Engineered acid alpha-glucosidase variants

Patent metadata

Field	Value
Publication number	US-11970722-B2
Application number	US-202017126647-A
Country	US
Kind code	B2
Filing date	Dec 18, 2020
Priority date	Dec 20, 2019
Publication date	Apr 30, 2024
Grant date	Apr 30, 2024

Abstract

The present invention provides engineered acid alpha-glucosidase (GAA) polypeptides and compositions thereof. In some embodiments, the engineered GAA polypeptides have been optimized to provide increased expression, stability at neutral pH, and activity in cell lysates. The invention also provides methods for utilization of the compositions comprising the engineered GAA polypeptides for therapeutic and other purposes.

First claim

What is claimed is:

1. A recombinant acid alpha glucosidase comprising an amino acid sequence comprising at least at least 85% sequence identity to the sequence of residues 20 to 944 of SEQ ID NO: 2, wherein the amino acid sequence comprises at least a substitution S135Q/P, V913G/H/L/Q/R/W/E, P39Q/D/H, Q50V, or T266N, or a substitution at amino acid position 150, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: 2.

2. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution S135Q/P.

3. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution T150G/S.

4. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises residues 20-944 of SEQ ID NO: 6, 8, 12, 14, 16, 18, 20, 946, 1956, 2496, 2880, or 3104.

5. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is more thermostable than the acid alpha glucosidase of SEQ ID NO: 2.

6. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is more stable at pH 7 than the acid alpha glucosidase of SEQ ID NO: 2.

7. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is more stable at pH 4 than the acid alpha glucosidase of SEQ ID NO: 2.

8. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase exhibits increased expression than the acid alpha glucosidase of SEQ ID NO: 2.

9. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is more lysosomally stable than the acid alpha glucosidase of SEQ ID NO: 2.

10. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is more readily taken up by cells than the acid alpha glucosidase of SEQ ID NO: 2.

11. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase exhibits greater enzymatic activity in cell lysates than the acid alpha glucosidase of SEQ ID NO: 2.

12. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase exhibits reduced immunogenicity, as compared to the acid alpha glucosidase of SEQ ID NO: 2.

13. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase is purified.

14. The recombinant acid alpha glucosidase of claim 1 , wherein said recombinant acid alpha glucosidase 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 expression; v) increased uptake into cells; vi) increased enzymatic activity in cell lysates; vii) reduced immunogenicity; or a combination of any of i), ii), iii), iv), v), v), and/or vii), as compared to a reference acid alpha glucosidase of SEQ ID NO: 2.

15. A composition comprising at least one recombinant acid alpha glucosidase of claim 1 .

16. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution V913G/H/L/Q/R/W/E.

17. The recombinant acid alpha glucosidase of claim 16 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution the substitution V913Q/R/W/E.

18. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution P39Q/D/H.

19. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution Q50V.

20. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution T266N.

21. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises at least the substitution S135Q, T150S, V913R, P39Q/D/H, Q50V, or T266N, or combinations thereof.

22. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises residues 20-944 of SEQ ID NO: 8, 14, 20, 136, 138, 150, 278, 286, 290, 302, 304, 306, 310, 314, 316, 318, 320, 322, 324, 328, 332, 334, 336, 338, 340, 344, 346, 348, 350, 352, 354, 356, 358, 350, 362, 364, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 394, 396, 398, 400, 402, 406, 408, 410, 478, 480, 486, 488, 490, 514, 520, 522, 524, 526, 528, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, or 828.

23. The recombinant acid alpha glucosidase of claim 1 , wherein said amino acid sequence of said recombinant acid alpha glucosidase comprises residues 20-944 of SEQ ID NO: 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850.852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1

Assignees

• Codexis Inc

Inventors

• Hallows William Casey
• Botham Rachel Cathleen
• Zhu Yu
• Chng Chinping
• Dellas Nikki
• Huisman Gjalt W
• Alaoui Ismaili Moulay Hicham
• Homan David William
• Silverman Adam P
• Vroom Jonathan
• Lao Jessica P

Classifications

• C12N9/2428 Primary

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12Y302/01003

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12Y302/0102

  Alpha-glucosidase (3.2.1.20) · CPC title