Claims for Patent: 8,372,615

Summary for Patent: 8,372,615

Title:	Fusion proteins
Abstract:	The invention provides a single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a target cell; a Targeting Moiety that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of undergoing endocytosis to be incorporated into an endocome within the target cell; a protease cleaving site at which site the fusion protein is cleavable by the protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and the translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the target cell.
Inventor(s):	Foster; Keith Alan (Abingdon, GB), Chaddock; John (Abingdon, GB), Marks; Philip (Abingdon, GB), Stancombe; Patrick (Abingdon, GB), Durose; Lyndsey (Abingdon, GB)
Assignee:	Syntaxin, Limited (Abingdon, Oxfordshire, GB)
Application Number:	13/354,787
Patent Claims:	1. A single chain, polypeptide fusion protein, comprising: a) a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment cleaves a protein of the exocytic fusion apparatus of a target cell; b) a Targeting Moiety that binds to a Binding Site on the target cell, which Binding Site undergoes endocytosis to be incorporated into an endosome within the target cell, wherein the Targeting Moiety is a vasoactive intestinal peptide analog or vasoactive intestinal peptide agonist; c) a protease cleavage site at which site the fusion protein is cleaved by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and d) a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the target cell; wherein the Targeting Moiety is located between the protease cleavage site and the translocation domain. 2. The fusion protein according to claim 1, wherein the Targeting Moiety and the protease cleavage site are separated by at most 10 amino acid residues, by at most 5 amino acid residues, or by zero amino acid residues. 3. The fusion protein according to claim 1, wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain. 4. The fusion protein according to claim 1, wherein the translocation domain is the H.sub.N domain of a clostridial neurotoxin. 5. The fusion protein according to claim 1, wherein the Targeting Moiety comprises at most 50 amino acid residues, at most 40 amino acid residues, or at most 20 amino acid residues. 6. The fusion protein according to claim 1, wherein the Targeting Moiety comprises a ligand that binds to PTH-1, or a PTH peptide. 7. The fusion protein according to claim 1, wherein the fusion protein comprises one or more purification tags. 8. The fusion protein according to claim 7, wherein the one or more purification tags are present at the N-terminal and/or C-terminal end of the fusion protein. 9. The fusion protein according to claim 8, wherein the one or more purification tags are joined to the fusion protein by a peptide spacer molecule. 10. The fusion protein according to claim 7, wherein the one or more purification tags are joined to the fusion protein by a peptide spacer molecule. 11. The fusion protein according to claim 1, wherein the translocation domain is separated from the Targeting Moiety by a peptide spacer molecule. 12. A polypeptide fusion protein comprising a polypeptide sequence selected from the group consisting of SEQ ID NOs: 12, 30, and 33. 13. A nucleic acid encoding the polypeptide fusion protein of claim 1. 14. The nucleic acid of claim 13, wherein the nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 8, 11, 29 and 32. 15. A DNA vector, which comprises a promoter, the nucleic acid of claim 13, and a terminator, wherein said nucleic acid sequence is located downstream of the promoter, and said terminatoris located downstream of the nucleic acid. 16. A nucleic acid which is complementary to the nucleic acid of claim 13. 17. A method for preparing a single-chain polypeptide fusion protein, comprising expressing the nucleic acid sequence of claim 13 in a host cell. 18. A method of preparing a di-chain fusion protein, comprising: a) contacting the single-chain polypeptide fusion protein of claim 1 with a protease capable of cleaving the protease cleavage site; b) cleaving the protease cleavage site; and thereby forming the di-chain fusion protein. 19. A di-chain fusion protein obtained by the method of claim 18, wherein the di-chain fusion protein comprises a first chain and a second chain, and wherein a) the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment cleaves a protein of the exocytic fusion apparatus of a target cell; and, b) the second chain comprises the Targeting Moiety and the translocation domain, wherein the translocation domain translocates the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the target cell; and the first and second chains are disulphide linked together. 20. A composition comprising a fusion protein according to claim 1. 21. The fusion protein according to claim 1, wherein the Targeting Moiety binds to a cell selected from the group consisting of: a mucus secreting cell; a neuronal cell controlling or directing mucus secretion; an endocrine cell; and an exocrine cell. 22. The fusion protein according to claim 1, wherein the Targeting Moiety comprises a ligand selected from the group consisting of: a vasoactive intestinal peptide; a pituitary adenyl cyclase activating peptide; calcitonin gene related peptide; a parathyroid hormone peptide; a corticotrophin releasing hormone peptide; and a gastrin releasing peptide. 23. The fusion protein of claim 1, wherein the protease cleavage site is cleaved by a protease selected from the group consisting of enterokinasae, Factor X, TEV (Tobacco Etch Virus), Thrombin and PreScission. 24. The fusion protein of claim 1, wherein the protease cleavage site is integrated at a position within the fusion protein such that cleavage of said integrated protease cleavage site converts the single-chain fusion protein into a di-chain polypeptide in which the Targeting Moiety has a free amino-terminus that interacts directly with the Binding Site.

Details for Patent 8,372,615

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2024-12-01