Claims for Patent: 9,194,011

Summary for Patent: 9,194,011

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 (Givataim, IL), Azulay; Yaniv (Akko, IL), Shaaltiel; Yoseph (Kibbutz HaSolelim, IL), Kizhner; Tali (Yishuv Atzmon-Segev, IL)
Assignee:	Protalix Ltd. (Carmiel, IL)
Application Number:	13/582,482
Patent Claims:	1. A multimeric protein structure comprising two .alpha.-galactosidase monomers, the protein structure being a dimeric protein structure, said two .alpha.-galactosidase 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 upon subjecting said native .alpha.-galactosidase 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 decreases upon subjecting said native .alpha.-galactosidase 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 upon subjecting said native .alpha.-galactosidase 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 decreases upon subjecting said native .alpha.-galactosidase 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 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 immediately upon subjecting said native .alpha.-galactosidase 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 by at least 20% than said circulating half-life of said native .alpha.-galactosidase, wherein said .alpha.-galactosidase has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. 2. The multimeric protein structure of claim 1, wherein said .alpha.-galactosidase activity of said multimeric protein structure 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, further remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to lysosomal conditions for one week. 3. The multimeric protein structure of claim 1, characterized by an .alpha.-galactosidase activity in an organ upon administration of said multimeric protein structure to a vertebrate, said organ being selected from the group consisting of a spleen, a heart and a kidney. 4. The multimeric protein structure of claim 1, wherein said .alpha.-galactosidase is a plant recombinant .alpha.-galactosidase. 5. The multimeric protein structure of claim 1, wherein said linking moiety comprises 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 1, wherein said at least one linking moiety has a general formula: --X.sub.1--(CR.sub.1R.sub.2--CR.sub.3R.sub.4--Y)n-X.sub.2-- wherein each of X.sub.1 and X.sub.2 is a functional group that forms a covalent bond with at least one .alpha.-galactosidase monomer; C is a carbon atom; Y is an oxygen atom, a sulfur atom or NR.sub.5, wherein NR.sub.5 is a nitrogen atom attached to R.sub.5; n is an integer from 1 to 200; and each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol and thioalkoxy. 9. A multimeric protein structure comprising two .alpha.-galactosidase monomers, the protein structure being a dimeric protein structure, said two .alpha.-galactosidase monomers being covalently linked to one another via a linking moiety, and said .alpha.-galactosidase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. 10. The multimeric protein structure of claim 9, 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 upon subjecting said native .alpha.-galactosidase 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 decreases upon subjecting said native .alpha.-galactosidase 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 upon subjecting said native .alpha.-galactosidase 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 decreases upon subjecting said native .alpha.-galactosidase 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 immediately upon subjecting said native .alpha.-galactosidase 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 immediately upon subjecting said native .alpha.-galactosidase 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. 11. The multimeric protein structure of claim 10, wherein said .alpha.-galactosidase activity of said multimeric protein structure 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, further remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to lysosomal conditions for one week. 12. The multimeric protein structure of claim 10, wherein said circulating half-life of the multimeric protein structure which is higher than a circulating half-life of said native .alpha.-galactosidase, is higher by at least 20% than said circulating half-life of said native .alpha.-galactosidase. 13. The multimeric protein structure of claim 9, characterized by an .alpha.-galactosidase activity in an organ upon administration of said multimeric protein structure to a vertebrate, said organ being selected from the group consisting of a spleen, a heart and a kidney. 14. The multimeric protein structure of claim 9, wherein said .alpha.-galactosidase is a human .alpha.-galactosidase selected from the group consisting of agalsidase alpha and agalsidase beta. 15. The multimeric protein structure of claim 9, wherein said .alpha.-galactosidase is a plant recombinant .alpha.-galactosidase. 16. The multimeric protein structure of claim 9, wherein said .alpha.-galactosidase has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. 17. The multimeric protein structure of claim 9, wherein said linking moiety comprises a poly(alkylene glycol). 18. The multimeric protein structure of claim 17, 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. 19. The multimeric protein structure of claim 18, wherein said at least two functional groups are terminal groups of said poly(alkylene glycol). 20. The multimeric protein structure of claim 9, wherein said at least one linking moiety has a general formula: --X.sub.1--(CR.sub.1R.sub.2--CR.sub.3R.sub.4--Y)n-X.sub.2-- wherein each of X.sub.1 and X.sub.2 is a functional group that forms a covalent bond with at least one .alpha.-galactosidase monomer; C is a carbon atom; Y is an oxygen atom, a sulfur atom or NR.sub.5, wherein NR.sub.5 is a nitrogen atom attached to R.sub.5; n is an integer from 1 to 200; and each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol and thioalkoxy. 21. A pharmaceutical composition comprising the multimeric protein structure of claim 1 and a pharmaceutically acceptable carrier. 22. The pharmaceutical composition of claim 21, further comprising, as an additional ingredient, a galactose. 23. A pharmaceutical composition comprising the multimeric protein structure of claim 9 and a pharmaceutically acceptable carrier. 24. The pharmaceutical composition of claim 23, further comprising, as an additional ingredient, a galactose. 25. 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 1, thereby treating the Fabry disease. 26. 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 9, thereby treating the Fabry disease. 27. A process of preparing the multimeric protein structure of claim 1, the process comprising reacting .alpha.-galactosidase with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 28. The process of claim 27, comprising reacting dimeric .alpha.-galactosidase with said cross-linking agent. 29. The process of claim 27, wherein said reactive groups comprise a leaving group. 30. The process of claim 27, wherein said reactive group reacts with an amine group to form an amide bond. 31. The process of claim 27, wherein each of said reactive groups is capable of forming a covalent bond between said linking moiety and at least one .alpha.-galactosidase monomer. 32. The process of claim 27, wherein a molar ratio of said cross-linking agent to monomers of said .alpha.-galactosidase is in a range of from 5:1 to 500:1. 33. A process of preparing the multimeric protein structure of claim 9, the process comprising reacting .alpha.-galactosidase with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 34. The multimeric protein structure of claim 1, wherein said .alpha.-galactosidase is a recombinant .alpha.-galactosidase. 35. The multimeric protein structure of claim 1, wherein said linking moiety is at least 20 atoms long. 36. The multimeric protein structure of claim 8, wherein n is at least 25. 37. The multimeric protein structure of claim 9, wherein said .alpha.-galactosidase is a recombinant .alpha.-galactosidase. 38. The multimeric protein structure of claim 9, wherein said linking moiety is at least 20 atoms long. 39. The multimeric protein structure of claim 20, wherein n is at least 25. 40. The multimeric protein structure of claim 20, wherein said .alpha.-galactosidase has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, each of said functional groups forms an amide bond with an .alpha.-galactosidase monomer, and n is an integer from 40 to 70. 41. The multimeric protein structure of claim 16, wherein said linking moiety has the formula: ##STR00002## wherein a molecular weight of polyethylene glycol in said linking moiety is 2 kDa, and the terminal groups of said linking moiety each form an amide bond with an .alpha.-galactosidase monomer.

Details for Patent 9,194,011

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