FUSION PROTEINS AND METHODS FOR TREATING, PREVENTING OR AMELIORATING PAIN

Abstract

A single chain polypeptide fusion protein, comprising: a non-cytotoxic protease capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a galanin targeting moiety; a protease cleavage site at which site the fusion protein is cleavable by a protease; a translocation domain capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; a first spacer located between the non-cytotoxic protease and the protease cleavage site; and a second spacer located between the galanin targeting moiety and the translocation domain.

Claims

1. A single chain polypeptide fusion protein, comprising: a non-cytotoxic protease capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a galanin-targeting moiety that binds to a binding site on the nociceptive sensory afferent, the binding site capable of incorporating into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease; a translocation domain capable of translocating the protease from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory a first spacer located between the non-cytotoxic protease and the protease cleavage site, the first spacer comprising an amino acid sequence of from 4 to 25 amino acid residues; and a second spacer located between the galanin-targeting moiety and the translocation domain, the second spacer comprising an amino acid sequence of from 4 to 35 amino acid residues; wherein: the protease cleavage site is located between the non-cytotoxic protease and the galanin-targeting moiety, and the galanin-targeting moiety is located between the protease cleavage site and the translocation domain.

2. The fusion protein of claim 1, wherein the first spacer comprises an amino acid sequence of from 6 to 16 amino acid residues.

3. The fusion protein of claim 1, wherein the first spacer comprises amino acid residues selected from the group consisting of: glycine, threonine, arginine, serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamic acid, and lysine.

4. The fusion protein of claim 1, wherein the first spacer comprises amino acid residues selected from the group consisting of: glycine, serine, and alanine.

5. The fusion protein of claim 1, wherein the first spacer is a GS5, GS10, GS15, GS18 or GS20 spacer.

6. The fusion protein of claim 1, wherein the galanin-targeting moiety binds specifically to the GALR1, GALR2 and/or the GALR3 receptor.

7. The fusion protein of claim 1, wherein the galanin-targeting moiety comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.

8. The fusion protein of claim 1, wherein the galanin-targeting moiety comprises: the amino acid sequence of SEQ ID NO. 7 a fragment of the amino acid sequence of SEQ ID NO: 7 comprising at least 14 contiguous amino acid residues thereof, a variant amino acid sequence of the sequence of SEQ ID NO: 7 having a maximum of 5 or 6 conservative amino acid substitutions as compared to the sequence of SEQ ID NO: 7, or a variant amino acid sequence of the fragment of SEQ ID NO: 7 having a maximum of 5 or 6 conservative amino acid substitutions as compared to the fragment.

9. The fusion protein of claim 1, wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease.

10. The fusion protein of claim 1, wherein the translocation domain is the H.sub.N domain of a clostridial neurotoxin.

11. The fusion protein of claim 1 comprising 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 59.

12. A polynucleotide encoding the fusion protein of claim 1.

13. An expression vector comprising , a promoter, the polynucleotide of claim 12 located downstream of the promoter, and a terminator located downstream of the polynucleotide.

14. A method for preparing a single-chain polypeptide fusion protein, comprising: transfecting a host cell with the expression vector of claim 13, and culturing the host cell under conditions that promote the expression of the polypeptide fusion protein by the expression vector.

15. A method of preparing a non-cytotoxic agent, comprising: contacting a single-chain polypeptide fusion protein of claim 1 with a protease capable of cleaving the protease cleavage site; cleaving the protease cleavage site, 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 comprising a first and second chain joined together by a disulphide bond; the first chain comprises the non-cytotoxic protease; and the second chain comprises the galanin-targeting moiety and the translocation domain.

17. A method of treating, preventing or ameliorating pain in a subject, comprising administering to the subject a therapeutically effective amount of the fusion protein of claim 1.

18. The method of 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 the subject a therapeutically effective amount of a polypeptide of claim 16.

20. The method of claim 19, wherein the pain is chronic pain selected from: neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.

Description

[0305] There now follows a brief description of the Figures, which illustrate aspects and/or embodiments of the present invention.

