Claims for Patent: 10,280,414

Summary for Patent: 10,280,414

Title:	Stabilized .alpha.-galactosidase and uses thereof
Abstract:	Multimeric protein structures comprising at least two alpha-galactosidase monomers being covalently linked to one another via a linking moiety are disclosed herein, as well a process for preparing same, and methods of treating Fabry disease via administration of a multimeric protein structure. The disclosed multimeric protein structures exhibit an improved performance, in terms of enhanced activity and/or a longer lasting activity under both lysosomal conditions and in a serum environment.
Inventor(s):	Shulman; Avidor (Rakefet, IL), Ruderfer; Ilya (Carmiel, IL), Ben-Moshe; Tehila (Koranit, IL), Shekhter; Talia (Petach-Tikva, IL), Azulay; Yaniv (Akko, IL), Kizhner; Tali (Atzmon-Segev, IL), Shaaltiel; Yoseph (Timrat, IL)
Assignee:	Protalix Ltd. (Carmiel, IL)
Application Number:	15/636,753
Patent Claims:	1. A multimeric protein structure comprising at least two .alpha.-galactosidase A monomers being covalently linked to one another via a linking moiety, the multimeric protein structure featuring a characteristic selected from the group consisting of: (a) an .alpha.-galactosidase activity upon subjecting the multimeric protein structure to human plasma conditions for one hour, which is at least 10% higher than an activity of native .alpha.-galactosidase A upon subjecting said native .alpha.-galactosidase A to said human plasma conditions for one hour; (b) an .alpha.-galactosidase activity which decreases upon subjecting the multimeric protein structure to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of said native .alpha.-galactosidase A decreases upon subjecting said native .alpha.-galactosidase A to said human plasma conditions for one hour; (c) an .alpha.-galactosidase activity which remains substantially unchanged upon subjecting the multimeric protein structure to human plasma conditions for one hour; (d) an .alpha.-galactosidase activity, upon subjecting the multimeric protein structure to lysosomal conditions for one week, which is at least 10% higher than an activity of native .alpha.-galactosidase A upon subjecting said native .alpha.-galactosidase A to said lysosomal conditions for one week; (e) an .alpha.-galactosidase activity which decreases upon subjecting the multimeric protein structure to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of said native .alpha.-galactosidase A decreases upon subjecting said native .alpha.-galactosidase A to said lysosomal conditions for one day; (f) an .alpha.-galactosidase activity which remains substantially unchanged upon subjecting the multimeric protein structure to lysosomal conditions for one day; (g) an .alpha.-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native .alpha.-galactosidase A immediately upon subjecting said native form of said protein to said lysosomal conditions; (h) an .alpha.-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37.degree. C., which is at least 10% higher than an activity of native .alpha.-galactosidase A immediately upon subjecting said native .alpha.-galactosidase A to said aqueous solution having a pH of 7 and a temperature of 37.degree. C.; and (i) a circulating half-life in a physiological system which is higher by at least 20% than said circulating half-life of said native .alpha.-galactosidase A. 2. A process of preparing the multimeric protein structure of claim 1, the process comprising reacting .alpha.-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 3. A multimeric protein structure comprising at least two .alpha.-galactosidase A monomers being covalently linked to one another via a linking moiety, wherein said linking moiety is not present in native .alpha.-galactosidase A. 4. A process of preparing the multimeric protein structure of claim 3, the process comprising reacting .alpha.-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 5. A multimeric protein structure comprising two .alpha.-galactosidase monomers, said at least two .alpha.-galactosidase monomers being .alpha.-galactosidase A monomers, the protein structure being a dimeric protein structure, said two .alpha.-galactosidase monomers being covalently linked to one another via a linking moiety comprising a poly(alkylene glycol). 6. The multimeric protein structure of claim 5, wherein said poly(alkylene glycol) comprises at least two functional groups, each functional group forming a covalent bond with one of the .alpha.-galactosidase monomers. 7. The multimeric protein structure of claim 6, wherein said at least two functional groups are terminal groups of said poly(alkylene glycol). 8. The multimeric protein structure of claim 6, wherein at least one of said functional groups forms an amide bond with an .alpha.-galactosidase monomer. 9. The multimeric protein structure of claim 5, wherein said poly(alkylene glycol) comprises at least 5 alkylene glycol units. 10. The multimeric protein structure of claim 5, wherein said poly(alkylene glycol) is poly(ethylene glycol). 11. The multimeric protein structure of claim 5, featuring a characteristic selected from the group consisting of: (a) an .alpha.-galactosidase activity, upon subjecting the multimeric protein structure to human plasma conditions for one hour, which is at least 10% higher than an activity of native .alpha.-galactosidase A upon subjecting said native .alpha.-galactosidase A to said human plasma conditions for one hour; (b) an .alpha.-galactosidase activity which decreases upon subjecting the multimeric protein structure to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of said native .alpha.-galactosidase A decreases upon subjecting said native .alpha.-galactosidase A to said human plasma conditions for one hour; (c) an .alpha.-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to human plasma conditions for one hour; (d) an .alpha.-galactosidase activity, upon subjecting the multimeric protein structure to lysosomal conditions for one week, which is at least 10% higher than an activity of native .alpha.-galactosidase A upon subjecting said native .alpha.-galactosidase A to said lysosomal conditions for one week; (e) an .alpha.-galactosidase activity which decreases upon subjecting the multimeric protein structure to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of said native .alpha.-galactosidase A decreases upon subjecting said native .alpha.-galactosidase A to said lysosomal conditions for one day; (f) an .alpha.-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to lysosomal conditions for one day; (g) an .alpha.-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native .alpha.-galactosidase A immediately upon subjecting said native .alpha.-galactosidase A to said lysosomal conditions; (h) an .alpha.-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37.degree. C., which is at least 10% higher than an activity of native .alpha.-galactosidase A immediately upon subjecting said native .alpha.-galactosidase A to said aqueous solution having a pH of 7 and a temperature of 37.degree. C.; and (i) a circulating half-life in human plasma which is higher than a circulating half-life of said native .alpha.-galactosidase A. 12. The multimeric protein structure of claim 5, wherein said .alpha.-galactosidase is a human .alpha.-galactosidase selected from the group consisting of agalsidase alpha and agalsidase beta. 13. The multimeric protein structure of claim 5, wherein said .alpha.-galactosidase is a plant recombinant .alpha.-galactosidase. 14. The multimeric protein structure of claim 5, wherein said .alpha.-galactosidase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. 15. The multimeric protein structure of claim 5, wherein said .alpha.-galactosidase has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 15. 16. A pharmaceutical composition comprising the multimeric protein structure of claim 5 and a pharmaceutically acceptable carrier. 17. A method of treating Fabry disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the multimeric protein structure of claim 5, thereby treating the Fabry disease. 18. The method of claim 17, wherein said administering is effected by intravenous infusion. 19. A process of preparing the multimeric protein structure of claim 5, the process comprising reacting .alpha.-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 20. The process of claim 19, wherein said reactive groups comprise a leaving group.

Details for Patent 10,280,414

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genzyme Corporation	FABRAZYME	agalsidase beta	For Injection	103979	04/24/2003		2029-11-17
Genzyme Corporation	FABRAZYME	agalsidase beta	For Injection	103979	10/10/2003		2029-11-17