Activatable recombinant neurotoxins

Abstract

Compositions comprising activatable recombinant neurotoxins and polypeptides derived therefrom. The invention also comprises nucleic acids encoding such polypeptides, and methods of making such polypeptides and nucleic acids.

1 . A single-chain polypeptide comprising:
 a) a first domain comprising a binding element, wherein the binding element comprising a nerve growth factor peptide able to preferentially interact with a nerve growth factor receptor under physiological conditions, a leukemia inhibitory factor peptide able to preferentially interact with a leukemia inhibitory factor receptor under physiological conditions, a brain-derived neurotrophic factor peptide able to preferentially interact with a brain-derived neurotrophic factor receptor under physiological conditions, a neurotrophin-3 peptide able to preferentially interact with a neurotrophin-3 receptor under physiological conditions, a hydra head activator peptide able to preferentially interact with a hydra head activator peptide receptor under physiological conditions, a transforming growth factor peptide able to preferentially interact with a transforming growth factor receptor under physiological conditions, a ciliary neurotrophic factor peptide able to preferentially interact with a ciliary neurotrophic factor receptor under physiological conditions, a tumour necrosis factor peptide able to preferentially interact with a tumour necrosis factor receptor under physiological conditions, an interleukin-1 peptide able to preferentially interact with an interleukin-1 receptor under physiological conditions, an interleukin-8 peptide able to preferentially interact with an interleukin-8 receptor under physiological conditions, an endorphin peptide able to preferentially interact with an endorphin receptor under physiological conditions, a methionine-enkephalin peptide able to preferentially interact with a methionine-enkephalin receptor under physiological conditions, a D-ala2-D-leu5-enkephalin peptide able to preferentially interact with a D-ala2-D-leu5-enkephalin receptor under physiological conditions, or a bradykinin peptide able to preferentially interact with a bradykinin receptor under physiological conditions;   b) a second domain comprising a translocation element comprising a Clostridial neurotoxin heavy chain able to facilitate the transfer of said single-chain polypeptide across a vesicular membrane;   c) a third domain comprising a therapeutic element comprising a Clostridial neurotoxin light chain having biological activity when released into the cytoplasm of said target cell; and   d) a fourth domain comprising a non-native Clostridial neurotoxin protease cleavage site;   
     
     
         2 . The polypeptide of  claim 1 , wherein said fourth domain intervenes between said first domain and said second domain. 
     
     
         3 . The polypeptide of  claim 1 , wherein said fourth domain intervenes between said second domain and said third domain. 
     
     
         4 . The polypeptide of  claim 1 , wherein said polypeptide comprises a fifth domain comprising a target-binding portion of a binding tag. 
     
     
         5 . The polypeptide of  claim 4 , wherein said target-binding portion comprises a His 6 , a monoclonal antibody, a maltose binding protein, a glutathione-S-transferase, a protein A or a calmodulin binding protein. 
     
     
         6 . The polypeptide of  claim 1 , wherein said translocation element is a  Clostridium botulinum  neurotoxin heavy chain. 
     
     
         7 . The polypeptide of  claim 6 , wherein said is a  Clostridium botulinum  neurotoxin heavy chain translocation element is selected from the group consisting of a  Clostridium botulinum  serotype A neurotoxin heavy chain, a  Clostridium botulinum  serotype B neurotoxin heavy chain, a  Clostridium botulinum  serotype C1 neurotoxin heavy chain, a  Clostridium botulinum  serotype D neurotoxin heavy chain, a  Clostridium botulinum  serotype E neurotoxin heavy chain, a  Clostridium botulinum  serotype F neurotoxin heavy chain and a  Clostridium botulinum  serotype G neurotoxin heavy chain. 
     
     
         8 . The polypeptide of  claim 1 , wherein said translocation element is a  Clostridium tetani  neurotoxin heavy chain. 
     
     
         9 . The polypeptide of  claim 1 , wherein said therapeutic element is a  Clostridium botulinum  neurotoxin light chain. 
     
     
         10 . The polypeptide of  claim 9 , wherein said is a  Clostridium botulinum  neurotoxin light chain therapeutic element is selected from the group consisting of a  Clostridium botulinum  serotype A neurotoxin light chain, a  Clostridium botulinum  serotype B neurotoxin light chain, a  Clostridium botulinum  serotype C1 neurotoxin light chain, a  Clostridium botulinum  serotype D neurotoxin light chain, a  Clostridium botulinum  serotype E neurotoxin light chain, a  Clostridium botulinum  serotype F neurotoxin light chain and a  Clostridium botulinum  serotype G neurotoxin light chain. 
     
     
         11 . The polypeptide of  claim 1 , wherein said therapeutic element is a  Clostridium tetani  neurotoxin light chain. 
     
     
         12 . The polypeptide of  claim 1 , wherein said protease cleavage site comprises a bovine enterokinase, a tobacco etch virus protease, a protease derived from  Bacillus subtilus , a protease derived from  Bacillus amyliquifaciens , a protease derived from a rhinovirus, a papain, an insect papain homolog, or a crustacian papain homolog. 
     
     
         13 . The polypeptide of  claim 1 , wherein said protease cleavage site comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, or SEQ ID NO: 31. 
     