[0306] FIG. 1—Purification of a LC/A-Spacer-Galanin-Spacer-H.sub.N/A Fusion Protein

[0307] Using the methodology outlined in Example 3, a LC/A-GS18-galanin-GS20-H.sub.N/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 (Panel A) and Western blotting (Panel B). Anti-galanin antisera (obtained from Abcam) and Anti-histag antisera (obtained from Qiagen) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes of Panel A marked [−] and [+] respectively. Panel A, Lane 1=Benchmark ladder; 2=soluble fraction; 3=1.sup.st His product; 4=activated purfied protein; 5=second His product; 6=final purified protein 5 μl; 7=final purified protein 10 μl; 8=final purified protein 20 μl; 9=final purified protein 5 μl+DTT; 10=final purified protein 10 μl+DTT. Panel B Lane 1=Benchmark ladder; 2=soluble fraction; 3=1.sup.st His product; 4=activated purfied protein; 5=second His product; 6=final purified protein 2 μl; 7=final purified protein 5 μl; 8=final purified protein 10 μl; 9=final purified protein 2 μl+DTT; 10=final purified protein 5 μl+DTT.

[0308] FIG. 2—Purification of a LC/C-Spacer-Galanin-Spacer-H.sub.N/C Fusion Protein

[0309] Using the methodology outlined in Example 3, an LC/C-galanin-H.sub.N/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 (Panel A) and Western blotting (Panel B). Anti-galanin antisera (obtained from Abcam) and Anti-histag antisera (obtained from Qiagen) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent in Panel A is identified in the lanes marked [−] and [+] respectively. Panel A, Lane 1=Benchmark ladder; 2=soluble fraction; 3=product 1.sup.st column; 4=enterokinase activated protein; 5=final product 0.1 mg/ml (5 μl); 6=final product 0.1 mg/ml+DTT (5 μl); 7=final product 0.1 mg/ml (10 μl); 8=final product 0.1 mg/ml+DTT (10 μl). Panel B, Lane 1=Magic mark; 2=soluble fraction; 3=product 1.sup.st His-tag column; 4=activated fusion; 5=purified@0.1 mg/ml (5 μl); 6=purified@0.1 mg/ml+DTT (5 μl); 7 purified@0.1 mg/ml+100 mm DTT (10 μl); 8=purified@0.1 mg/ml+100 mm DTT (10 μl)+DTT.

[0310] FIG. 3—Comparison of SNARE Cleavage Efficacy of a LC-Spacer-Galanin-Spacer-H.sub.N Fusion Protein and a LC-H.sub.N-Galanin Fusion Protein

[0311] Panels A & B: The ability of galanin fusions to cleave SNAP-25 in a CHO GALR1 SNAP25 cells was assessed. 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. It is clear from the data that the LC-spacer-galanin-spacer-H.sub.N fusion (Fusion 1) is more potent than the LC-H.sub.N-galanin fusion and the unliganded LC/A-H.sub.N/A control molecule.

[0312] FIG. 4—GALR1 Receptor Activation Studies in the CHO-GALCHO-GALR1 SNAP-25 Cleavage Assay with Galanin Fusion Proteins of the Present Invention having Different Serotype Backbones

[0313] 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 labelled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody and incubating at room temperature for 24 hours. Then samples are 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.

[0314] FIG. 5—Cleavage of SNARE Protein by Galanin (GA16 and GA30) Fusion Proteins in CHO-GALR1 SNAP-25 Cleavage Assay

[0315] 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.

[0316] FIG. 6—Results of in Vivo Paw Guarding Assay Employing Galanin Fusion Proteins

[0317] 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 300-380g 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 antinociceptive effect. The data demonstrated the antinociceptive effect of the galanin fusion proteins of the present invention.

[0318] FIG. 7—Galanin Fusion Protein Efficacy in Capsaicin-Induced Thermal Hyperalgesia Assay

[0319] 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 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 antinociceptive effect. No. 1=LC.H.sub.N-GA16; No. 2=LC-H.sub.N-GA30; No. 3=LC-GS5-EN-CPGA16-GS20-H.sub.N-HT; No. 4=LC-GS18-EN-CPGA16-GS20-H.sub.N-HT; No. 5=BOTOX; No. 6=morphine. The data demonstrated the enhanced antinociceptive effect of the galanin fusion proteins of the present invention compared to fusion proteins with a C-terminally presented ligand.

