Novel agent for controlling cell activity
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 Translation 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 protein conjugate, comprising three Domains B, T and E covalently linked together, wherein:
Domain B is a binding Domain which binds the conjugate to a Binding Site on the cell which undergoes endocytosis to be incorporated into an endosome;
Domain T is a Translocation Domain which translocates Domain E (with or without the other domains of the agent and/or the Binding Site) from within the endosome across the endosomal membrane into the cytosol of the cell; and
Domain E is the Effector Domain comprising a domain or domain fragment of the Light chain of Clostridial neurotoxin having Zn 2 + dependent metalloprotease activity;
with the proviso that said conjugate is not capable of ADP-ribosylation of a G-protein, and with the proviso that said conjugate does not possess adenylate cyclase activity.
2 . A protein conjugate according to claim 1 in which Domain T is a domain or domain fragment of Clostridial neurotoxin Heavy Chain.
3 . A protein conjugate according to claim 1 in which Domain E is obtained from botulinum neurotoxin.
4 . A protein conjugate according to claim 1 , wherein the three Domains B, T and E linked are together by way of at least one covalently linked spacer molecule.
5 . A protein conjugate according to claim 1 in which Domain B comprises a ligand to a cell surface acceptor on the target cell capable of undergoing endocytosis to be incorporated into an endosome.
6 . A protein conjugate according to claim 1 in which Domain B comprises a ligand to a cell surface receptor on the target cell capable of undergoing endocytosis to be incorporated into an endosome.
7 . A protein conjugate according to claim 1 in which Domain B comprises a monoclonal antibody or is derived from a monoclonal antibody to a cell surface antigen on the target cell capable of undergoing endocytosis to be incorporated into an endosome.
8 . A protein conjugate according to claim 1 in which Domain B, the Binding Domain, binds to a Binding Site which is characteristic of a particular cell type.
9 . A protein conjugate according to claim 1 which affects the rate of glucose uptake by adipose cells in response to insulin in which Domain B is a ligand to a binding site on adipose cells capable of undergoing endocytosis to be incorporated into an endosome.
10 . A protein conjugate according to claim 1 which affects the responsiveness of neutrophils to complement fragment C3b in which Domain B is a ligand to a binding site on neutrophils capable of undergoing endocytosis to be incorporated into an endosome.
11 . A protein conjugate according to claim 1 which affects the expression by neutrophils of the adhesion molecule Mac-1 in which Domain B is a ligand to a binding site on neutrophils capable of undergoing endocytosis to be incorporated into an endosome.
12 . A protein conjugate according to claim 1 which affects the presentation of antigen by antigen-presenting cells in which domain B is a ligand to a binding site on antigen presenting cells capable of undergoing endocytosis to be incorporated into an endosome.
13 . A protein conjugate according to claim 1 in which domain B is a ligand to the insulin like growth factor II receptor.
14 . A protein conjugate according to claim 1 in which Domain B is a monoclonal antibody or is derived from a monoclonal antibody to the adhesion molecule p150, 95.
15 . A protein conjugate according to claim 1 in which Domain B is a monoclonal antibody or is derived from a monoclonal antibody to a surface antigen on antigen presenting cells capable of undergoing endocytosis to be incorporated into an endosome.
16 . A protein conjugate according to claim 1 in which Domain B is insulin like growth factor II.
17 . A protein conjugate according to claim 1 in which Domain B is, or is derived from, the monoclonal antibody SHCL3.
18 . A protein conjugate according to claim 1 in which Domain B is, or is derived from, monoclonal antibody LL2.
19 . A protein conjugate according to claim 1 , wherein said conjugate does not possess ricin B chain or wheat germ agglutinin.
20 . A protein conjugate according to claim 1 , wherein said conjugate is in the form of a fusion protein.
21 . A nucleic acid encoding the protein conjugate according to claim 20 .
22 . A method of obtaining the protein conjugate according to claim 1 which comprises constructing a genetic construct according to claim 21 , incorporating said construct into a host organism and expressing the construct to produce the agent.