Stabilized alpha-galactosidase and uses thereof
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.
1. A multimeric protein structure comprising two α-galactosidase monomers, the protein structure being a dimeric protein structure, said two α-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 α-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 α-galactosidase upon subjecting said native α-galactosidase to said human plasma conditions for one hour;
(b) an α-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 α-galactosidase decreases upon subjecting said native α-galactosidase to said human plasma conditions for one hour;
(c) an α-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 α-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 α-galactosidase upon subjecting said native α-galactosidase to said lysosomal conditions for one week;
(e) an α-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 α-galactosidase decreases upon subjecting said native α-galactosidase to said lysosomal conditions for one day;
(f) an α-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 α-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native α-galactosidase immediately upon subjecting said native form of said protein to said lysosomal conditions;
(h) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37° C., which is at least 10% higher than an activity of native α-galactosidase immediately upon subjecting said native α-galactosidase to said aqueous solution having a pH of 7 and a temperature of 37° 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 α-galactosidase,
wherein said α-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 α-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 α-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 α-galactosidase is a plant recombinant α-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 α-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 1 —(CR 1 R 2 —CR 3 R 4 —Y) n -X 2 —
wherein each of X 1 and X 2 is a functional group that forms a covalent bond with at least one α-galactosidase monomer;
C is a carbon atom;
Y is an oxygen atom, a sulfur atom or NR 5 , wherein NR 5 is a nitrogen atom attached to R 5 ;
n is an integer from 1 to 200; and
each of R 1 , R 2 , R 3 , R 4 and R 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 α-galactosidase monomers, the protein structure being a dimeric protein structure, said two α-galactosidase monomers being covalently linked to one another via a linking moiety, and said α-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 α-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 α-galactosidase upon subjecting said native α-galactosidase to said human plasma conditions for one hour;
(b) an α-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 α-galactosidase decreases upon subjecting said native α-galactosidase to said human plasma conditions for one hour;
(c) an α-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 α-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 α-galactosidase upon subjecting said native α-galactosidase to said lysosomal conditions for one week;
(e) an α-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 α-galactosidase decreases upon subjecting said native α-galactosidase to said lysosomal conditions for one day;
(f) an α-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 α-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native α-galactosidase immediately upon subjecting said native α-galactosidase to said lysosomal conditions;
(h) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37° C., which is at least 10% higher than an activity of native α-galactosidase immediately upon subjecting said native α-galactosidase to said aqueous solution having a pH of 7 and a temperature of 37° C.; and
(i) a circulating half-life in human plasma which is higher than a circulating half-life of said native α-galactosidase.
11. The multimeric protein structure of claim 10 , wherein said α-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 α-galactosidase, is higher by at least 20% than said circulating half-life of said native α-galactosidase.
13. The multimeric protein structure of claim 9 , characterized by an α-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 α-galactosidase is a human α-galactosidase selected from the group consisting of agalsidase alpha and agalsidase beta.
15. The multimeric protein structure of claim 9 , wherein said α-galactosidase is a plant recombinant α-galactosidase.
16. The multimeric protein structure of claim 9 , wherein said α-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 α-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 1 —(CR 1 R 2 —CR 3 R 4 —Y) n -X 2 —
wherein each of X 1 and X 2 is a functional group that forms a covalent bond with at least one α-galactosidase monomer;
C is a carbon atom;
Y is an oxygen atom, a sulfur atom or NR 5 , wherein NR 5 is a nitrogen atom attached to R 5 ;
n is an integer from 1 to 200; and
each of R 1 , R 2 , R 3 , R 4 and R 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 α-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 α-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 α-galactosidase monomer.
32. The process of claim 27 , wherein a molar ratio of said cross-linking agent to monomers of said α-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 α-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 α-galactosidase is a recombinant α-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 α-galactosidase is a recombinant α-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 α-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 α-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:
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 α-galactosidase monomer.