[0320] FIG. 8—Galanin Fusion Protein Efficacy in Capsaicin-Induced Thermal Hyperalgesia Assay

[0321] 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 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 antinociceptive effect. The data demonstrated the antinociceptive effect of the galanin fusion proteins of the present invention having different serotype backbones (i.e. A, B, C and D).

[0322] FIG. 9—Activation of Galanin Fusion Proteins with Single and Double-Spacers 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 (Panels A and B). Panel C demonstrates the enhanced activation of galanin fusion proteins of the present invention having both first (spacer 1) and second (spacer 2) spacers. Panels A&B: 1) Benchmark ladder; 2) Unactivated control; 3) Unactivated control+DTT; 4) Protease activated protein+0.0 mM ZnCl2; 5) Protease activated protein+0.0 mM ZnCl2+DTT; 6) Protease activated protein+0.2 mM ZnCl2; 7) Protease activated protein+0.2 mM ZnCl2+DTT; 8) Protease activated protein+0.4 mM ZnCl2; 9) Protease activated protein+0.4 mM ZnCl2+DTT; 10) Protease activated protein+0.8 mM ZnCl2; 11) Protease activated protein+0.8 mM ZnCl2+DTT. Panel C: 1) Benchmark ladder; 2) Unactivated control 25° C.; 3) Unactivated control 25° C.+DTT; 4) Protease activated protein 25° C.; 5) Protease activated protein 25° C.+DTT; 6) Benchmark ladder.

SEQ ID NOs

[0323] 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.

[0324] SEQ ID NO 1 DNA sequence of the LC/A

[0325] SEQ ID NO 2 DNA sequence of the H.sub.N/A

[0326] SEQ ID NO 3 DNA sequence of the LC/B

[0327] SEQ ID NO 4 DNA sequence of the H.sub.N/B

[0328] SEQ ID NO 5 DNA sequence of the LC/C

[0329] SEQ ID NO 6 DNA sequence of the H.sub.N/C

[0330] SEQ ID NO7 Protein sequence of galanin GA30

[0331] SEQ ID NO8 Protein sequence of galanin GA16

[0332] SEQ ID NO9 DNA sequence of LC/A-GS18-EN-CPGA16-GS20-H.sub.N/A-HT

[0333] SEQ ID NO10 Protein sequence of LC/A-GS18-EN-CPGA16-GS20-H.sub.N/A-HT

[0334] SEQ ID NO11 Protein sequence of LC/A-GS18-EN-CPGA16-GS20-H.sub.N/A

[0335] SEQ ID NO12 DNA sequence of LC/A-GS5-EN-CPGA16-GS20-H.sub.N/A-HT

[0336] SEQ ID NO13 Protein sequence of LC/A-GS5-EN-CPGA16-GS20-H.sub.N/A-HT

[0337] SEQ ID NO14 Protein sequence of LC/A-GS5-EN-CPGA16-H.sub.N/A-GS20

[0338] SEQ ID NO15 DNA sequence of LC/A-GS5-EN-CPGA30-GS20-H.sub.N/A-HT

[0339] SEQ ID NO16 Protein sequence of LC/A-GS5-EN-CPGA30-GS20-H.sub.N/A-HT

[0340] SEQ ID NO17 Protein sequence of LC/A-GS5-EN-CPGA30-GS20-H.sub.N/A

[0341] SEQ ID NO18 DNA sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B(K191A)-HT

[0342] SEQ ID NO19 Protein sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B(K191A)-HT

[0343] SEQ ID NO20 Protein sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B(K191A)

[0344] SEQ ID NO21 DNA sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B-HT

[0345] SEQ ID NO22 Protein sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B-HT

