HUMAN ALPHA-GALACTOSIDASE VARIANTS
The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved thermostability, serum stability, improved cellular uptake, stability under both acidic (pH<4) and basic (pH>7) conditions, reduced immunogenicity, and improved globotriaosylceramide removal from cells. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.
1 . A recombinant alpha galactosidase A and/or biologically active recombinant alpha galactosidase A fragment comprising an amino acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 374, 704, and/or 1022.
2 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8, or a functional fragment thereof, and wherein said recombinant alpha galactosidase A comprises at least one substitution or substitution set at one or more positions selected from 2, 4, 5, 24/59, 24/143/144, 24/143/202/333, 24/143/202/352/390/391, 24/143/333/352/387/390/391, 24/143/390/391, 24/202, 24/202/271, 24/202/333/352, 24/271/352, 24/352/387/390/391, 24/387/391, 31, 40, 59, 59/143, 59/143/202, 59/143/202/271/333, 59/143/271, 59/143/333, 59/202, 59/202/333, 59/271/387/390, 73, 76, 80, 83, 84, 91/215/361, 122, 123, 143, 143/202, 143/271, 143/271/352/390, 143/333, 143/333/387/390, 143/387/391, 147, 155, 164, 165, 179, 186, 202, 202/333, 210, 215/218, 218, 218/361, 218/361/398, 218/398, 246, 254/398, 271, 271/333, 271/333/390/391, 271/333/391, 271/352/391, 273, 275, 277, 278, 280, 281, 283, 284, 287, 300, 303, 304, 325, 331, 332, 333/352, 333/390/391, 333/391, 334, 335, 336, 338, 339, 340, 341, 343, 359, 360, 361, 362, 367, 369, 371, 373, 375, 377, 382, 382/398, 385, 387/391, 390, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 704.
3 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8, or a functional fragment thereof, and wherein said recombinant alpha galactosidase A comprises at least one substitution or substitution set at one or more positions selected from 10, 39, 44, 47, 92, 166, 206, 217, 247, 261, 271, 302, 316, 322, 337, 368, and 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374.
4 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:58, or a functional fragment thereof, and wherein said recombinant alpha galactosidase A comprises at least one substitution or substitution set at one or more positions selected from 10, 10/392, 31, 31/39/44/166/302, 31/47, 31/283/284, 39, 39/44, 39/44/47, 39/44/47/261/283/284, 39/44/283, 39/44/339, 39/47/261, 39/92, 39/206, 39/284, 44, 44/284/302, 84, 84/92, 84/284/302/392, 84/316, 84/368/392, 92, 92/206/217, 92/206/275, 92/206/284, 92/206/302/368, 92/271, 92/271/277, 92/275/284, 92/283, 92/283/392, 92/284, 92/302, 92/316, 92/368, 155, 155/217, 155/368, 166, 166/283/284, 166/302, 206, 206/217, 206/334, 261, 261/283, 271, 271/368, 275, 283, 283/284, 283/392, 284, 302, 316, 334, 339, 368, 368/392, and 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022.
5 . The recombinant alpha galactosidase A of claim 1 , wherein said alpha galactosidase A comprises at least one mutation in at least one position as provided in Tables 11.1, 12.1, and/or 13.1.
6 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is derived from a human alpha galactosidase A.
7 . A recombinant alpha galactosidase A comprising the polypeptide sequence of SEQ ID NO: 374, 704, and/or 1022.
8 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more thermostable than the alpha galactosidase A of SEQ ID NO: 2, 374,704, and/or 1022.
9 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more stable at pH 7 than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
10 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more stable at pH 4 than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
11 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more stable to exposure to serum than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
12 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more lysosomally stable than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
13 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is more readily taken up by cells than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
14 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A depletes more globotriaosylceramide from cells than the alpha galactosidase A of SEQ ID NO: 2, 374, 704, and/or 1022.
15 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A is purified.
16 . The recombinant alpha galactosidase A of claim 1 , wherein said recombinant alpha galactosidase A exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7; iii) increased tolerance to pH 4; iv) increased tolerance to serum; v) increased uptake into cells; vi) increased depletion of globotriaosylceramide from cells; vii) reduced immunogenicity; or a combination of any of i), ii), iii), iv), v), vi), and/or vii), as compared to a reference sequence.
17 . The recombinant alpha galactosidase A of claim 16 , wherein said reference sequence is selected from SEQ ID NO: 374, 704, and/or 1022.
18 . A composition comprising at least one recombinant alpha galactosidase A of claim 1 .
19 . A recombinant polynucleotide sequence encoding at least one recombinant alpha galactosidase A set forth in claim 1 .
20 . The recombinant polynucleotide sequence of claim 19 , wherein said polynucleotide sequence is selected from DNA, RNA, and mRNA.
21 . The recombinant polynucleotide sequence of claim 20 , wherein said polynucleotide sequence is codon-optimized.
22 . An expression vector comprising the recombinant polynucleotide sequence of claim 19 .
23 . The expression vector of claim 22 , wherein said recombinant polynucleotide sequence is operably linked to one or more control sequences.
24 . The expression vector of claim 23 , wherein said control sequence is a promoter.
25 . The expression vector of claim 24 , wherein said promoter is a heterologous promoter.
26 . A host cell comprising the expression vector of claim 22 .
27 . The host cell of claim 26 , wherein said host cell is selected from eukaryotes and prokaryotes.
28 . The host cell of claim 26 , wherein said host cell is a mammalian cell.
29 . A method of producing an alpha galactosidase A variant, comprising culturing said host cell of claim 26 , under conditions that said alpha galactosidase A encoded by said recombinant polynucleotide is produced.
30 . The method of claim 29 , further comprising the step of recovering said alpha galactosidase A.
31 . The method of claim 30 , further comprising the step of purifying said alpha galactosidase A.
32 . A pharmaceutical composition for the treatment of Fabry disease, comprising the composition of claim 18 .
33 . The pharmaceutical composition of claim 32 , further comprising a pharmaceutically acceptable carrier and/or excipient.
34 . The pharmaceutical composition of claim 32 , wherein said composition is suitable for parenteral injection or infusion to a human.
35 . A pharmaceutical composition comprising the recombinant polynucleotide of claim 19 .
36 . A method for treating and/or preventing the symptoms of Fabry disease in a subject, comprising providing a subject having Fabry disease, and providing the pharmaceutical composition of claim 32 , to said subject.
37 . The method of claim 36 , wherein said symptoms of Fabry disease are ameliorated.
38 . The method of claim 36 , wherein said subject is able to eat a diet that is less restricted in its fat content than diets required by subjects exhibiting the symptoms of Fabry disease.
39 . The method of claim 36 , wherein said subject is an infant or child.
40 . The method of claim 36 , wherein said subject is an adult or young adult.