RECOMBINANT FACTOR VIII VARIANTS

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing submitted concurrently herewith with a file name of “CX7-224US2_ST26.xml,” a creation date of Jan. 5, 2023, and a size of 11.4 megabytes, is part of the specification and is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure provides recombinant factor VIII polypeptide variants and compositions thereof. The recombinant factor VIII polypeptides have been engineered to have improved properties, such as improved expression, thermostability, serum stability, plasma stability, stability under basic conditions, reduced immunogenicity, improved cofactor potency, and improved blood clotting. The present disclosure also relates to the use of the compositions comprising the recombinant factor VIII polypeptides for therapeutic purposes.

BACKGROUND

Human factor VIII (“FVIII”), also known as clotting factor VIII or anti-hemophilic factor, is a protein cofactor that functions as a necessary part of the blood coagulation pathway which in response to injury initiates a chain of enzymatic and chemical reactions that results in a blood clot. Factor VIII directly supports Factor IX as a cofactor, improving its enzymatic activity by 10⁵-fold (see e.g., Mazurkiewicz-Pisarek et al., Acta Biochimica Polonica, 2016, 63.1:11-16). Hemophilia A, also referred to as haemophilia A, is an X-linked genetic deficiency in factor VIII that results in increased bleeding and lack of blood clots. There are three generally recognized forms of hemophilia A. Patients who have mild hemophilia A have excessive bleeding after trauma or surgery but are otherwise healthy. Patients who have moderate hemophilia A also suffer from joint and muscle bleeding after mild trauma. Patients who have severe hemophilia A suffer from spontaneous joint and muscle bleeding even without trauma. All patients with hemophilia A require treatment, but patients with the severe form and some patients with the moderate form require regular and frequent infusions of factor VIII to prevent excessive blood loss and death (see, e.g., Peters et al., Nature Reviews Drug Discovery, 2018, 17(7):493-508). This disease is estimated to affect 1 in 5,000 males, but also occurs in females. Treatment is generally supportive and there is no cure, thus there remains a need for a safe and effective treatment.

SUMMARY

The present disclosure provides recombinant factor VIII polypeptide variants and compositions thereof. The recombinant factor VIII polypeptide variants have been engineered to have improved properties, including increased activity, thermostability, serum stability, plasma stability, reduced immunogenicity, and/or improved blood clotting. The disclosure also relates to the use of the recombinant factor VIII polypeptides for therapeutic purposes.

In one aspect, the present disclosure provides a recombinant factor VIII polypeptide, or abiologically active fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or to a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534F/I/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T, or 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at amino acid position 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377, or 1439, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G/H/N/P/T/W, or 1439A/E/H/L/S/T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1148, 85/708/1148, 798, 706, 171, 335, 63, 344, 428, 879, 59, 429, 205, 1264, 797, 1248, 873, 792, 708, 57, 96, 362, 1292, 346, 433, 65, 795, 684/798, 353, 332, 1259, 356, 436, 1245, 1145, 716, 56, 773, 734, 358, 729, 665, 359, 61, 1250, 726, 37/96/171/429/708/799/1148, 171/429/732/1148, 96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/799, 171/429/708/1422, 171/429, 321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643/797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349, 257, 646, 435, or 58, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments of the foregoing, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or an amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T, or 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054, 755, 1041, 748, 1049, 1221, 1051, 612, 1037, 616, 387, 489, 750, 1224, 618, 1050, 1053, 896, 897, 749, 260, 481, 613, 756, 63, 265, 752, 1218, 1378, 614, 1440, 1439, 1205, 1222, 1389, 1269, 390, 397/1218, 757, 1373, 346, 63/1248, 349, 306/355/1422, 1422, 306/346/708, 1148, 171/346/1248, 63/797/799/1248/1422, 171/799, 362, 1248, 171/306/346/999/1248, 171/1148, 734/799/1248/1422, 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/349, 63/171/1148/1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349/354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/1248, 344/1248/1250, 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248, or 354/503, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or to the reference sequence corresponding to SEQ ID NO: 504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306/749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/387/750/1051/1148, 171, 750/1051/1148, 171/260/756, 171/1248, 171/1148, 59, 1440, 332/750/1248, 756/1248, 1248, 171/387/1248/1440, 750/1440, 59/171/750, 171/1193/1248, 750/1148/1193/1440, 1148, 171/387/1051, 748, 306, 306/387/390/749/1220, 390, 306/390/1037, 306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612/749/1037, or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or to the reference sequence corresponding to SEQ ID NO: 706, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816, 824, 826, 393, 825, 698, 332/387/390/1148, 328/387/749, 749, 306/328/332/390/1051/1193/1218, 63/306/332/1051/1148, 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306/332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306/387/749/1051/1148, 387/749, 306/945/1051/1193/1221, 306/390/749/1051, 63/306/332/390, 306/387/390/749/1148/1193/1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922, 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392, or 397, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840, or to the reference sequence corresponding to SEQ ID NO: 840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 840, or relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555/684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747, 753, 393/732/751, 63/797, 622, 617, 907, 1040, 611, 488, 1192, 1194, 619, 16, 802, 211, 555, 536, 527, 841, 320, 837, 558, 914, 312, 681, 838, 15, 1427, 18, 683, 24, 534, 840, 1160, 551, 393, 393/747/752/811/819/923/1148, 393/751/775, 63/393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/747/751, 393/747/753/775/1148, 752/923/1051, 63/775, 63/393/752/775/793/1051/1053, 752/753/775/793/819, 63/1051, 63/747/752/753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/752/753/793/811, 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148, or 747/753, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant factor VIII polypeptide of comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200, or to the reference sequence corresponding to SEQ ID NO: 1200, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1200, or relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 681, 622, 617, 558, 536, 211, 320, 839, 797, 533, 751, 303, 951, 164, 818, 807, 799, 735, 794, 926, 755, 921, 801, 1094, 968, 929, 924, 698, 168, 581, 916, 1428, 805, 1114, 311, 737, 693, 809, 757, 1099, 676, 740, 791, 211/536/622/681, 536/558, 211/536/555/617/732, 211/681/732, 211/536/555, 555, 211/536, 211/212, 211/536/617/681/732, 211/536/558/617/681, 555/732, 536/555/558/617/732, 536/617/681/732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619/681/839, 797/839, 212/312/320/533/534/617/681, 558/619/681, 320/558/681, 212/320/534/619/681/797/839, 320/558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/619/681, 211/534/558/611/681, 211/320/617/619/681, 558/617/681/797, 320/534/619/732, 211/619/681, 211/212/534/558/619/732/839, 212/320/619/681/797/839, 212/320/611/839, 320/558/619/681, 312/533/617/681/797/839, 558/611/681, 312, 211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681, 211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681, 211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558/681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538, or 1109, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514, or to the reference sequence corresponding to SEQ ID NO: 1514, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1514, or to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050, 1210, 104, 615, 610, 536, 618, 621, 614, 110, 301, 1078, 812, 786, 127, 175, 1093, 1088, 650, 535, 806, 738, 700, 132, 1076, 1084, 498, 756, 1108, 746, 160, 543, 310, 584, 918, 1452, 790, 292, 800, 167, 892, 304, 1116, 752, 281, 917, 712, 1080, 806/1352, 1095, 744, 1098, 948, 119, 785, 1113, 667, 158, 580, 707, 731, 1100, 1089, 1092, 939, 1104, 736, 1297, 1127, 686, 1129, 1138, 786/821, 680, 1101, 808, 739, 308, 536/555/619/732/799/805, 555/619/801, 555/619/799, 619/799/801/805, 536/555/619/732/799, 799/805, 801, 246/619/799/801, 536/555/619, 619/839, 619/799/805, 536/801/805, 555/619/799/805, 619/801/805, 555/619, 536/619/801, 801/805, 536/619/799/805, 555/619/732/799/801/805, 749, 616, 611, 370/614, 1156, 1158, 555/681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/799/916/1109, 555/619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799, 555, 732, 619/799, 619, 681, 555/916, 839, or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072, or to the reference sequence corresponding to SEQ ID NO: 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318/700/1076, 167/318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/752/1093, 167/700, 167/700/738/739/1093, 167/700/738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/739/809, 738/739/809/1093/1116, 738/739/1093, 738/739/752/1093, 738/739/799/800/1093, 738/739/1116, 738/800, 738/1093, 738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/1093, 739, 739/800/809, 800, 800/805/809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738/809, 318/1093, 318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/543/1093, 318/700/738/1093/1414, 681, 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/739/1116, 681/738/800, 681/800/805/809, 681/809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/1116, 681/700/809, 681/700/1093, 681/700/752/808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/1076/1116, 178/318/800/809, 178/318/681/800/808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116, 543, 543/738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/809/1093, 543/809/1093/1116, 543/681/738/739/752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/809/1093, 543/681/700/738/739/800/1093/1116, 543/681/700/738/739/805/808/809, 543/681/700/752, 543/681/700/752/809/1093, 543/1093, 543/700, 543/700/738/739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809/1116, 543/700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809, 700/809/1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700/752/805/809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809, or 805/809/1116, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least one substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least a substitution or substitution set of a variant set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of the sequence of an even numbered SEQ ID NO. set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, or comprising the sequence of an even numbered SEQ ID NO. set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1.

In the embodiments herein, the recombinant factor VIII polypeptide has clotting activity. In some embodiments, the recombinant factor VIII polypeptide displays an improved property as compared to a reference factor VIII polypeptide. In some embodiments, the reference factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the reference factor VIII polypeptide has the sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or the sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide described herein exhibits at least one improved property selected from: i) increased activity/potency; ii) increased clotting activity/potency; iii) increased stability to plasma or serum; iv) increased thermostability; v) reduced immunogenicity; vi) increased expression and/or secretion from cells that are transfected with an expression vector; vii) increased transduction and/or secretion from cells that are transduced with a gene therapy vector (e.g., AAV, mRNA, or lipid nanoparticle), or a combination of any of i), ii), iii), iv), v), vi), and vii), as compared to reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or having a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the reference factor VIII polypeptide has the sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or has the sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide comprises a “deimmunized” recombinant factor VIII polypeptide, such as provided in Tables 12-1 to 12-8 and 15-2.

In some further embodiments, the recombinant factor VIII is purified. In a further aspect, the present disclosure also provides functional or biologically active fragments of the recombinant factor VIII polypeptides disclosed herein.

In another aspect, the present disclosure further provides a recombinant polynucleotide encoding at least one recombinant factor VIII polypeptide, or a biologically active fragment thereof, described herein. In some embodiments, the polynucleotide is DNA, RNA, or mRNA.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 1-2345 and 2359-2869, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 1-2345 and 2349-2869, wherein the recombinant polynucleotide encodes a recombinant factor VIII polypeptide.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence codon-optimized for expression in a host cell. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence codon-optimized for expression in a mammalian cell, particularly a human cell.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 58-4371 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 1-2345 and 2359-2869, or comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 1-2345 and 2349-2869.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071, or comprising SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071.

In a further aspect, the present disclosure provides expression vectors comprising the recombinant polynucleotide encoding a recombinant factor VIII polypeptide as described herein. In some embodiments, the recombinant polynucleotide is operably linked to one or more control sequences. In some embodiments, the control sequence comprises a promoter. In some further embodiments, the promoter is a heterologous promoter.

In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a recombinant factor VIII polypeptide. In some embodiments, the host cell is an eukaryotic cell or prokaryotic cell. In some embodiments, the host cell is a mammalian cell, particularly a human cell, such as a cultured human cell. In some embodiments, the host cell is a human stem cell.

In a further aspect, the present disclosure provides methods of producing a recombinant factor VIII polypeptide described herein, comprising culturing a host cell comprising a recombinant polynucleotide encoding a recombinant factor VIII polypeptide disclosed herein, under conditions such that the recombinant factor VIII polypeptide is produced. In some embodiments, the method further comprises the step of recovering the expressed recombinant factor VIII polypeptide, such as from the media or cells. In some embodiments, the method further comprises the step of purifying the expressed recombinant factor VIII polypeptide.

In another aspect, the present disclosure provides compositions comprising at least one recombinant factor VIII polypeptide described herein. In some embodiments, the recombinant factor VIII polypeptides are provided as a pharmaceutical composition. In some embodiments, the pharmaceutical compositions comprises a recombinant factor VIII polypeptide, and a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion, particularly for administration to a human subject.

In some embodiments, the pharmaceutical compositions comprise at least one recombinant polynucleotide encoding a recombinant factor VIII polypeptide, particularly for use in gene therapy. In some embodiments, a composition for gene therapy comprises a recombinant polynucleotide encoding at least one of the recombinant factor VIII polypeptide described herein. In some embodiments, the gene therapy composition comprises a viral vector comprising a recombinant polynucleotide encoding the recombinant factor VIII polypeptide described herein.

In some embodiments, a gene therapy composition comprises a recombinant polynucleotide encoding at least one recombinant factor VIII polypeptides described herein, wherein the polynucleotide is formulated in liposomes, cationic polymers, or with dendrimers or attached to a cell penetrating peptide. In some embodiments, the recombinant polynucleotide of the gene therapy composition is provided as an expression vector for expression in mammalian cells.

In another aspect, the recombinant factor VIII polypeptides and the corresponding recombinant polynucleotides encoding the recombinant factor VIII polypeptides are useful in therapeutic applications for treating conditions or diseases characterized by a deficiency in factor VIII. In some embodiments, a method for treating and/or preventing symptoms of a deficiency in factor VIII in a subject comprises administering to a subject having a deficiency in factor VIII an effective amount of a recombinant factor VIII polypeptide or a pharmaceutical composition comprising the recombinant factor VIII polypeptide. In some embodiments, a method for treating and/or preventing symptoms of deficiency in factor VIII in a subject comprises providing to a subject having a deficiency in factor VIII a gene therapy composition comprising a recombinant polynucleotide encoding a recombinant factor VIII polypeptide described herein.

In a further aspect, the present disclosure also provides use of a recombinant factor VIII polypeptide, or a pharmaceutical thereof, or a gene therapy composition comprising a recombinant polynucleotide encoding the recombinant factor VIII polypeptide, for the treatment of a deficiency in factor VIII in a subject. In some embodiments, use of the recombinant factor VIII polypeptide, or a gene therapy composition comprising a recombinant polynucleotide encoding the recombinant factor VIII polypeptide, is for the preparation of a medicament for treating a deficiency in factor VIII in a subject. In some embodiments, the method or use is for treating hemophilia A in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows percentage of 50 donors whose peripheral blood mononuclear cells (PBMCs) generated a CD4 T-cell response when incubated with peptides of recombinant factor VIII in a T-cell assay for examining immuogenicity.

FIG. 2 shows the results of a study in which recombinant factor VIII polypeptides were expressed using either Expi293F or HepG2 cultures and assessed for activity using a chromogenic assay (BIOPHEN™ FVIII:C, Aniara), before or after incubation with Hemophilia A patient plasma (George King Bio-Medical, Inc.). Activities are shown relative to SEQ ID NO: 1514.

FIG. 3 shows the results of a study in which truncated recombinant factor VIII polypeptides were expressed using either Expi293F cultures and assessed for activity using a chromogenic assay (BIOPHEN™ FVIII:C, Aniara). Activities are shown relative to SEQ ID NO: 2 on the left axis. Numbers of amino acids removed relative to SEQ ID NO: 2 are shown on the right axis.

FIG. 4 shows expression of recombinant factor VIII polypeptide variants in Expi293F cells as measured by ELISA.

FIG. 5 shows expression of recombinant factor VIII polypeptide variants in HepG2 cells as measured by ELISA.

FIG. 6 shows activity of recombinant factor VIII polypeptide variants expressed in Expi293F cells as measured by chromogenic assay of the conditioned media, before normalization with protein concentration determined by ELISA.

FIG. 7 shows activity of recombinant factor VIII polypeptide variants expressed in HepG2 cells as measured by chromogenic assay of the conditioned media, before normalization with protein concentration determined by ELISA.

FIG. 8 shows activity of recombinant factor VIII polypeptide variants expressed in Expi293F cells as measured by chromogenic assay of the conditioned media, after normalization with protein concentration determined by ELISA.

FIG. 9 shows activity of recombinant factor VIII polypeptide variants expressed in HepG2 cells as measured by chromogenic assay of the conditioned media, after normalization with protein concentration determined by ELISA.

FIG. 10 shows clotting activity measured using activated partial thromboplastin time assay of condition media of Expi293F cells expressing recombinant factor VIII polypeptide variants.

FIG. 11 shows residual activity as measured using a chromogenic assay of recombinant factor VIII polypeptide variants following exposure of the recombinant factor VIII polypeptide variants to plasma obtained from a Hemophilia A patient for different time periods.

DESCRIPTION OF THE INVENTION

The present disclosure relates to recombinant factor VIII polypeptide (FVIII) variants and compositions thereof. The recombinant factor VIII polypeptide variants have been engineered to exhibit improved properties, such as improved activity, including blood clotting activity, improved cofactor potency, improved expression, increased thermostability, increased serum stability, increased plasma stability, and/or reduced immunogenicity. The present disclosure also provides use of the compositions comprising the recombinant factor VIII polypeptide variants or the polynucleotides encoding the recombinant factor VIII polypeptide variants for therapeutic applications.

It is to be understood that both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure.

Abbreviations and Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole.

Also, as used herein, the singular “a”, “an,” and “the” include the plural references, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

The term “about” means an acceptable error for a particular value. In some instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

It is also to be understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

“ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

“NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

1 “Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

“Mature protein” or “mature polypeptide” refers to the final processed biological protein or polypeptide or product.

“Pro-protein,” “pro-polypeptide,” or “pro-peptide” refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification, to form a biologically active protein, polypeptide, or peptide. In some embodiments, the post translational modification is a cleavage reaction to form the protein, polypeptide, or peptide.

“Pre-pro-protein,” “pre-pro-polypeptide,” or “pre-pro-peptide” refers to a precursor protein, polypeptide, or peptide that includes a signal sequence or signal peptide, and which can be processed by posttranslational modification, in particular a cleavage reaction, to generate a pro-protein, pro-polypeptide, or pro-peptide. Generally, a cleavage reaction removes a signal sequence or signal peptide to generate a pro-protein, pro-polypeptide, or pro-peptide.

“Full-length” in context of a protein or polypeptide refers to the protein or polypeptide which is not processed to alter the amino acid sequence of the entire protein or polypeptide. For example, a full-length protein is the entire protein encoded in the corresponding mRNA.

“Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about α-carbon (C_α). For example, whereas “Ala” designates alanine without specifying the configuration about the α-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

“Polynucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides, or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for the genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2′-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2′-deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5′ to 3′ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid.

“Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.

“Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

“Coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein or polypeptide.

1 “Percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., 1977, Nucleic Acids Res., 1977, 3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

“Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant factor VIII polypeptide, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

“Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

“Amino acid difference” or “residue difference” refers to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X59 as compared to SEQ ID NO: 2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 59 of SEQ ID NO: 2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a phenylalanine at position 59, then a “residue difference at position X59 as compared to SEQ ID NO: 2” an amino acid substitution of any residue other than phenylalanine at the position of the polypeptide corresponding to position 59 of SEQ ID NO: 2. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., as shown in in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1), the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide.

In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence, as described below. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/” (e.g., X61Q/X61S or X61Q/S or 61Q/S).

“Amino acid substitution set” and “substitution set” refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant polypeptides listed in any of the Tables in the Examples. In substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., Q96R;L171P or abbreviated 96R;171P) or slash (“/”; e.g., Q96R/L171P or abbreviated 96R/171P). In some embodiments, the “substitution” comprises the deletion of an amino acid, and can be denoted by “−” symbol.

“Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining activity and/or retaining the improved properties of an engineered polypeptide. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

“Functional fragment” or a “biologically active fragment” used interchangeably herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length recombinant factor VIII of the present disclosure) and that retains substantially all of the activity of the full-length polypeptide. In some embodiments, “functional fragment” or a “biologically active fragment” encompasses recombinant factor VIII processed into polypeptides by natural biological processes, such as cleavage into polypeptide fragments that form dimers and activated trimers.

“Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant factor VIII polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant factor VIII polypeptides can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure factor VIII composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant factor VIII polypeptides are substantially pure polypeptide compositions.

“Improved protein property” refers to a recombinant factor VIII polypeptide that exhibits an improvement in any protein property as compared to a reference factor VIII polypeptide and/or as a wild-type factor VIII polypeptide or another recombinant factor VIII polypeptide. Improved properties include, but are not limited to, such properties as increased gene expression, increased protein production, increased thermoactivity, increased thermostability, increased activity at various pH levels, increased stability, increased cofactor potency, increased specific activity, increased resistance to substrate and/or product inhibition, increased chemical stability, improved solvent stability, increased tolerance to acidic, neutral, or basic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, increased secretion from polypeptide producing cells, etc.

