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 stabilized form of α-galactosidase comprising at least two native α-galactosidase monomers covalently linked to one another via a linking moiety, wherein said α-galactosidase is a human α-galactosidase.
2. The stabilized form of α-galactosidase of claim 1 comprising two native α-galactosidase monomers covalently linked to one another via a linking moiety.
3. The stabilized form of α-galactosidase of claim 1 , wherein the linking moiety is a non-peptidic moiety.
4. The stabilized form of α-galactosidase of claim 3 , wherein the linking moiety comprises poly(alkylene glycol) and at least two functional groups, wherein each functional group forms a covalent bond with one of the native α-galactosidase monomers.
5. The stabilized form of α-galactosidase of claim 4 , wherein the poly(alkylene glycol) comprises ethylene glycol and/or propylene glycol units linked together.
6. The stabilized form of α-galactosidase of claim 4 , wherein the linking moiety has the following 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 5 to 150; 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.
7. The stabilized form of α-galactosidase of claim 6 , wherein at least one of said functional groups forms an amide bond with an α-galactosidase monomer.
8. The stabilized form of α-galactosidase of claim 6 , wherein said linking moiety is at least 20 atoms long.
9. The stabilized form of α-galactosidase of claim 6 , wherein n is at least 25.
10. The stabilized form of α-galactosidase of claim 6 , wherein n is an integer from 40 to 70.
11. The stabilized form of α-galactosidase of claim 6 , wherein each of R 1 , R 2 , R 3 , R 4 and R 5 is independently selected from the group consisting of hydrogen and oxo.
12. The stabilized form of α-galactosidase of claim 1 , featuring a characteristic selected from the group consisting of:
(a) an α-galactosidase activity upon subjecting the stabilized form of α-galactosidase to human plasma conditions for one hour which is at least 10% higher than an activity of naturally occurring α-galactosidase upon subjecting said naturally occurring α-galactosidase to said human plasma conditions for one hour;
(b) an α-galactosidase activity which decreases upon subjecting the stabilized form of α-galactosidase to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of naturally occurring α-galactosidase decreases upon subjecting said naturally occurring 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 stabilized form of α-galactosidase to human plasma conditions for one hour;
(d) an α-galactosidase activity upon subjecting the stabilized form of α-galactosidase to lysosomal conditions for one week, which is at least 10% higher than an activity of naturally occurring α-galactosidase upon subjecting said naturally occurring α-galactosidase to said lysosomal conditions for one week;
(e) an α-galactosidase activity which decreases upon subjecting the stabilized form of α-galactosidase to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of naturally occurring α-galactosidase decreases upon subjecting said naturally occurring 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 stabilized form of α-galactosidase to lysosomal conditions for one day;
(g) an α-galactosidase activity immediately upon subjecting the stabilized form of α-galactosidase to lysosomal conditions, which is at least 10% higher than an activity of naturally occurring α-galactosidase immediately upon subjecting said naturally occurring α-galactosidase to said lysosomal conditions;
(h) an α-galactosidase activity immediately upon subjecting the stabilized form of α-galactosidase 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 naturally occurring α-galactosidase immediately upon subjecting said naturally occurring 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 naturally occurring α-galactosidase.
13. The stabilized form of α-galactosidase of claim 1 , wherein said human α-galactosidase is selected from the group consisting of agalsidase alpha and agalsidase beta.
14. The stabilized form of α-galactosidase of claim 1 , wherein said α-galactosidase is a plant recombinant α-galactosidase.
15. A pharmaceutical composition comprising the stabilized form of α-galactosidase of claim 1 and a pharmaceutically acceptable carrier.
16. A method of treating Fabry disease in a subject in need thereof, said method comprising administering to said subject the stabilized form of α-galactosidase of claim 1 , such that said Fabry disease in said subject is treated.
17. The method of claim 16 , wherein said administering is by an intravenous infusion.
18. A process of preparing the stabilized form of α-galactosidase of claim 1 , said process comprising reacting α-galactosidase comprising at least two native α-galactosidase monomers with a cross-linking agent, wherein said cross-linking agent comprises a linking moiety and at least two reactive groups.
19. The process of claim 18 , wherein said reactive groups comprise a leaving group.
20. The stabilized form of α-galactosidase of claim 1 , wherein said human α-galactosidase comprises an amino acid sequence having at least 99% sequence homology to an amino acid sequence of an α-galactosidase naturally occurring in humans.