chain. The term non-cytotoxic protease embraces functionally equivalent fragments and derivatives of said non-cytotoxic protease(s). A particularly preferred non-cytotoxic protease component is a botulinum neurotoxin (BoNT) L-chain.
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 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 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 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-chain.
The galanin TM component of the present invention is responsible for binding the fusion protein of the present invention to a Binding Site on a target cell. Thus, the galanin TM component is a ligand through which the fusion proteins of the present invention bind 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-fiber such as an Aδ-fiber or a C-fiber). Thus, the fusion proteins of the present invention are capable of 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 fusion proteins reduce or prevent the transmission of sensory afferent signals (e.g., neurotransmitters or neuromodulators) from peripheral to central pain fibers, and 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 which tissue or cells representative of the nociceptive sensory afferent (for example DRGs) are exposed to labeled (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, 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 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. Hulme, Oxford University Press.
The fusion proteins of the present invention generally demonstrate a reduced binding affinity (in the region of up to 10-fold) for the galanin receptor (e.g., GALR1) when compared with the corresponding ‘free’ TM (e.g., gal16). However, despite this observation, the fusion proteins of the present invention surprisingly demonstrate good efficacy. This can be attributed to two principal features. First, the non-cytotoxic protease component is catalytic—thus, the therapeutic effect of a few such molecules is rapidly amplified. Secondly, the galanin 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 fusion proteins of the present invention may be administered at a dosage that is much lower than 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 administered at much lower dosages, typically at least 10-fold lower, and more typically at 100-fold lower.
The galanin TM of the invention can also be a molecule that acts as an “agonist” at one or more of the galanin receptors present on a nociceptive sensory afferent, more particularly on a primary nociceptive afferent. Conventionally, an agonist has 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, 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 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. 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-cytotoxic toxin molecule.
In one embodiment, the fusion proteins according to the present invention demonstrate preferential receptor binding and/or internalization properties. This, in turn, may result in more efficient delivery of the protease component to a pain-sensing target cell.
Use of an agonist as a TM is self-limiting with respect to side-effects. In more detail, binding of an agonist TM to a pain-sensing target cell increases exocytic fusion, which may exacerbate the sensation of pain. However, the exocytic process that is stimulated by agonist binding is subsequently reduced or inhibited by the protease component of the fusion protein.
The agonist properties of a TM that binds to a receptor on a nociceptive afferent can be confirmed using the methods described in Example 9.
The Targeting Moiety of the present invention comprises or consists of galanin and/or derivatives of galanin. Galanin receptors (e.g., GALR1, GALR2 and GALR3) are found pre- and post-synaptically in dorsal root ganglia (DRGs) (Liu and Hokfelt, (2002) Trends Pharm. Sci., 23(10):468-474), and are enhanced in expression during neuropathic pain states. Xu et al., (2000) Neuropeptides, 34(3-4):137-147 provides further information in relation to galanin. All of the above cited references are incorporated by reference herein.
In one embodiment of the invention, the target for the galanin TM is the GALR1, GALR2 and/or the GALR3 receptor. These receptors are members of the G-protein-coupled class of receptors, and have a seven transmembrane domain structure.
In one embodiment, the galanin TM is a molecule that binds (preferably that specifically binds) to the GALR1, GALR2 and/or the GALR3 receptor. More preferably, the galanin TM is an “agonist” of the GALR1, GALR2 and/or the GALR3 receptor. The term “agonist” in this context is defined as above.
Wild-type human galanin peptide is a 30 amino acid peptide, abbreviated herein as “GA30” (represented by SEQ ID NO: 7). In one embodiment, the galanin TM comprises or consists of SEQ ID NO: 7.
The invention also encompasses fragments, variants, and derivatives of the galanin TM described above. These fragments, variants, and derivatives substantially retain the properties that are ascribed to said galanin TM (i.e., are functionally equivalent). For example, the fragments, variants, and derivatives may retain the ability to bind to the GALR1, GALR2 and/or GALR3 receptor. In one embodiment, the galanin TM of the invention comprises or consists of a 16 amino acid fragment of full-length galanin peptide and is referred to herein as GA16 (represented by SEQ ID NO: 8).
In one embodiment, the galanin TM comprises or consists of an amino acid sequence having at least 70%, preferably at least 80% (such as at least 82, 84, 85, 86, 88 or 89%), more preferably at least 90% (such as at least 91, 92, 93 or 94%), and most preferably at least 95% (such as at least 96, 97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 7 or SEQ ID NO: 8.
In one embodiment the galanin TM comprises or consists of an amino acid sequence having at least 70% (such as at least 80, 82, 84, 85, 86, 88 or 89%), more preferably at least 90% (such as at least 91, 92, 93 or 94%), and most preferably at least 95% (such as at least 96, 97, 98, 99 or 100%) amino acid sequence acid identity to full-length amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a fragment of SEQ ID NO: 7 or SEQ ID NO: 8 comprising or consisting of at least 10 (such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) contiguous amino acid residues thereof.
In one embodiment, the galanin Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO: 7 or a fragment comprising or consisting of at least 16 (such as at least 10, 11, 12, 13, 14 or 15) contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 7 or said fragment having a maximum of 6 (such as a maximum of 5, 4, 3, 2 or 1) conservative amino acid substitutions.
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 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 galanin TM binds via a domain or amino acid sequence that is located away from the C-terminus of the galanin 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 galanin TM, more preferably at or near to the N-terminus.
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 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 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:
In one embodiment, the protease cleavage site is an enterokinase cleavage site (DDDDK↓). In one embodiment, enterokinase protease is used to cleave the enterokinase cleavage site and activate the fusion protein.
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.
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 fusion protein. This arrangement ensures that the N-terminal component (or intra domain) of the TM may interact directly with a Binding Site on a target cell.
In one 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 at most 5 amino acid residues, and most preferably by zero amino acid residues. In one embodiment, the TM and the protease cleavage site are distanced apart in the fusion protein by 0-10 (such as 0-9,0-8, 0-7,0-6, 0-5,0-4, 0-3, 0-2) and preferably 0-1 amino acid residues Thus, following cleavage of the protease cleavage site, a fusion is provided with a TM that has an N-terminal domain that is substantially free from the remainder of the fusion. This arrangement ensures that the N-terminal component of the Targeting Moiety may interact directly with a Binding Site on a target cell.
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 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.
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 components is reversed vis-à-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 et al., (2000) Phys. Rev. 80:717-766; Turton et al., (2002) Trends Biochem. Sci. 27:552-558).
The single chain fusion protein of the present invention comprises a first spacer located between the non-cytotoxic protease and the protease cleavage site, wherein said first spacer comprises (or consists of) an amino acid sequence of from 4 to 25 (such as from 6 to 25, 8 to 25, 10 to 25, 15 to 25 or from 4 to 21, 4 to 20, 4 to 18, 4 to 15, 4 to 12 or 4 to 10) amino acid residues. In one embodiment, the first spacer comprises (or consists of) an amino acid sequence of at least 4 (such as at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acid residues. In one embodiment, the first spacer comprises (or consists of) an amino acid sequence of at most 25 (such as at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10) amino acid residues. Said first spacer enables cleavage of the fusion protein at the protease cleavage site.
Without a first spacer of the present invention, protease cleavage and activation of the fusion protein is markedly poor. Without wishing to be bound by theory, it is hypothesized that the galanin Targeting Moiety may sterically block or interact with the protease cleavage site resulting in poor activation of fusion proteins lacking a first spacer of the present invention. The present inventors believe that it is the flexibility afforded by the first spacer which provides for the enhanced/improved activation properties of the presently claimed fusion proteins. Rigid linkers such as alpha-helical linkers do not afford the necessary flexibility. This is also true for galanin fusion proteins having ‘natural’ spacer sequences containing a protease cleavage site, which may replicate undesirable rigid alpha-helical linker structures. Flexibility and mobility of polypeptide domains can be ascertained by a number of methods including determining the X-ray crystallographic B-factor (see e.g., Smith et al., (2003) Protein Sci. 12:1060-1072; incorporated by reference herein). The specifically selected spacer sequences of the present invention provide for enhanced activation over and above any ‘natural’ spacer sequences. Activation in this context means that said first spacer enables cleavage of the fusion protein at the protease cleavage site. Particularly preferred amino acid residues for use in the first spacer include glycine, threonine, arginine, serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamic acid and/or lysine. The aforementioned amino acids are considered to be the most flexible amino acids—see Smith et al., (2003) Protein Sci. 12:1060-1072.
In one embodiment, the amino acid residues of the first spacer are selected from the group consisting of glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine and/or valine. In one embodiment, the amino acid residues of the first spacer are selected from the group consisting of glycine, serine, alanine, leucine and/or valine. In one embodiment, the amino acid residues of the first spacer are selected from the group consisting of glycine, serine and/or alanine Glycine and serine are particularly preferred. In one embodiment, the first spacer comprises or consists of one or more pentapeptides having glycine, serine, and or threonine residues. One way of assessing whether the first spacer possesses the requisite flexibility in the presently claimed fusion proteins is by performing a simple protease cleavage assay. It would be routine for a person skilled in the art to assess cleavage/activation of a fusion protein—standard methodology is described, for example, in Example 1.