“Increased activity”, “increased cofactor potency”, or “enhanced activity” refers to an improved property of the recombinant factor VIII polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein in combination with factor IX (FIX)) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of factor VIII in combination with FIX) as compared to the reference factor VIII polypeptide. Exemplary methods to determine protein activity are provided in the Examples. Any property relating to protein activity may be affected. Improvements in protein activity can be from about 1.1 fold the protein activity of the corresponding wild-type protein or a parent protein or polypeptide, to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more activity than the naturally occurring factor VIII or another recombinant factor VIII from which the factor VIII polypeptides were derived (e.g., B-domain deleted factor VIII).

“Improved tolerance to basic pH” means that a recombinant factor VIII according to the invention will have increased stability (e.g., higher retained activity at >pH 7 after exposure to basic pH for a specified period of time (1 hour, up to 24 hours)) as compared to a reference factor VIII or another recombinant factor VIII polypeptide.

“Physiological pH” as used herein means the pH range generally found in a subject's (e.g., human) blood. In some embodiments, “physiological pH” is about pH 7 to about 7.4.

“Basic pH” (e.g., used with reference to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH range >pH 7, e.g., about 7 to 11.

“Acidic pH” (e.g., used with reference to improved stability to acidic pH conditions or increased tolerance to acidic pH) means a pH range of pH 7>, e.g., about 1.5 to 4.5.

“Deimmunized” as used herein, refers to the manipulation of a protein sequence to create a variant that is predicted to be not as immunogenic as the wild-type or reference protein. In some embodiments, the predicted deimmunization is complete, in that the variant protein is predicted to not stimulate an immune response in patients to whom the variant protein is administered. This response can be measured by various methods including but not limited to, the presence or abundance of anti-drug antibodies, the presence or abundance of neutralizing antibodies, the presence of an anaphylactic response, peptide presentation on major histocompatibility complex-II (MHC-II) proteins, or the prevalence or intensity of cytokine release upon administration of the protein. In some embodiments, the variant protein is less immunogenic than the wild-type or reference protein. In some embodiments, deimmunization involves modifications to subsequences of proteins (e.g., epitopes) that are recognized by human leukocyte antigen (HLA) receptors. In some embodiments, these epitopes are removed by changing their amino acid sequences to produce a deimmunized variant protein in which such subsequences are no longer recognized by the HLA receptors. In some other embodiments, these epitopes retain binding affinity to HLA receptors, but are not presented. In some embodiments, the deimmunized protein shows lower levels of response in biochemical and cell-biological predictors of human immunological responses including dendritic-cell T-cell activation assays, or (HLA) peptide binding assays. In some embodiments, these epitopes are removed by changing their amino acid sequence to produce a deimmunized variant protein in which the epitopes are no longer recognized by T-cell receptors. In still other embodiments the deimmunized protein induces anergy in its corresponding T-cells, activates T regulatory cells, or results in clonal deletion of recognizing B-cells. In some embodiments, a total immunogenicity score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity).

“Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. “High stringency hybridization” refers generally to conditions that are about 10° C. or less from the thermal melting temperature T_mas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v) SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited herein.

“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein such that the encoded protein is more efficiently expressed in the organism and/or cells of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the factor VIII polypeptides may be codon optimized for optimal production from the host organism(s) and/or cell type(s) selected for expression, accounting for GC content, cryptic splice sites, transcription termination signals, motifs that may affect RNA stability, and nucleic acid secondary structures, as well as any other factors of interest.

“Control sequence” refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. In some instances, at a minimum the control sequences include a promoter, and transcriptional and translational stop signals.

“Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

“Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or protein sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

“Suitable reaction conditions” refers to those conditions in the reaction solution (e.g., ranges of protein loading, temperature, pH, buffers, co-solvents, etc.) under which a factor VIII polypeptide of the present disclosure is capable of acting as a cofactor for factor IX to convert a substrate to the desired product compound. Exemplary “suitable reaction conditions” are provided in the present application and illustrated in the Examples.

“Protein loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. “Substrate” in the context of a reaction process involving factor VIII refers to the compound or molecule acted on by the factor VIII-factor IX polypeptide complex.

“Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the factor VIII-factor IX polypeptide complex on a substrate.

“Culturing” as used herein refers to the growing of a population of microbial, mammalian, or other suitable cells under any suitable conditions (e.g., using a liquid, gel or solid medium).

“Vector” refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector having a polynucleotide of interest operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the polynucleotide sequence of interest. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

“Gene therapy vector” as used herein refers to vehicles or carriers suitable for delivery of polynucleotide sequences to cells. In some embodiments, the vectors encapsulate genes (e.g., therapeutic genes) or polynucleotide sequences for delivery to cells or tissues, including but not limited to adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and non-viral vectors, such as liposomes. It is not intended that the present invention be limited to any specific gene therapy vector, as any vehicle suitable for a given setting finds use herein. The gene therapy vector may be designed to deliver genes to a specific species or host, or may find more general applicability.

“Expression” as used herein includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

“Produces” as used herein refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

“Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., the polynucleotides encoding the recombinant factor VIII variants). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

“Analogue” as used herein means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, “analogues” means polypeptides that contain one or more non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

“Therapeutic” as used herein refers to a compound or composition having beneficial or desirable medical effects administered to a subject who shows signs or symptoms of a disease or condition or pathology.

“Pharmaceutical composition” as used herein refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of a recombinant factor VIII polypeptide and an acceptable carrier and/or excipient.

“Composition” and “formulation” encompass products comprising at least one recombinant factor VIII of the present disclosure, intended for any suitable use (e.g., pharmaceutical compositions, etc.).

“Gene therapy” refers to the delivery of a gene, polydeoxyribonucleotide, or polynucleotide sequence(s) with a gene therapy vector to cells or tissues for the modification of those cells or tissues for the treatment or prevention of a disease. Gene therapy may include replacing a mutated gene that causes disease with a healthy copy of the gene; inactivating, or “knocking out,” a mutated gene that is functioning improperly; or providing a functional copy of a gene sufficient to treat and/or prevent the disease or condition. In some embodiments, gene therapy is used in the treatment of disease in patients.

“mRNA therapy” refers to the delivery of an mRNA polyribonucleotide to cells or tissues for the modification of those cells or tissues for the treatment or prevention of a disease. In some embodiments, the mRNA polynucleotides for delivery to cells or tissue, are formulated, for instance, but not limited to, in liposomes. In some embodiments, mRNA therapy is used in the treatment of a disease in patients.

“Cell therapy” refers to the delivery of living cells that have been modified exogenously to patients to provide a missing gene for the treatment or prevention of a disease. The modified cells are then reintroduced into the body.

“Effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

“Subject” encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the term encompass females as well as males.

“Patient” means any subject that is being assessed for, treated for, or is experiencing disease. In some embodiment, the patient is a human patient.

“Infant” refers to a child in the period of the first month afterbirth to approximately one (1) year of age. As used herein, the term “newborn” refers to child in the period from birth to the 28th day of life. The term “premature infant” refers to an infant born after the twentieth completed week of gestation, yet before full term, generally weighing ˜500 to ˜2499 grams at birth. A “very low birth weight infant” is an infant weighing less than 1500 g at birth.

“Child” refers to a person who has not attained the legal age for consent to treatment or research procedures. In some embodiments, the term refers to a person between the time of birth and adolescence.

“Adult” refers to a person who has attained legal age for the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term “young adult” refers to a person less than 18 years of age, but who has reached sexual maturity.

“Administration” and “administering” a composition mean providing a composition of the present invention to a subject (e.g., to a person suffering from the effects of deficiency in factor VIII, such as hemophilia A).

“Pharmaceutically acceptable” means a material that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.

“Excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the recombinant factor VIII polypeptides of the present disclosure). Excipients are typically included for formulation and/or administration purposes.

“Carrier” when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

“Therapeutically effective amount” when used in reference to symptoms of disease/condition refers to the amount and/or concentration of a compound (e.g., recombinant factor VIII polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease/condition or prevents or delays the onset of symptom(s). A “therapeutically effective amount” when used in reference to a disease or condition refers to the amount and/or concentration of a composition (e.g., recombinant factor VIII polypeptides) that ameliorates, attenuates, or eliminates the disease/condition. In some embodiments, the term is use in reference to the amount of a composition that elicits the biological (e.g., medical) response by a tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other clinician.

“Treating” or “treatment” of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As such, the terms “treating,” “treat” and “treatment” encompass preventative (e.g., prophylactic), as well as palliative treatment. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable by one of ordinary skill in the art.

Recombinant Factor VIII Polypeptides

As discussed above, blood clotting factor VIII (FVIII) is the cofactor to the activated clotting factor IX (e.g., FIXa), which in its proteolytically activated form interacts with FIXa to form a tight noncovalent complex that binds to and activates factor X (FX). Hemophilia A is a deficiency in factor VIII caused by missing or defective FVIII protein. Treatment of hemophilia A patients may include enzyme replacement therapy with preparations of factor VIII protein. Standard enzyme replacement therapy uses recombinantly expressed forms of the wild-type human factor VIII or B-domain deleted form of human factor VIII (e.g., XYNTHA® antihemophilic factor (recombinant); Wyeth Pharmaceuticals LLC). It is known that intravenously administered FVIII circulates in the bloodstream and restores blood clotting functions. However, these drugs do not completely relieve patient symptoms, as additional doses are needed after trauma or before surgery. In addition, the protein is relatively unstable and is cleared from the bloodstream within days. Thus, issues remain with available enzyme replacement treatments. In addition, patients may develop an immune response (IgG and IgE antibodies targeting the administered drug), and suffer severe allergic (anaphylactic) reactions, severe infusion reactions, and even death.

The recombinant factor VIII polypeptides, or biologically active fragments thereof disclosed herein, are intended to provide more stable and efficacious proteins suitable for treatment of hemophilia A, yet with reduced side effects and improved outcomes, as compared to currently available treatments. In some embodiments, the recombinant factor VIII polypeptides are characterized by increased stability in blood (pH 7.4), which the enzyme encounters upon introduction into the bloodstream and where the enzyme is active during therapy. The recombinant factor VIII polypeptides also display, among other properties, improved clotting function, and in some embodiments, reduced predicted immunogenicity (deimmunized).

The recombinant factor VIII polypeptides of the present disclosure are derived from a recombinant factor VIII with a deletion of the B-domain (SEQ ID NO: 2; see, e.g., US Patent Publication. No. 2017/0360900), where the B-domain deleted factor VIII is based on the naturally occurring factor VIII amino acid sequence. In brief, for purposes of explanation and not limitation, the factor VIII polypeptide is composed of five domains within the polypeptide: the signal peptide, the A1 domain, the A2 domain, the B domain, and the Light Chain domain. The recombinant factor VIII with a deletion of the B-domain represented by SEQ ID NO: 2 has identical signal peptide, A1, A2, and Light Chain domains to the wild-type human factor VIII, but has reduced the B domain to fourteen residues. In some embodiments, in the present disclosure, when a particular factor VIII variant is referred to by reference to amino acid differences in particular amino acids residues in the sequence of a B-domain deleted factor VIII or reference factor VIII polypeptide, it is to be understood that variants of another factor VIII polypeptide modified in the equivalent position(s) (as determined from the optional amino acid sequence alignment between the respective amino acid sequences) are encompassed herein.

Furthermore, the present disclosure also provides biologically active fragments of the recombinant factor VIII polypeptides variants disclosed herein. It is understood that factor VIII is biologically processed into polypeptide fragments. By way of example and not limitation, a factor VIII with the B-domain deletion may be processed by removal of signal sequence (e.g., amino acid residues 1-19) to yield a processed factor VIII (e.g., amino acid residues of about 20-1457). The factor VIII may next be processed to produce a polypeptide encoding dimerized human factor VIII (or variant thereof), the cleavage or cleavages occurring between residues 760 to 773 of SEQ ID NO: 2 and the polypeptide being devoid of some or all of an amino acid sequence corresponding to amino acids 760 to 773 of SEQ ID NO: 2. The factor VIII may be further processed to produce a polypeptide encoding trimerized human factor VIII (or variant thereof), the additional cleavage occurring between residues 391 and 392 of SEQ ID NO: 2 and the polypeptide being devoid of some or all of an amino acid sequence corresponding to amino acids 760 to 773 of SEQ ID NO: 2. Recombinant factor VIII polypeptide variants may or may not be processed the same as the B-domain deleted factor VIII having the naturally occurring sequences.

Accordingly, a reference to “biologically active” or “functionally active” fragment of the recombinant factor VIII polypeptide herein is meant to include the polypeptides formed by natural biological processing upon expression in a mammalian cell or patient, or upon administration of the recombinant factor VIII polypeptide to a patient, whether the “biologically active” or “functionally active” fragments are products from processing similar to the B-domain deleted factor VIII having the naturally occurring sequence or fragments that are the result of processing different from that of the B-domain deleted factor VIII having the naturally occurring sequence.

In one aspect, the present disclosure provides a recombinant factor VIII polypeptide, or a biologically active fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or to a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence.