[0346] SEQ ID NO23 Protein sequence of LC/B-GS5-EN-CPGA16-GS20-H.sub.N/B

[0347] SEQ ID NO24 DNA sequence of LC/C-GS5-EN-CPGA16-GS20-H.sub.N/C-HT

[0348] SEQ ID NO25 Protein sequence of LC/C-GS5-EN-CPGA16-GS20-H.sub.N/C-HT

[0349] SEQ ID NO26 Protein sequence of LC/C-GS5-EN-CPGA16-GS20-H.sub.N/C

[0350] SEQ ID NO27 DNA sequence of LC/D-GS5-EN-CPGA16-GS20-H.sub.N/D-HT

[0351] SEQ ID NO28 Protein sequence of LC/D-GS5-EN-CPGA16-GS20-H.sub.N/D-HT

[0352] SEQ ID NO29 Protein sequence of LC/D-GS5-EN-CPGA16-H.sub.N/D-GS20

[0353] SEQ ID NO30 DNA sequence of LC/A-GS5-EN-CPGA16-HX27-H.sub.N/A-HT

[0354] SEQ ID NO31 Protein sequence of LC/A-GS5-EN-CPGA16-HX27-H.sub.N/A-HT

[0355] SEQ ID NO32 Protein sequence of LC/A-GS5-EN-CPGA16-HX27-H.sub.N/A-

[0356] SEQ ID NO33 Protein sequence of LC/A-GS10-EN-CPGA16-H.sub.N/A-GS20-HT

[0357] SEQ ID NO34 Protein sequence of LC/A-GS10-EN-CPGA16-GS20-H.sub.N/A

[0358] SEQ ID NO35 Protein sequence of LC/A-GS5-EN-CPGA16-GS15-H.sub.N/A-HT

[0359] SEQ ID NO36 Protein sequence of LC/A-GS5-EN-CPGA16-GS15-H.sub.N/A

[0360] SEQ ID NO37 Protein sequence of LC/A-GS5-EN-CPGA16-GS10-H.sub.N/A-HT

[0361] SEQ ID NO38 Protein sequence of LC/A-GS5-EN-CPGA16-GS10-H.sub.N/A

[0362] SEQ ID NO39 Protein sequence of LC/A-GS18-EN-CPGA16-HX27-H.sub.N/A-HT

[0363] SEQ ID NO40 Protein sequence of LC/A-GS18-EN-CPGA16-HX27-H.sub.N/A

[0364] SEQ ID NO41 Protein sequence of LC/A-GS18-EN-CPGA16-GS15-H.sub.N/A-HT

[0365] SEQ ID NO42 Protein sequence of LC/A-GS18-EN-CPGA16-GS15

[0366] SEQ ID NO43 Protein sequence of LC/A-GS18-EN-CPGA16-GS10-HT

[0367] SEQ ID NO44 Protein sequence of LC/A-GS18-EN-CPGA16-GS10

[0368] SEQ ID NO45 Protein sequence of LC/A-GS10-EN-CPGA16-HX27-HT

[0369] SEQ ID NO46 Protein sequence of LC/A-GS10-EN-CPGA16-HX27

[0370] SEQ ID NO47 Protein sequence of LC/A-GS10-EN-CPGA16-GS15-H.sub.N/A-HT

[0371] SEQ ID NO48 Protein sequence of LC/A-GS10-EN-CPGA16-GS15-H.sub.N/A

[0372] SEQ ID NO49 Protein sequence of LC/A-GS10-EN-CPGA16-GS10-H.sub.N/A-HT

[0373] SEQ ID NO50 Protein sequence of LC/A-GS10-EN-CPGA16-GS10-H.sub.N/A

[0374] SEQ ID NO51 DNA sequence of the IgA protease

[0375] SEQ ID NO52 DNA sequence of the IgA-GS5-CPGA16-GS20-H.sub.N/A fusion

[0376] SEQ ID NO53 Protein sequence of the IgA-GS5-CPGA16-GS20-H.sub.N/A fusion

[0377] SEQ ID NO54 DNA sequence of DT translocation domain

[0378] SEQ ID NO55 DNA sequence of LC/A-GS5-GA16-GS20-DT

[0379] SEQ ID NO56 Protein sequence of LC/A-GS5-GA16-GS20-DT

[0380] SEQ ID NO57 DNA sequence of TeNT LC

[0381] SEQ ID NO58 DNA sequence of TeNT LC-GS5-CPGA16-GS20-H.sub.N/A

[0382] SEQ ID NO59 Protein sequence of TeNT LC-GS5-EN-CPGA16-GS20-H .sub.N/A

EXAMPLES

Example 1

Construction and Activation of Galanin Fusion Proteins

[0383] Preparation of a LC/A and H.sub.A/A Backbone Clones

[0384] The following procedure creates the LC and H.sub.N 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 NO1 and SEQ ID NO2), 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 NO3 and SEQ ID NO4) and serotype C (SEQ ID NO5 and SEQ ID NO6)].