In one embodiment, the first spacer may be selected from a GS5, GS10, GS15, GS18, GS20, FL3 and/or FL4 spacers. The sequence of said spacers is provided in Table 1, below.
In one embodiment, the first spacer enables at least 45% (such as at least 50, 55, 60, 65, 70, 75, 80, 90, 95, 98, 99 or 100%) activation of the fusion protein by protease cleavage. In one embodiment, the first spacer enables at least 70% activation of the fusion protein by protease cleavage.
In one embodiment, the first spacer is not a naturally-occurring spacer sequence. In one embodiment, the first spacer does not comprise or consist of an amino acid sequence native to the natural (i.e., wild-type) clostridial neurotoxin, such as botulinum neurotoxin. In other words, the first spacer may be a non-clostridial sequence (i.e., not found in the native clostridial neurotoxin). In one embodiment, the fusion protein does not comprise or consist of the amino acid sequence GIITSK (BoNT/A) (SEQ ID NO:74); VK (BoNT B); AIDGR (BoNT/C) (SEQ ID NO:75); LTK (BoNT/D); IVSVK (BoNT/E) (SEQ ID NO:76); VIPR (BoNT/F) (SEQ ID NO:77); VMYK (BoNT/G) (SEQ ID NO:78) and/or IIPPTNIREN (TeNT) (SEQ ID NO:79) as the first spacer.
In one embodiment, the first spacer begins on the third amino acid residue following the conserved cysteine residue in the clostridial neurotoxin L-chain (see Table 3 below). In one embodiment, the first spacer begins after the VD amino acid residues of a non-cytotoxic protease clostridial L-chain engineered with a SalI site following the conserved cysteine residue. In one embodiment, the first spacer ends with the amino acid residue marking the beginning of the protease cleavage sites mentioned above.
In one embodiment, the single chain fusion protein comprises a second spacer, which is located between the galanin Targeting Moiety and the translocation domain. Said second spacer may comprise (or consist of) an amino acid sequence of from 4 to 35 (such as from 6 to 35, 10 to 35, 15 to 35, 20 to 35 or from 4 to 28, 4 to 25, 4 to 20 or 4 to 10) amino acid residues. The present inventors have unexpectedly found that the fusion proteins of the present invention may demonstrate an improved binding activity when the size of the second 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 50-100 angstroms, and more preferably by 50-90 angstroms.
Suitable second spacers may be routinely identified and obtained according to Crasto and Feng, (2000) Protein Eng. 13(5):309-312. In one embodiment, the second spacer is selected from a GS5, GS10, GS15, GS18, GS20 or HX27 spacer. The sequence of said spacers is provided in Table 2, below.
The Inventors have surprisingly found, that the presently claimed fusion proteins having said first and second spacer features display enhanced activation properties and increased yield during recombinant expression. In addition, the presently claimed fusion proteins display enhanced potency compared to fusion proteins wherein the galanin TM is C-terminal of the translocation domain component.
In one embodiment, the invention provides a single-chain polypeptide fusion protein comprising (or consisting of) an amino acid sequence having at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the amino acid sequence of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56 and/or 59.
In one embodiment, the invention provides a single-chain polypeptide fusion protein comprising (or consisting of) an amino acid sequence having at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the full-length amino acid sequence of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56 and/or 59.
In one embodiment, in the single chain polypeptide, the non-cytotoxic protease component and the translocation component are linked together by a disulphide 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 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.
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 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.
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 the 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 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.
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 residues are distanced apart by between 8 and 27 amino acid residues—taken from Popoff and Marvaud, 1999, Structural and genomic features of clostridial neurotoxins, Chapter 9, in The Comprehensive Sourcebook of Bacterial Protein Toxins. eds. Alouf and Freer, Elsevier:
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.
Whilst any purification tag may be employed, the following are preferred:
His-tag (e.g., 6× histidine), preferably as a C-terminal and/or N-terminal tag
MBP-tag (maltose binding protein), preferably as an N-terminal tag
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.
According to a further embodiment of the present invention, one or more additional 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 component of the present invention; and/or between a C-terminal purification tag and a translocation component of the present invention.
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 of a DNA vector, wherein the vector comprises a promoter and terminator.
In a preferred embodiment, the vector has a promoter selected from:
The DNA construct of the present invention is preferably designed in silico, and then synthesized by conventional DNA synthesis techniques.
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 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.).
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;
- 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 has a structure wherein:
-
- a. the first chain comprises the non-cytotoxic protease, which protease is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;
- b. the second chain comprises the galanin TM and the translocation domain that is capable of translocating the protease 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.
In use, the single chain or di-chain polypeptide of the invention treat, prevent or ameliorate pain.
In use, a therapeutically effective amount of a single chain or di-chain polypeptide of the invention is administered to a patient.
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 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 compounds described here may be used to treat a patient suffering from one or more types of chronic pain including neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
To “treat,” as used here, means to deal with medically. It includes, for example, administering a compound of the invention to prevent pain or to lessen its severity.
The term “pain,” as used here, means any unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a clinician. Pain is of two types: chronic and acute. An “acute pain” is a pain of short duration having a sudden onset. One type of acute pain, for example, is cutaneous pain felt on injury to the skin or other superficial tissues, such as caused by a cut or a burn. Cutaneous nociceptors terminate just below the skin, and due to the high concentration of nerve endings, produce a well-defined, localized pain of short duration. “Chronic pain” is a pain other than an acute pain. Chronic pain includes neuropathic pain, inflammatory pain, headache pain, somatic pain visceral pain and referred pain.
I. Neuropathic Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following neuropathic pain conditions. “Neuropathic pain” means abnormal sensory input, resulting in discomfort, from the peripheral nervous system, central nervous systems, or both.
A. Symptoms of Neuropathic Pain
Symptoms of neuropathic pain can involve persistent, spontaneous pain, as well as allodynia (a painful response to a stimulus that normally is not painful), hyperalgesia (an accentuated response to a painful stimulus that usually causes only a mild discomfort, such as a pin prick), or hyperpathia (where a short discomfort becomes a prolonged severe pain).
B. Causes of Neuropathic Pain
Neuropathic pain may be caused by any of the following.
1. A traumatic insult, such as, for example, a nerve compression injury (e.g., a nerve crush, a nerve stretch, a nerve entrapment or an incomplete nerve transsection); a spinal cord injury (e.g., a hemisection of the spinal cord); a limb amputation; a contusion; an inflammation (e.g., an inflammation of the spinal cord); or a surgical procedure.
2. An ischemic event, including, for example, a stroke and heart attack.
3. An infectious agent
4. Exposure to a toxic agent, including, for example, a drug, an alcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrial agent (e.g., a solvent, fumes from a glue) or nitrous oxide.
5. A disease, including, for example, an inflammatory disorder, a neoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lyme disease, a leprosy, a metabolic disease, a peripheral nerve disorder, like neuroma, a mononeuropathy or a polyneuropathy.
C. Types of Neuropathic Pain
1. Neuralgia.
A neuralgia is a pain that radiates along the course of one or more specific nerves usually without any demonstrable pathological change in the nerve structure. The causes of neuralgia are varied. Chemical irritation, inflammation, trauma (including surgery), compression by nearby structures (for instance, tumors), and infections may all lead to neuralgia. In many cases, however, the cause is unknown or unidentifiable. Neuralgia is most common in elderly persons, but it may occur at any age. A neuralgia, includes, without limitation, a trigeminal neuralgia, a post-herpetic neuralgia, a postherpetic neuralgia, a glossopharyngeal neuralgia, a sciatica and an atypical facial pain.
Neuralgia is pain in the distribution of a nerve or nerves. Examples are trigeminal neuralgia, atypical facial pain, and postherpetic neuralgia (caused by shingles or herpes). The affected nerves are responsible for sensing touch, temperature and pressure in the facial area from the jaw to the forehead. The disorder generally causes short episodes of excruciating pain, usually for less than two minutes and on only one side of the face. The pain can be described in a variety of ways such as “stabbing,” “sharp,” “like lightning,” “burning,” and even “itchy”. In the atypical form of TN, the pain can also present as severe or merely aching and last for extended periods. The pain associated with TN is recognized as one the most excruciating pains that can be experienced.
Simple stimuli such as eating, talking, washing the face, or any light touch or sensation can trigger an attack (even the sensation of a gentle breeze). The attacks can occur in clusters or as an isolated attack.
Symptoms include sharp, stabbing pain or constant, burning pain located anywhere, usually on or near the surface of the body, in the same location for each episode; pain along the path of a specific nerve; impaired function of affected body part due to pain, or muscle weakness due to concomitant motor nerve damage; increased sensitivity of the skin or numbness of the affected skin area (feeling similar to a local anesthetic such as a Novacaine shot); and any touch or pressure is interpreted as pain. Movement may also be painful.
Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve of the face, the trigeminal nerve (“trigeminal” literally means “three origins”, referring to the division of the nerve into 3 branches). This condition involves sudden and short attacks of severe pain on the side of the face, along the area supplied by the trigeminal nerve on that side. The pain attacks may be severe enough to cause a facial grimace, which is classically referred to as a painful tic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is a blood vessel or small tumor pressing on the nerve. Disorders such as multiple sclerosis (an inflammatory disease affecting the brain and spinal cord), certain forms of arthritis, and diabetes (high blood sugar) may also cause trigeminal neuralgia, but a cause is not always identified. In this condition, certain movements such as chewing, talking, swallowing, or touching an area of the face may trigger a spasm of excruciating pain.
A related but rather uncommon neuralgia affects the glosso-pharyngeal nerve, which provides sensation to the throat. Symptoms of this neuralgia are short, shock-like episodes of pain located in the throat.
Neuralgia may occur after infections such as shingles, which is caused by the varicella-zoster virus, a type of herpesvirus. This neuralgia produces a constant burning pain after the shingles rash has healed. The pain is worsened by movement of or contact with the affected area. Not all of those diagnosed with shingles go on to experience postherpetic neuralgia, which can be more painful than shingles. The pain and sensitivity can last for months or even years. The pain is usually in the form of an intolerable sensitivity to any touch but especially light touch. Postherpetic neuralgia is not restricted to the face; it can occur anywhere on the body but usually occurs at the location of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.
Postherpetic neuralgia may be debilitating long after signs of the original herpes infection have disappeared. Other infectious diseases that may cause neuralgia are syphilis and Lyme disease.
Diabetes is another common cause of neuralgia. This very common medical problem affects almost 1 out of every 20 Americans during adulthood. Diabetes damages the tiny arteries that supply circulation to the nerves, resulting in nerve fiber malfunction and sometimes nerve loss. Diabetes can produce almost any neuralgia, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness of the hand and wrist), and meralgia paresthetica (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Strict control of blood sugar may prevent diabetic nerve damage and may accelerate recovery in patients who do develop neuralgia.
Other medical conditions that may be associated with neuralgias are chronic renal insufficiency and porphyria—a hereditary disease in which the body cannot rid itself of certain substances produced after the normal breakdown of blood in the body. Certain drugs may also cause this problem.
2. Deafferentation.
Deafferentation indicates a loss of the sensory input from a portion of the body, and can be caused by interruption of either peripheral sensory fibers or nerves from the central nervous system. A deafferentation pain syndrome, includes, without limitation, an injury to the brain or spinal cord, a post-stroke pain, a phantom pain, a paraplegia, a brachial plexus avulsion injuries, lumbar radiculopathies.
3. Complex Regional Pain Syndromes (CRPSs)
CRPS is a chronic pain syndrome resulting from sympathetically-maintained pain, and presents in two forms. CRPS 1 currently replaces the term “reflex sympathetic dystrophy syndrome”. It is a chronic nerve disorder that occurs most often in the arms or legs after a minor or major injury. CRPS 1 is associated with severe pain; changes in the nails, bone, and skin; and an increased sensitivity to touch in the affected limb. CRPS 2 replaces the term causalgia, and results from an identified injury to the nerve. A CRPS, includes, without limitation, a CRPS Type I (reflex sympathetic dystrophy) and a CRPS Type II (causalgia).
4. Neuropathy.
A neuropathy is a functional or pathological change in a nerve and is characterized clinically by sensory or motor neuron abnormalities.
Central neuropathy is a functional or pathological change in the central nervous system.
Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. The peripheral nerves relay information from your central nervous system (brain and spinal cord) to muscles and other organs and from your skin, joints, and other organs back to your brain. Peripheral neuropathy occurs when these nerves fail to carry information to and from the brain and spinal cord, resulting in pain, loss of sensation, or inability to control muscles. In some cases, the failure of nerves that control blood vessels, intestines, and other organs results in abnormal blood pressure, digestion problems, and loss of other basic body processes. Risk factors for neuropathy include diabetes, heavy alcohol use, and exposure to certain chemicals and drugs. Some people have a hereditary predisposition for neuropathy. Prolonged pressure on a nerve is another risk for developing a nerve injury. Pressure injury may be caused by prolonged immobility (such as a long surgical procedure or lengthy illness) or compression of a nerve by casts, splints, braces, crutches, or other devices. Polyneuropathy implies a widespread process that usually affects both sides of the body equally. The symptoms depend on which type of nerve is affected. The three main types of nerves are sensory, motor, and autonomic. Neuropathy can affect any one or a combination of all three types of nerves. Symptoms also depend on whether the condition affects the whole body or just one nerve (as from an injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. The specific antigens, immune processes, and triggering factors are variable and in many cases are unknown. It may occur in association with other conditions such as HIV, inflammatory bowel disease, lupus erythematosis, chronic active hepatitis, and blood cell abnormalities.
Peripheral neuropathy may involve a function or pathological change to a single nerve or nerve group (mononeuropathy) or a function or pathological change affecting multiple nerves (polyneuropathy).
Peripheral Neuropathies
Hereditary disorders
-
- Charcot-Marie-Tooth disease
- Friedreich's ataxia
Systemic or metabolic disorders
-
- Diabetes (diabetic neuropathy)
- Dietary deficiencies (especially vitamin B-12)
- Excessive alcohol use (alcoholic neuropathy)
- Uremia (from kidney failure)
- Cancer
Infectious or Inflammatory Conditions
-
- AIDS
- Hepatitis
- Colorado tick fever
- diphtheria
- Guillain-Barre syndrome
- HIV infection without development of AIDS
- leprosy
- Lyme
- polyarteritis nodosa
- rheumatoid arthritis
- sarcoidosis
- Sjogren syndrome
- syphilis
- systemic lupus erythematosus
- amyloid
Exposure to Toxic Compounds
-
- sniffing glue or other toxic compounds
- nitrous oxide
- industrial agents—especially solvents
- heavy metals (lead, arsenic, mercury, etc.)
- Neuropathy secondary to drugs like analgesic nephropathy
Miscellaneous Causes
-
- ischemia (decreased oxygen/decreased blood flow)
- prolonged exposure to cold temperature
a. Polyneuropathy
Polyneuropathy is a peripheral neuropathy involving the loss of movement or sensation to an area caused by damage or destruction to multiple peripheral nerves. Polyneuropathic pain, includes, without limitation, post-polio syndrome, postmastectomy syndrome, diabetic neuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, 2′,3′-didexoycytidine (ddC) treatment, Guillain-Barré syndrome or Fabry's disease.
b. Mononeuropathy
Mononeuropathy is a peripheral neuropathy involving loss of movement or sensation to an area caused by damage or destruction to a single peripheral nerve or nerve group. Mononeuropathy is most often caused by damage to a local area resulting from injury or trauma, although occasionally systemic disorders may cause isolated nerve damage (as with mononeuritis multiplex). The usual causes are direct trauma, prolonged pressure on the nerve, and compression of the nerve by swelling or injury to nearby body structures. The damage includes destruction of the myelin sheath (covering) of the nerve or of part of the nerve cell (the axon). This damage slows or prevents conduction of impulses through the nerve. Mononeuropathy may involve any part of the body. Mononeuropathic pain, includes, without limitation, a sciatic nerve dysfunction, a common peroneal nerve dysfunction. a radial nerve dysfunction, an ulnar nerve dysfunction, a cranial mononeuropathy VI, a cranial mononeuropathy VII, a cranial mononeuropathy III (compression type), a cranial mononeuropathy III (diabetic type), an axillary nerve dysfunction, a carpal tunnel syndrome, a femoral nerve dysfunction, a tibial nerve dysfunction, a Bell's palsy, a thoracic outlet syndrome, a carpal tunnel syndrome and a sixth (abducent) nerve palsy
c. Generalized Peripheral Neuropathies
Generalized peripheral neuropathies are symmetrical, and usually due to various systematic illnesses and disease processes that affect the peripheral nervous system in its entirety. They are further subdivided into several categories:
i. Distal axonopathies are the result of some metabolic or toxic derangement of neurons. They may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. Distal axonopathy (aka dying back neuropathy) is a type of peripheral neuropathy that results from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of nerves to metabolic or toxic disturbances, and as such may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.
ii. Myelinopathies are due to a primary attack on myelin causing an acute failure of impulse conduction. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; aka Guillain-Barré syndrome), though other causes include chronic inflammatory demyelinating syndrome (CIDP), genetic metabolic disorders (e.g., leukodystrophy), or toxins. Myelinopathy is due to primary destruction of myelin or the myelinating Schwann cells, which leaves the axon intact, but causes an acute failure of impulse conduction. This demyelination slows down or completely blocks the conduction of electrical impulses through the nerve. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, better known as Guillain-Barré syndrome), though other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophy or Charcot-Marie-Tooth disease), or toxins.
iii. Neuronopathies are the result of destruction of peripheral nervous system (PNS) neurons. They may be caused by motor neuron diseases, sensory neuronopathies (e.g., Herpes zoster), toxins or autonomic dysfunction. Neurotoxins may cause neuronopathies, such as the chemotherapy agent vincristine. Neuronopathy is dysfunction due to damage to neurons of the peripheral nervous system (PNS), resulting in a peripheral neuropathy. It may be caused by motor neuron diseases, sensory neuronopathies (e.g., Herpes zoster), toxic substances or autonomic dysfunction. A person with neuronopathy may present in different ways, depending on the cause, the way it affects the nerve cells, and the type of nerve cell that is most affected.