In some embodiments, the recombinant factor VIII polypeptide, or a biologically active fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or to a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide, or a biologically active fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide, or biologically active fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide, or biologically active fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2360-2870, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2350-2870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution L13G/Y, L14G/M/V, R15G/S, F16A/D/G/P/V, C17S/V, F18L/R, S19G, A20L, T21M, Y24V, Q37H, S56E/P, F57A/L/S/T/V, P58K, F59D/G/L/M/Q/R/S/V/W, T61Q/S, V63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, V64D/G, Y65A, P85Q, Q96I/L/R/V/W, V104S, M110H, A119G/V, A127V, E132P/R, Q158G, L160E/T, G164E, A167G/H/R, S168G/R, L171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, Y175A/G/H, L178C/M/Q/V/W, D186A, L187C/G, K205R, T210P, L211G/K/P/Q/R, H212A/F/G/L/K/M/N/P/R/S/V/Y, I215E/P/S, K225R, A246D, V257I, S260E/K/T, T265R/S/W, T281A/S, G292D, T294G, R301K/M, A303G/T, S304A/E/G, E306A/D/G/P/S/T, S308P, I310G/V, T311E/G, F312L, L318C/G/K/M/Q/R/T/V, M320G/I/L/P/Q/R/S/T/V, D321Y, F325Y, F328G/Q/S, S332A/F/G/H/N/P/Q/R/T/V/W/Y, H334G, Q335E/P, K344A/E/L, D346R/V, S347N, P349G, E351S, Q353S, L354R, R355C, M356L/N, N358K/S, N359E/G, A362E/G/N/P/T/V/W, T370A, F387A/G/I/L/Q/R/S/T/V/W, 1388A/P, I390A/E/G/L/M/P/S/T/V, S392A, V393A/F/G/H/M/Q/R/W/Y, H397G/N, A407V, S428F, Q429L/S, N433A/G, P435Q, Q436D/G, L481V, Q487N, A488G, S489E/F/Q/R, P491E, G498A, 1499C/L, R503S, V514L, I527A, T533R/V/Y, V534I/F/L/M, T535C/G/N, V536A/G/K/L/M/P/R/W, D538V, S543G/L/P/W, R550G, Y551W, F555E/G/R/T, M558A/E/G/L/P/Q/R/S/T/V/W, P569T, Q580S/Y, R581K/V, Q584A, I610H/K/L/R/S/T, Q611D/E, R612G/S/T, F613S, L614A/F/H/I/N/T, P615D/N, N616D/E/G/Q/W, P617D/E/G/N, A618C/E/G/P/Y, G619E, V620N/P, Q621K/M/R/T/W, L622D, S643N, L646I, L650M, T665G, F667I, T676Q, K680L/R, M681A/C/E/F/G/L/N/R/Q/S/T/W, V682R, Y683W, E684G/K, T686W, S693T, T696E, F698D/S/W/Y, S700A/E/Q/T/V, L706A/G/M/Y, W707H, I708A/K/T/V, H712G/L/M/Q/S/T/Y, F716K/L/W, L724I, K726A/L, S729T, D731I/L/R, K732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, T734I/R/S, G735D/SN, D736F/G, Y737F/G/K/L/M/N/S/V, Y738A/D/E/F/H/K/N/P/R/S/T, E739G/P/R, D740G, D744A/R, A746P, A747D, Y748F/G/K/N/S/V, L749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, L750A/D/G/K/M/N/P/Q/R/S/T/V/W, A751A/D/E/Y, K752D/E/F/G/L/M/Q/R/S/V, N753D, N754W, A755G/L/P/Q/S/Y, I756G/P/R/T, E757A/G/K/T, R759S, R770W, R773H, I775E/G/K/N/P/S/V, E785V, E786C/D/L, I787W, D790A/G/I/M/P/W, D791M/T, T792H/W/Y, I793G, S794R, Y795C/N/S/T, M797C/G/P/Q/T/W/Y, K798C/R/T, K799A/E/G/M/P/Q/R/S/V, E800C/G/I/L/M/N/P/Q/R/T/V/W/Y, D801A/G/I/R, F802SN, Y805C/G/H/K/L/M/P/Q/R/SN/W, D806E/G/R/S, E807C/L/P/R, D808K/S, E809A/C/G/I/K/L/N/Q/R/S/T/V/W, N810V, Q811F, S812G/I/V, S815I, F816V/W, Q817A/D, K818A/E/G/P/S/V/W, K819V, R821Q, H822S/T, F824W, I825L, A826G, V828K, R830M/V, L831W, M836G/R/V/W, S837G, S838G/T, S839A/G/K/P, P840G, H841W, R844H, G866A, Y873L/Q/T, E879Q, V892T, 1896L/V, M897G/K/R, Q903E, P907D, L914V, S916C/E/I/K/L/M/Q/T, Y917G, E918M/S, E919G, Q921R/W, R922W, Q923M, G924I/M/P/Q/W, E926T/V, K929A/Q/V, K938R/V, T939E/S, Q945K, M948S, T951V, L968A/P, Q999R, F1037H/M/N/P, N1040E, Y1041G/H/N, R1042A/E/T, F1043E/R, Y1049G/W, I1050E/P/Q/S/T, M1051E/H/K/R/S/W, T1053A/R, L1054C, E1076G/S, I1078S, S1080G/V, S1084L/N, F1088G/S, T1089F/S, K1092G, K1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, E1094S, E1095G, M1098G, A1099G, L1100I/S, Y1101G, Y1104C/E, F1108M/S/V/W, E1109G, M1113F, L1114A/M, S1116A/G/I/L/R, I1127L/V, E1129P, L1138Q, K1145R, T1148A/C/E/L/M/N/Q/R/S, G1151S, H1156D/G/N/R, R1158N, F1160R, A1186E, P1192D, F1193A/E/G/I/L, S1194P, I1205L, K1210M/T/V/W, F1218A/G/L/M/P, S1220D/E/L/T, L1221G/S/T/V, Y1222F, S1224P, M1229D, T1245R, L1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, V1250A/P/S/T, G1259T, I1264L/R/W, I1269F, I1270V, I1274N/V, Y1281H/K/P/Q/R/S, I1283E/H/L/S/T, M1292Y, N1297A, G1339C, K1352E, V1373S, L1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, T1378A/C/D/E, M1380A/G/W, S1389W, 51414A/C/D/E/G/H/N/P/T/W, L1422I, L1427Y, T1428G, V1439A/E/H/L/S/T, H1440A/C/E/G/S/T, I1442T, or E1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or amino acid residue 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P, 387I, 390A, 393G, 534I, 558W, 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D, or 1439E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution V63Q, L171P, H212A, S260T, E306G, L317G, M320R, S332P, F387I, 1390A, V393G, V534I, M558W, M681G, K732W, Y738E, L749F, L750D, 1775G, T1148N, L1248D, L1377D, or V1439E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO:2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at position 1377. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution 1377D.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position 1439 or 1377/1439. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 1439E or 1377D/1439E.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position(s) 171, 260, 387, 750, 1248 or 171/260/387/750/1248. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 171P, 260T, 387I, 750D, 1248D or 171P/260T/387I/750D/1248D.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position(s) 306, 332, 390, 749 or 306/332/390/749. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 306G, 332P, 390A, 749F or 306G/332P/390A/749F.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position(s) 63, 393, 732, 775, 1148 or 63/393/732/775/1148. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 63Q, 393G, 732W, 775G, 1148N or 63Q/393G/732W/775G/1148N.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position(s) 212, 320, 534, 558, 681, or 212/320/534/558/681. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 212A, 320R, 534I, 558W, 681G, or 212A/320R/534I/558W/681G.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at position(s) 317, 738, or 317/738. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 317G, 738E, or 317G/738E.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 1148N, 85Q/708T/1148N, 798T, 706G, 706A, 171Q, 335E, 63R, 344A, 428F, 879Q, 59L, 429S, 205R, 1264W, 797P, 1248S, 1264R, 873T, 792Y, 708K, 344L, 57V, 96L, 362W, 63G, 873L, 1292Y, 1148Q, 346V, 797G, 706Y, 1248R, 433A, 65A, 59W, 873Q, 795C, 684K/798R, 57L, 353S, 332Q, 708A, 1259T, 63M, 356L, 1248G, 1248P, 436G, 57A, 59S, 1245R, 59R, 96W, 797Y, 1145R, 436D, 716L, 56E, 773H, 734I, 358S, 335P, 59Q, 729T, 716K, 665G, 362T, 57T, 359G, 61S, 1250A, 362V, 359E, 716W, 332W, 708V, 63H, 797C, 61Q, 356N, 1250S, 798C, 433G, 795N, 96I, 56P, 63N, 792W, 1264L, 1250P, 332P, 59V, 726L, 37H/96R/171P/429L/708T/799R/1148N, 171P, 171P/429L/732R/1148N, 96R/171P, 171P/919G/1151S, 171P/792Y/797Q/799R/1148N, 171P/569T/1148N, 96R/171P/429L/792Y/797T/799R, 171P/429L/708T/1422I, 171P/429L, 321Y/362P/732E/797T/799R, 171P/1100I, 708T/1148N/1186E, 429L/724I/732E/1148N, 171P/362E/795T/797Q/799M, 171P/429L/732E, 96R/171P/643N/797T/799R, 171P/792Y/797T/799M, 503S, 171P/732E, 1148N/1193L, 96R/171P/362E/429L/732R/795T/797Q, 726A, 59M, 734R, 362N, 334G, 732G, 63Q, 351S, 1377D, 795S, 358K, 999R, 1250T, 306G, 349G, 1377G, 332F, 1377E, 257I, 646I, 346R, 435Q, 96V, 1377R, 1377S, 59G, 171M, 792H, 1248D, 306S, 706M, 171H, 1377A, 1377V, 734S, 1148R, 57S, 59D, 344E, 362G, or 58K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set T1148N, P85Q/1708T/T1148N, K798T, L706G, L706A, L171Q, Q335E, V63R, K344A, S428F, E879Q, F59L, Q429S, K205R, I1264W, M797P, L1248S, I1264R, Y873T, T792Y, I708K, K344L, F57V, Q96L, A362W, V63G, Y873L, M1292Y, T1148Q, D346V, M797G, L706Y, L1248R, N433A, Y65A, F59W, Y873Q, V795C, E684K/K798R, F57L, Q353S, S332Q, I708A, G1259T, V63M, M356L, L1248G, L1248P, Q436G, F57A, F59S, T1245R, F59R, Q96W, M797Y, K1145R, Q436D, F716L, S56E, R773H, T734I, N358S, Q335P, F59Q, S729T, F716K, T665G, A362T, F57T, N359G, T61S, V1250A, A362V, N359E, F716W, S332W, I708V, V63H, M797C, T61Q, M356N, V1250S, K798C, N433G, V795N, Q96I, S56P, V63N, T792W, I1264L, V1250P, S332P, F59V, K726L, Q37H/Q96R/L171P/Q429L/I708T/K799R/T1148N, L171P, L171P/Q429L/K732R/T1148N, Q96R/L171P, L171P/E919G/G1151S, L171P/T792Y/M797Q/K799R/T1148N, L171P/P569T/T1148N, Q96R/L171P/Q429L/T792Y/M797T/K799R, L171P/Q429L/I708T/L1422I, L171P/Q429L, D321Y/A362P/K732E/M797T/K799R, L171P/L1100I, I708T/T1148N/A1186E, Q429L/L724I/K732E/T1148N, L171P/A362E/V795T/M797Q/K799M, L171P/Q429L/K732E, Q96R/L171P/S643N/M797T/K799R, L171P/T792Y/M797T/K799M, R503S, L171P/K732E, T1148N/F1193L, Q96R/L171P/A362E/Q429L/K732R/V795T/M797Q, K726A, F59M, T734R, A362N, H334G, K732G, V63Q, E351S, L1377D, V795S, N358K, Q999R, V1250T, E306G, P349G, L1377G, S332F, L1377E, V257I, L646I, D346R, P435Q, Q96V, L1377R, L1377S, F59G, L171M, T792H, L1248D, E306S, L706M, L171H, L1377A, L1377V, T734S, T1148R, F57S, F59D, K344E, A362G, or P58K, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1377/1380, 388/1377, 1043/1377, 610/1377, 1274/1377, 1220/1377, 1377/1442, 1193/1377, 621/1377, 1054/1377, 755/1377, 1041/1377, 748/1377, 1049/1377, 1221/1377, 1051/1377, 612/1377, 1037/1377, 616/1377, 387/1377, 489/1377, 750/1377, 1224/1377, 618/1377, 1050/1377, 1053/1377, 896/1377, 897/1377, 749/1377, 260/1377, 481/1377, 613/1377, 756/1377, 63/1377, 265/1377, 752/1377, 1218/1377, 1377/1378, 614/1377, 1378/1377, 1440/1377, 1377/1440, 1377/1439, 1205/1377, 1222/1377, 1377/1389, 1269/1377, 390/1377, 397/1218/1377, 757/1377, 1373/1377, 346/1377, 63/1248/1377, 349/1377, 306/355/1377/1422, 1377/1422, 306/346/708/1377, 1148/1377, 171/346/1248/1377, 63/797/799/1248/1377/1422, 171/799/1377, 362/1377, 1248/1377, 171/306/346/999/1248/1377, 171/1148/1377, 734/799/1248/1377/1422, 63/732/1377, 63/171/306/346/732/1377, 171/1377, 171/1377/1422, 63/362/732/799/1248/1377, 999/1148/1377/1422, 346/349/1377, 306/349/1377, 63/171/1148/1377/1151, 306/1377, 344/349/354/1250/1377, 1250/1377, 63/306/344/349/354/503/1248/1377, 59/63/332/349/503/706/708/1377, 63/306/344/349/354/706/1248/1377, 59/63/1377, 332/706/708/1248/1250/1377, 503/1377, 59/63/349/354/1377, 59/349/708/1377, 59/306/344/1248/1377, 344/1248/1250/1377, 708/1377, 63/344/349/1377, 59/1377, 63/344/349/354/1377, 706/1377, 59/306/770/1377, 63/347/354/1248/1377, or 354/503/1377, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 1377D/1380W, 388A/1377D, 1043E/1377D, 1043R/1377D, 610R/1377D, 1274N/1377D, 1220T/1377D, 1377D/1442T, 1193L/1377D, 621T/1377D, 1054C/1377D, 755Q/1377D, 1041G/1377D, 748S/1377D, 1049G/1377D, 1221V/1377D, 1051E/1377D, 612G/1377D, 1037H1/1377D, 616E/1377D, 1041H/1377D, 387S/1377D, 489R/1377D, 621M/1377D, 489Q/1377D, 750G/1377D, 1224P/1377D, 618C/1377D, 616G/1377D, 612T/1377D, 1050Q/1377D, 1053A/1377D, 616W/1377D, 896V/1377D, 489F/1377D, 1050P/1377D, 897G/1377D, 749T/1377D, 1274V/1377D, 1220L/1377D, 260E/1377D, 481V/1377D, 613S/1377D, 387G/1377D, 621R/1377D, 756R/1377D, 63R/1377D, 265S/1377D, 1221T/1377D, 1377D/1380G, 752G/1377D, 748K/1377D, 755L/1377D, 1218A/1377D, 748N/1377D, 1377D/1378E, 614I/1377D, 749F/1377D, 387V/1377D, 750T/1377D, 1193G/1377D, 749V/1377D, 1378A/1377D, 750V/1377D, 387T/1377D, 387Q/1377D, 750A/1377D, 1037N/1377D, 1377D/1440A, 1051S/1377D, 1221G/1377D, 1377D/1378D, 1377D/1440S, 387R/1377D, 1193A/1377D, 387I/1377D, 1218M/1377D, 265W/1377D, 1377D/1440E, 1377D/1439L, 1377D/1440T, 612S/1377D, 1049W/1377D, 1221S/1377D, 1220D/1377D, 749G/1377D, 749S/1377D, 1218G/1377D, 1205L/1377D, 1222F/1377D, 1193E/1377D, 1050S/1377D, 1377D/1389W, 1377D/1439E, 260T/1377D, 1037P/1377D, 1051K/1377D, 1269F/1377D, 265R/1377D, 1051R/1377D, 1218L/1377D, 897R/1377D, 388P/1377D, 748G/1377D, 756P/1377D, 748F/1377D, 390A/1377D, 610L/1377D, 755G/1377D, 749R/1377D, 748V/1377D, 1377D/1380A, 1377D/1440C, 397N/F1218M/1377D, 1037M/1377D, 1218P/1377D, 757K/1377D, 1373S/1377D, 1377D/1440G, 1193I/1377D, 1377D/1439T, 750D/1377D, 390G/1377D, 756G/1377D, 1220E/1377D, 1377D/1378C, 618G/1377D, 346R/1377D, 63Q/1248D/1377D, 349G/1377D, 306G/355C/1377D/1422I, 1377D/1422I, 306G/346R/708T/1377D, 1148N/1377D, 171P/346R/1248D/1377D, 63Q/797T/799R/1248D/1377D/1422I, 171P/799R/1377D, 362P/1377D, 1248D/1377D, 63Q/1377D, 171P/306G/346R/999R/1248D/1377D, 171P/1148N/1377D, 734S/799R/1248D/1377D/1422I, 63Q/732E/1377D, 63Q/171P/306G/346R/732G/1377D, 171P/1377D, 171P/1377D/1422I, 63Q/362P/732G/799R/1248D/1377D, 999R/1148N/1377D/1422I, 346R/349G/1377D, 306G/349G/1377D, 63Q/171P/1148N/1377D/1151S, 306G/1377D, 344E/349G/354R/1250T/1377D, 63G/1377D, 1250T/1377D, 63R/306G/344E/349G/354R/503S/1248R/1377D, 59D/63S/332P/349G/503S/706M/708K/1377D, 63S/306G/344A/349G/354R/706M/1248R/1377D, 1248R/1377D, 59D/63G/1377D, 332P/706M/708K/1248R/1250T/1377D, 503S/1377D, 59D/63R/349G/354R/1377D, 59D/349G/708K/1377D, 59D/306G/344E/1248R/1377D, 344E/1248R/1250T/1377D, 708K/1377D, 63R/344E/349G/1377D, 59D/1377D, 63S/344A/349G/354R/1377D, 706M/1377D, 59D/306G/770W/1377D, 63G/347N/354R/1248R/1377D, or 354R/503S/1377D, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 750/1377/1439, 1051/1377/1439, 260/1377/1439, 756/1377/1439, 1193/1377/1439, 332/1377/1439, 897/1377/1439, 387/1377/1439, 1037/1377/1439, 306/749/1377/1439, 1270/1377/1439, 489/1377/1439, 487/1377/1439, 488/1377/1439, 903/1377/1439, 171/1248/1377/1439/1440, 260/1148/1377/1378/1439, 171/260/387/750/1248/1377/1439, 171/260/1377/1439, 171/260/387/750/1051/1148/1377/1439, 171/1377/1439, 750/1051/1148/1377/1439, 171/260/756/1377/1439, 171/1248/1377/1439, 171/1148/1377/1439, 59/1377/1439, 1377/1439/1440, 332/750/1248/1377/1439, 756/1248/1377/1439, 1248/1377/1439, 171/387/1248/1377/1440/1439, 750/1377/1439/1440, 59/171/750/1377/1439, 171/1193/1248/1377/1439, 750/1148/1193/1377/1439/1440, 1148/1377/1439, 171/387/1051/1377/1439, 748/1377/1439, 306/1377/1439, 306/387/390/749/1220/1377/1439, 390/1377/1439, 306/390/1037/1377/1439, 306/612/749/1377/1439, 387/390/749/1377/1439, 63/390/503/749/1377/1439, 1220/1377/1439, 63/306/1377/1439, 749/1377/1439, 306/390/749/1037/1377/1439, 306/749/1220/1377/1439, 306/387/390/612/749/1037/1377/1439, or 328/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 750D/1377D/1439E, 1051K/1377D/1439E, 1051S/1377D/1439E, 260T/1377D/1439E, 756P/1377D/1439E, 1193A/1377D/1439E, 332P/1377D/1439E, 897R/1377D/1439E, 387Q/1377D/1439E, 1037N/1377D/1439E, 387R/1377D/1439E, 306G/749F/1377D/1439E, 1270V/1377D/1439E, 489E/1377D/1439E, 487N/1377D/1439E, 488G/1377D/1439E, 903E/1377D/1439E, 171P/1248D/1377D/1439E/1440T, 260T/1148N/1377D/1378D/1439E, 171P/260T/387I/750D/1248D/1377D/1439E, 171P/260T/1377D/1439E, 171P/260T/387I/750D/1051S/1148N/1377D/1439E, 171P/1377D/1439E, 750D/1051S/1148N/1377D/1439E, 171P/260T/756P/1377D/1439E, 171P/1248D/1377D/1439E, 171P/1148N/1377D/1439E, 387I/1377D/1439E, 59D/1377D/1439E, 1377D/1439E/1440S, 332P/750D/1248D/1377D/1439E, 756P/1248D/1377D/1439E, 1248D/1377D/1439E, 171P/387I/1248D/1377D/1440S/1439E, 750D/1377D/1439E/1440S, 59D/171P/750D/1377D/1439E, 171P/1193A/1248D/1377D/1439E, 750D/1148N/1193A/1377D/1439E/1440S, 1148N/1377D/1439E, 171P/387I I051K/1377D/1439E, 748G/1377D/1439E, 306G/1377D/1439E, 306G/387R/390A/749F/1220D/1377D/1439E, 390A/1377D/1439E, 306G/390A/1037N/1377D/1439E, 306G/612S/749F/1377D/1439E, 387V/390A/749F/1377D/1439E, 63S/390A/503S/749F/1377D/1439E, 387R/390A/749R/1377D/1439E, 1220D/1377D/1439E, 63S/306G/1377D/1439E, 749F/1377D/1439E, 306G/390A/749F/1037N/1377D/1439E, 387V/1377D/1439E, 306G/749F/1220D/1377D/1439E, 306G/387V/390A/612S/749F/1037N/1377D/1439E, or 328S/1377D/1439E wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 171/260/387/750/1051/1248/1377/1439, 171/260/387/750/1221/1248/1377/1439, 171/260/387/750/1218/1248/1377/1439, 171/260/387/750/1248/1377/1439, 171/260/387/749/750/1218/1248/1377/1439, 171/260/332/387/750/1248/1377/1439, 171/260/387/750/897/1248/1377/1439, 171/260/387/696/750/1248/1377/1439, 171/260/387/750/816/1248/1377/1439, 171/260/387/750/824/1248/1377/1439, 171/260/387/750/826/1248/1377/1439, 171/260/387/393/750/1248/1377/1439, 171/260/387/750/825/1248/1377/1439, 171/260/387/698/750/1248/1377/1439, 171/260/332/387/390/750/1148/1248/1377/1439, 171/260/328/387/749/750/1248/1377/1439, 171/260/387/749/750/1248/1377/1439, 171/260/306/328/332/387/390/750/1051/1193/1218/1248/1377/1439, 63/171/260/306/332/387/750/1051/1148/1248/1377/1439, 63/171/260/306/332/387/750/1051/1248/1377/1439, 171/260/387/390/750/1051/1248/1377/1439, 171/186/260/306/328/332/387/390/749/750/1051/1148/1193/1248/1377/1439, 171/260/387/390/750/756/1051/1248/1377/1439, 171/260/306/387/390/750/1051/1248/1377/1439, 171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/387/390/750/1148/1248/1377/1439, 171/260/306/328/332/387/390/749/750/1051/1148/1193/1248/1377/1439, 171/260/332/387/390/749/750/1148/1248/1377/1439, 171/260/387/390/749/750/1248/1377/1439, 171/260/306/387/749/750/1051/1148/1248/1377/1439, 171/260/306/387/750/945/1051/1193/1221/1248/1377/1439, 171/260/306/387/390/749/750/1051/1248/1377/1439, 63/171/260/306/332/387/390/750/1248/1377/1439, 171/260/306/387/390/749/750/1148/1193/1221/1248/1377/1439, 171/260/306/332/387/390/749/750/1218/1248/1377/1439, 63/171/260/306/332/387/390/750/1051/1148/1193/1218/1248/1377/1439, 171/260/387/750/924/1248/1377/1439, 171/260/387/750/755/1248/1377/1439, 171/260/387/750/752/1248/1377/1439, 171/260/387/750/751/1248/1377/1439, 171/260/387/750/923/1248/1377/1439, 171/260/387/750/797/1248/1377/1439, 171/260/387/750/754/1248/1377/1439, 171/260/387/750/793/1248/1377/1439, 171/260/387/750/753/1248/1377/1439, 171/260/387/750/922/1248/1377/1439, 171/260/387/750/896/1248/1377/1439, 171/260/387/750/938/1248/1377/1439, 171/260/387/750/775/1248/1377/1439, 171/260/387/750/732/1248/1377/1439, 171/260/387/750/810/1248/1377/1439, 171/260/387/750/747/1248/1377/1439, 171/260/387/393/407/750/1248/1377/1439, 171/260/387/750/819/1248/1377/1439, 171/260/387/750/787/1248/1377/1439, 171/260/387/750/802/1248/1377/1439, 171/260/387/732/750/1248/1377/1439, 171/260/387/750/811/1248/1377/1439, 171/260/387/392/750/1248/1377/1439, or 171/260/387/397/750/1248/1377/1439, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 171P/260T/387I/750D/1051K/1248D/1377D/1439E, 171P/260T/387I/750D/1221G/1248D/1377D/1439E, 171P/260T/387I/750D/1218G/1248D/1377D/1439E, 171P/260T/387R/750D/1248D/1377D/1439E, 171P/260T/387I/750D/1051R/1248D/1377D/1439E, 171P/260T/387I/749F/750D/1218G/1248D/1377D/1439E, 171P/260T/387T/750D/1248D/1377D/1439E, 171P/260T/332P/387I/750D/1248D/1377D/1439E, 171P/260T/387I/750D/897K/1248D/1377D/1439E, 171P/260T/387I/696E/750D/1248D/1377D/1439E, 171P/260T/387I/750D/816W/1248D/1377D/1439E, 171P/260T/387I/750D/824W/1248D/1377D/1439E, 171P/260T/387I/750D/826G/1248D/1377D/1439E, 171P/260T/387I/393W/750D/1248D/1377D/1439E, 171P/260T/387I/750D/825L/1248D/1377D/1439E, 171P/260T/387I/750D/897R/1248D/1377D/1439E, 171P/260T/387I/750D/816V/1248D/1377D/1439E, 171P/260T/387I/698W/750D/1248D/1377D/1439E, 171P/260T/387I/393Q/750D/1248D/1377D/1439E, 171P/260T/332P/387R/390A/750D/1148N/1248D/1377D/1439E, 171P/260T/328S/387R/749F/750D/1248D/1377D/1439E, 171P/260T/387I/749F/750D/1248D/1377D/1439E, 171P/260T/306G/328S/332P/387I/390A/750D/1051R/1193A/1218G/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/750D/1051R/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387T/750D/1051K/1248D/1377D/1439E, 171P/260T/387T/390A/750D/1051R/1248D/1377D/1439E, 171P/186A/260T/306G/328S/332P/387I/390A/749F/750D/1051K/1148N/1193A/1248D/1377D/1439E, 171P/260T/387T/390A/750D/756P/1051R/1248D/1377D/1439E, 171P/260T/306G/387I/390A/750D/1051R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/387R/390A/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/328S/332P/387T/390A/749F/750D/1051R/1148N/1193A/1248D/1377D/1439E, 171P/260T/332P/387R/390A/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/387R/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/387T/749F/750D/1051K/1148N/1248D/1377D/1439E, 171P/260T/387R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/387I/750D/945K/1051R/1193A/1221G/1248D/1377D/1439E, 171P/260T/306G/387I/390A/749F/750D/1051K/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/750D/1248D/1377D/1439E, 171P/260T/306G/387T/390A/749F/750D/1148N/1193A/1221G/1248D/1377D/1439E, 171P/260T/306G/332P/387R/390A/749F/750D/1218M/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/750D/1051R/1148N/1193A/1218G/1248D/1377D/1439E, 171P/260T/387I/750D/924I/1248D/1377D/1439E, 171P/260T/387I/750D/755S/1248D/1377D/1439E, 171P/260T/387I/750D/752F/1248D/1377D/1439E, 171P/260T/387I/750D/S751Y/1248D/1377D/1439E, 171P/260T/387I/750D/923M/1248D/1377D/1439E, 171P/260T/387I/750D/797Y/1248D/1377D/1439E, 171P/260T/387I/750D/754W/1248D/1377D/1439E, 171P/260T/387I/750D/793G/1248D/1377D/1439E, 171P/260T/387I/750D/753D/1248D/1377D/1439E, 171P/260T/387I/750D/922W/1248D/1377D/1439E, 171P/260T/387I/750D/896L/1248D/1377D/1439E, 171P/260T/387I/750D/938R/1248D/1377D/1439E, 171P/260T/387I/750D/775G/1248D/1377D/1439E, 171P/260T/387I/750D/751E/1248D/1377D/1439E, 171P/260T/387I/750D/732W/1248D/1377D/1439E, 171P/260T/387I/750D/752G/1248D/1377D/1439E, 171P/260T/387I/750D/810V/1248D/1377D/1439E, 171P/260T/387I/750D/938V/1248D/1377D/1439E, 171P/260T/387I/750D/747D/1248D/1377D/1439E, 171P/260T/387I/750D/755G/1248D/1377D/1439E, 171P/260T/387I/393G/407V/750D/1248D/1377D/1439E, 171P/260T/387I/750D/797W/1248D/1377D/1439E, 171P/260T/387I/750D/819V/1248D/1377D/1439E, 171P/260T/387I/393G/750D/1248D/1377D/1439E, 171P/260T/387I/750D/787W/1248D/1377D/1439E, 171P/260T/387I/393R/750D/1248D/1377D/1439E, 171P/260T/387I/750D/802V/1248D/1377D/1439E, 171P/260T/387I/750D/775E/1248D/1377D/1439E, 171P/260T/387I/750D/924P/1248D/1377D/1439E, 171P/260T/387I/732G/750D/1248D/1377D/1439E, 171P/260T/387I/750D/811F/1248D/1377D/1439E, 171P/260T/387I/750D/802S/1248D/1377D/1439E, 171P/260T/387I/750D/752L/1248D/1377D/1439E, 171P/260T/387I/392A/750D/1248D/1377D/1439E, or 171P/260T/387I/397G/750D/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 171/260/306/332/387/390/620/749/750/1248/1377/1439, 171/260/306/332/387/390/491/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/1229/1248/1377/1439, 171/260/306/332/387/390/615/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/1248/1283/1377/1439, 171/260/306/332/387/390/749/750/822/1248/1377/1439, 171/260/306/328/332/387/390/749/750/1248/1377/1439, 171/212/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/818/1248/1377/1439, 171/260/306/332/387/390/555/684/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/828/1248/1377/1439, 171/260/306/332/387/390/550/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/830/1248/1377/1439, 171/260/306/332/387/390/749/750/839/1248/1377/1439, 13/171/260/306/332/387/390/749/750/1248/1377/1439, 14/171/260/306/332/387/390/749/750/1248/1377/1439, 17/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/836/1248/1377/1439, 171/260/306/332/387/390/749/750/1210/1248/1377/1439, 171/260/306/332/387/390/749/750/1248/1281/1377/1439, 64/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/682/749/750/1248/1377/1439, 171/260/306/318/332/387/390/749/750/1248/1377/1439, 20/171/260/306/332/387/390/749/750/1248/1377/1439, 21/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/325/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/533/749/750/1248/1377/1439, 19/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/817/1248/1377/1439, 171/260/306/332/387/390/749/750/831/1248/1377/1439, 171/225/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/1156/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/753/1248/1377/1439, 171/260/306/332/387/390/393/732/749/750/751/1248/1377/1439, 63/171/260/306/332/387/390/749/750/797/1248/1377/1439, 171/260/306/332/387/390/622/749/750/1248/1377/1439, 171/260/306/332/387/390/617/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/907/1248/1377/1439, 171/260/306/332/387/390/749/750/1040/1248/1377/1439, 171/260/306/332/387/390/611/749/750/1248/1377/1439, 171/260/306/332/387/390/488/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/1192/1248/1377/1439, 171/260/306/332/387/390/749/750/1194/1248/1377/1439, 171/260/306/332/387/390/619/749/750/1248/1377/1439, 16/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/802/1248/1377/1439, 171/211/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/836/750/1248/1377/1439, 171/260/306/332/387/390/555/749/750/1248/1377/1439, 171/260/306/332/387/390/536/749/750/1248/1377/1439, 171/260/306/332/387/390/527/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/841/1248/1377/1439, 171/260/306/320/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/837/1248/1377/1439, 171/260/306/332/387/390/558/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/914/1248/1377/1439, 171/260/306/312/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/681/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/838/1248/1377/1439, 15/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/1248/1377/1427/1439, 18/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/683/749/750/1248/1377/1439, 24/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/534/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/840/1248/1377/1439, 171/260/306/332/387/390/749/750/1160/1248/1377/1439, 171/260/306/332/387/390/551/749/750/1248/1377/1439, 171/260/306/332/387/390/393/749/750/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/752/811/819/923/1148 1248/1377/1439, 171/260/306/332/387/390/393/749/750/751/775/1248/1377/1439, 63/171/260/306/332/387/390/393/732/749/750/775/1148/1248/1377/1439, 171/260/306/332/387/390/393/732/749/750/1148/1248/1377/1439, 171/260/306/332/387/390/749/750/775/1148/1248/1377/1439, 171/260/306/332/387/390/393/749/750/752/753/775/793/797/1248/1377/1439, 171/260/306/332/387/390/393/749/750/923/1248/1377/1439, 171/260/306/332/387/390/393/749/750/923/1148/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/751/1248/1377/1439, 171/260/306/332/387/390/393/747/749/750/753/775/1148/1248/1377/1439, 171/260/306/332/387/390/749/750/752/923/1051/1248/1377/1439, 63/171/260/306/332/387/390/749/750/775/1248/1377/1439, 63/171/260/306/332/387/390/393/749/750/752/775/793/1051/1053 1248/1377/1439, 171/260/306/332/387/390/749/750/752/753/775/793/819/1248/1377/1439, 63/171/260/306/332/387/390/749/750/1051/1248/1377/1439, 63/171/260/306/332/387/390/747/749/750/752/753/775/811/819/923/1051 1248/1377/1439, 171/260/306/332/387/390/393/749/750/752/787/1248/1377/1439, 171/260/306/332/387/390/749/750/751/1248/1377/1439, 63/171/260/306/332/387/390/749/750/1148/1248/1377/1439, 63/171/260/306/332/387/390/747/749/750/752/753/819/923/1148 1248/1377/1439, 171/260/306/332/387/390/749/750/1148/1248/1377/1439, 171/260/306/332/387/390/747/749/750/752/753/793/811/1248/1377/1439, 63/171/260/306/332/387/390/749/750/1248/1377/1439, 171/260/306/332/387/390/749/750/797/1148/1248/1377/1439, 171/260/306/332/387/390/749/750/751/752/753/1051/1248/1377/1439, 63/171/260/306/332/387/390/732/749/750/1051/1248/1377/1439, 171/260/306/332/387/390/749/750/752/797/815/923/1051/1148 1248/1377/1439, or 171/260/306/332/387/390/747/749/750/753/1248/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 171P/260T/306G/332P/387I/390A/620N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/491E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/491/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/615N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1283 S/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/822S/1248D/1377D/1439E, 171P/260T/306G/328G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/212L/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/818W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1283L/1377D/1439E, 171P/260T/306G/332P/387I/390A/555G/684G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/828K/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/550G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/830M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839A/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/822T/1248D/1377D/1439E, 13Y/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 14G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 17V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836V/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839P/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1210W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1281R/1377D/1439E, 64D/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/212G/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/682R/749F/750D/1248D/1377D/1439E, 14M/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/318C/332P/387I/390A/749F/750D/1248D/1377D/1439E, 17S/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 20L/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/818G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836G/1248D/1377D/1439E, 21M/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/836R/1248D/1377D/1439E, 171P/260T/306G/325Y/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/533V/749F/750D/1248D/1377D/1439E, 19G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 14V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/817D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/831W/1248D/1377D/1439E, 171P/225R/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1156R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/830V/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/A747D/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/753D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/S751Y/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/797W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/622D/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/907D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1040E/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617D/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/611E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/620P/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/617G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/488G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1192D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1194P/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/619E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/615D/749F/750D/1248D/1377D/1439E, 16A/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/802S/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/818V/1248D/1377D/1439E, 171P/L211K/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/836W/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/527A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/841W/1248D/1377D/1439E, 171P/260T/306G/320Q/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/837G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555G/749F/750D/1248D/1377D/1439E, 171P/211R/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 64G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/320R/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/533Y/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/914V/1248D/1377D/1439E, 16V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1283E/1377D/1439E, 171P/260T/306G/332P/387I/390A/536M/749F/750D/1248D/1377D/1439E, 171P/260T/306G/312L/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681N/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/838T/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536R/749F/750D/1248D/1377D/1439E, 15G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536P/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558W/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1427Y/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839G/1248D/1377D/1439E, 18L/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 13G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1210M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1156G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/558L/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/839K/1248D/1377D/1439E, 171P/211P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/211G/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/211Q/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 16D/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/M320G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/683W/749F/750D/1248D/1377D/1439E, 24V/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/534I/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/840G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1160R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681C/749F/750D/1248D/1377D/1439E, 171P/260T/306G/328Q/332P/387I/390A/749F/750D/1248D/1377D/1439E, 15S/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536K/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/533R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681E/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/536W/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1156N/1248D/1377D/1439E, 16P/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/817A/1248D/1377D/1439E, 18R/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/551W/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/555T/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681G/749F/750D/1248D/1377D/1439E, 171P/212A/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 16G/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/838G/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/681R/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/747D/749F/750D/752G/811F/819V/923M/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/751Y/775G/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/775G/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/752L/753D/775G/793G/797W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/923M/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/923M/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/747D/749F/750D/751Y/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/747D/749F/750D/753D/775G/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/752L/923M/1051R/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/775G/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/749F/750D/752L/775G/793G/1051R/1053R 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/752G/753D/775G/793G/819V/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/1051R/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/747D/749F/750D/752G/753D/775G/811F/819V/923M/1051R 1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/393G/749F/750D/752G/787W/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/751Y/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/1148N/1248D/1377D/1439E, 63H1/171P/260T/306G/332P/387I/390A/747D/749F/750D/752G/753D/819V/923M/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/747D/749F/750D/752L/753D/793G/811F/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/749F/750D/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/797W/1148N/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/751Y/752L/753D/1051R/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/732W/749F/750D/1051R/1248D/1377D/1439E, 171P/260T/306G/332P/387I/390A/749F/750D/752G/797W/815I/923M/1051R/1148N 1248D/1377D/1439E, or 171P/260T/306G/332P/387I/390A/747D/749F/750D/753D/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 63Q/171P/260T/306G/332P/387I/390A/393G/681A/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/622D/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/617G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558L/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211Q/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/533R/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751A/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751T/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/951V/775G/1148N/1248D/1377D/1439E, 63Q/164E/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/807L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/735S/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/794R/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/926V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/755P/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/921W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1094S/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/968A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/929Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/924W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/807P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/698D/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/168G/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/929V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818S/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/581K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/698S/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/751G/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916C/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1428G/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/924M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1114M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/311G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737N/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801I/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/751D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/693T/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818E/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/929A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/311E/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737L/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/809W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757A/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1099G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916I/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737F/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737M/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/807C/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916E/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/676Q/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/581V/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/968P/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/755Y/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/735D/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/740G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/698Y/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757G/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/791T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1114A/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/735V/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536P/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/622D/681A/732W/749F/750D/775G/1148N/1248 D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/558L/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536R/555G/617G/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/681R/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/555E/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536R/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/555E/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536P/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/212P/260T/306G/332P/387I/390A/393 G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536K/617G/681A/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211R/260T/306G/332P/387I/390A/393G/536P/558L/617G/681A/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/555G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536P/555E/558L/617G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/617G/681A/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536K/555G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/536P/622D/681A/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320Q/332P/387I/390A/393G/533R/617E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/312L/332P/387I/390A/393G/533R/534I/617D/619E/681N/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/212A/260T/306G/320Q/332P/387I/390A/393G/619E/681G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/312L/320Q/332P/387I/390A/393G/533R/534I/617E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320Q/332P/387I/390A/393G/534I/619E/681G/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/558W/619E/749F/750D/775G/839K/1148N/1248D/1377 D/1439E, 63Q/171P/211K/212A/260T/306G/312L/332P/387I/390A/393G/534I/558W/619E/681N/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/534I/558R/619E/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/617D/619E/681N/732W/749F/750D/775G/1148N/1248 D/1377D/1439E, 63Q/171P/211Q/260T/306G/332P/387I/390A/393G/534I/558W/611E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/320R/332P/387I/390A/393G/617E/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/617D/681G/732W/749F/750D/775G/797W/1148N/1248 D/1377D/1439E, 63Q/171P/260T/306G/320R/332P/387I/390A/393G/534I/619E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/332P/387I/390A/393G/619E/681N/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/211K/212A/260T/306G/332P/387I/390A/393G/534I/558W/619E/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/619E/681N/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/611E/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/558W/619E/681N/732W/749F/750D/775G/1148N/1248 D/1377D/1439E, 63Q/171P/260T/306G/312L/332P/387I/390A/393G/533R/617D/681N/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/611E/681N/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/260T/306G/312L/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211Q/260T/306G/320R/332P/387I/390A/393G/558W/619E/681G/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/558W/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/533R/558W/611E/619E/716L/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/312L/332P/387I/390A/393G/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/320Q/332P/387I/390A/393G/534I/619E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/320Q/332P/387I/390A/393G/533R/536L/619E/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/558W/617E/681N/732W/749F/750D/775G/839K/1148N/1248 D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/533R/534I/681N/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/332P/387I/390A/393G/534I/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/617E/619E/681G/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/211Q/212A/260T/306G/320R/332P/387I/390A/393G/558W/617E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/797W/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/211K/260T/306G/312L/332P/387I/390A/393G/619E/732W/749F/750D/775G/1148N/1248D/1377 D/1439E, 63Q/171P/212A/260T/306G/312L/332P/387I/390A/393G/619E/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/210P/212A/260T/306G/320R/332P/387I/390A/393G/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/312L/332P/387I/390A/393G/534I/681N/732W/749F/750D/775G/797W/839K/1148N/1248D/1377D/1439E, 63Q/171P/211K/260T/306G/320R/332P/387I/390A/393G/558W/732W/749F/750D/775G/839K/1148N/1248 D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/924Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916L/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/757T/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737K/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/818V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/260T/294G/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799E/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/791M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737V/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737S/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916M/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/759S/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/799V/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/538V/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1109G/1148N/1248D/1377D/1439E, 63Q/168R/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/921R/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/926T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/916T/1148N/1248D/1377D/1439E, 63Q/171P/260T/306G/332P/387I/390A/393G/732W/737G/749F/750D/775G/1148N/1248D/1377D/1439E, or 63Q/171P/260T/306G/332P/387I/390A/393G/732W/749F/750D/775G/801R/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1042/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1041/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1051/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1283/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1281/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/916/1148/1248/1377/1439, 63/171/187/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/215/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1050/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1210/1248/1377/1439, 63/104/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/615/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/610/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/618/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/621/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/614/681/732/749/750/775/1148/1248/1377/1439, 63/110/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/301/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1078/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/812/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/786/1148/1248/1377/1439, 63/127/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/175/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1088/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/650/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/535/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/806/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/700/732/749/750/775/1148/1248/1377/1439, 63/132/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1084/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/498/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/756/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1108/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/746/749/750/775/1148/1248/1377/1439, 63/160/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/310/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/584/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/918/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439/1452, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/790/1148/1248/1377/1439, 63/171/212/260/292/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/892/1148/1248/1377/1439, 63/171/212/260/304/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/281/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/917/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/712/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1080/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/806/1148/1248/1352/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1095/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/744/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1098/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/948/1148/1248/1377/1439, 63/119/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/785/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1113/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/667/681/732/749/750/775/1148/1248/1377/1439, 63/158/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/580/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/707/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/731/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1100/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1089/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1092/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/939/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1104/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/736/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1297/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1127/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/686/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1129/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1138/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/786/821/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/680/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/132/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1101/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/808/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/739/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/308/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/801/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/801/1148/1248/1377/1439, 