[0385] Preparation of Cloning and Expression Vectors

[0386] pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHI-Sa/I-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.

[0387] Preparation of protease (e.g. LC/A) insert

[0388] The LC/A (SEQ ID NO1) 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 v2.0 (Entelechon)]. BamHI/Sa/I recognition sequences are incorporated at the 5′ and 3′ ends respectively of the sequence, maintaining the correct reading frame. The

[0389] 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 optimised 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.

[0390] The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and Sa/I restriction enzyme sequences incorporated into the 5′ and 3′ PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair has the ability to hybridize to the opposite strands (3′ ends pointing “towards” each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the 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 (50-200 μM) and a buffer appropriate for the enzyme optimised for Mg.sup.2+ concentration (0.5-5 mM).

[0391] 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.)].

[0392] Preparation of Translocation (e.g. H.sub.N) Insert

[0393] The H.sub.N/A (SEQ ID NO2) is created by one of two ways:

[0394] The DNA sequence is designed by back translation of the H.sub.N/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 v2.0 (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 optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

[0395] The alternative method is to use PCR amplification from an existing DNA sequence with PstI and XbaI-stop codon-HindlIl 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.)].

[0396] Preparation of LC/A-GS18-EN-CPGA16-GS20-H.sub.N/A Fusion

[0397] In order to create the LC/A-GS18-EN-CPGA16-GS20-H.sub.N/A construct, an A serotype linker with the addition of an Enterokinase site for activation, arranged as BamHI-Sa/I-GS18 -protease site-GS20-PstI-XbaI-stop codon-HindIII is synthesised. The pCR 4 vector encoding the linker is cleaved with BamHI+Sa/I restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID NO1) also cleaved with BamHI+Sa/I. 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 H.sub.N/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-H.sub.N/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 NO10.

[0398] Activation Assay

[0399] NuPAGE 4-12% Bis-Tris gels (10, 12 and 15 well pre-poured gel) were used to analyze activation of fusion proteins after treatment with protease. Protein samples were prepared with NuPAGE 4X 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 protesin concentration. The samples were mixed and then heated in the heat block at 95° C. for 5 min before loading onto the gel. 5-20 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 dH.sub.2O 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 0.5-2 h to stain. The gel was then destained by pouring off the Safestain and rinsing the gel with dH.sub.2O. The gels were left in dH.sub.2O 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 LCIA-GS18-EN-CPGA16-GS20-H.SUB.N./A Fusion Protein Family with Variable Spacer Length

[0400] Using the same strategy as employed in Example 1, a range of DNA linkers were prepared that encoded galanin 16 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 v2.0 (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-Sa/I-Spacer 1-protease site-CPGA16-Nhel-spacer 2-Spel-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 on 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 optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

[0401] The spacer-linkers that were created included:

TABLE-US-00007 Spacer 1 - protease SEQ ID NO site-GA16- Spacer 2 of the linker GS5-EN-CPGA16-GS20 12, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 GS10-EN-CPGA16-GS20 33, 34 GS5-EN-CPGA16-HX27 30, 31, 32 GS5-EN-CPGA16-GS15 35, 36 GS5-EN-CPGA16-GS10 37, 38 GS18-EN-CPGA16-HX27 39, 40 GS18-EN-CPGA16-GS15 41, 42 GS18-EN-CPGA16-GS10 43, 44 GS10-EN-CPGA16-HX27 45, 46 GS10-EN-CPGA16-GS15 47, 48 GS10-EN-CPGA16-GS10 49, 50