     
         14 . A single-chain polypeptide comprising:
 a) a first amino acid sequence region comprising
 i) a first domain comprising a binding element, wherein the binding element comprising a nerve growth factor peptide able to preferentially interact with a nerve growth factor receptor under physiological conditions, a leukemia inhibitory factor peptide able to preferentially interact with a leukemia inhibitory factor receptor under physiological conditions, a brain-derived neurotrophic factor peptide able to preferentially interact with a brain-derived neurotrophic factor receptor under physiological conditions, a neurotrophin-3 peptide able to preferentially interact with a neurotrophin-3 receptor under physiological conditions, a hydra head activator peptide able to preferentially interact with a hydra head activator peptide receptor under physiological conditions, a transforming growth factor peptide able to preferentially interact with a transforming growth factor receptor under physiological conditions, a ciliary neurotrophic factor peptide able to preferentially interact with a ciliary neurotrophic factor receptor under physiological conditions, a tumour necrosis factor peptide able to preferentially interact with a tumour necrosis factor receptor under physiological conditions, an interleukin-1 peptide able to preferentially interact with an interleukin-1 receptor under physiological conditions, an interleukin-8 peptide able to preferentially interact with an interleukin-8 receptor under physiological conditions, an endorphin peptide able to preferentially interact with an endorphin receptor under physiological conditions, a methionine-enkephalin peptide able to preferentially interact with a methionine-enkephalin receptor under physiological conditions, a D-ala2-D-leu5-enkephalin peptide able to preferentially interact with a D-ala2-D-leu5-enkephalin receptor under physiological conditions, or a bradykinin peptide able to preferentially interact with a bradykinin receptor under physiological conditions; 
 ii) a second domain comprising a translocation element comprising a Clostridial neurotoxin heavy chain able to facilitate the transfer of said single-chain polypeptide across a vesicular membrane; and 
   b) a second amino acid sequence region comprising a therapeutic element comprising a Clostridial neurotoxin light chain having biological activity when released into the cytoplasm of said target cell;   c) a third amino acid sequence region comprising a non-native Clostridial neurotoxin protease cleavage site;
 wherein said first and second amino acid sequence regions are separated by said third amino acid sequence region. 
   
     
     
         15 . The polypeptide of  claim 14 , wherein said polypeptide comprises a fourth amino acid sequence region comprising a target-binding portion of a binding tag. 
     
     
         16 . The polypeptide of  claim 15 , wherein said target-binding portion comprises a His 6 , a monoclonal antibody, a maltose binding protein, a glutathione-S-transferase, a protein A or a calmodulin binding protein. 
     
     
         17 . The polypeptide of  claim 14 , wherein said translocation element is a  Clostridium botulinum  neurotoxin heavy chain. 
     
     
         18 . The polypeptide of  claim 17 , wherein said is a  Clostridium botulinum  neurotoxin heavy chain translocation element is selected from the group consisting of a  Clostridium botulinum  serotype A neurotoxin heavy chain, a  Clostridium botulinum  serotype B neurotoxin heavy chain, a  Clostridium botulinum  serotype C1 neurotoxin heavy chain, a  Clostridium botulinum  serotype D neurotoxin heavy chain, a  Clostridium botulinum  serotype E neurotoxin heavy chain, a  Clostridium botulinum  serotype F neurotoxin heavy chain and a  Clostridium botulinum  serotype G neurotoxin heavy chain. 
     
     
         19 . The polypeptide of  claim 14 , wherein said translocation element is a  Clostridium tetani  neurotoxin heavy chain. 
     
     
         20 . The polypeptide of  claim 14 , wherein said therapeutic element is a  Clostridium botulinum  neurotoxin light chain. 
     
     
         21 . The polypeptide of  claim 20 , wherein said is a  Clostridium botulinum  neurotoxin light chain therapeutic element is selected from the group consisting of a  Clostridium botulinum  serotype A neurotoxin light chain, a  Clostridium botulinum  serotype B neurotoxin light chain, a  Clostridium botulinum  serotype C1 neurotoxin light chain, a  Clostridium botulinum  serotype D neurotoxin light chain, a  Clostridium botulinum  serotype E neurotoxin light chain, a  Clostridium botulinum  serotype F neurotoxin light chain and a  Clostridium botulinum  serotype G neurotoxin light chain. 
     
     
         22 . The polypeptide of  claim 14 , wherein said therapeutic element is a  Clostridium tetani  neurotoxin light chain. 
     
     
         23 . The polypeptide of  claim 14 , wherein said protease cleavage site comprises a bovine enterokinase, a tobacco etch virus protease, a protease derived from  Bacillus subtilus , a protease derived from  Bacillus amyliquifaciens , a protease derived from a rhinovirus, a papain, an insect papain homolog, or a crustacian papain homolog. 
     
     
         24 . The polypeptide of  claim 14 , wherein said protease cleavage site comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, or SEQ ID NO: 31. 
     
     
         25 . A nucleotide sequence encoding a single-chain polypeptide according to any one of  claims 1 - 24 . 
     
     
         26 . The nucleotide sequence of  claim 25 , further comprising an expression vector. 
     
     
         27 . A method of making a single-chain polypeptide comprising:
 a) inserting a nucleotide sequence of  claims 26  into a suitable host cell;   b) growing said host cell in culture; and   c) permitting or inducing the host cell to express the single chain polypeptide encoded by said nucleotide sequence.   
     
     
         28 . A method of purifying a single chain-polypeptide comprising:
 a) lysing a host cell containing a nucleotide sequence expressing a single-chain polypeptide to produce a cell lysate, said single-chain polypeptide according to any one of  claims 1 - 24 ;   b) contacting said cell lysate with a target compound so as to form a specific binding complex capable of being immobilized comprising said binding tag and said target compound; and   c) separating said binding complex from said cell lysate.