63/171/212/246/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/801/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/839/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/681/732/749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/619/681/732/749/750/775/801/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/619/681/732/749/750/775/799/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/749/750/775/799/801/805/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/616/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/611/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/370/387/390/393/534/558/614/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1156/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1158/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/681/732/737/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/619/681/732/749/750/775/809/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/555/558/619/681/732/737/749/750/775/799/809/839/916/921/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/916/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/536/558/619/681/732/749/750/775/799/916/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/732/749/750/775/807/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/619/681/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/916/1109/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/681/732/749/750/775/921/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/916/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/619/681/732/749/750/775/799/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/555/558/681/732/749/750/775/916/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/839/1148/1248/1377/1439, or 63/171/212/260/306/320/332/387/390/393/534/558/681/732/749/750/775/799/1148/1248/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1042E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1041N/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1051H/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1283H/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1283T/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281Q/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281P/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/916E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1051W/1148N/1248D/1377D/1439E, 63Q/171P/187C/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/215E/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/215P/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/215S/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/178C/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1050T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1210T/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281K/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1042A/1148N/1248D/1377D/1439E, 63Q/104S/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/615D/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610S/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610K/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610T/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/618Y/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/621W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/618P/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614F/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/110H/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/301K/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1078S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/812G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/786C/1148N/1248D/1377D/1439E, 63Q/127V/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/175G/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093W/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1088G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/650M/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/301M/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/535N/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/806R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/132R/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1076G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1084L/1148N/1248D/1377D/1439E, 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63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093N/1148N/1248D/1377D/1439E, 63Q/119G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148 N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1116R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/580Y/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/304A/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712Q/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/806S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/790G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1108V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/731L/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1084N/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1127L/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/308P/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093P/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093Y/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739R/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739P/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/310V/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/712T/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167H/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/555E/558W/619E/681G/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/555E/558W/619E/681G/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/246D/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/555E/558W/619E/681G/732W/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/619E/681G/732W/749F/750D/775G/801G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/619E/681G/732W/749F/750D/775G/799S/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681G/749F/750D/775G/799S/801G/805Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281H/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/750D/775G/1148N/1248 D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614H/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/616D/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/621K/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1050E/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/610H/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/611D/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1042T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/616Q/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614N/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/370A/387I/390A/393G/534I/558W/614A/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1156D/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1210V/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/614T/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1158N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1281S/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/618E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/681N/732W/737K/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536P/558W/619E/681G/732W/749F/750D/775G/809G/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536K/555G/558W/619E/681G/732W/737K/749F/750D/775G/799Q/809G/839K/916T/921R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/536R/558W/619E/681N/732W/749F/750D/775G/799Q/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555G/558W/619E/681N/732W/749F/750D/775G/807R/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/619E/681N/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681N/732W/749F/750D/775G/799S/916T/1109G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555G/558W/681G/732W/749F/750D/775G/921R/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681N/732W/749F/750D/775G/916T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555G/558W/619E/681N/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555E/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/619E/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/555G/558W/681G/732W/749F/750D/775G/916T/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/839K/1148N/1248D/1377D/1439E, or 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/799Q/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1076/1093/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/752/775/800/805/808/809/1116/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/800/809/1076/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/800/805/808/809/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/805/808/809/1076/1116/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1076/1148/1248/1377/1439, 63/167/171/212/260/306/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1076/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/808/809/1148/1248/1377/1439, 63/167/171/L178/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1093/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/752/775/1093/1148/1248/1377/1439, 63/167/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/752/775/805/808/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/799/800/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/752/775/799/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/799/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/799/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/739/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/739/749/750/775/800/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/800/805/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/809/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1076/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1076/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/739/749/750/775/805/808/809/1076/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/499/534/558/681/700/732/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543P/558/681/732/738/739/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543/558/681/732/738/749/750/752/775/1116/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/543/558/681/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/320/332/387/390/393/534/558/681/700/732/749/750/775/1093/1148/1248/1377/1414/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/739/749/750/752/775/799/800/809/1116/1148/1248/1339/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/808/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/844/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/805/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/800/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/800/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/866/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/1116/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/739/749/750/752/775/809/1076/1116/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/800/809/1148/1248/1377/1439, 63/171/178/212/260/306/320/332/387/390/393/534/558/681/732/738/749/750/775/800/808/809/1076/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732/749/750/775/1116/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/752/775/809/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/800/805/808/809/1148/1248/1377/1439, 63/171/178/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/805/809/866/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/739/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/739/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/749/750/775/800/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/749/750/775/809/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/739/749/750/752/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/739/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/739/749/750/752/775/800/805/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/739/749/750/775/800/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/739/749/750/775/805/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/739/749/750/752/775/800/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/775/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/800/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/809/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/543/558/681/700/732/738/749/750/752/775/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/800/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/739/749/750/775/799/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/775/800/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1093/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/799/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/752/775/805/809/1093/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/799/800/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/1116/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/700/732/738/749/750/775/805/808/809/1076/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/514/534/543/558/681/732/738/749/750/775/805/808/809/1148/1248/1377/1439, 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/805/808/809/1148/1248/1377/1439, or 63/171/212/260/306/318/320/332/387/390/393/534/558/681/732/738/749/750/775/805/809/1116/1148/1248/1377/1439, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1076S/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/752E/775G/800V/805Q/808S/809G/1116I/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/809G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/805Q/808S/809G/1076S/1116I/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/800V/808S/809G/1148N/1248D/1377D/1439E, 63Q/167G/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/167G/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/752E/775G/805Q/808S/809G/1093A/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/799S/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/739G/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/752E/775G/799S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/799S/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/749F/750D/775G/799S/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/739G/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/739G/749F/750D/775G/800V/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/805Q/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/809G/1116111148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1076S/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/739G/749F/750D/775G/805Q/808S/809G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/I499L/534I/558W/681G/700E/732W/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/739G/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681N/732W/738E/749F/750D/752E/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/1093A/1148N/1248D/1377D/1414N/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/739G/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/739G/749F/750D/752E/775G/799S/800V/809G/1116I/1148N/1248D/1339C/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/739G/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/800V/805Q/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/752E/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/739G/749F/750D/775G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/752E/775G/808S/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/844H/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/700E/732W/738E/749F/75 OD/775G/805Q/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/800V/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/866A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/739G/749F/750D/752E/775G/809G/1076S/1116I/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/800V/809G/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/800V/808S/809G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681N/732W/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/752E/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/750D/775G/800V/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/178Q/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/805Q/809G/866A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/739G/749F/75 OD/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/749F/750D/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/739G/749F/750D/775G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/75 OD/775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/75 OD/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/75 OD/775G/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/732W/739G/749F/75 OD/752E/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/732W/739G/749F/75 OD/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/732W/738E/739G/749F/750D/752E/775G/800V/805Q/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/739G/749F/750D/775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/739G/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/738E/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681N/700E/732W/738E/749F/750D/752E/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/732W/738E/749F/75 OD/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/739G/749F/750D/752E/775G/800V/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/809G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/543P/558W/681G/700E/732W/738E/749F/750D/752E/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/739G/749F/750D/775G/799S/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/800V/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/749F/750D/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/752E/775G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/752E/775G/808S/809G/1093G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/752E/775G/809G/1093G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/752E/775G/799S/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/752E/775G/805Q/809G/1093A/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/799S/800V/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/1116I/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/700E/732W/738E/749F/75 OD/775G/805Q/808S/809G/1076S/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/514L/534I/543P/558W/681N/732W/738E/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/1439E, 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/805Q/808S/809G/1148N/1248D/1377D/1439E, or 63Q/171P/212A/260T/306G/318G/320R/332P/387I/390A/393G/534I/558W/681G/732W/738E/749F/750D/775G/805Q/809G/1116I/1148N/1248D/1377D/1439E, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises a substitution at an amino acid position set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least one substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at the position(s) set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set of a variant set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, with the proviso that the recombinant factor VIII polypeptide does not include the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or an amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, or 1442T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or an amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G/H/N/P/T/W, or 1439A/E/H/L/S/T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or an amino acid residue 63Q, 171P, 212A, 260T, 306G, 317G, 320R, 332P, 387I, 390A, 393G, 534I, 558W, 681G, 732W, 7338E, 749F, 750D, 775G, 1148N, 1248D, 1377D, or 1439E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 1380W, 388A, 1043E, 1043R, 610R, 1274N, 1220T, 1442T, 1193L, 621T, 1054C, 755Q, 1041G, 748S, 1049G, 1221V, 1051E, 612G, 1037H, 616E, 1041H, 387S, 489R, 621M, 489Q, 750G, 1224P, 618C, 616G, 612T, 1050Q, 1053A, 616W, 896V, 489F, 1050P, 897G, 749T, 1274V, 1220L, 260E, 481V, 613S, 387G, 621R, 756R, 63R, 265S, 1221T, 1380G, 752G, 748K, 755L, 1218A, 748N, 1378E, 614I, 749F, 387V, 750T, 1193G, 749V, 1378A, 750V, 387T, 387Q, 750A, 1037N, 1440A, 1051S, 1221G, 1378D, 1440S, 387R, 1193A, 387I, 1218M, 265W, 1440E, 1439L, 1440T, 612S, 1049W, 1221S, 1220D, 749G, 749S, 1218G, 1205L, 1222F, 1193E, 1050S, 1389W, 1439E, 260T, 1037P, 1051K, 1269F, 265R, 1051R, 1218L, 897R, 388P, 748G, 756P, 748F, 390A, 610L, 755G, 749R, 748V, 1380A, 1440C, 397N/1218M, 1037M, 1218P, 757K, 1373S, 1440G, 1193I, 1439T, 750D, 390G, 756G, 1220E, 1378C, 618G, 346R, 63Q/1248D, 349G, 306G/355C/1422I, 1422I, 306G/346R/708T, 1148N, 171P/346R/1248D, 63Q/797T/799R/1248D/1422I, 171P/799R, 362P, 1248D, 63Q, 171P/306G/346R/999R/1248D, 171P/1148N, 734S/799R/1248D/1422I, 63Q/732E, 63Q/171P/306G/346R/732G, 171P, 171P/1422I, 63Q/362P/732G/799R/1248D, 999R/1148N/1422I, 346R/349G, 306G/349G, 63Q/171P/1148N/1151S, 306G, 344E/349G/354R/1250T, 63G, 1250T, 63R/306G/344E/349G/354R/503S/1248R, 59D/63S/332P/349G/503S/706M/708K, 63S/306G/344A/349G/354R/706M/1248R, 1248R, 59D/63G, 332P/706M/708K/1248R/1250T, 503S, 59D/63R/349G/354R, 59D/349G/708K, 59D/306G/344E/1248R, 344E/1248R/1250T, 708K, 63R/344E/349G, 59D, 63S/344A/349G/354R, 706M, 59D/306G/770W, 63G/347N/354R/1248R, or 354R/503S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set M1380W, I388A, F1043E, F1043R, I610R, I1274N, S1220T, I1442T, F1193L, Q621T, L1054C, A755Q, Y1041G, Y748S, Y1049G, L1221V, M1051E, R612G, F1037H, N616E, Y1041H, F387S, S489R, Q621M, S489Q, L750G, S1224P, A618C, N616G, R612T, I1050Q, T1053A, N616W, I896V, S489F, I1050P, M897G, L749T, I1274V, S1220L, S260E, L481V, F613S, F387G, Q621R, I756R, V63R, I265S, L1221T, M1380G, K752G, Y748K, A755L, F1218A, Y748N, T1378E, L614I, L749F, F387V, L750T, F1193G, L749V, T1378A, L750V, F387T, F387Q, L750A, F1037N, H1440A, M1051S, L1221G, T1378D, H1440S, F387R, F1193A, F387I, F1218M, I265W, H1440E, V1439L, H1440T, R612S, Y1049W, L1221S, S1220D, L749G, L749S, F1218G, I1205L, Y1222F, F1193E, I1050S, S1389W, V1439E, S260T, F1037P, M1051K, I1269F, I265R, M1051R, F1218L, M897R, I388P, Y748G, I756P, Y748F, I390A, I610L, A755G, L749R, Y748V, M1380A, H1440C, H397N/F1218M, F1037M, F1218P, E757K, V1373S, H1440G, F1193I, V1439T, L750D, I390G, I756G, S1220E, T1378C, A618G, D346R, V63Q/L1248D, P349G, E306G/R355C/L1422I, L1422I, E306G/D346R/I708T, T1148N, L171P/D346R/L1248D, V63Q/M797T/K799R/L1248D/L1422I, L171P/K799R, A362P, L1248D, V63Q, L171P/E306G/D346R/Q999R/L1248D, L171P/T1148N, T734S/K799R/L1248D/L1422I, V63Q/K732E, V63Q/L171P/E306G/D346R/K732G, L171P, L171P/L1422I, V63Q/A362P/K732G/K799R/L1248D, Q999R/T1148N/L1422I, D346R/P349G, E306G/P349G, V63Q/L171P/T1148N/G1151S, E306G, K344E/P349G/L354R/V1250T, V63G, V1250T, V63R/E306G/K344E/P349G/L354R/R503S/L1248R, F59D/V63S/S332P/P349G/R503S/L706M/I708K, V63S/E306G/K344A/P349G/L354R/L706M/L1248R, L1248R, F59D/V63G, S332P/L706M/I708K/L1248R/V1250T, R503S, F59D/V63R/P349G/L354R, F59D/P349G/I708K, F59D/E306G/K344E/L1248R, K344E/L1248R/V1250T, I708K, V63R/K344E/P349G, F59D, V63S/K344A/P349G/L354R, L706M, F59D/E306G/R770W, V63G/S347N/L354R/L1248R, or L354R/R503S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or to the reference sequence corresponding to SEQ ID NO: 504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 750D, 1051K, 1051S, 260T, 756P, 1193A, 332P, 897R, 387Q, 1037N, 387R, 306G/749F, 1270V, 489E, 487N, 488G, 903E, 171P/1248D/1440T, 260T/1148N/1378D, 171P/260T/387I/750D/1248D, 171P/260T, 171P/260T/387I/750D/1051S/1148N, 171P, 750D/1051S/1148N, 171P/260T/756P, 171P/1248D, 171P/1148N, 387I, 59D, 1440S, 332P/750D/1248D, 756P/1248D, 1248D, 171P/387I/1248D/1440S, 750D/1440S, 59D/171P/750D, 171P/1193A/1248D, 750D/1148N/1193A/1440S, 1148N, 171P/387I/1051K, 748G, 306G, 306G/387R/390A/749F/1220D, 390A, 306G/390A/1037N, 306G/612S/749F, 387V/390A/749F, 63S/390A/503S/749F, 387R/390A/749R, 1220D, 63S/306G, 749F, 306G/390A/749F/1037N, 387V, 306G/749F/1220D, 306G/387V/390A/612S/749F/1037N, or 328S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set L750D, M1051K, M1051S, S260T, I756P, F1193A, S332P, M897R, F387Q, F1037N, F387R, E306G/L749F, I1270V, S489E, Q487N, A488G, Q903E, L171P/L1248D/H1440T, S260T/T1148N/T1378D, L171P/S260T/F387I/L750D/L1248D, L171P/S260T, L171P/S260T/F387I/L750D/M1051S/T1148N, L171P, L750D/M1051S/T1148N, L171P/S260T/I756P, L171P/L1248D, L171P/T1148N, F387I, F59D, H1440S, S332P/L750D/L1248D, I756P/L1248D, L1248D, L171P/F387I/L1248D/H1440S, L750D/H1440S, F59D/L171P/L750D, L171P/F1193A/L1248D, L750D/T1148N/F1193A/H1440S, T1148N, L171P/F387I/M1051K, Y748G, E306G, E306G/F387R/I390A/L749F/S1220D, I390A, E306G/I390A/F1037N, E306G/R612S/L749F, F387V/I390A/L749F, V63S/I390A/R503S/L749F, F387R/I390A/L749R, S1220D, V63S/E306G, L749F, E306G/I390A/L749F/F1037N, F387V, E306G/L749F/S1220D, E306G/F387V/I390A/R612S/L749F/F1037N, or F328S, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or to the reference sequence corresponding to SEQ ID NO: 706, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least a substitution or substitution set 1051K, 1221G, 1218G, 387R, 1051R, 749F/1218G, 387T, 332P, 897K, 696E, 816W, 824W, 826G, 393W, 825L, 897R, 816V, 698W, 393Q, 332P/387R/390A/1148N, 328S/387R/749F, 749F, 306G/328S/332P/390A/1051R/1193A/1218G, 63Q/306G/332P/1051R/1148N, 63Q/306G/332P/387T/1051K, 387T/390A/1051R, 186A/306G/328S/332P/390A/749F/1051K/1148N/1193A, 387T/390A/756P/1051R, 306G/390A/1051R, 306G/332P/390A/749F, 306G/387R/390A/1148N, 306G/328S/332P/387T/390A/749F/1051R/1148N/1193A, 332P/387R/390A/749F/1148N, 387R/390A/749F, 306G/387T/749F/1051K/1148N, 387R/749F, 306G/945K/1051R/1193A/1221G, 306G/390A/749F/1051K, 63Q/306G/332P/390A, 306G/387T/390A/749F/1148N/1193A/1221G, 306G/332P/387R/390A/749F/1218M, 63Q/306G/332P/390A/1051R/1148N/1193A/1218G, 924I, 755S, 752F, 751Y, 923M, 797Y, 754W, 793G, 753D, 922W, 896L, 938R, 775G, 751E, 732W, 752G, 810V, 938V, 747D, 755G, 393G/407V, 797W, 819V, 393G, 787W, 393R, 802V, 775E, 924P, 732G, 811F, 802S, 752L, 392A, or 397G, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set M1051K, L1221G, F1218G, I387R, M1051R, L749F/F1218G, I387T, S332P, M897K, T696E, F816W, F824W, A826G, V393W, I825L, M897R, F816V, F698W, V393Q, S332P/I387R/I390A/T1148N, F328S/I387R/L749F, L749F, E306G/F328S/S332P/I390A/M1051R/F1193A/F1218G, V63Q/E306G/S332P/M1051R/T1148N, V63Q/E306G/S332P/I387T/M1051K, I387T/I390A/M1051R, D186A/E306G/F328S/S332P/I390A/L749F/M1051K/T1148N/F1193A, I387T/I390A/I756P/M1051R, E306G/I390A/M1051R, E306G/S332P/I390A/L749F, E306G/I387R/I390A/T1148N, E306G/F328S/S332P/I387T/I390A/L749F/M1051R/T1148N/F1193A, S332P/I387R/I390A/L749F/T1148N, I387R/I390A/L749F, E306G/I387T/L749F/M1051K/T1148N, I387R/L749F, E306G/Q945K/M1051R/F1193A/L1221G, E306G/I390A/L749F/M1051K, V63Q/E306G/S332P/I390A, E306G/I387T/I390A/L749F/T1148N/F1193A/L1221G, E306G/S332P/I387R/I390A/L749F/F1218M, V63Q/E306G/S332P/I390A/M1051R/T1148N/F1193A/F1218G, G924I, A755S, K752F, S751Y, Q923M, M797Y, N754W, I793G, N753D, R922W, I896L, K938R, I775G, S751E, K732W, K752G, N810V, K938V, A747D, A755G, V393G/A407V, M797W, K819V, V393G, I787W, V393R, F802V, I775E, G924P, K732G, Q811F, F802S, K752L, S392A, or H397G, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840, or to the reference sequence corresponding to SEQ ID NO: 840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 840, or relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 620N, 491E, 1229D, 615N, 1283S, 822S, 328G, 212L, 818W, 1283L, 555G/684G, 828K, 550G, 830M, 839A, 822T, 13Y, 14G, 17V, 836V, 839P, 1210W, 1281R, 64D, 212G, 682R, 14M, 318C, 17S, 20L, 818G, 836G, 21M, 836R, 325Y, 533V, 19G, 14V, 817D, 831W, 225R, 1156R, 830V, 393G/747D, 753D, 393G/732W/751Y, 63Q/797W, 622D, 617E, 617N, 907D, 1040E, 617D, 611E, 620P, 617G, 488G, 1192D, 1194P, 619E, 615D, 16A, 802S, 818V, 211K, 836W, 555R, 536A, 527A, 555E, 841W, 320Q, 837G, 558R, 555G, 211R, 64G, 320R, 533Y, 914V, 16V, 1283E, 536M, 312L, 681N, 838T, 536R, 15G, 536P, 558W, 558E, 1427Y, 839G, 18L, 13G, 1210M, 1156G, 558L, 839K, 211P, 211G, 211Q, 16D, 320G, 683W, 24V, 534I, 681A, 840G, 1160R, 681C, 328Q, 15S, 536K, 536G, 533R, 681E, 536W, 1156N, 16P, 817A, 18R, 551W, 555T, 681G, 212A, 16G, 838G, 681R, 393G, 393G/747D/752G/811F/819V/923M/1148N, 393G/751Y/775G, 63Q/393G/732W/775G/1148N, 393G/732W/1148N, 775G/1148N, 393G/752L/753D/775G/793G/797W, 393G/923M, 393G/923M/1148N, 393G/747D/751Y, 393G/747D/753D/775G/1148N, 752L/923M/1051R, 63Q/775G, 63Q/393G/752L/775G/793G/1051R/1053R, 752G/753D/775G/793G/819V, 63Q/1051R, 63Q/747D/752G/753D/775G/811F/819V/923M/1051R, 393G/752G/787W, 751Y, 63Q/1148N, 63H/747D/752G/753D/819V/923M/1148N, 1148N, 747D/752L/753D/793G/811F, 63Q, 797W/1148N, 751Y/752L/753D/1051R, 63Q/732W/1051R, 752G/797W/815I/923M/1051R/1148N, or 747D/753D, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set V620N, P491E, M1229D, P615N, I1283S, H822S, F328G, H212L, K818W, I1283L, F555G/E684G, V828K, R550G, R830M, S839A, H822T, L13Y, L14G, C17V, M836V, S839P, K1210W, Y1281R, V64D, H212G, V682R, L14M, L318C, C17S, A20L, K818G, M836G, T21M, M836R, F325Y, T533V, S19G, L14V, Q817D, L831W, K225R, H1156R, R830V, V393G/A747D, N753D, V393G/K732W/S751Y, V63Q/M797W, L622D, P617E, P617N, P907D, N1040E, P617D, Q611E, V620P, P617G, A488G, P1192D, S1194P, G619E, P615D, F16A, F802S, K818V, L211K, M836W, F555R, V536A, I527A, F555E, H841W, M320Q, S837G, M558R, F555G, L211R, V64G, M320R, T533Y, L914V, F16V, I1283E, V536M, F312L, M681N, S838T, V536R, R15G, V536P, M558W, M558E, L1427Y, S839G, F18L, L13G, K1210M, H1156G, M558L, S839K, L211P, L211G, L211Q, F16D, M320G, Y683W, Y24V, V534I, M681A, P840G, F1160R, M681C, F328Q, R15S, V536K, V536G, T533R, M681E, V536W, H1156N, F16P, Q817A, F18R, Y551W, F555T, M681G, H212A, F16G, S838G, M681R, V393G, V393G/A747D/K752G/Q811F/K819V/Q923M/T1148N, V393G/S751Y/I775G, V63Q/V393G/K732W/I775G/T1148N, V393G/K732W/T1148N, I775G/T1148N, V393G/K752L/N753D/I775G/I793G/M797W, V393G/Q923M, V393G/Q923M/T1148N, V393G/A747D/S751Y, V393G/A747D/N753D/I775G/T1148N, K752L/Q923M/M1051R, V63Q/I775G, V63Q/V393G/K752L/I775G/I793G/M1051R/T1053R, K752G/N753D/I775G/I793G/K819V, V63Q/M1051R, V63Q/A747D/K752G/N753D/I775G/Q811F/K819V/Q923M/M1051R, V393G/K752G/I787W, S751Y, V63Q/T1148N, V63H/A747D/K752G/N753D/K819V/Q923M/T1148N, T1148N, A747D/K752L/N753D/I793G/Q811F, V63Q, M797W/T1148N, S751Y/K752L/N753D/M1051R, V63Q/K732W/M1051R, K752G/M797W/S815I/Q923M/M1051R/T1148N, or A747D/N753D, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200, or to the reference sequence corresponding to SEQ ID NO: 1200, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1200, or relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 681A, 622D, 617G, 558L, 536K, 681G, 211Q, 320R, 558W, 839K, 211K, 797W, 533R, 751A, 303T, 951V, 164E, 818G, 807L, 799A, 735S, 794R, 926V, 755P, 921W, 801A, 1094S, 968A, 929Q, 924W, 807P, 698D, 168G, 929V, 818A, 818S, 581K, 818P, 916K, 698S, 303G, 916C, 1428G, 805L, 924M, 1114M, 311G, 737N, 805V, 801I, 751D, 693T, 818E, 929A, 311E, 737L, 809W, 757A, 1099G, 799G, 916I, 805G, 737F, 737M, 799P, 807C, 916E, 676Q, 581V, 968P, 755Y, 916Q, 735D, 740G, 698Y, 757G, 791T, 1114A, 735V, 536P, 211R/536K/622D/681A, 536K/558L, 211R/536R/555G/617G/732K, 211R/681R/732K, 211R/536K/555E, 536R, 555E, 211R/536P, 211R/212P, 211R/536K, 211R/536K/617G/681A/732K, 211R, 211R/536P/558L/617G/681A, 555G/732K, 536P/555E/558L/617G/732K, 536K/617G/681A/732K, 536K/555G, 536P/622D/681A, 732K, 212A/320Q/533R/617E/681G, 211K/312L/533R/534I/617D/619E/681N/839K, 212A, 211K/212A/320Q/619E/681G/839K, 797W/S839K, 212A/312L/320Q/533R/534I/617E/681G, 558W/619E/681G, 320Q/558W/681G, 212A/320Q/534I/619E/681G/797W/839K, 320R/558W/619E/732K/839K, 211K/212A/312L/534I/558W/619E/681N/732K/839K, 320R/534I/558R/619E/732K/797W, 558W/617D/619E/681N, 211Q/534I/558W/611E/681N, 211K/320R/617E/619E/681G, 558W/617D/681G/797W, 320R/534I/619E/732K, 211K/619E/681N, 211K/212A/534I/558W/619E/732K/839K, 212A/320R/619E/681N/797W/839K, 212A/320R/611E/839K, 320Q/558W/619E/681N, 312L/533R/617D/681N/797W/839K, 558W/611E/681N, 312L, 211Q/320R/558W/619E/681G/732K/797W/839K, 320Q/558W/732K/797W, 533R/558W/611E/619E/716L/732K/839K, 312L/681G, 320Q/534I/619E/681N, 211K/320Q/533R/536L/619E, 558W/617E/681N/839K, 212A/320R/534I/558W/681G, 533R/534I/681N/839K, 212A/534I/619E/681G, 617E/619E/681G, 211Q/212A/320R/558W/617E/681N, 212A/534I/558W/681N/797W, 212A/320R/534I, 211K/312L/619E, 212A/312L/619E/839K, 210P/212A/320R/558W/681G, 212A/312L/534I/681N/797W/839K, 211K/320R/558W/839K, 924Q, 916L, 757T, 737K, 818V, 801G, 294G, 799S, 799E, 809G, 791M, 737V, 805Q, 799Q, 737S, 916M, 759S, 799V, 538V, 1109G, 168R, 921R, 926T, 916T, 737G, or 801R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set M681A, L622D, P617G, M558L, V536K, M681G, L211Q, M320R, M558W, S839K, L211K, M797W, T533R, S751A, A303T, T951V, G164E, K818G, E807L, K799A, G735S, S794R, E926V, A755P, Q921W, D801A, E1094S, L968A, K929Q, G924W, E807P, F698D, S168G, K929V, K818A, K818S, R581K, K818P, S916K, F698S, A303G, S916C, T1428G, Y805L, G924M, L1114M, T311G, Y737N, Y805V, D801I, S751D, S693T, K818E, K929A, T311E, Y737L, E809W, E757A, A1099G, K799G, S916I, Y805G, Y737F, Y737M, K799P, E807C, S916E, T676Q, R581V, L968P, A755Y, S916Q, G735D, D740G, F698Y, E757G, D791T, L1114A, G735V, V536P, L211R/V536K/L622D/M681A, V536K/M558L, L211R/V536R/F555G/P617G/W732K, L211R/M681R/W732K, L211R/V536K/F555E, V536R, F555E, L211R/V536P, L211R/H212P, L211R/V536K, L211R/V536K/P617G/M681A/W732K, L211R, L211R/V536P/M558L/P617G/M681A, F555G/W732K, V536P/F555E/M558L/P617G/W732K, V536K/P617G/M681A/W732K, V536K/F555G, V536P/L622D/M681A, W732K, H212A/M320Q/T533R/P617E/M681G, L211K/F312L/T533R/V534I/P617D/G619E/M681N/S839K, H212A, L211K/H212A/M320Q/G619E/M681G/S839K, M797W/S839K, H212A/F312L/M320Q/T533R/V534I/P617E/M681G, M558W/G619E/M681G, M320Q/M558W/M681G, H212A/M320Q/V534I/G619E/M681G/M797W/S839K, M320R/M558W/G619E/W732K/S839K, L211K/H212A/F312L/V534I/M558W/G619E/M681N/W732K/S839K, M320R/V534I/M558R/G619E/W732K/M797W, M558W/P617D/G619E/M681N, L211Q/V534I/M558W/Q611E/M681N, L211K/M320R/P617E/G619E/M681G, M558W/P617D/M681G/M797W, M320R/V534I/G619E/W732K, L211K/G619E/M681N, L211K/H212A/V534I/M558W/G619E/W732K/S839K, H212A/M320R/G619E/M681N/M797W/S839K, H212A/M320R/Q611E/S839K, M320Q/M558W/G619E/M681N, F312L/T533R/P617D/M681N/M797W/S839K, M558W/Q611E/M681N, F312L, L211Q/M320R/M558W/G619E/M681G/W732K/M797W/S839K, M320Q/M558W/W732K/M797W, T533R/M558W/Q611E/G619E/F716L/W732K/S839K, F312L/M681G, M320Q/V534I/G619E/M681N, L211K/M320Q/T533R/V536L/G619E, M558W/P617E/M681N/S839K, H212A/M320R/V534I/M558W/M681G, T533R/V534I/M681N/S839K, H212A/V534I/G619E/M681G, P617E/G619E/M681G, L211Q/H212A/M320R/M558W/P617E/M681N, H212A/V534I/M558W/M681N/M797W, H212A/M320R/V534I, L211K/F312L/G619E, H212A/F312L/G619E/S839K, T210P/H212A/M320R/M558W/M681G, H212A/F312L/V534I/M681N/M797W/S839K, L211K/M320R/M558W/S839K, G924Q, S916L, E757T, Y737K, K818V, D801G, T294G, K799S, K799E, E809G, D791M, Y737V, Y805Q, K799Q, Y737S, S916M, R759S, K799V, D538V, E1109G, S168R, Q921R, E926T, S916T, Y737G, or D801R, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 1200.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514, or to the reference sequence corresponding to SEQ ID NO: 1514, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1514, or relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 1042E, 1041N, 1051H, 1283H, 1283T, 1281Q, 1281P, 916E, 1051W, 187C, 215E, 215P, 215S, 178C, 1050T, 1210T, 1281K, 1042A, 104S, 615D, 610S, 610K, 610T, 536P, 618Y, 621W, 618P, 614F, 110H, 301K, 1078S, 812G, 786C, 127V, 175G, 1093W, 1088G, 1093T, 650M, 301M, 535N, 806R, 738E, 700E, 132R, 1076G, 1084L, 498A, 786L, 738F, 535G, 806E, 756G, 1108S, 1093V, 746P, 160E, 160T, 178V, 543L, 310G, 700Q, 1093A, 584A, 918S, 1452L, 738P, 790W, 790I, 292D, 800M, 1108M, 1088S, 167G, 892T, 790P, 800R, 738K, 304G, 1116G, 178Q, 1116A, 752G, 1108W, 738D, 281A, 917G, 712M, 1080V, 806G/1352E, 1095G, 1093G, 744A, 543G, 1098G, 281S, 756T, 948S, 119V, 1116L, 785V, 1113F, 700A, 786D, 175A, 790M, 667I, 158G, 580S, 707H, 731I, 918M, 1100S, 1089S, 712Y, 175H, 1092G, 939E, 1104E, 800G, 736F, 543W, 1297A, 712L, 1127V, 939S, 178M, 731R, 686W, 543P, 736G, 1129P, 1138Q, 812V, 786D/821Q, 535C, 680R, 1116I, 1089F, 304E, 178W, 1104C, 744R, 738R, 1093S, 712S, 187G, 132P, 812I, 1080G, 800C, 1101G, 808K, 808S, 790A, 800V, 680L, 167R, 752E, 1093N, 119G, 739G, 1116R, 580Y, 304A, 712Q, 806S, 712G, 790G, 1108V, 731L, 1084N, 1127L, 308P, 1076S, 1093P, 1093Y, 739R, 739P, 310V, 712T, 167H, 1093R, 536P/555E/619E/732K/799S/805Q, 555E/619E/801G, 555E/619E/799S, 619E/799S/801G/805Q, 536P/555E/619E/732K/799S, 799S/805Q, 801G, 246D/619E/799S/801G, 536P/555E/619E, 619E/839K, 619E/799S/805Q, 536P/801G/805Q, 555E/619E/799S/805Q, 619E/801G/805Q, 555E/619E, 536P/619E/801G, 801G/805Q, 536P/619E/799S/805Q, 555E/619E/732K/799S/801G/805Q, 1281H, 749L, 614H, 616D, 621K, 1050E, 610H, 611D, 1042T, 616Q, 614N, 370A/614A, 1156D, 1210V, 614T, 1158N, 1281S, 618E, 555E/681N/737K/799Q, 536P/619E/809G/1109G, 536K/555G/619E/737K/799Q/809G/839K/916T/921R, 916T/1109G, 536R/619E/681N/799Q/916T/1109G, 555G/619E/681N/807R/1109G, 555E/619E/681N/737K/799Q, 619E/681N/799S/916T/1109G, 619E/681N, 555G/921R, 619E/681N/916T, 555G/619E/681N/799S, 555E, 732K, 619E/799S, 619E, 681N, 555G/916T, 839K, or 799Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set R1042E, Y1041N, M1051H, I1283H, I1283T, Y1281Q, Y1281P, S916E, M1051W, L187C, I215E, I215P, I215S, L178C, I1050T, K1210T, Y1281K, R1042A, V104S, P615D, I610S, I610K, I610T, V536P, A618Y, Q621W, A618P, L614F, M110H, R301K, I1078S, S812G, E786C, A127V, Y175G, K1093W, F1088G, K1093T, L650M, R301M, T535N, D806R, Y738E, S700E, E132R, E1076G, S1084L, G498A, E786L, Y738F, T535G, D806E, I756G, F1108S, K1093V, S746P, L160E, L160T, L178V, S543L, 1310G, S700Q, K1093A, Q584A, E918S, E1452L, Y738P, D790W, D790I, G292D, E800M, F1108M, F1088S, A167G, V892T, D790P, E800R, Y738K, S304G, S1116G, L178Q, S1116A, K752G, F1108W, Y738D, T281A, Y917G, H712M, S1080V, D806G/K1352E, E1095G, K1093G, D744A, S543G, M1098G, T281S, I756T, M948S, A119V, S1116L, E785V, M1113F, S700A, E786D, Y175A, D790M, F667I, Q158G, Q580S, W707H, D731I, E918M, L1100S, T1089S, H712Y, Y175H, K1092G, T939E, Y1104E, E800G, D736F, S543W, N1297A, H712L, 11127V, T939S, L178M, D731R, T686W, S543P, D736G, E1129P, L1138Q, S812V, E786D/R821Q, T535C, K680R, S1116I, T1089F, S304E, L178W, Y1104C, D744R, Y738R, K1093S, H712S, L187G, E132P, S812I, S1080G, E800C, Y1101G, D808K, D808S, D790A, E800V, K680L, A167R, K752E, K1093N, A119G, E739G, S1116R, Q580Y, S304A, H712Q, D806S, H712G, D790G, F1108V, D731L, S1084N, I1127L, S308P, E1076S, K1093P, K1093Y, E739R, E739P, I310V, H712T, A167H, K1093R, V536P/F555E/G619E/W732K/K799S/Y805Q, F555E/G619E/D801G, F555E/G619E/K799S, G619E/K799S/D801G/Y805Q, V536P/F555E/G619E/W732K/K799S, K799S/Y805Q, D801G, A246D/G619E/K799S/D801G, V536P/F555E/G619E, G619E/S839K, G619E/K799S/Y805Q, V536P/D801G/Y805Q, F555E/G619E/K799S/Y805Q, G619E/D801G/Y805Q, F555E/G619E, V536P/G619E/D801G, D801G/Y805Q, V536P/G619E/K799S/Y805Q, F555E/G619E/W732K/K799S/D801G/Y805Q, Y1281H, F749L, L614H, N616D, Q621K, I1050E, I610H, Q611D, R1042T, N616Q, L614N, T370A/L614A, H1156D, K1210V, L614T, R1158N, Y1281S, A618E, F555E/G681N/Y737K/K799Q, V536P/G619E/E809G/E1109G, V536K/F555G/G619E/Y737K/K799Q/E809G/S839K/S916T/Q921R, S916T/E1109G, V536R/G619E/G681N/K799Q/S916T/E1109G, F555G/G619E/G681N/E807R/E1109G, F555E/G619E/G681N/Y737K/K799Q, G619E/G681N/K799S/S916T/E1109G, G619E/G681N, F555G/Q921R, G619E/G681N/S916T, F555G/G619E/G681N/K799S, F555E, W732K, G619E/K799S, G619E, G681N, F555G/S916T, S839K, or K799Q, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072, or to the reference sequence corresponding to SEQ ID NO: 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set 167G, 167G/738Y/1076S/1093G, 167G/738Y/752E/800V/805Q/808S/809G/1116I, 167G/800V/809G/1076S, 167G/318L, 167G/318L/800V/805Q/808S/809G, 167G/318L/681N, 167G/318L/681N/700E/805Q/808S/809G/1076S/1116I, 167G/318L/700E, 167G/318L/700E/1076S, 167G/318L/700E/738Y/739G/1076S, 167G/681N/738Y/800V/808S/809G, 167G/178Q/700E/752E/1093G, 167G/700E, 167G/700E/738Y/739G/1093G, 167G/700E/738Y/752E/1093G, 167G/700E/738Y/752E/805Q/808S/809G/1093A/1116I, 808S/809G, 1076S, 738Y, 738Y/808S/809G, 738Y/1076S, 738Y/739G, 738Y/739G/809G, 738Y/739G/809G/1093G/1116I, 738Y/739G/1093G, 738Y/739G/752E/1093G, 738Y/739G/799S/800V/1093G, 738Y/739G/1116I, 738Y/800V, 738Y/1093G, 738Y/752E, 738Y/752E/1093G, 738Y/752E/799S, 738Y/799S/809G/1093G, 738Y/799S/1093G, 739G, 739G/800V/809G, 800V, 800V/805Q/809G, 809G, 809G/1093G, 809G/1093G/1116I, 809G/1116I, 318L, 318L/1076S, 318L/1076S/1116I, 318L/739G/805Q/808S/809G/1076S, 318L/499L/700E/738Y/809G, 318L/1093A, 318L/752E/1093A, 318L/1116I, 318L/543P/739G, 318L/543P/681N/752E/1116I, 318L/543P/1093A, 318L/700E/738Y/1093A/1414N, 681N, 681N/738Y, 681N/738Y/739G/1093G, 681N/738Y/739G/752E/799S/800V/809G/1116I/1339C, 681N/738Y/739G/1116I, 681N/738Y/800V, 681N/800V/805Q/809G, 681N/809G/1093G, 681N/752E/1093A, 681N/752E/1093G, 681N/700E, 681N/700E/738Y/739G/1093G/1116I, 681N/700E/809G, 681N/700E/1093G, 681N/700E/752E/808S/809G/1093G/1116I, 681N/700E/752E/1093G, 681N/700E/844H, 681N/700E/805Q/809G, 1093A, 1093G, 1093G/1116I, 752E, 752E/1076S, 752E/800V/809G, 752E/800V/1093G, 752E/809G, 752E/866A, 752E/1093G, 752E/1116I, 178Q/318L/739G/752E/809G/1076S/1116I, 178Q/318L/800V/809G, 178Q/318L/681N/800V/808S/809G/1076S, 178Q/681N, 178Q/681N/738Y/1116I, 178Q/752E/809G, 178Q/700E/800V/805Q/808S/809G, 178Q/805Q/809G/866A, 1116I, 543P, 543P/738Y/739G/809G, 543P/738Y/809G/1093G, 543P/739G/1093A, 543P/800V/1093G/1116I, 543P/809G/1093G, 543P/809G/1093G/1116I, 543P/681N/738Y/739G/752E/809G/1093G, 543P/681N/738Y/739G/752E/1093G, 543P/681N/739G/752E/800V/805Q/809G/1093G, 543P/681N/700E/738Y/739G/800V/1093G/1116I, 543P/681N/700E/738Y/739G/805Q/808S/809G, 543P/681N/700E/752E, 543P/681N/700E/752E/809G/1093G, 543P/1093G, 543P/700E, 543P/700E/738Y/739G/752E/800V/1093G/1116I, 543P/700E/809G, 543P/700E/1093G, 543P/700E/752E, 543P/700E/752E/800V/1093G, 543P/700E/752E/809G/1116I, 543P/700E/752E/1093G, 700E, 700E/738Y/739G/800V/1093G, 700E/738Y/739G/799S/1093G, 700E/738Y/800V/1093G, 700E/738Y/1116I, 700E/809G, 700E/809G/1093G, 700E/1093G, 700E/1093G/1116I, 700E/752E, 700E/752E/808S/809G/1093G/1116I, 700E/752E/809G/1093G, 700E/752E/799S/1116I, 700E/752E/805Q/809G/1093A, 700E/799S/800V, 700E/1116I, 700E/805Q/808S/809G/1076S, 514L/543P/681N/805Q/808S/809G, 805Q/808S/809G, or 805Q/809G/1116I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set A167G, A167G/E738Y/E1076S/K1093G, A167G/E738Y/K752E/E800V/Y805Q/D808S/E809G/S1116I, A167G/E800V/E809G/E1076S, A167G/G318L, A167G/G318L/E800V/Y805Q/D808S/E809G, A167G/G318L/G681N, A167G/G318L/G681N/S700E/Y805Q/D808S/E809G/E1076S/S1116I, A167G/G318L/S700E, A167G/G318L/S700E/E1076S, A167G/G318L/S700E/E738Y/E739G/E1076S, A167G/G681N/E738Y/E800V/D808S/E809G, A167G/L178Q/S700E/K752E/K1093G, A167G/S700E, A167G/S700E/E738Y/E739G/K1093G, A167G/S700E/E738Y/K752E/K1093G, A167G/S700E/E738Y/K752E/Y805Q/D808S/E809G/K1093A/S1116I, D808S/E809G, E1076S, E738Y, E738Y/D808S/E809G, E738Y/E1076S, E738Y/E739G, E738Y/E739G/E809G, E738Y/E739G/E809G/K1093G/S1116I, E738Y/E739G/K1093G, E738Y/E739G/K752E/K1093G, E738Y/E739G/K799S/E800V/K1093G, E738Y/E739G/S1116I, E738Y/E800V, E738Y/K1093G, E738Y/K752E, E738Y/K752E/K1093G, E738Y/K752E/K799S, E738Y/K799S/E809G/K1093G, E738Y/K799S/K1093G, E739G, E739G/E800V/E809G, E800V, E800V/Y805Q/E809G, E809G, E809G/K1093G, E809G/K1093G/S1116I, E809G/S1116I, G318L, G318L/E1076S, G318L/E1076S/S1116I, G318L/E739G/Y805Q/D808S/E809G/E1076S, G318L/I499L/S700E/E738Y/E809G, G318L/K1093A, G318L/K752E/K1093A, G318L/S1116I, G318L/S543P/E739G, G318L/S543P/G681N/K752E/S1116I, G318L/S543P/K1093A, G318L/S700E/E738Y/K1093A/S1414N, G681N, G681N/E738Y, G681N/E738Y/E739G/K1093G, G681N/E738Y/E739G/K752E/K799S/E800V/E809G/S1116I/G1339C, G681N/E738Y/E739G/S1116I, G681N/E738Y/E800V, G681N/E800V/Y805Q/E809G, G681N/E809G/K1093G, G681N/K752E/K1093A, G681N/K752E/K1093G, G681N/S700E, G681N/S700E/E738Y/E739G/K1093G/S1116I, G681N/S700E/E809G, G681N/S700E/K1093G, G681N/S700E/K752E/D808S/E809G/K1093G/S1116I, G681N/S700E/K752E/K1093G, G681N/S700E/R844H, G681N/S700E/Y805Q/E809G, K1093A, K1093G, K1093G/S1116I, K752E, K752E/E1076S, K752E/E800V/E809G, K752E/E800V/K1093G, K752E/E809G, K752E/G866A, K752E/K1093G, K752E/S1116I, L178Q/G318L/E739G/K752E/E809G/E1076S/S1116I, L178Q/G318L/E800V/E809G, L178Q/G318L/G681N/E800V/D808S/E809G/E1076S, L178Q/G681N, L178Q/G681N/E738Y/S1116I, L178Q/K752E/E809G, L178Q/S700E/E800V/Y805Q/D808S/E809G, L178Q/Y805Q/E809G/G866A, S1116I, S543P, S543P/E738Y/E739G/E809G, S543P/E738Y/E809G/K1093G, S543P/E739G/K1093A, S543P/E800V/K1093G/S1116I, S543P/E809G/K1093G, S543P/E809G/K1093G/S1116I, S543P/G681N/E738Y/E739G/K752E/E809G/K1093G, S543P/G681N/E738Y/E739G/K752E/K1093G, S543P/G681N/E739G/K752E/E800V/Y805Q/E809G/K1093G, S543P/G681N/S700E/E738Y/E739G/E800V/K1093G/S1116I, S543P/G681N/S700E/E738Y/E739G/Y805Q/D808S/E809G, S543P/G681N/S700E/K752E, S543P/G681N/S700E/K752E/E809G/K1093G, S543P/K1093G, S543P/S700E, S543P/S700E/E738Y/E739G/K752E/E800V/K1093G/S1116I, S543P/S700E/E809G, S543P/S700E/K1093G, S543P/S700E/K752E, S543P/S700E/K752E/E800V/K1093G, S543P/S700E/K752E/E809G/S1116I, S543P/S700E/K752E/K1093G, S700E, S700E/E738Y/E739G/E800V/K1093G, S700E/E738Y/E739G/K799S/K1093G, S700E/E738Y/E800V/K1093G, S700E/E738Y/S1116I, S700E/E809G, S700E/E809G/K1093G, S700E/K1093G, S700E/K1093G/S1116I, S700E/K752E, S700E/K752E/D808S/E809G/K1093G/S1116I, S700E/K752E/E809G/K1093G, S700E/K752E/K799S/S1116I, S700E/K752E/Y805Q/E809G/K1093A, S700E/K799S/E800V, S700E/S1116I, S700E/Y805Q/D808S/E809G/E1076S, V514L/S543P/G681N/Y805Q/D808S/E809G, Y805Q/D808S/E809G, or Y805Q/E809G/S1116I, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising at least a substitution at an amino position set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising at least one substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising at least a substitution or substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising at least a substitution or substitution of a variant set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2360-2870, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2350-2870, wherein optionally the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions or deletions. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments of the foregoing, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, or 1, 2, 3, 4, or 5 substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-316, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 4-316. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 318-410 or 412-666, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 318-410 or 412-666. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 668-700 or 702-780, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 668-700 or 702-780. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 782-818 or 820-934, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 782-818 or 820-934. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 936-1028 or 1030-1250, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 936-1028 or 1030-1250. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1252-1408 or 1410-1588, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 1252-1408 or 1410-1588. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1590-1648 or 1650-2068, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 1590-1648 or 1650-2068. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2070-2346, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 2070-2346. In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2360-2870, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 2350-2870. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions or deletions. In some embodiments, the amino acid sequence of the recombinant factor VIII polypeptide optionally includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments of the foregoing, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, or 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