Focal entrapment neuropathies (e.g., carpal tunnel syndrome).
II. Inflammatory Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following inflammatory conditions
A. Arthritic Disorder
Arthritic disorders include, for example, a rheumatoid arthritis; a juvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); a gouty arthritis; a scleroderma; an osteoarthritis; a psoriatic arthritis; an ankylosing spondylitis; a Reiter's syndrome (reactive arthritis); an adult Still's disease; an arthritis from a viral infection; an arthritis from a bacterial infection, such as, e.g., a gonococcal arthritis and a non-gonococcal bacterial arthritis (septic arthritis); a Tertiary Lyme disease; a tuberculous arthritis; and an arthritis from a fungal infection, such as, e.g., a blastomycosis
B. Autoimmune Diseases
Autoimmune diseases include, for example, a Guillain-Barré syndrome, a Hashimoto's thyroiditis, a pernicious anemia, an Addison's disease, a type I diabetes, a systemic lupus erythematosus, a dermatomyositis, a Sjogren's syndrome, a lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a Reiter's syndrome and a Grave's disease.
C. Connective Tissue Disorder
Connective tissue disorders include, for example, a spondyloarthritis a dermatomyositis, and a fibromyalgia.
D. Injury
Inflammation caused by injury, including, for example, a crush, puncture, stretch of a tissue or joint, may cause chronic inflammatory pain.
E. Infection
Inflammation caused by infection, including, for example, a tuberculosis or an interstitial keratitis may cause chronic inflammatory pain.
F. Neuritis
Neuritis is an inflammatory process affecting a nerve or group of nerves. Symptoms depend on the nerves involved, but may include pain, paresthesias, paresis, or hypesthesia (numbness).
-
- Examples include:
- a. Brachial neuritis
- b. Retrobulbar neuropathy, an inflammatory process affecting the part of the optic nerve lying immediately behind the eyeball.
- c. Optic neuropathy, an inflammatory process affecting the optic nerve causing sudden, reduced vision in the affected eye. The cause of optic neuritis is unknown. The sudden inflammation of the optic nerve (the nerve connecting the eye and the brain) leads to swelling and destruction of the myelin sheath. The inflammation may occasionally be the result of a viral infection, or it may be caused by autoimmune diseases such as multiple sclerosis. Risk factors are related to the possible causes.
- d. Vestibular neuritis, a viral infection causing an inflammatory process affecting the vestibular nerve.
G. Joint Inflammation
Inflammation of the joint, such as that caused by bursitis or tendonitis, for example, may cause chronic inflammatory pain.
III. Headache Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following headache conditions. A headache (medically known as cephalgia) is a condition of mild to severe pain in the head; sometimes neck or upper back pain may also be interpreted as a headache. It may indicate an underlying local or systemic disease or be a disorder in itself.
A. Muscular/Myogenic Headache
Muscular/myogenic headaches appear to involve the tightening or tensing of facial and neck muscles; they may radiate to the forehead. Tension headache is the most common form of myogenic headache.
A tension headache is a condition involving pain or discomfort in the head, scalp, or neck, usually associated with muscle tightness in these areas. Tension headaches result from the contraction of neck and scalp muscles. One cause of this muscle contraction is a response to stress, depression or anxiety. Any activity that causes the head to be held in one position for a long time without moving can cause a headache. Such activities include typing or use of computers, fine work with the hands, and use of a microscope. Sleeping in a cold room or sleeping with the neck in an abnormal position may also trigger this type of headache. A tension-type headache, includes, without limitation, an episodic tension headache and a chronic tension headache.
B. Vascular Headache
The most common type of vascular headache is migraine. Other kinds of vascular headaches include cluster headaches, which cause repeated episodes of intense pain, and headaches resulting from high blood pressure
1. Migraine
-
- A migraine is a heterogeneous disorder that generally involves recurring headaches. Migraines are different from other headaches because they occur with other symptoms, such as, e.g., nausea, vomiting, or sensitivity to light. In most people, a throbbing pain is felt only on one side of the head. Clinical features such as type of aura symptoms, presence of prodromes, or associated symptoms such as vertigo, may be seen in subgroups of patients with different underlying pathophysiological and genetic mechanisms. A migraine headache, includes, without limitation, a migraine without aura (common migraine), a migraine with aura (classic migraine), a menstrual migraine, a migraine equivalent (acephalic headache), a complicated migraine, an abdominal migraine and a mixed tension migraine.
2. Cluster Headache
-
- Cluster headaches affect one side of the head (unilateral) and may be associated with tearing of the eyes and nasal congestion. They occur in clusters, happening repeatedly every day at the same time for several weeks and then remitting.
D. High Blood Pressure Headache
E. Traction and Inflammatory Headache
Traction and inflammatory headaches are usually symptoms of other disorders, ranging from stroke to sinus infection.
F. Hormone Headache
G. Rebound Headache
Rebound headaches, also known as medication overuse headaches, occur when medication is taken too frequently to relieve headache. Rebound headaches frequently occur daily and can be very painful.
H. Chronic Sinusitis Headache
Sinusitis is inflammation, either bacterial, fungal, viral, allergic or autoimmune, of the paranasal sinuses. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include: nasal congestion; facial pain; headache; fever; general malaise; thick green or yellow discharge; feeling of facial ‘fullness’ worsening on bending over. In a small number of cases, chronic maxillary sinusitis can also be brought on by the spreading of bacteria from a dental infection. Chronic hyperplastic eosinophilic sinusitis is a non-infective form of chronic sinusitis.
I. An Organic Headache
J. Ictal Headaches
Ictal headaches are headaches associated with seizure activity.
IV. Somatic Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions. Somatic pain originates from ligaments, tendons, bones, blood vessels, and even nerves themselves. It is detected with somatic nociceptors. The scarcity of pain receptors in these areas produces a dull, poorly-localized pain of longer duration than cutaneous pain; examples include sprains and broken bones. Additional examples include the following.
A. Excessive Muscle Tension
Excessive muscle tension can be caused, for example, by a sprain or a strain.
B. Repetitive Motion Disorders
Repetitive motion disorders can result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles.
C. Muscle Disorders
Muscle disorders causing somatic pain include, for example, a polymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgia rheumatica, and a rhabdomyolysis.
D. Myalgia
Myalgia is muscle pain and is a symptom of many diseases and disorders. The most common cause for myalgia is either overuse or over-stretching of a muscle or group of muscles. Myalgia without a traumatic history is often due to viral infections. Longer-term myalgias may be indicative of a metabolic myopathy, some nutritional deficiencies or chronic fatigue syndrome.
E. Infection
Infection can cause somatic pain. Examples of such infection include, for example, an abscess in the muscle, a trichinosis, an influenza, a Lyme disease, a malaria, a Rocky Mountain spotted fever, Avian influenza, the common cold, community-acquired pneumonia, meningitis, monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome, trichinosis, typhoid fever, and upper respiratory tract infection.
F. Drugs
Drugs can cause somatic pain. Such drugs include, for example, cocaine, a statin for lowering cholesterol (such as atorvastatin, simvastatin, and lovastatin), and an ACE inhibitor for lowering blood pressure (such as enalapril and captopril)
V. Visceral Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following visceral pain conditions. Visceral pain originates from body's viscera, or organs. Visceral nociceptors are located within body organs and internal cavities. The even greater scarcity of nociceptors in these areas produces pain that is usually more aching and of a longer duration than somatic pain. Visceral pain is extremely difficult to localize, and several injuries to visceral tissue exhibit “referred” pain, where the sensation is localized to an area completely unrelated to the site of injury. Examples of visceral pain include the following:
A. Functional Visceral Pain
Functional visceral pain includes, for example, an irritable bowel syndrome and a chronic functional abdominal pain (CFAP), a functional constipation and a functional dyspepsia, a non-cardiac chest pain (NCCP) and a chronic abdominal pain.
B. Chronic Gastrointestinal Inflammation
Chronic gastrointestinal inflammation includes, for example, a gastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease, an ulcerative colitis, a microscopic colitis, a diverticulitis and a gastroenteritis; an interstitial cystitis; an intestinal ischemia; a cholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, a nephrolithiasis, an urinary tract infection, a pancreatitis and a hernia.
C. Autoimmune Pain
Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.
D. Organic Visceral Pain
Organic visceral pain includes, for example, pain resulting from a traumatic, inflammatory or degenerative lesion of the gut or produced by a tumor impinging on sensory innervation.
