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 cleavable single-chain polypeptide comprising:
 a) a first region comprising a botulinum neurotoxin heavy chain binding element able to preferentially interact with a target cell surface marker under physiological conditions;   b) a second region comprising a botulinum neurotoxin heavy chain translocation element able to facilitate the transfer of said single-chain polypeptide across a vesicular membrane;   c) a third region comprising a botulinum neurotoxin light chain therapeutic element having SNARE-protein sequence-specific endopeptidase activity, and   d) a fourth region comprising a protease cleavage site, wherein the protease cleavage site is cleaved by a non-human 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, and a crustacean papain homolog,   wherein said fourth domain intervenes between said first domain and said second domain, between said second domain and said third domain, or between said first domain and said third domain.   
     
     
         2 . The polypeptide of  claim 1 , wherein said polypeptide comprises a fifth region comprising a target-binding portion of a binding tag. 
     
     
         3 . The polypeptide of  claim 2 , 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. 
     
     
         4 . The polypeptide of  claim 1 , wherein said botulinum neurotoxin heavy chain binding element is a botulinum neurotoxin serotype A neurotoxin heavy chain, a botulinum neurotoxin serotype B neurotoxin heavy chain, a botulinum neurotoxin serotype C 1  neurotoxin heavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, a botulinum neurotoxin serotype E neurotoxin heavy chain, a botulinum neurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxin serotype G neurotoxin heavy chain. 
     
     
         5 . The polypeptide of  claim 1 , wherein said botulinum neurotoxin heavy chain translocation element is a botulinum neurotoxin serotype A neurotoxin heavy chain, a botulinum neurotoxin serotype B neurotoxin heavy chain, a botulinum neurotoxin serotype C 1  neurotoxin heavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, a botulinum neurotoxin serotype E neurotoxin heavy chain, a botulinum neurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxin serotype G neurotoxin heavy chain. 
     
     
         6 . The polypeptide of  claim 1 , wherein said botulinum neurotoxin light chain therapeutic element is a botulinum neurotoxin serotype A neurotoxin light chain, a botulinum neurotoxin serotype B neurotoxin light chain, a botulinum neurotoxin serotype C1 neurotoxin light chain, a botulinum neurotoxin serotype D neurotoxin light chain, a botulinum neurotoxin serotype E neurotoxin light chain, a botulinum neurotoxin serotype F neurotoxin light chain and a botulinum neurotoxin serotype G neurotoxin light chain. 
     
     
         7 . The polypeptide of  claim 1 , wherein said protease cleavage site is SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 23. 
     
     
         8 . A composition comprising an active di-chain form of a cleavable single-chain polypeptide as defined in  claim 1 . 
     
     
         9 . A cleavable single-chain polypeptide comprising:
 a) a first region comprising a binding element peptide able to preferentially interact with a sensory afferent neuron cell surface marker under physiological conditions;   b) a second region comprising a botulinum neurotoxin heavy chain translocation element able to facilitate the transfer of said single-chain polypeptide across a vesicular membrane;   c) a third region comprising a botulinum neurotoxin light chain therapeutic element having SNARE-protein sequence-specific endopeptidase activity; and   d) a fourth region comprising a protease cleavage site, wherein the protease cleavage site is cleaved by a non-human 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, and a crustacean papain homolog,   wherein said fourth domain intervenes between said first domain and said second domain, between said second domain and said third domain, or between said first domain and said third domain.   
     
     
         10 . The polypeptide of  claim 9 , wherein said polypeptide comprises a fifth region comprising a target-binding portion of a binding tag. 
     
     
         11 . The polypeptide of  claim 10 , 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. 
     
     
         12 . The polypeptide of  claim 9 , wherein said botulinum neurotoxin heavy chain binding element is a botulinum neurotoxin serotype A neurotoxin heavy chain, a botulinum neurotoxin serotype B neurotoxin heavy chain, a botulinum neurotoxin serotype C 1  neurotoxin heavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, a botulinum neurotoxin serotype E neurotoxin heavy chain, a botulinum neurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxin serotype G neurotoxin heavy chain. 
     
     
         13 . The polypeptide of  claim 9 , wherein said botulinum neurotoxin heavy chain translocation element is a botulinum neurotoxin serotype A neurotoxin heavy chain, a botulinum neurotoxin serotype B neurotoxin heavy chain, a botulinum neurotoxin serotype C 1  neurotoxin heavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, a botulinum neurotoxin serotype E neurotoxin heavy chain, a botulinum neurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxin serotype G neurotoxin heavy chain. 
     
     
         14 . The polypeptide of  claim 9 , wherein said botulinum neurotoxin light chain therapeutic element is a botulinum neurotoxin serotype A neurotoxin light chain, a botulinum neurotoxin serotype B neurotoxin light chain, a botulinum neurotoxin serotype C1 neurotoxin light chain, a botulinum neurotoxin serotype D neurotoxin light chain, a botulinum neurotoxin serotype E neurotoxin light chain, a botulinum neurotoxin serotype F neurotoxin light chain and a botulinum neurotoxin serotype G neurotoxin light chain. 
     
     
         15 . The polypeptide of  claim 9 , wherein said protease cleavage site is SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 23. 
     
     
         16 . A composition comprising an active di-chain form of a cleavable single-chain polypeptide as defined in  claim 9 .