[0402] By way of example, in order to create the LC/A-GS5-EN-CPGA16-GS20-H.sub.N/A fusion construct (SEQ ID NO12), the pCR 4 vector encoding BamHI-Sa/I-GS5-protease site-GS20-PstI-XbaI-stop codon-HindIII the linker is cleaved with BamHI+Sa/I restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID NO1) also cleaved with BamHI+Sa/I. 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, Sa/I, PstI, and HindIII such as the pMAL vector (NEB) or the pET vector (Novagen). The H.sub.N/A DNA (SEQ ID NO2) 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-H.sub.N/A ORF for expression as a protein of the sequence illustrated in SEQ ID NO13.

Example 3

Purification Method for Galanin Fusion Protein

[0403] Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO.sub.4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measure the OD of the dialysed fusion protein. Add 1 μg of enterokinase (1 mg/ml) per 100 μg of purified fusion protein and 10 μl of factor Xa per mg of purified fusion protein if the fusion protesin contains a maltose binding protein. Incubate at 25° C. static overnight. Load onto a 0.1 M NiSO.sub.4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at −20° C.

Example 4

Preparation of a LC/C-GAI6-H.SUB.N./C Fusion Protein with a Serotype A Activation Sequence

[0404] Following the methods used in Examples 1 and 2, the LC/C (SEQ ID NO5) and H.sub.N/C (SEQ ID NO6) are created and inserted into the A serotype linker arranged as BamHI-Sa/l-Spacer 1-protease site-GA16-Nhel-spacer 2-SpeI-PstI-XbaI-stop codon-HindIII. The final construct contains the LC-spacer 1-GA16-spacer 2-H.sub.N ORF for expression as a protein of the sequence illustrated in SEQ ID NO25.

Example 5

Preparation of an IgA Protease-GA16-H.SUB.N./A Fusion Protein

[0405] The IgA protease amino acid sequence was obtained from freely available database sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325(6103), 458-62). Using

[0406] Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli expression was determined. A BamHI recognition sequence was incorporated at the 5′ end and a codon encoding a cysteine amino acid and Sa/I 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 are 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 optimised DNA sequence (SEQ ID NO51) containing the IgA open reading frame (ORF) is then commercially synthesized.

[0407] The IgA (SEQ ID NO51) is inserted into the LC-GS5-CPGA16-GS20-H.sub.N ORF using BamHI and Sa/I restriction enzymes to replace the LC with the IgA protease DNA. The final construct contains the IgA-GS5-CPGA16-GS20-H.sub.N ORF for expression as a protein of the sequence illustrated in SEQ ID NO53.

Example 6

Preparation of a Galanin Targeted Endopeptidase Fusion Protein Containing a LC Domain Derived from Tetanus

[0408] The 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 v2.0 (Entelechon)]. BamHI/Sa/I recognition sequences are incorporated at the 5′ and 3′ ends respectively of the sequence maintaining the correct reading frame (SEQ ID NO57). 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 optimised 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 pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHI and Sa/I. The BamHI-Sa/I fragment is then inserted into the LC/A-GA16-H.sub.N/A vector that has also been cleaved by BamHI and Sa/I. The final construct contains the TeNT LC-GS5-GA16-GS20-H.sub.N ORF sequences for expression as a protein of the sequence illustrated in SEQ ID NO58.

Example 7

Construction of CHO-K1 GALR1 & GALR2 Receptor Activation Assay and SNAP-25 Cleavage Assay

[0409] Cell-Line Creation

[0410] CHO-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 ug/m1 Zeocin was added after a further 24 hours to begin selection of SNAP-25 expressing cells, and 5 ug/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 30-70% 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 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 they had expanded sufficiently each clone was seeded to 24 wells of a 96 well plate, plus a frozen stock vial created. Galanin fusion proteins of the invention and LC/A-H.sub.NA 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-H.sub.NA cleavage levels was selected.

[0411] GALR1 Receptor Activation Assay

[0412] The 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 labelled cAMP tracer (Europium-streptavadin/biotin-cAMP) and fluorescently (Alexa) labelled 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-labelled 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.

[0413] Using Gilson pipettes and Sigmacoted 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.