E. Treatment-Induced Visceral Pain
Treatment-induced visceral pain includes, for example, a pain attendant to chemotherapy therapy or a pain attendant to radiation therapy.
VI. Referred Pain
The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following referred pain conditions.
Referred pain arises from pain localized to an area separate from the site of pain stimulation. Often, referred pain arises when a nerve is compressed or damaged at or near its origin. In this circumstance, the sensation of pain will generally be felt in the territory that the nerve serves, even though the damage originates elsewhere. A common example occurs in intervertebral disc herniation, in which a nerve root arising from the spinal cord is compressed by adjacent disc material. Although pain may arise from the damaged disc itself, pain will also be felt in the region served by the compressed nerve (for example, the thigh, knee, or foot). Relieving the pressure on the nerve root may ameliorate the referred pain, provided that permanent nerve damage has not occurred. Myocardial ischaemia (the loss of blood flow to a part of the heart muscle tissue) is possibly the best known example of referred pain; the sensation can occur in the upper chest as a restricted feeling, or as an ache in the left shoulder, arm or even hand.
The present invention addresses a wide range of pain conditions, in particular 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.
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 mammal, more preferably to a human.
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.
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 the judgment of the attending physician.
Suitable daily dosages are in the range 0.0001 to 1 mg/kg, preferably 0.0001 to 0.5 mg/kg, more preferably 0.002 to 0.5 mg/kg, and particularly preferably 0.004 to 0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30 mg, but typically will be in the region of 0.01 to 1 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 as the 1× dose. In this regard, preferred dosages are in the range 1×-100× (i.e., 2.5 to 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 present invention have a considerably greater molecular weight 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 routes of administration.
Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
Fluid unit dosage forms are typically prepared utilizing a pyrogen-free sterile vehicle. The active ingredients, depending on the vehicle and concentration 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 sterilized by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilized by autoclaving.
Advantageously additives such as buffering, solubilizing, stabilizing, preservative 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-sterilized drug substance and other ingredients into a sterile container using aseptic technique in a sterile area.
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.
Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilized after isolation, e.g., by gamma irradiation.
Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.
DEFINITIONS SECTIONTargeting Moiety (TM) means any chemical structure associated with an agent that functionally interacts with a Binding Site to cause a physical association between the 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 internalization (e.g., endosome formation)—also referred to as receptor-mediated endocytosis. The TM may possess an endosomal membrane translocation function, in which case separate TM 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 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 galanin receptors, such as GALR1, GALR2 and/or GALR3 receptors, with a binding affinity (Ka) of 106M−1 or greater, preferably 107M−1 or greater, more preferably 108 M−1 or greater, and most preferably, 109 M−1 or greater.
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 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.
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-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 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues of the protein in question.
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.
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, 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 components such as lipid, and/or polysaccharide, and/or polypeptide components may be included.
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 exocytic fusion apparatus in eukaryotic cells.
The non-cytotoxic protease of the present invention is preferably a bacterial protease. In one embodiment, the non-cytotoxic protease is selected from the genera Clostridium or Neisseria (e.g., a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae). The term protease embraces functionally equivalent fragments and molecules thereof.
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-mentioned protease activity.
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, synaptobrevin/VAMP, or syntaxin).
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.
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 through 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 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 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 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, (1987) Eur. J. Biochem 167(1):175-180, describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K+ and/or labeled NAD, which may be readily monitored.
A further example is provided by Blaustein, (1987) FEBS Letts. 226:115-120, which describes a simple in 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.
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.
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.
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 translocation domain of an enzyme, such as a bacterial toxin or viral protein.
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.
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 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 HC function may be removed by deletion of the HC amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the He 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.
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)
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)
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, reference is made to Henderson et at (1997) in The Clostridia: 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 demonstrate the above-mentioned translocation function.
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., (1992) Proc. Natl. Acad. Sci. USA 89:6202-6206; Silverman et al., (1993) J. Biol. Chem. 269:22524-22532; and London, (1992) Biochem. Biophys. Acta. 1112:25-51), the translocation domain of Pseudomonas exotoxin type A (Prior et al., (1992) Biochemistry 31:3555-3559), the translocation domains of anthrax toxin (Blanke et al., (1996) Proc. Natl. Acad. Sci. USA 93:8437-8442), a variety of fusogenic or hydrophobic peptides of translocating function (Plank et al., (1994) J. Biol. Chem. 269:12918-12924; and Wagner et al., (1992) Proc. Natl. Acad. Sci. USA 89:7934-7938), and amphiphilic peptides (Murata et al., (1992) Biochemistry, 31:1986-1992). 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.
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), (both supra) describe the translocation (i.e., membrane fusion and vesiculation) function of a 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 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 E1 protein of SFV and the G protein of the G protein of VSV.
Use of the Translocation Domains listed in Table 4 (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 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.
Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.
- SEQ ID NO: 1 DNA SEQUENCE OF THE LC/A
- SEQ ID NO: 2 DNA SEQUENCE OF THE HN/A
- SEQ ID NO: 3 DNA SEQUENCE OF THE LC/B
- SEQ ID NO: 4 DNA SEQUENCE OF THE HN/B
- SEQ ID NO: 5 DNA SEQUENCE OF THE LC/C
- SEQ ID NO: 6 DNA SEQUENCE OF THE HN/C
- SEQ ID NO: 7 PROTEIN SEQUENCE OF GALANIN GA30
- SEQ ID NO: 8 PROTEIN SEQUENCE OF GALANIN GA16
- SEQ ID NO: 9 DNA SEQUENCE OF LHA-GS18-EN-CPGA16-GS20-HT
- SEQ ID NO: 10 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS20-HT
- SEQ ID NO: 11 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS20
- SEQ ID NO: 12 DNA SEQUENCE OF LHA-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 13 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 14 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS20
- SEQ ID NO: 15 DNA SEQUENCE OF LHA-GS5-EN-CPGA30-GS20-HT
- SEQ ID NO: 16 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA30-GS20-HT
- SEQ ID NO: 17 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA30-GS20
- SEQ ID NO: 18 DNA SEQUENCE OF LHBK191A-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 19 PROTEIN SEQUENCE OF LHBK191A-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 20 PROTEIN SEQUENCE OF LHBK191A-GS5-EN-CPGA16-GS20
- SEQ ID NO: 21 DNA SEQUENCE OF LHB-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 22 PROTEIN SEQUENCE OF LHB-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 23 PROTEIN SEQUENCE OF LHB-GS5-EN-CPGA16-GS20
- SEQ ID NO: 24 DNA SEQUENCE OF LHC-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 25 PROTEIN SEQUENCE OF LHC-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 26 PROTEIN SEQUENCE OF LHC-GS5-EN-CPGA16-GS20
- SEQ ID NO: 27 DNA SEQUENCE OF LHD-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 28 PROTEIN SEQUENCE OF LHD-GS5-EN-CPGA16-GS20-HT
- SEQ ID NO: 29 PROTEIN SEQUENCE OF LHD-GS5-EN-CPGA16-GS20
- SEQ ID NO: 30 DNA SEQUENCE OF LHA-GS5-EN-CPGA16-HX27-HT
- SEQ ID NO: 31 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-HX27-HT
- SEQ ID NO: 32 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-HX27
- SEQ ID NO: 33 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS20-HN/A
- SEQ ID NO: 34 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS20
- SEQ ID NO: 35 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS15-HT
- SEQ ID NO: 36 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS15
- SEQ ID NO: 37 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS10-HT
- SEQ ID NO: 38 PROTEIN SEQUENCE OF LHA-GS5-EN-CPGA16-GS10
- SEQ ID NO: 39 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-HX27-HN/A
- SEQ ID NO: 40 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-HX27
- SEQ ID NO: 41 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS15-HT
- SEQ ID NO: 42 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS15
- SEQ ID NO: 43 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS10-HT
- SEQ ID NO: 44 PROTEIN SEQUENCE OF LHA-GS18-EN-CPGA16-GS10
- SEQ ID NO: 45 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-HX27-HT
- SEQ ID NO: 46 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-HX27
- SEQ ID NO: 47 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS15-HT
- SEQ ID NO: 48 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS15
- SEQ ID NO: 49 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS10-HT
- SEQ ID NO: 50 PROTEIN SEQUENCE OF LHA-GS10-EN-CPGA16-GS10
- SEQ ID NO: 51 DNA SEQUENCE OF THE IGA PROTEASE
- SEQ ID NO: 52 DNA SEQUENCE OF THE IGA-GS5-GAL16-GS18-HN/A FUSION
- SEQ ID NO: 53 PROTEIN SEQUENCE OF THE IGA-GS5-GAL16-GS18-HN/A FUSION
- SEQ ID NO: 54 DNA SEQUENCE OF DT TRANSLOCATION DOMAIN
- SEQ ID NO: 55 DNA SEQUENCE OF LC/A-GS5-GAL16-GS20-DT-A
- SEQ ID NO: 56 PROTEIN SEQUENCE OF LC/A-GS5-GAL16-GS20-DT-A
- SEQ ID NO: 57 DNA SEQUENCE OF TENT LC
- SEQ ID NO: 58 DNA SEQUENCE OF TENT LC-GS5-CPGAL16-GS20-HN/A
- SEQ ID NO: 59 PROTEIN SEQUENCE OF TENT LC-GS5-EN-CPGA16-GS20-HN/A
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 ID NO: 1 and SEQ ID NO: 2), though the procedures and methods are equally applicable to the other serotypes (i.e., A, B, C, D and E serotypes) as illustrated by the sequence listing for serotype B (SEQ ID NO: 3 and SEQ ID NO: 4) and serotype C (SEQ ID NO: 5 and SEQ ID NO: 6).