In some embodiments, the recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or an amino acid sequence comprising SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, insertions or deletions. In some embodiments, optionally the amino acid sequence of the recombinant factor VIII polypeptide includes 1, 2, 3, 4, or 5 amino acid substitutions, insertions, or deletions. In some embodiments of the foregoing, optionally the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions, or 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

In some embodiments, the recombinant factor VIII polypeptide described herein has clotting activity. In some embodiments, the recombinant factor VIII polypeptide has an improved property compared to a reference factor VIII polypeptide, wherein the reference factor VIII polypeptide has a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide has increased activity compared to the reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the increased activity is clotting activity, particularly increased potency, compared to the reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide has increased stability to serum and/or plasma exposure, particularly serum or plasma from a hemophilia A patient, compared to the reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide is more stable at basic pH, e.g., about pH 7.4, than the reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide has increased thermostability compared to the reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide is characterized by increased expression and/or secretion from cells that are transfected with an expression vector as compared to a reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant factor VIII polypeptide is characterized by increased transduction and/or secretion from cells that are transduced with a gene therapy composition (e.g., AAV, mRNA, or lipid nanoparticle) as compared to a reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the improved property is in comparison to a reference factor VIII polypeptide having the sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or the sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide of the present disclosure exhibits at least one improved property selected from: i) increased activity/potency; ii) increased clotting activity/potency; iii) increased stability to plasma or serum; iv) increased thermostability; v) reduced immunogenicity; vi) increased expression and/or secretion from cells that are transfected with an expression vector; vii) increased transduction and/or secretion from cells that are transduced with a gene therapy vector (e.g., AAV, mRNA, or lipid nanoparticle), or a combination of any of i), ii), iii), iv), v), vi, and/or vii), as compared to a reference factor VIII polypeptide having a sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the reference recombinant factor VIII polypeptide has the sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or the sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant factor VIII polypeptide is a “deimmunized” recombinant factor VIII polypeptide (e.g., as presented in the Examples in Tables 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, and 15.2). In some embodiments, the recombinant factor VIII polypeptide is characterized by a reduction of Total Immunogenicity Score (TIS) and/or Immunogenic Hit Count (IHC) as compared to a reference factor VIII polypeptide, wherein the reference factor VIII polypeptide has a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, such as provided in Tables 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, and 15.2 of the Examples.