[0414] 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 ul 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.

[0415] LANCE Eu-W8044 labelled 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

[0416] MTS 2/4 orbital shaker set to maximum speed). Plate sealers were removed prior to reading on the Envision.

[0417] GALR2 Receptor Activation Assay

[0418] The GALR2 receptor activation assay measures the potency and intrinsic efficacy of ligands at GALR2 receptor in transfected CHO-K1 cells by measuring the calcium mobilisation that occurs when the receptor is activated. The transfected cells are pre-loaded with a calcium sensitive dye (FLIPR) before treatment. When read using Flexstation 3 microplate reader (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 mobilisation. This will be measured as an increase in the relative fluorescence units (RFU) at 525 nM.

[0419] Culture of Cells for Receptor Activation Assay:

[0420] Cells were seeded and cultured in T175 flasks containing Ham F12 with Glutamax, 10% Foetal bovine serum, 5 μg ml-1 Blasticidin and 100 μg ml-1 Zeocin. The flasks were incubated at 37° C. in a humidified environment containing 5% CO.sub.2 until 60-80% confluent. On the day of harvest the media was removed and the cells 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 in assay), 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 vapour phase of a liquid nitrogen storage tank.

[0421] FIG. 4 demonstrates that galanin fusion proteins of the present invention having different galanin ligands (i.e. galanin-16 and galanin-30) and different serotype backbones (i.e. LC/A-H.sub.N/A, LC/B-H.sub.N/B, LC/C-H.sub.N/C and LC/D-H.sub.N/D) activate GALR1 receptors.

[0422] CHO-KI GALRI SNAP-25 Cleavage Assays

[0423] 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).

[0424] Plating Cells

[0425] Prepare cells at 2×10e5 cells/ml and seed 125 μl per well of 96 well plate. Use the following media: 500 ml Gibco Ham F12 with Glutamax (product code 31765068), 50 ml FBS, 5 ug/ml Blasticidin (250 μl aliquot from box in freezer, G13) (Calbiochem #203351, 10 ml at 10 mg/ml), 100 ug/ml Zeocin (500 μl from box in freezer, G35). (Invitrogen from Fisher, 1 g in 8×1.25 ml tubes at 100 mg/ml product code VXR25001). Allow cells to grow for 24 hrs (37° C., 5% CO.sub.2, humidified atmosphere).

[0426] Cell Treatment

[0427] Prepare dilutions of test protein for a dose range of each test proteins (make up double (2×) the desired final concentrations because 125 μl will be applied directly onto 125 μl of media already in each well). Filter sterilize CHO GALR1 feeding medium (20 ml syringe, 0.2 μm syringe filter) to make the dilutions. Add the filtered medium into 5 labelled bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette or multi-stepper. Dilute the stock test protein to 2000 nM (working stock solution 1) and 600 nM (working stock solution 2). Using a Gilson pipette prepare 10-fold serial dilutions of each working stock, by adding 100 μl to the next concentration in the series. Pipette up and down to mix thoroughly. Repeat to obtain 4 serial dilutions for solution 1, and 3 serial dilutions for solution 2. A 0 nM control (filtered feeding medium only) should also be prepared as a negative control for each plate. Repeat the above for each test protein. In each experiment a ‘standard’ batch of material must be included as control/reference material , this is unliganded LC/A-H.sub.N/A.

[0428] Apply Diluted Sample to CHO GALR1 Plates

[0429] Apply 125 μl of test sample (double concentration) per well. Each test sample should be applied to triplicate wells and each dose range should include a 0 nM control. Incubate for 24 hrs (37° C., 5% CO.sub.2, humidified atmosphere).

[0430] Cell Lysis

[0431] Prepare fresh lysis buffer (20 mls per plate) with 25% (4×) NuPAGE LDS sample buffer, 65% dH.sub.2O and 10% 1 M DTT. Remove medium from the CHO GALR1 plate by inverting over a waste receptacle. Drain the remaining media from each well using a fine-tipped pipette. Lyse the cells by adding 125 μl of lysis buffer per well using a multi-stepper pipette. After a minimum of 20 mins, remove the buffer from each well to a 1.5 ml microcentrifuge tube. Tubes must be numbered to allowing 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-Al2 down to H10-H12 numbered 73-96. Vortex each sample and heat at 90° C. for 5-10 mins in a prewarmed heat block. Store at −20° C. or use on the same day on an SDS gel.