Preparation of Cloning and Expression VectorspCR 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-SalI-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilizable plasmid and a variety of different fusion tags have been inserted to increase purification options.
Preparation of Protease (e.g. LC/A) Insert
The LC/A (SEQ ID NO: 1) is created by one of two ways:
The DNA sequence is designed by back translation of the LC/A amino acid 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 v 2.0 (Entelechon)). BamHI/SalI recognition sequences are incorporated at the 5′ and 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 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 Sep. 2004). This optimized DNA sequence containing the 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 SalI restriction enzyme sequences incorporated into the 5′ and 3′ PCR primers respectively. Complementary oligonucleotide primers are chemically synthesized 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 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 temperature of the reaction tube automatically, cycling between approximately 94° C. (for denaturation), 55° C. (for oligonucleotide annealing), and 72° C. (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 dNTP building blocks of DNA in equimolar amounts (50 to 200 μM) and a buffer appropriate for the enzyme optimized for Mg2+ concentration (0.5 to 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 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.)).
Preparation of Translocation (e.g. HN) Insert
The HN/A (SEQ ID NO: 2) 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)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v 2.0 (Entelechon)). A PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII to the C-terminus ensuring the correct reading frame is maintained. The DNA sequence is screened (using software such as MapDraw, 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 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 Sep. 2004). This optimized DNA sequence 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 PstI and XbaI-stop codon-HindIII 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 compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of LHA-GS18-EN-CPGA16-GS20 FusionIn order to create the LC/A-GS18-EN-CPGA16-GS20-HN/A construct, an A serotype linker with the addition of an Enterokinase (EN) site for activation, arranged as BamHI-SalI-GS18-protease site-GS20-PstI-XbaI-stop codon-HindIII is synthesized. The pCR 4 vector encoding the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID NO: 1) also cleaved with BamHI+SalI. This construct is then cleaved with BamHI+HindIII and inserted into an expression vector such as the pMAL plasmid (NEB) or pET based plasmid (Novagen). The resulting plasmid DNA is then cleaved with PstI+XbaI restriction enzymes and the HN/A DNA (SEQ ID NO2) is then cleaved with PstI+XbaI restriction enzymes and inserted into the a similarly cleaved pMAL vector to create pMAL-LC/A-GS18-EN-CPGA16-GS20-HN/A-XbaI-His-tag-stop codon-HindIII. The final construct contains the GS18-EN-CPGA16-GS20 spacer ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 10.
Activation AssayNuPAGE 4-12% Bis-Tris gels (10, 12 and 15 well pre-poured gels) were used to analyze activation of fusion proteins after treatment with protease. Protein samples were prepared with NuPAGE 4×LDS sample buffer, typically to a final volume of 100 μl Samples were either diluted or made up neat (75 μl of sample, 25 μl of sample buffer) depending on protein concentration. The samples were mixed and then heated in a heat block at 95° C. for 5 min before loading onto the gel. 5 to 20 μl of sample was loaded along with 5 μl of the protein marker (Benchmark™ protein marker from Invitrogen). The gels were typically run for 50 min at 200 V. The gel was immersed in dH2O and microwaved for 2 min on full power. The gel was rinsed and the microwave step was repeated. The gel was transferred to a staining box and immersed in Simply Blue SafeStain (Invitrogen). It was microwaved for 1 minute on full power and left for 0.5 to 2 h to stain. The gel was then destained by pouring off the Safestain and rinsing the gel with dH2O. The gels were left in dH2O to destain overnight and an image was taken on a GeneGnome (Syngene) imager. Total activated protein was calculated by comparing the density of the band that corresponded to full-length fusion protein (after protease treatment) in non-reduced and reduced conditions.
Example 2 Preparation of an LHA-GS18-EN-CPGA16-GS20 Fusion Protein Family with Variable Spacer LengthUsing the same strategy as employed in Example 1, a range of DNA linkers were prepared that encoded galanin16 and variable spacer content. Using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v 2.0 (Entelechon)), the DNA sequence encoding the Spacer 1-Protease site-ligand-spacer 2 region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-SalI-Spacer 1-protease site-CPGA16-NheI-spacer 2-SpeI-PstI-XbaI-stop codon-HindIII. It is important to ensure the correct reading frame is maintained for the spacer, GA16 and restriction sequences and that the XbaI sequence is not preceded by the bases TC which would result in DAM methylation. The DNA sequence is screened for restriction sequence incorporation 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 codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimized DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
By way of example, and in order to create the LC/A-GS5-EN-CPGA16-GS20-HN/A fusion construct (SEQ ID NO: 12), the pCR 4 vector encoding BamHI-SalI-GS5-protease site-GS20-PstI-XbaI-stop codon-HindIII the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID NO: 1) also cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB) or the pET vector (Novagen). The HN/A DNA (SEQ ID NO: 2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-GS5-EN-CPGA16-GS20-HN/A ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 13.
Example 3 Purification Method for Galanin Fusion ProteinA falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste was defrosted. The thawed cell paste was made up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicated on ice for 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring that the sample remained cool. The lysed cells were centrifuged at 18,000 rpm, 4° C. for 30 minutes. The supernatant was loaded onto a 0.1 M NiSO4 charged Chelating column (a 20-30 ml column was sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. A step gradient of 10 and 40 mM imidazole was used, the non-specific bound protein was washed and the fusion protein with eluted with 100 mM imidazole. The eluted fusion protein was dialyzed against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and the OD of the dialysed fusion protein was measured. 1 μg of enterokinase (1 mg/ml) was added per 100 μg of purified fusion protein along with 10 μl of factor Xa per mg of purified fusion protein if the fusion protein contained a maltose binding protein. Incubation was at 25° C. static overnight. Dialysate was loaded onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. The column was washed to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Then, using a step gradient of 10 and 40 mM imidazole the non-specific bound protein was washed away and the fusion protein was eluted with 100 mM imidazole. The eluted fusion protein was dialyzed against 5 L of 50 mM
HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and the fusion protein was concentrated to about 2 mg/ml, aliquoted, and frozen at −20° C.
Example 4 Preparation of a LC/C-GA16-HN/C Fusion Protein with a Serotype A Activation Sequence Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO: 5) andHN/C (SEQ ID NO: 6) are created and inserted into the A serotype linker arranged as BamHI-SalI-Spacer 1-protease site-GA16-NheI-spacer 2-SpeI-PstI-XbaI-stop codon-HindIII. The final construct contains the LC-spacer 1-GA16-spacer 2-HN ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 25.
Example 5 Preparation of an IgA Protease-GA16-HN/A Fusion ProteinThe 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., (1987) Nature 325(6103):458-462). Using Backtranslation tool v 2.0 (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 SalI recognition sequence were incorporated at the 3′ end of the IgA DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that were found to be common to those required for 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 optimized DNA sequence (SEQ ID NO: 51) containing the IgA open reading frame (ORF) was then commercially synthesized.
The IgA (SEQ ID NO: 51) was inserted into the LC-GS5-GA16-GS18-HN ORF using BamHI and SalI restriction enzymes to replace the LC with the IgA protease DNA. The final construct contains the IgA-GS5-GA16-GS18-HN ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 53.
Example 6 Preparation of a Galanin Targeted Endopeptidase Fusion Protein Containing a LC Domain Derived from TetanusThe DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v 2.0 (Entelechon)). BamHI/SalI recognition sequences are incorporated at the 5′ and 3′ ends respectively of the sequence maintaining the correct reading frame (SEQ ID NO: 57). 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 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 Sep. 2004). This optimized DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR4 vector (Invitrogen). The pCR4 vector encoding the TeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment is then inserted into the LC/A-GA16-HN/A vector that has also been cleaved by BamHI and SalI. The final construct contains the TeNT LC-GS5-GA16-GS20-HN ORF sequences for expression as a protein of the sequence illustrated in SEQ ID NO: 58.
Example 7 Construction of CHO-K1 GALR1 & GALR2 Receptor Activation Assay and SNAP-25 Cleavage Assay Cell-Line CreationCHO-K1 cells stably expressing either the human galanin 1 receptor (CHO-K1-Gal-1R; product number ES-510-C) or human galanin 2 receptor (CHO-K1-Gal-2R; product number ES-511-C) were purchased from Perkin-Elmer (Bucks, UK).