In some embodiments, a “deimmunized” recombinant factor VIII polypeptide comprises an amino acid sequence comprising residues 20-1457 or residues 1-1457 of a sequence selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 64, 66, 68, 72, 74, 76, 78, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 130, 132, 134, 136, 138, 140, 142, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 242, 244, 248, 252, 260, 262, 266, 270, 274, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, 302, 310, 312, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 424, 426, 428, 430, 432, 434, 436, 438, 442, 444, 446, 448, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 574, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 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, 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, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 868, 876, 878, 880, 882, 884, 886, 890, 892, 896, 900, 904, 906, 908, 910, 912, 914, 922, 932, 934, 936, 938, 940, 942, 944, 946, 948, 952, 956, 958, 960, 962, 964, 966, 970, 974, 976, 978, 980, 982, 986, 988, 990, 996, 998, 1002, 1006, 1008, 1012, 1014, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1096, 1098, 1100, 1102, 1104, 1108, 1110, 1112, 1114, 1116, 1120, 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, 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, 1286, 1300, 1306, 1310, 1314, 1316, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1344, 1352, 1356, 1368, 1370, 1372, 1380, 1384, 1386, 1388, 1390, 1392, 1394, 1400, 1406, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1540, 1550, 1552, 1554, 1564, 1568, 1570, 1584, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1654, 1658, 1660, 1662, 1664, 1666, 1670, 1676, 1690, 1694, 1698, 1706, 1710, 1712, 1714, 1720, 1724, 1726, 1728, 1732, 1734, 1740, 1746, 1748, 1750, 1752, 1754, 1756, 1760, 1762, 1772, 1778, 1782, 1784, 1786, 1788, 1792, 1794, 1796, 1798, 1806, 1810, 1814, 1820, 1826, 1846, 1854, 1858, 1860, 1868, 1870, 1872, 1878, 1880, 1882, 1898, 1906, 1908, 1910, 1920, 1924, 1926, 1930, 1932, 1940, 1942, 1950, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1990, 1992, 1994, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2038, 2040, 2042, 2044, 2046, 2050, 2052, 2054, 2056, 2058, 2060, 2064, 2066, 2068, 2070, 2098, 2102, 2104, 2106, 2122, 2138, 2142, 2190, 2204, 2206, 2210, 2212, 2214, 2216, 2220, 2226, 2228, 2234, 2236, 2242, 2250, 2252, 2254, 2266, 2268, 2272, 2276, 2278, 2280, 2282, 2284, 2288, 2290, 2302, 2306, 2308, 2310, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2330, 2334, 2336, 2338, 2340, 2370, 2372, 2374, 2380, 2382, 2386, 2390, 2392, 2394, 2396, 2398, 2402, 2406, 2408, 2412, 2420, 2422, 2432, 2436, 2440, 2446, 2448, 2450, 2454, 2456, 2460, 2464, 2466, 2468, 2472, 2476, 2478, 2484, 2486, 2488, 2494, 2496, 2498, 2500, 2504, 2506, 2510, 2518, 2520, 2526, 2530, 2534, 2542, 2544, 2554, 2556, 2560, 2562, 2564, 2570, 2574, 2576, 2578, 2586, 2588, 2590, 2592, 2594, 2598, 2600, 2602, 2604, 2610, 2614, 2618, 2622, 2628, 2632, 2638, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2662, 2664, 2672, 2674, 2676, 2682, 2684, 2688, 2690, 2694, 2702, 2704, 2708, 2710, 2712, 2714, 2728, 2734, 2736, 2738, 2742, 2748, 2750, 2754, 2760, 2762, 2764, 2766, 2768, 2770, 2774, 2776, 2778, 2780, 2782, 2784, 2790, 2794, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2818, 2820, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2840, 2842, 2844, 2848, 2850, 2852, 2854, 2856, 2858, 2860, 2862, 2864, 2866, 2868, or 2870.

