Clostridial toxin derivatives able to modify peripheral sensory afferent functions

Abstract

A novel agent for the targeted control of a mammalian cell activity can be 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 from within the endosome across the endosomal membrane into the cytosol of the cell; and 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 non-cytotoxic agent that binds to a primary sensory afferent neuron, which comprises:
 a Targeting Moiety (TM) coupled to a modified clostridial neurotoxin in which the TM comprises a ligand to a cell-surface binding site present on a primary sensory afferent neuron, and is capable of functionally interacting with a binding site causing a physical association between the agent and the surface of a primary sensory afferent neuron;   and the heavy chain (H-chain) of the clostridial neurotoxin is removed or io modified by chemical derivatisation, mutation or proteolysis to reduce or remove its native binding affinity for motor neurons;   and the light chain (L-chain) of the clostridial neurotoxin or a fragment thereof retains a protease activity specific for components of the neurosecretory machinery;   the TM and the modified H-chain, if present, forming a molecule that introduces the L-chain or fragment thereof into the cytosol of a primary sensory afferent neuron, thereby inhibiting the transmission of signals between a primary sensory afferent neuron and a projection neuron by controlling the release of at least one neurotransmitter or neuromodulator from the primary sensory afferent neuron.   
     
     
         2 . An agent according to  claim 1 , which comprises a Targeting Moiety (TM) coupled to a clostridial neurotoxin in which the H C  part of the H-chain is removed or modified. 
     
     
         3 . An agent according to  claim 1  in which the modified H-chain is the H N -fragment of a clostridial neurotoxin. 
     
     
         4 . An agent according to  claim 1  in which the clostridial neurotoxin component is obtained from botulinum neurotoxin. 
     
     
         5 . An agent according to  claim 4  in which the clostridial neurotoxin component is obtained from botulinum neurotoxin selected from the group consisting of botulinum neurotoxin type A, botulinum neurotoxin type B, and botulinum neurotoxin type C. 
     
     
         6 . An agent according to  claim 1  in which the H-chain is obtained from a different clostridial neurotoxin from that from which the L-chain is obtained. 
     
     
         7 . An agent according to  claim 1  in which the TM binds to a binding site that is characteristic of a particular defined population of primary sensory afferent neurons. 
     
     
         8 . An agent according to  claim 1  in which said cell-surface binding site present on a primary sensory afferent is selected from the group consisting of a growth factor receptor, a neuropeptide receptor, a cytokine receptor, and a hormone receptor. 
     
     
         9 . An agent according to  claim 1  in which the TM comprises a monoclonal antibody or is derived from a monoclonal antibody to a surface antigen on a nociceptive afferent neuron. 
     
     
         10 . An agent according to  claim 1 , which binds specifically to a primary sensory afferent neuron. 
     
     
         11 . An agent according to  claim 1 , wherein the TM is a growth factor. 
     
     
         12 . An agent according to  claim 1 , wherein the TM is a cytokine. 
     
     
         13 . An agent according to  claim 1 , wherein the TM is an endorphin or endorphin receptor antibody. 
     
     
         14 . An agent according to  claim 1 , wherein the TM is bradykinin or a bradykinin receptor antibody. 
     
     
         15 . An agent according to  claim 1 , wherein the TM is an enkephalin or enkephalin receptor antibody. 
     
     
         16 . A method for obtaining an agent according to  claim 1 , which comprises constructing a genetic construct that codes for a modified clostridial neurotoxin or a fragment of a clostridial neurotoxin, incorporating said construct into a host organism, expressing the construct to produce the modified clostridial neurotoxin or fragment of a clostridial neurotoxin, and covalently attaching said clostridial neurotoxin or fragment thereof to a TM. 
     
     
         17 . A method of preventing or alleviating pain, which comprises administering an effective dose of the agent according to  claim 1 . 
     
     
         18 . A method according to  claim 17 , wherein said agent is injected locally. 
     
     
         19 . A method according to  claim 17 , wherein said agent is injected spinally at he level of the spinal segment involved in the innervation of an affected organ. 
     
     
         20 . A method for preparing an agent according to  claim 1  in the form of a fusion protein, said method comprising expressing in a host organism a genetic construct which codes for the agent. 
     
     
         21 . A method according to  claim 20 , which further comprises constructing the genetic construct and transforming the host with said construct. 
     
     
         22 . A method according to  claim 20 , wherein the nucleic acid sequence of the genetic construct is modified in accordance with the codon bias of the host cell. 
     
     
         23 . A method according to  claim 20 , wherein the genetic construct incorporates a nucleic acid sequence encoding an affinity tag to facilitate purification of the assembled toxin. 
     
     
         24 . An agent that has been obtained in the form of a fusion protein by the method according to  claim 20 .