Fusion proteins

Abstract

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.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
         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 H N  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.