In some embodiments, for any of the recombinant factor VIII polypeptide disclosed herein, the recombinant factor VIII polypeptide is purified or is a purified preparation.

In some embodiments, the recombinant factor VIII polypeptide comprises a full length recombinant factor VIII polypeptide of any of the recombinant factor VIII polypeptide described herein. In some embodiments, the recombinant factor VIII polypeptide comprises a “pre-pro-protein” or “pre-pro-polypeptide” of any of the recombinant factor VIII polypeptide described herein. In some embodiments, the signal sequence of the full length recombinant factor VIII polypeptide or “pre-pro-protein” or “pre-pro-polypeptide” thereof can comprise any compatible signal sequence, including synthetic, mouse (e.g., IgG), or human signal sequence. In some embodiments, the signal sequence present in the exemplary recombinant factor VIII polypeptide is replaced with a compatible signal sequence, e.g., synthetic or mouse. In some embodiments, the signal sequence comprises the signal sequence in the naturally occurring human factor VIII polypeptide.

In some embodiments, the recombinant factor VIII polypeptide is processed or cleaved to a pro-polypeptide or pro-protein form, for example, lacking the signal sequence. In some embodiments, the recombinant factor VIII polypeptide is processed or cleaved to a mature form of the recombinant factor VIII polypeptide, such as described herein. In some embodiments, the mature form of the recombinant factor VIII polypeptide comprises the active form of the recombinant factor VIII polypeptide, particularly the polypeptides (e.g., equivalent form for the light and heavy chain of factor VIII) that interact with FIX (see, e.g., Orlova et al., Acta Naturae, 2013, 5(2): 19-39:3983-3996; Lenting et al., Blood, 1998, 92(11); incorporated herein by reference).

Thus, for each and every recombinant factor VIII polypeptide described herein, the present disclosure provides a pre-pro-polypeptide, pro-polypeptide, or mature, biologically active form of the recombinant factor VIII polypeptide, including each recombinant factor VIII polypeptides disclosed in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1.

In some embodiments, the present disclosure further provides a functional or biologically active fragment of a recombinant factor VIII polypeptide described herein. In some embodiments, functional or biologically active fragments have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the recombinant factor polypeptide from which is was derived (i.e., the parent engineered factor VIII). In some embodiments, a functional or biologically active fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the recombinant factor VIII polypeptide. In some embodiments the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids. Exemplary biologically active or functional fragments of the recombinant factor VIII polypeptide include amino acid sequences comprising SEQ ID NO: 2350, 2352, 2354, 2356, or 2358, whose activities are shown in FIG. 3.

In some embodiments, the functional or biologically active fragment of the recombinant factor VIII polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of the recombinant factor VIII described herein. Accordingly, in some embodiments, the functional or biologically active fragments of the recombinant factor VIII displays the enhanced or improved property associated with the mutation or mutation set in the parent recombinant factor VIII polypeptide.

In some further embodiments, as discussed herein, a functional fragment or a biologically active fragment encompasses biologically processed polypeptides of a recombinant factor VIII polypeptide, such as functional or biologically active fragments produced upon expression in a mammalian cell or in a patient, or when a recombinant factor VIII polypeptide in administered to a patient. In some embodiments, a biologically processed functional or biologically active fragments of a recombinant factor VIII polypeptide resulting from cleavage or cleavages occurring between residues 760 to 773, and in some embodiments, additional cleavage or cleavages occurring between residues 391 and 392, wherein the positions are relative to the reference sequence of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the functional or biologically active fragment is derived from a recombinant factor VIII polypeptide presented in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1. In some embodiments, the functional or biologically active fragment is derived from a recombinant factor VIII polypeptide comprising an amino acid sequence of an even-numbered SEQ ID NO. selected from SEQ ID NOs: 4-2346 and 2350-2870.

Polynucleotides Encoding Recombinant Polypeptides, Expression Vectors, and Host Cells

In another aspect, the present disclosure provides polynucleotides encoding the engineered factor VIII polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous or homologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the recombinant factor VIII polypeptides can be introduced into appropriate host cells to express the corresponding recombinant factor VIII polypeptide.

As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode the recombinant factor VIII polypeptide. Thus, having knowledge of a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the variants provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1.

In some embodiments, the codons are preferably selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used for expression in bacteria, preferred codons used in fungi are typically used for expression in fungi, and preferred codons used in mammals are used for expression in mammals and mammalian cells. In some embodiments, codon optimized polynucleotides encoding the engineered factor polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full length coding region. In some embodiments, the present invention provides recombinant polynucleotide sequences in which the codons are optimized for expression in human cells or tissues.

As discussed above, it is to be understood that the present disclosure provides recombinant polynucleotides encoding each and every recombinant factor VIII polypeptide, or a biologically or functionally active fragment thereof, described herein.

Accordingly, by way of example and not limitation, in some embodiments, a recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII, or biologically active recombinant factor VIII fragments thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or to a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2360-2870, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2350-2870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution at position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or amino acid residue, 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534F/I/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T, or 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377, or 1439, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or an amino acid residue 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 212A/F/G/L/K/M/N/P/R/S/V/Y, 260E/K/T, 306A/D/G/P/S/T, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 387A/G/I/L/Q/R/S/T/V/W, 390A/E/G/L/M/P/S/T/V, 393A/F/G/H/M/Q/R/W/Y, 499C/L, 534I/F/L/M, 558A/E/G/L/P/Q/R/S/T/V/W, 681A/C/E/F/G/L/N/R/Q/S/T/W, 700A/E/Q/T/V, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 738A/D/E/F/H/K/N/P/R/S/T/Y, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 752D/E/F/G/L/M/Q/R/S/V, 775E/G/K/N/P/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 805C/G/H/K/L/M/P/Q/R/S/V/W, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1148A/C/E/L/M/N/Q/R/S, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1414A/C/D/E/G/H/N/P/T/W, or 1439A/E/H/L/S/T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 1148, 85/708/1148, 798, 706, 171, 335, 63, 344, 428, 879, 59, 429, 205, 1264, 797, 1248, 873, 792, 708, 57, 96, 362, 1292, 346, 433, 65, 795, 684/798, 353, 332, 1259, 356, 436, 1245, 1145, 716, 56, 773, 734, 358, 729, 665, 359, 61, 1250, 726, 37/96/171/429/708/799/1148, 171/429/732/1148, 96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/799, 171/429/708/1422, 171/429, 321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643/797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349, 257, 646, 435, or 58, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising a substitution at an amino acid position set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least one substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at the position(s) set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set of a variant set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2360-2870, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2350-2870.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or an amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T, or 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position 63, 171, 212, 260, 306, 317, 320, 332, 387, 390, 393, 534, 558, 681, 732, 738, 749, 750, 775, 1148, 1248, 1377, or 1439, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054, 755, 1041, 748, 1049, 1221, 1051, 612, 1037, 616, 387, 489, 750, 1224, 618, 1050, 1053, 896, 897, 749, 260, 481, 613, 756, 63, 265, 752, 1218, 1378, 614, 1440, 1439, 1205, 1222, 1389, 1269, 390, 397/1218, 757, 1373, 346, 63/1248, 349, 306/355/1422, 1422, 306/346/708, 1148, 171/346/1248, 63/797/799/1248/1422, 171/799, 362, 1248, 171/306/346/999/1248, 171/1148, 734/799/1248/1422, 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/349, 63/171/1148/1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349/354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/1248, 344/1248/1250, 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248, or 354/503, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or to the reference sequence corresponding to SEQ ID NO: 504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306/749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/387/750/1051/1148, 171, 750/1051/1148, 171/260/756, 171/1248, 171/1148, 59, 1440, 332/750/1248, 756/1248, 1248, 171/387/1248/1440, 750/1440, 59/171/750, 171/1193/1248, 750/1148/1193/1440, 1148, 171/387/1051, 748, 306, 306/387/390/749/1220, 390, 306/390/1037, 306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612/749/1037, or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or to the reference sequence corresponding to SEQ ID NO: 706, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816, 824, 826, 393, 825, 698, 332/387/390/1148, 328/387/749, 749, 306/328/332/390/1051/1193/1218, 63/306/332/1051/1148, 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306/332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306/387/749/1051/1148, 387/749, 306/945/1051/1193/1221, 306/390/749/1051, 63/306/332/390, 306/387/390/749/1148/1193/1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922, 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392, or 397, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840, or to the reference sequence corresponding to SEQ ID NO: 840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 840, or relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555/684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747, 753, 393/732/751, 63/797, 622, 617, 907, 1040, 611, 488, 1192, 1194, 619, 16, 802, 211, 555, 536, 527, 841, 320, 837, 558, 914, 312, 681, 838, 15, 1427, 18, 683, 24, 534, 840, 1160, 551, 393, 393/747/752/811/819/923/1148, 393/751/775, 63/393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/747/751, 393/747/753/775/1148, 752/923/1051, 63/775, 63/393/752/775/793/1051/1053, 752/753/775/793/819, 63/1051, 63/747/752/753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/752/753/793/811, 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148, or 747/753, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200, or to the reference sequence corresponding to SEQ ID NO: 1200, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1200, or relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 681, 622, 617, 558, 536, 211, 320, 839, 797, 533, 751, 303, 951, 164, 818, 807, 799, 735, 794, 926, 755, 921, 801, 1094, 968, 929, 924, 698, 168, 581, 916, 1428, 805, 1114, 311, 737, 693, 809, 757, 1099, 676, 740, 791, 211/536/622/681, 536/558, 211/536/555/617/732, 211/681/732, 211/536/555, 555, 211/536, 211/212, 211/536/617/681/732, 211/536/558/617/681, 555/732, 536/555/558/617/732, 536/617/681/732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619/681/839, 797/839, 212/312/320/533/534/617/681, 558/619/681, 320/558/681, 212/320/534/619/681/797/839, 320/558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/619/681, 211/534/558/611/681, 211/320/617/619/681, 558/617/681/797, 320/534/619/732, 211/619/681, 211/212/534/558/619/732/839, 212/320/619/681/797/839, 212/320/611/839, 320/558/619/681, 312/533/617/681/797/839, 558/611/681, 312, 211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681, 211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681, 211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558/681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538, or 1109, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514, or to the reference sequence corresponding to SEQ ID NO: 1514, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1514, or relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least a substitution or substitution set at amino acid position(s) 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050, 1210, 104, 615, 610, 536, 618, 621, 614, 110, 301, 1078, 812, 786, 127, 175, 1093, 1088, 650, 535, 806, 738, 700, 132, 1076, 1084, 498, 756, 1108, 746, 160, 543, 310, 584, 918, 1452, 790, 292, 800, 167, 892, 304, 1116, 752, 281, 917, 712, 1080, 806/1352, 1095, 744, 1098, 948, 119, 785, 1113, 667, 158, 580, 707, 731, 1100, 1089, 1092, 939, 1104, 736, 1297, 1127, 686, 1129, 1138, 786/821, 680, 1101, 808, 739, 308, 536/555/619/732/799/805, 555/619/801, 555/619/799, 619/799/801/805, 536/555/619/732/799, 799/805, 801, 246/619/799/801, 536/555/619, 619/839, 619/799/805, 536/801/805, 555/619/799/805, 619/801/805, 555/619, 536/619/801, 801/805, 536/619/799/805, 555/619/732/799/801/805, 749, 616, 611, 370/614, 1156, 1158, 555/681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/799/916/1109, 555/619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799, 555, 732, 619/799, 619, 681, 555/916, 839, or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072, or to the reference sequence corresponding to SEQ ID NO: 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318/700/1076, 167/318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/752/1093, 167/700, 167/700/738/739/1093, 167/700/738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/739/809, 738/739/809/1093/1116, 738/739/1093, 738/739/752/1093, 738/739/799/800/1093, 738/739/1116, 738/800, 738/1093, 738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/1093, 739, 739/800/809, 800, 800/805/809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738/809, 318/1093, 318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/543/1093, 318/700/738/1093/1414, 681, 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/739/1116, 681/738/800, 681/800/805/809, 681/809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/1116, 681/700/809, 681/700/1093, 681/700/752/808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/1076/1116, 178/318/800/809, 178/318/681/800/808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116, 543, 543/738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/809/1093, 543/809/1093/1116, 543/681/738/739/752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/809/1093, 543/681/700/738/739/800/1093/1116, 543/681/700/738/739/805/808/809, 543/681/700/752, 543/681/700/752/809/1093, 543/1093, 543/700, 543/700/738/739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809/1116, 543/700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809, 700/809/1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700/752/805/809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809, or 805/809/1116, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising a substitution at an amino position set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least one substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising at least a substitution or substitution of a variant set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising residues 20-1457 of the sequence of an even numbered SEQ ID NO. set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, or comprising the sequence of an even numbered SEQ ID NO. set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2360-2870, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2350-2870.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the recombinant factor VIII polypeptide comprising an amino acid sequence comprising residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or comprising SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the full length polypeptide, or the pre-pro-protein or the pre-pro-polypeptide of the recombinant factor VIII polypeptide described herein.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the pro-protein or pro-polypeptide, or the mature form of the recombinant factor VIII polypeptide described herein.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the biologically or functionally active fragment of a recombinant VIII polypeptide described herein. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the biologically or functionally active fragment having a cleavage or cleavages between amino acid residues 760 to 773, and the polypeptide being devoid of some or all of an amino acid sequence corresponding to amino acids 760-773, wherein the positions are relative to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the biologically or functionally active fragment having a cleavage between amino acid residues 391 and 392, wherein the positions are relative to the reference sequence of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-2345 and 2359-2869, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-2345 and 2349-2869, wherein the recombinant polynucleotide encodes a recombinant factor VIII polypeptide.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071, wherein the recombinant polynucleotide encodes a recombinant factor VIII polypeptide.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant factor VIII polypeptide described herein, wherein the polynucleotide sequence is codon-optimized for expression of the encoded recombinant factor VIII polypeptide. In some embodiments, the polynucleotide sequence is codon optimized for expression in prokaryotic cells or eukaryotic cells. In some embodiments, the polynucleotide sequence is codon optimized for expression in mammalian cells. In some embodiments, the polynucleotide sequence is codon optimized for expression in human cells.

