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US 9,849,163 B2Granted
Modified non-cytotoxic proteases
AssigneeIpsen Bioinnovation Limited
InventorsJohn Andrew Chaddock, Keith Alan Foster
Life of this patent
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Abstract
The present invention relates to a modified polypeptide comprising a non-cytotoxic protease, a translocation domain, a destructive protease cleavage site and a Targeting Moiety that binds to a Binding Site on a nerve cell, wherein after cleavage of the destructive cleavage site the polypeptide has reduced potency. The destructive cleavage site is recognized and cleaved by a protease present at or in an off-site target cell, and, in one embodiment, the polypeptide is a modified clostridial neurotoxin. The present invention also relates to the use of said polypeptides for treating a range of conditions, and to nucleic acids encoding said polypeptides.
Claims5
A polypeptide comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence selected from the group consisting of:
- SEQ ID NOs:
- 4, 6, 10, 11, 13, 15, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, and 39, wherein the amino acid sequence comprises, in N-C order:
- a non-cytotoxic protease that is capable of cleaving a SNARE protein, wherein the non-cytotoxic protease is the L-chain of a botulinum neurotoxin;
- a translocation domain that is capable of translocating the non-cytotoxic protease from within an endosome of a motor neuron of a neuromuscular junction, across the endosomal membrane thereof and into the cytosol of the motor neuron, wherein the translocation domain is the HN of a botulinum neurotoxin;
- and
- a targeting moiety that binds to a Binding Site on the motor neuron, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the motor neuron;
- wherein the amino acid sequence comprises a cleavage site that is cleavable by a second protease and not by the non-cytotoxic protease, wherein the cleavage site is located in the non-cytotoxic protease or translocation domain;
- and
- wherein, when the targeting moiety of the polypeptide binds to a Binding Site on the motor neuron, the translocation domain can translocate the non-cytotoxic protease into the cytosol of the motor neuron such that the non-cytotoxic protease can cleave the SNARE protein;
- and
- wherein after cleavage of the cleavage site by the second protease, the polypeptide has reduced potency as measured by a reduced ability to cleave said SNARE protein and/or a reduced ability to translocate said non-cytotoxic protease.
8 more elements
A polypeptide comprising:
- (a) a non-cytotoxic protease that is capable of cleaving a SNARE protein, the non-cytotoxic protease being selected from the group consisting of:
- the light chain of BoNT/A, the light chain of BoNT/B, the light chain of BoNT/C1, the light chain of BoNT/D, the light chain of BoNT/E, the light chain of BoNT/F, and the light chain of BoNT/G;
- b) a translocation domain that is capable of translocating the non-cytotoxic protease from within an endosome of a motor neuron of a neuromuscular junction, across the endosomal membrane thereof and into the cytosol of the motor neuron, the translocation domain being selected from the group consisting of:
- the translocation domain of BoNT/A, the translocation domain of BoNT/B, the translocation domain of BoNT/C1, the translocation domain of BoNT/D, the translocation domain of BoNT/E, the translocation domain of BoNT/F, and the translocation domain of BoNT/G;
- c) a cleavage site that is cleavable by a second protease and not by the non-cytotoxic protease, and
- d) a Targeting Moiety that binds to a Binding Site on the motor neuron, the Binding Site being capable of undergoing endocytosis to be incorporated into an endosome within the motor neuron;
- wherein:
- when the Targeting Moiety binds to a motor neuron of the neuromuscular junction, the translocation domain transports the non-cytotoxic protease into the cytosol of the motor neuron and inhibits the exocytic fusion process of the motor neuron;
- after cleavage of the polypeptide by the second protease, the polypeptide has reduced potency as measured by a reduced ability to cleave the SNARE protein and/or a reduced ability to translocate the non-cytotoxic protease;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/A, the cleavage site is inserted within the sequence of SEQ ID NO:
- 52, 64 or 71;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/B, the cleavage site is inserted within the sequence of SEQ ID NO:
- 53, 65 or 72;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/C1, the cleavage site is inserted within the sequence of SEQ ID NO.:
- 54, 66 or 73;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/D, the cleavage site is inserted within the sequence of SEQ ID NO.:
- 55, 67 or 73;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/E, the cleavage site is inserted within the sequence SEQ ID NO.:
- 56, 68 or 74;
- when the polypeptide comprises the light chain or the translocation domain of BoNT/F, the cleavage site is inserted within the sequence SEQ ID NO.:
- 57, 69 or 75;
- and
- when the polypeptide comprises the light chain or the translocation domain of BoNT/G, the cleavage site is inserted within the sequence SEQ ID NO.:
- 58, 70 or 76.
21 more elements
Dependencies are inferred from the claim text.