Clostridial toxin derivatives able to modify peripheral sensory afferent functions
View Patent ↗This invention describes a novel agent for the targeted control of a mammalian cell activity, in particular the agent is used to control the interaction of particular cell types with their external environment. The agent has applications as a pharmaceutical for the treatment of a variety of disorders. An agent according to the invention comprises three Domains B, T and E linked together in the following manner: Domain B-Domain T-Domain E where Domain B is the Binding Domain which binds the agent to a Binding Site on the cell which undergoes endocytosis to produce an endosome, Domain T is the Translocation Domain which translocates the agent (with or without the Binding Site) from within the endosome across the endosomal membrane into the cytosol of the cell, Domain E is the Effector Domain which inhibits the ability of the Recyclable Membrane Vesicles to transport the Integral Membrane Proteins to the surface of the cell.
1. A single chain polypeptide fusion protein comprising:
(a) a targeting moiety (TM) that binds the polypeptide to a nociceptive afferent neuron by binding of said TM directly to a nociceptive afferent neuron or by binding of said TM to a ligand or to ligands that bind to a nociceptive afferent neuron;
(b) a clostridial neurotoxin light chain (L-chain) or fragment thereof that is selected from a first clostridial neurotoxin serotype, wherein said L-chain or fragment thereof has protease activity specific for components of the neurosecretory machinery;
(c) a clostridial neurotoxin heavy chain (H-chain) or fragment thereof that is selected from a second clostridium neurotoxin serotype; and
(d) a site for cleavage by a proteolytic enzyme, wherein said cleavage site allows proteolytic cleavage between said L-chain or fragment thereof and H-chain or fragment thereof;
wherein said H-chain or fragment thereof introduces said L-chain or fragment thereof into the cytosol of a nociceptive afferent neuron; and
wherein said H-chain or fragment thereof lacks a functional C-terminal part of a clostridial neurotoxin heavy chain designated H C from one or both of said first or second clostridial neurotoxin serotypes.
2. A polypeptide according to claim 1 wherein the L-chain or fragment thereof is from botulinum neurotoxin.
3. A polypeptide according to claim 1 wherein the H-chain or fragment thereof is from botulinum neurotoxin.
4. A polypeptide according to claim 1 wherein said first and second serotypes are different.
5. A polypeptide according to claim 1 wherein said first and second serotypes are the same.
6. A polypeptide according to claim 1 , which prevents the release of a neurotransmitter or neuromodulator from a nociceptive afferent neuron.
7. A polypeptide according to claim 1 , wherein said L-chain or fragment thereof exhibits endopeptidase activity specific for a substrate selected from one or more of SNAP-25, synaptobrevin/VAMP and syntaxin.
8. A polypeptide according to claim 1 , wherein said cleavage site is not present in a native clostridial neurotoxin.
9. A polypeptide according to claim 1 , wherein said cleavage site allows proteolytic cleavage of said L-chain or fragment thereof and H-chain or fragment thereof, and when so cleaved said L-chain or fragment thereof exhibits greater protease activity specific for components of the neurosecretory machinery than does the polypeptide prior to said proteolytic cleavage.