[0432] Gel Electrophoresis

[0433] If the sample has been stored o/n or longer, put in a heat block prewarmed to 90° C. for 5-10 mins. Set up SDS page gels, use 1 gel per 12 samples, prepare running buffer (1×, Invitrogen NuPAGE MOPS SDS Running Buffer (20×) (NP0001))≈800 ml/gel tank. Add 500 μl of NuPAGE antioxidant to the upper buffer chamber. Load 15 μl samples onto gel lanes from left to right as and load 2.5 ul of Invitrogen Magic Marker XP and 5 ul Invitrogen See Blue Plus 2 pre-stained standard and 15 ul of non-treated control. It is important to maximize the resolution of separation during SDS_PAGE. This can be achieved by running 12% bis-tris gels at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) marker reaches the bottom of the tank).

[0434] Western Blotting

[0435] Complete a Semi-dry transfer: using an Invitrogen iBlot (use iBlot Programme 3 for 6 minutes). Put the nitrocellulose membranes in individual small trays. Incubate the membranes 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. Apply primary antibody (Anti-SNAP-25 1:1000 dilution) and incubate the membranes with primary antibody (diluted in blocking buffer) for 1 hour on a rocker at room temperature. Wash the membranes by rinsing 3 times with PBS/Tween (0.1%). Then apply the secondary (Anti-Rabbit-HRP conjugate diluted 1:1000) and incubate the membranes with secondary antibody (diluted in blocking buffer) at room temperature, on a rocker, for 1 hour. Wash the membranes by rinsing 3 times with PBS/Tween (0.1%), leave membrane a minimum of 20 mins for the last wash. Detect the bound antibody using Syngene: Drain blots of PBS/Tween, mix WestDura reagents 1:1 and add to blots for 5 minutes. Ensure enough solution is added to the membranes to completely cover them. Place membrane in Syngene tray, set up Syngene software for 5 min expose time.

[0436] FIGS. 3 and 5 demonstrate that galanin fusion proteins of the invention effectively cleave SNAP-25.

Example 8

Assessment of in Vivo Efficacy of a Galanin Fusion

[0437] 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 antinociceptive effect. The data demonstrated the antinociceptive effect of the galanin fusion proteins of the present invention as a percentage (FIG. 6)

[0438] The ability of different galanin fusion proteins of the invention to inhibit capsaicin-induced thermal hyperalgesia was evaluated (FIGS. 7 and 8). 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 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 antinociceptive effect. The data demonstrated the enhanced antinociceptive effect of the galanin fusion proteins of the present invention compared to fusion proteins with a C-terminally presented ligand.

Example 9

Confirmation of TM Agonist Activity by Measuring Release of Substance P from Neuronal Cell Cultures

[0439] Materials

[0440] Substance P EIA is obtained from R&D Systems, UK.

[0441] Methods

[0442] Primary neuronal cultures of eDRG are established as described previously (Duggan et al., 2002). Substance P release from the cultures is assessed by EIA, essentially as described previously (Duggan et al., 2002). The TM of interest is added to the neuronal cultures (established for at least 2 weeks prior to treatment); control cultures are performed in parallel by addition of vehicle in place of TM. Stimulated (100 mM KCl) and basal release, together with total cell lysate content, of substance P are obtained for both control and TM treated cultures. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK) according to manufacturers' instructions.

[0443] The amount of Substance P released by the neuronal cells in the presence of the TM of interest is 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 is an “agonist ligand” as defined in this specification. If desired the stimulation of Substance P release by the TM of interest can be compared to a standard Substance P release-curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris).

Example 10

[0444] A 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).

[0445] Patient A

[0446] A 73 year old woman suffering from severe pain caused by posthepatic 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.

[0447] Patient B

[0448] A 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.

[0449] Patient C

[0450] A 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.

[0451] Patient D A 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.

[0452] 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.