Where required, cells were transfected with SNAP-25 DNA using Lipofectamine™ 2000 and incubated for 4 hours before media replacement. After 24 hours, cells were transferred to a T175 flask. 100 μg/ml Zeocin was added after a further 24 hours to begin selection of SNAP-25 expressing cells, and 5 μg/ml Blasticidin added to maintain selective pressure for the receptor. Cells were maintained in media containing selection agents for two weeks, passaging cells every two to three days to maintain 30 to 70% confluence. Cells were then diluted in selective media to achieve 0.5 cell per well in a 96 well microplate. After a few days, the plates were examined under a microscope, and those wells containing single colonies were marked. Media in these wells was changed weekly. As cells became confluent in the wells, they were transferred to T25 flasks. When the cells had expanded sufficiently each clone was seeded to 24 wells of a 96 well plate, plus a frozen stock vial was created. Galanin fusion proteins of the invention and LC/A-HNA (LHA) were applied to the cells for 24 hours, and then Western blots performed to detect SNAP-25 cleavage. Clones from which SNAP-25 bands were strong and cleavage levels were high with fusion were maintained for further investigation. Full dose curves were run on these, and the clone with the highest differential between galanin fusion protein and LC/A-HNA cleavage levels was selected.
GALR1 Receptor Activation AssayThe GALR1 receptor activation assay measures the potency and intrinsic efficacy of ligands at the GALR1 receptor in transfected CHO-K1 cells by quantifying the reduction of forskolin-stimulated intracellular cAMP using a FRET-based cAMP (Perkin Elmer LANCE cAMP kit). After stimulation, a fluorescently labeled cAMP tracer (Europium-streptavadin/biotin-cAMP) and fluorescently (Alexa) labeled anti-cAMP antibody are added to the cells in a lysis buffer. cAMP from the cells competes with the cAMP tracer for antibody binding sites. When read, a light pulse at 320 nm excites the fluorescent portion (Europium) of the cAMP tracer. The energy emitted from the europium is transferred to the Alexa fluor-labeled antibodies bound to the tracer, generating a TR-FRET signal at 665 nm (Time-resolved fluorescence resonance energy transfer is based on the proximity of the donor label, europium, and the acceptor label, Alexa fluor, which have been brought together by a specific binding reaction). Residual energy from the europium produces light at 615 nm. In agonist treated cells there will be less cAMP to compete with the tracer so a dose dependant increase in signal at 665 nm will be observed compared with samples treated with forskolin alone. The signal at 665 nm signal is converted to cAMP concentration by interpolation to a cAMP standard curve which is included in each experiment.
Using Gilson pipettes and Sigmacote® treated or lo-bind tips, test materials and standards were diluted to the appropriate concentrations in the wells of the first two columns of an Eppendorf 500 μl deep-well lo-bind plate, in assay buffer containing 10 μM forskolin. The chosen concentrations in columns one and two were half a log unit apart. From these, serial 1:10 dilutions were made across the plate (using an electronic eight channel pipette with Sigmacote® or lo-bind tips) until eleven concentrations at half log intervals had been created. In the twelfth column, assay buffer only was added as a ‘basal’. Using a 12 channel digital pipette, 10 μl of sample from the lo-bind plate was transferred to the Optiplate™ 96 well microplate.
To wells containing the standard curve, 10 μl of assay buffer was added using a multichannel digital pipette. To wells containing the test materials, 10 μl of cells in assay buffer at the appropriate concentration were added. Plates were sealed and incubated for 120 min at room temperature, for the first hour on an IKA MTS 2/4 orbital shaker set to maximum speed.
LANCE Eu-W8044 labeled streptavidin (Eu-SA) and Biotin-cAMP (b-cAMP) were diluted in cAMP Detection Buffer (both from Perkin Elmer LANCE cAMP kit) to create sub-stocks, at dilution ratios of 1:17 and 1:5, respectively. The final detection mix was prepared by diluting from the two sub stocks into detection buffer at a ratio of 1:125. The mixture was incubated for 15-30 min at room temperature before addition of 1:200 Alexa Fluor® 647-anti cAMP Antibody (Alexa-Fluor Ab). After briefly vortex mixing, 20 μl was immediately added to each well using a digital multichannel pipette. Microplate sealers were applied and plates incubated for 24 h at room temperature (for the first hour on an IKA MTS 2/4 orbital shaker set to maximum speed). Plate sealers were removed prior to reading on a microplate reader (EnVision®).
GALR2 Receptor Activation AssayThe GALR2 receptor activation assay measures the potency and intrinsic efficacy of ligands at GALR2 receptor in transfected CHO-K1 cells by measuring the calcium mobilization that occurs when the receptor is activated. The transfected cells are pre-loaded with a calcium sensitive dye (FLIPR) before treatment. When read using a microplate reader (Flexstation 3, Molecular Devices) a light pulse at 485 nm excites the fluorescent dye and causes an emission at 525 nm. This provides real-time fluorescence data from changes in intracellular calcium. In agonist treated cells there will be activation of the receptor, leading to an increase in calcium mobilization. This will be measured as an increase in the relative fluorescence units (RFU) at 525 nM.
Culture of Cells for Receptor Activation Assay:Cells were seeded and cultured in T175 flasks containing Ham F12 with Glutamax, 10% Fetal bovine serum, 5 μg/ml Blasticidin and 100 μg/ml Zeocin. The flasks were incubated at 37° C. in a humidified environment containing 5% CO2 until 60 to 80% confluent. On the day of harvest the media was removed and the cells were washed twice with 25 ml PBS. The cells were removed from the flask by addition of 10 ml of Tryple Express, and incubation at 37° C. for 10 min followed by gentle tapping of the flask. The dislodged cells were transferred to a 50 ml centrifuge tube and the flask washed twice with 10 ml media which was added to the cell suspension. The tube was centrifuged at 1300×g for 3 min and the supernatant removed. Cells were gently re-suspended in 10 ml media (if freezing cells) or assay buffer (if using ‘fresh’ cells), and a sample was removed for counting using a Nucleocounter® (ChemoMetec). Cells for use ‘fresh’ in an assay were diluted further in assay buffer to the appropriate concentration. Cells harvested for freezing were re-centrifuged (1300×g; 3 min), the supernatant removed and cells re-suspended in Synth-a-freeze at 4° C. to 3×106 cells/ml. Cryovials containing 1 ml suspension each were placed in a chilled Nalgene Mr. Frosty freezing container (−1° C./minute cooling rate), and left overnight in a −80° C. freezer. The following day vials were transferred to the vapor phase of a liquid nitrogen storage tank.
Cultures of cells were exposed to varying concentrations of galanin fusion protein for 24 hours. Cellular proteins were separated by SDS-PAGE and Western blotted with anti-SNAP-25 antibody to facilitate assessment of SNAP-25 cleavage. SNAP-25 cleavage calculated by densitometric analysis (Syngene).
Plating CellsCells at 2×105 cells/ml were prepared and seeded at 125 μl per well of a 96 well plate using 500 ml Gibco Ham F12 with Glutamax, 50 ml FBS, 5 μg/ml Blasticidin (Calbiochem, 10 ml at 10 mg/ml), 100 μg/ml Zeocin. Cells were allowed to grow for 24 hrs (37° C., 5% CO2, humidified atmosphere).
Cell TreatmentDilutions of test protein were prepared for a dose range of each test proteins. CHO GALR1 feeding medium was filter sterilized (20 ml syringe, 0.2 μm syringe filter) to make the dilutions. Filtered medium was added into 5 labeled Bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette or multi-stepper. The stock test protein was diluted to 2000 nM (working stock solution 1) and 600 nM (working stock solution 2). Using a Gilson pipette a 10-fold serial dilutions of each working stock was prepared, by adding 100 μl to the next concentration in the series. Each dilution was pipetted up and down to mix thoroughly. This step was repeated to obtain 4 serial dilutions for solution 1, and 3 serial dilutions for solution 2. A 0 nM control (filtered feeding medium only) was also prepared as a negative control for each plate. The above steps were repeated for each test protein. In each experiment a ‘standard’ batch of material was included as control/reference material, this is also called unliganded LC/A-HN/A.
Apply Diluted Sample to CHO GALR1 PlatesTest sample (125 μl, double concentration) was applied per well. Each test sample was applied to triplicate wells and each dose range tested included a 0 nM control. Samples were incubated for 24 hrs (37° C., 5% CO2, humidified atmosphere).
Cell LysisFresh lysis buffer (20 mls per plate) was prepared with 25% (4×) NuPAGE LDS sample buffer, 65% dH20 and 10% 1 M DTT. Medium was removed from the CHO GALR1 plate by inverting over a waste receptacle. The remaining media was drained from each well using a fine-tipped pipette. The cells were lysed by adding 125 μl of lysis buffer per well using a multi-stepper pipette. After a minimum of 20 min, the buffer was removed from each well to a 1.5 ml microcentrifuge tube. Tubes were numbered to allow tracking of the CHO GALR1 treatments throughout the blotting procedure. A1-A3 down to H1-H3 numbered 1-24, A4-A6 down to H4-H6 numbered 25-48, A7-A9 down to H7-H93 numbered 49-72, A10-A12 down to H10-H12 numbered 73-96. Each sample was mixed by vortex and heated at 90° C. for 5 to 10 min in a pre-warmed heat block. The samples were either stored at −20° C. or separated on the same day on an SDS gel.
Gel ElectrophoresisIf the sample had been stored over night or longer, the sample was pre-warmed in a heat block to 90° C. for 5 to 10 min. 12% bis-tris SDS page gels were used with running buffer (lx, Invitrogen NuPAGE MOPS SDS Running Buffer (20×). 500 μl of NuPAGE antioxidant was added to the upper buffer chamber. 15 μl samples were loaded onto the gel lanes from left to right. 2.5 μl Magic Marker XP (Invitrogen), 5 μl See Blue Plus 2 pre-stained standard (Invitrogen) and 15 μl of non-treated control were also loaded onto gel lanes. The gels were run at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) marker reached the bottom of the tank).
Western BlottingSemi-dry transfer was carried out using an Invitrogen iBlot (iBlot Programme 3 for 6 minutes). Nitrocellulose membranes were put in individual small trays and incubated with blocking buffer solution (5 g Marvel milk powder per 100 ml 0.1% PBS/Tween) at room temperature on a rocker for 1 hour. Primary antibody (Anti-SNAP-25 1:1000 dilution) was applied and the membranes were incubated with primary antibody (diluted in blocking buffer) for 1 hour on a rocker at room temperature. Membranes were washed by rinsing 3 times with PBS/Tween (0.1%). The secondary (Anti-Rabbit-HRP conjugate diluted 1:1000) was then applied and the membranes were incubated with secondary antibody (diluted in blocking buffer) at room temperature on a rocker for 1 hour. Membranes were again washed by rinsing 3 times with PBS/Tween (0.1%) and the membranes were left for a minimum of 20 min for the last wash. Bound antibody was detected using Syngene: Blots were drained of PBS/Tween, and WestDura reagents were mixed 1:1 and added to completely cover the blots for 5 minutes. The membranes were placed in a Syngene tray, and the Syngene software was set for a 5 min expose time.
The nociceptive flexion reflex (also known as paw guarding assay) is a rapid withdrawal movement that constitutes a protective mechanism against possible limb damage. It can be quantified by assessment of electromyography (EMG) response in anesthetized rat as a result of low dose capsaicin, electrical stimulation or the capsaicin-sensitized electrical response. Intraplantar pretreatment (24 hour) of fusion proteins of the present invention into 300-380 g male Sprague-Dawley rats. Induction of paw guarding was achieved by 0.006% capsaicin, 10 μl in PBS (7.5% DMSO), injected in 10 seconds. This produced a robust reflex response from biceps feroris muscle. A reduction/inhibition of the nociceptive flexion reflex indicates that the test substance demonstrates an anti-nociceptive effect. The data demonstrated the anti-nociceptive effect of the galanin fusion proteins of the present invention as a percentage (
The ability of different galanin fusion proteins of the invention to inhibit capsaicin-induced thermal hyperalgesia was evaluated (
Substance P EIA was obtained from R&D Systems, UK.
MethodsPrimary neuronal cultures of eDRG were established as described previously (Duggan et al., (2002) J. Biol. Chem. 277:34846-34852). Substance P released from the cultures was assessed by EIA, essentially as described previously (Duggan et al. (2002), J. Biol. Chem. 277:34846-34852). The TM of interest was added to the neuronal cultures (established for at least 2 weeks prior to treatment); control cultures were 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 were obtained for both control and TM treated cultures. Substance P immunoreactivity was measured using Substance P Enzyme 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 TM of interest was compared to the release obtained in the presence and absence of 100 mM KCl. Stimulation of Substance P release by the TM of interest above the basal release, establishes that the TM of interest was an “agonist ligand” as defined in this specification. If desired the stimulation of Substance P released by the TM of interest could be compared to a standard Substance P release-curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris).
Example 10A method of treating, preventing or ameliorating pain in a subject, comprising administration to said patient a therapeutic effective amount of fusion protein, wherein said pain is selected from the group consisting of: chronic pain arising from malignant disease, chronic pain not caused by malignant disease (peripheral neuropathies).
Patient AA 73 year old woman suffering from severe pain caused by [postherpatic?] neuralgia is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 2 hours of said injection.
Patient BA 32 year old male suffering from phantom limb pain after having his left arm amputated following a car accident is treated by peripheral injection with fusion protein to reduce the pain. The patient experiences good analgesic effect within 1 hour of said injection.
Patient CA 55 year male suffering from diabetic neuropathy is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection.
Patient DA 63 year old woman suffering from cancer pain is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection.
All documents, books, manuals, papers, patents, published patent applications, guides, abstracts and other reference materials cited herein are incorporated by reference in their entirety. While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
Claims
1. A single chain, polypeptide fusion protein, comprising:
- a. a non-cytotoxic protease, which protease cleaves a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;
- b. a galanin Targeting Moiety that binds to a Binding Site on the nociceptive sensory afferent, which Binding Site endocytoses 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 and the galanin Targeting Moiety;
- d. a translocation domain that translocates the protease from within an endosome, across the endosomal 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;
- e. a first spacer located between the non-cytotoxic protease and the protease cleavage site, wherein said first spacer comprises an amino acid sequence of from 4 to 25 amino acid residues;
- f. a second spacer located between the galanin Targeting Moiety and the translocation domain, wherein said second spacer comprises an amino acid sequence of from 4 to 35 amino acid residues.
2. The fusion protein according to claim 1, wherein the first spacer comprises an amino acid sequence of from 6 to 16 amino acid residues.
3. The fusion protein according to claim 1, wherein said amino acid residues of said first spacer are selected from the group consisting of glycine, threonine, arginine, serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamic acid and/or lysine.
4. The fusion protein according to claim 1, wherein the amino acid residues of the first spacer are selected from the group consisting of glycine, serine and alanine.
5. The fusion protein according to claim 1, wherein the first spacer is selected from a GS5, GS10, GS15, GS18 or GS20 spacer.
6. The fusion protein according to claim 1, wherein the galanin Targeting Moiety binds specifically to the GALR1, GALR2 and/or the GALR3 receptor.
7. The fusion protein according to claim 1, wherein the galanin Targeting Moiety comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.
8. The fusion protein according to claim 1, wherein the galanin Targeting Moiety comprises an amino acid sequence according to SEQ ID NO: 7 or a fragment comprising or consisting of at least 14 or 16 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 7 or said fragment having a maximum of 5 or 6 conservative amino acid substitutions.
9. The fusion protein according to claim 1, wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease.
10. The fusion protein according to claim 1, wherein the translocation domain is the HN domain of a clostridial neurotoxin.
11. The fusion protein according to claim 1, wherein said fusion protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56 and/or 59.
12. A polynucleotide molecule encoding the polypeptide fusion protein according to claim 1.
13. An expression vector, which comprises a promoter, the polynucleotide molecule according to claim 12, wherein said polynucleotide molecule is located downstream of the promoter, and a terminator located downstream of the polynucleotide molecule.
14. A method for preparing a single-chain polypeptide fusion protein, comprising:
- a. transfecting a host cell with the expression vector of claim 13, and
- b. culturing said host cell under conditions promoting expressing of the polypeptide fusion protein by the expression vector.
15. A method of preparing a non-cytotoxic agent, comprising:
- a. contacting a single-chain polypeptide fusion protein according to claim 1 with a protease capable of cleaving the protease cleavage site;
- b. cleaving the protease cleavage site; and thereby forming a di-chain fusion protein.
16. A non-cytotoxic polypeptide, obtained by the method of claim 15, wherein the polypeptide is a di-chain polypeptide, and wherein:
- a. the first chain comprises the non-cytotoxic protease, which protease is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;
- b. the second chain comprises the galanin TM and the translocation domain that is capable of translocating the protease 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.
17. A method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of the fusion protein according to claim 1.
18. A method according to claim 17, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
19. A method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a polypeptide according to claim 16.
20. A method according to claim 19, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
Type: Application
Filed: Aug 27, 2012
Publication Date: Feb 27, 2014
Applicants: ALLERGAN, INC. (Irvine, CA), SYNTAXIN LIMITED (Abingdon)
Inventors: Peter James (Eastleigh), Keith Foster (Salisbury), John Chaddock (Salisbury), Kei Roger Aoki (Coto de Caza, CA), Lance Steward (Irvine, CA), Joseph Francis (Laguna Niguel, CA)
Application Number: 13/595,927
International Classification: C12N 9/48 (20060101); A61P 29/00 (20060101); A61K 38/48 (20060101); C12N 15/57 (20060101); C12N 15/63 (20060101);