In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 58-4371 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-2345 and 2359-2869, or comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-2345 and 2349-2869.

In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reference polynucleotide encoding a recombinant factor VIII described herein, e.g., a recombinant polynucleotide provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, or a reverse complement thereof. In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reverse complement of a reference polynucleotide encoding a recombinant factor VIII polypeptide described herein, wherein the recombinant polynucleotide hybridizing under stringent conditions encodes a recombinant factor VIII polypeptide comprising an amino acid sequence having one or more residue differences as compared to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072 at residue positions selected from any positions as set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1.

In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues nucleotide residues 58-4371 of SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071, or to a reference sequence corresponding to SEQ ID NO: SEQ ID NO: 1, 251, 503, 705, 839, 1199, 1513, or 2071. In some additional embodiments, the recombinant polynucleotide hybridizing under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one polynucleotide reference sequence corresponding to nucleotide residues 58-4371 of a polynucleotide sequence provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1. or a polynucleotide sequence provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1. wherein the recombinant polynucleotide hybridizing under stringent conditions encodes a recombinant factor VIII polypeptide.

In some embodiments, an isolated polynucleotide encoding any of the recombinant factor VIII polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among others, promoter sequences, Kozak sequence, leader sequences, polyadenylation sequences, pro-peptide sequences, signal peptide sequences, DNA based regulatory elements for gene therapy retention and transcription terminators. As known in the art, suitable promoters can be selected based on the host cells used. Exemplary promoters for filamentous fungal host cells, include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (FVIIIA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see e.g., Romanos et al., 1992, Yeast 8:423-488). Exemplary promoters for use in mammalian cells include, but are not limited to those from cytomegalovirus (CMV), chicken @-actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor-la or glyceraldehyde-3-phosphate dehydrogenase, or from Gallus @-actin.

In some embodiments, the control sequence is a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

In some embodiments, the control sequence is a suitable leader sequence, 5′-cap modification, 5′ UTR, etc. In some embodiments, these regulatory sequence elements mediate binding to molecules involved in mRNA trafficking and translation, inhibit 5′-exonucleolytic degradation and confer resistance to de-capping. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). Suitable leaders for mammalian host cells include but are not limited to the 5′-UTR element present in orthopoxvirus mRNA.

In some embodiments, the control sequence comprises a 3′ untranslated nucleic acid region and polyadenylation tail nucleic acid sequence, sequences operably linked to the 3′ terminus of the protein coding nucleic acid sequence which mediate binding to proteins involved in mRNA trafficking and translation and mRNA half-life. Any polyadenylation sequence and 3′ UTR which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3′ UTR sequences for mammalian host cells include, but are not limited to, the 3′-UTRs of α- and β-globin mRNAs that harbor several sequence elements that increase the stability and translation of mRNA.

In some embodiments, the control sequence is a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the recombinant factor VIII polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to those from the genes for immunoglobulin gamma (IgG). In some embodiments, the signal peptide is a signal peptide of a protein expressed in human cells.

In another aspect, the present disclosure also provides a recombinant expression vector comprising a polynucleotide encoding a recombinant factor VIII polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described above are joined together to produce a recombinant expression vector which includes one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the recombinant factor VIII polypeptide at such sites. Alternatively, the polynucleotide sequence(s) of the present disclosure are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus including but not limited to adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and non-viral vectors, such as liposomes), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the recombinant factor VIII polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

In some embodiments, the expression vector preferably contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one recombinant factor VIII polypeptide described herein, the polynucleotide being operatively linked to one or more control sequences for expression of the recombinant factor VIII polypeptides(s) in the host cell. Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris, e.g., ATCC Accession No. 201178); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, CHO-K1, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat and Bowes melanoma cell lines). In some embodiments, the host cell is a human cell obtained or derived from a subject with hemophilia A.

Accordingly, in another aspect, the present disclosure provides a method for producing the recombinant factor VIII polypeptides, where the method comprises culturing a host cell capable of expressing a polynucleotide encoding the recombinant factor VIII polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of isolating the recombinant factor VIII polypeptide, such as from the culture media or cells. In some embodiments, the method further comprises the step of purifying the expressed recombinant factor VIII polypeptide, as described herein.

Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the recombinant factor VIII polypeptides may be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

The recombinant factor VIII polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or recombinant factor VIII polypeptide to mutagenesis and/or directed evolution methods known in the art, and as described herein. An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499, 7,904,249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066, 8,768,871, 9,593,326, and all related non-US counterparts; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet, 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., 1984, Cell, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 370:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; U.S. patent publication Nos. 2008/0220990, US 2009/0312196, US2014/0005057, US2014/0214391, US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and all related non-US counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO 00/42651, WO 01/75767, and WO 2009/152336; all of which are incorporated herein by reference).

In some embodiments, the protein variants obtained following mutagenesis treatment are screened by subjecting the protein variants to a defined temperature, pH, or other assay conditions, and measuring the amount of protein activity remaining after the treatments or other assay conditions. DNA containing the polynucleotide encoding the factor VIII polypeptide is then isolated from the host cell, sequenced to identify the nucleotide sequence changes (if any), and used to express the protein in a different or the same host cell. Measuring protein activity from the expression libraries can be performed using any suitable method known in the art, such as provided in the Examples.

For engineered polypeptides of known sequence, the polynucleotides encoding the polypeptide can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, polynucleotide fragments can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tetra. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods.

Accordingly, in some embodiments, a method for preparing the recombinant factor VIII polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, and 15.1, as well as SEQ ID NOs: 2-2346 and 2350-2870, and (b) expressing the recombinant factor VIII polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.

The expressed recombinant factor VIII polypeptide can be assessed for any desired improved property (e.g., activity/potency, selectivity, stability, serum/plasma stability, basic pH tolerance, protease sensitivity, etc.), using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

In some embodiments, any of the recombinant factor VIII polypeptides expressed in a host cell are recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.

Chromatographic techniques for isolation of the recombinant factor VIII polypeptides include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular polypeptide depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the improved variant factor VIII polypeptides. In some embodiments utilizing affinity chromatography purification, any antibody which specifically binds the variant factor VIII polypeptide finds use. In some embodiments utilizing affinity chromatography purification, proteins that bind to the glycans covalently attached to factor VIII find use. In still other embodiments utilizing affinity-chromatography purifications, any small molecule that binds to the factor VIII active site finds use. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with a factor VIII polypeptide (e.g., a factor VIII variant), or a fragment thereof. In some embodiments, the factor VIII polypeptide or fragment is attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.

In some embodiments, once the recombinant factor VIII polypeptides are recovered or isolated from the recombinant host cells or cell culture medium, they are further purified by any suitable method(s) known in the art. In some additional embodiments, the purified factor VIII polypeptides are combined with other ingredients and compounds to provide compositions and formulations comprising the recombinant factor VIII polypeptide as appropriate for different applications and uses (e.g., pharmaceutical compositions).

In some additional embodiments, the purified recombinant factor VIII polypeptides, or the formulated recombinant factor VIII polypeptides are lyophilized. In some embodiments, the recombinant factor VIII polypeptides are directly produced within a body (i.e., cells within a body, such as a human or another animal) and are not purified. However, in some alternative embodiments, the recombinant factor VIII polypeptides are produced within a body (i.e., cells within a body, such as a human or another animal) and are collected from the body using methods known in the art. In some additional embodiments, these collected recombinant factor VIII polypeptides are purified.

Pharmaceutical Compositions

In a further aspect, the present disclosure provides various compositions and formats comprising a recombinant factor VIII polypeptide, including but not limited to those described below. In some embodiments, the present disclosure provides recombinant polypeptides or recombinant polynucleotides suitable for use in pharmaceutical and other compositions.

Depending on the mode of administration, a composition comprising a therapeutically effective amount of a recombinant factor VIII described herein is in the form of a solid, semi-solid, or liquid. In some embodiments, the composition includes other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details on techniques for formulation and administration are well known in the art and described in the literature. In some embodiments, these compositions are produced directly in the human body after introduction as gene therapy.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant factor VIII polypeptide described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. Any suitable form for use in delivering the recombinant factor VIII polypeptides find use in the present disclosure, including but not limited to pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks, solutions, suspensions (including but not limited to oil-based suspensions, oil-in water emulsions, etc.), slurries, syrups, controlled release formulations, etc.

In some embodiments, the recombinant factor VIII polypeptides are provided in a form suitable for injection or infusion (i.e., in an injectable formulation). In some embodiments, the polynucleotide sequences for the recombinant factor VIII polypeptide is provided in a form suitable for injection or infusion.

In some embodiments, the recombinant factor VIII polypeptides are provided in biocompatible matrices such as sol-gels, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the recombinant factor VIII polypeptides are encapsulated. In some alternative embodiments, the recombinant factor VIII polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.).

In some embodiments, a composition comprising the recombinant factor VIII polypeptides of the present disclosure may include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and/or sorbitol), starches, cellulose (e.g., methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin, collagen, etc.).

In some embodiments, the recombinant factor VIII polypeptides are chemically modified by glycosylation, chemical crosslinking reagents, pegylation (i.e., modified with polyethylene glycol (PEG) or activated PEG, etc.) or other compounds (see e.g., Ikeda, Amino Acids, 2005, 29:283-287; U.S. Pat. Nos. 7,531,341, 7,534,595, and 7,560,263; US Pat. Publication Nos. 2013/0039898, 2012/0177722, etc.).

It is intended that the recombinant factor VIII polypeptides be administered by any suitable means known in the art, including but not limited to parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc. It is not intended that the present invention be limited to any particular delivery method and/or mechanism.

In some embodiments, the pharmaceutical composition comprises a recombinant polynucleotide encoding a recombinant factor VIII polynucleotide described herein, such as for gene therapy. In some embodiments, a gene therapy composition comprises a recombinant polynucleotide encoding any of the recombinant factor VIII polypeptides described herein. In some embodiments, the gene therapy composition comprises a viral vector comprising the recombinant polynucleotide encoding a recombinant factor VIII polypeptide. In some embodiments, viral delivery vectors, include but are not limited to adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), retrovirus, or herpes virus vectors. In some embodiments, the gene therapy compositions comprise non-viral delivery compositions, where the recombinant polynucleotide is formulated with liposomes, cationic polymers, dendrimers, or conjugated to cell-penetrating peptides. In some embodiments, the recombinant factor VIII polypeptides are provided for delivery to cells or tissues via mRNA therapy, such as by formulating a polyribonucleotide (e.g., mRNA) in an encapsulated delivery vehicle (e.g., liposomes).

In some additional embodiments, the recombinant factor VIII polypeptides are provided for delivery to cells or tissues via cell therapy, where the polynucleotide sequence encoding the recombinant factor VIII polypeptides is introduced into exogenous cell and that cell (or cells) are introduced into a recipient or subject (e.g., a patient exhibiting deficiency in factor VIII). In some embodiments, the cell is an stem cell, particularly a human stem cell.

Uses and Methods

In another aspect, the recombinant factor VIII polypeptides disclosed herein are used for therapeutic purposes. In some embodiments, the recombinant factor VIII polypeptides and/or recombinant polynucleotides encoding the recombinant factor VIII polypeptides are used in the treatment of conditions or diseases associated with a deficiency in factor VIII.

In some embodiments, a method for treating and/or preventing the symptoms of deficiency in factor VIII in a subject comprises administering to a subject having a deficiency in factor VIII a therapeutically effective amount of a recombinant factor VIII polypeptide described herein, or a pharmaceutical composition thereof. In some embodiments, administration of the recombinant factor VIII is provided as enzyme replacement therapy.

In some embodiments, a method for treating and/or preventing the symptoms of deficiency in factor VIII in a subject comprises providing to a subject having a deficiency in factor VIII a recombinant polynucleotide encoding a recombinant factor VIII polypeptide, such as the gene therapy composition comprising the recombinant polypeptide described above. In some embodiments, the recombinant polynucleotides are provided in a viral vector. In some embodiments, the viral vectors are expression vectors that provide for expression of the encoded recombinant factor VIII polypeptide when the vector is administered to a patient or inserted in a mammalian cell. In some embodiments, the viral vector is based on adenovirus, adeno-associated virus, lentivirus, retrovirus, or herpes virus vectors. In some embodiments, the recombinant polynucleotide is formulated in non-viral delivery compositions, as described above. In some embodiments, the recombinant polynucleotide is provided as gene replacement therapy, for example using CRISPR technology.

In some embodiments, the condition or disease associated with a deficiency in factor VIII and treated with the recombinant factor VIII polypeptide or a polynucleotide encoding the recombinant factor VIII polypeptide is hemophilia A. In some embodiments, the hemophilia is acquired hemophilia A. In some embodiments, the hemophilia A is from a genetic defect in the gene encoding factor VIII. In some embodiments, the symptoms of deficiency in factor VIII are ameliorated by the treatment.

EXAMPLES

The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

In the experimental disclosure below, the following abbreviations apply where applicable: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and l (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); ° C. (degrees Centigrade); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); FIOPC (fold improvements over positive control).

Example 1
FVIII Gene Acquisition and Construction of Expression Vectors

Synthetic genes (SEQ ID NO: 1) coding for the WT human FVIII sequence with B-domain deleted (SEQ ID NO: 2) were constructed as previously described (See e.g. U.S. Patent Publication. No. 2017/0360900). Oligonucleotides BamHI-pcDNA-FVIII-F and XhoI-pcDNA-FVIII-R were used to amplify a fragment for the coding sequence for the FVIII variants. The PCR product was ligated into the BamHI/XhoI linearized mammalian expression vector pcDNA3.1(+) (Invitrogen), or a vector containing a CMV promoter and BGH-pA (bovine growth hormone polyadenylation) sequence. Directed evolution techniques generally known by those skilled in the art were used to generate gene variants derived from SEQ ID NO: 2 within this plasmid construct (see e.g., U.S. Pat. No. 8,383,346 and International patent publications WO2010/144103).

Secreted expression of a mammalian cell codon-optimized human FVIII was achieved using the native FVIII signal peptide. Clones were expressed primarily from a pDH.1(+) vector in HEK293T cells, Expi293F™ (ThermoFisher Scientific) cells, and HepG2 cells. This approach provided supernatants with measurable activity in the chromogenic assay (See e.g., Aniara Diagnostica BIOPHEN™ FVIII:C) and the activated partial thromboplastin time (APTT) assay, using plasma from hemophilia A patients.

In some embodiments, to identify mutational diversity with similar stability and improved blood clotting function compared to SEQ ID NO: 2, a combinatorial library was constructed generating FVIII variants derived from SEQ ID NO:2. Equivalent volumes of supernatant were screened in an unchallenged condition (no incubation, pH 7.4) or following a one-hour to seventeen-hour incubation in a human plasma (physiological pH 7.1-8.2) environment at 37° C. to 55° C. FVIII variants with improved activity due to increased FVIII expression or FVIII specific activity were identified based on their fold improvement over the parent FVIII. FVIII variants with increased stability were identified by dividing the fold-improvement observed under challenged conditions by the fold-improvement observed under unchallenged conditions. This approach reduces the bias towards selecting variants based on increased expression but without changes in stability. Composite activity scores (the product of fold-improvements for all three conditions) and stability (the product of stability scores) were used to rank mutations in improved variants for inclusion in subsequent FVIII libraries. In additional embodiments, the additional methods and sequences described in the Examples were used.

Example 2
High-Throughput Growth
High-Throughput (HTP) Growth of FVIII and FVIII Variants

Expi293F™ (ThermoFisher Scientific) cells or HepG2 cells were transfected with pcDNA 3.1(+) having a CMV promoter and BGH-pA containing vectors encoding wild type FVIII with B-domain deleted, or variants, using the lipofection method with ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) or LIPOFECTAMINE® 3000 Reagent (ThermoFisher Scientific). Expi293F™ (ThermoFisher Scientific) cells were cultured in growth medium (Expi293™ Expression Medium, ThermoFisher Scientific). Immediately before transfection, cells were transferred to NUNC™ 96-Well DeepWell™ plates (ThermoFisher Scientific) at densities of 10⁸cells/well/400 μL in growth medium, and incubated at 37° C., 8% CO₂in an incubator. HepG2 cells were cultured in growth medium (EMEM with 10% fetal bovine serum, both from Corning). 24 hours before transfection, cells were seeded to NUNC® Edge 2.0 96-well plate (ThermoFisher Scientific) at densities of 10⁴cells/well/250 μL in growth medium, and incubated at 37° C., 5% CO₂in an incubator. Cells were incubated for 24-96 hours at specific conditions to allow for expression and secretion of FVIII variants. Conditioned media (50-100 μL) from the transfections were transferred into Corning 96-well solid black plates (Corning) for activity and/or stability analysis.

Example 3
In Vitro Characterization of Variants in HTP
HTP-Analysis of Supernatants (Chromogenic Assay)

FVIII variant activity was determined by measuring the release of paranitroaniline (pNa) from a synthetic peptide substrate. For an unchallenged assay, 25 μL of Expi293F or HepG2 conditioned media produced as described above were warmed to 37° C. and mixed with 25 μL of “Reagent 1” (BIOPHEN™ FVIII:C, Aniara) and 25 μL of “Reagent 2” (BIOPHEN™ FVIII:C, Aniara) in a 96-well, black, opaque bottom microtiter plate. The reactions were mixed briefly and incubated at 37° C. for 5 minutes. 25 μL of “Reagent 3” (BIOPHEN™ FVIII:C, Aniara) was then added, and the reactions were mixed briefly and incubated at 37° C. for 5 minutes prior to quenching with 25 μL pf 2% citric acid. Release of pNA was analyzed using an EnVision® 2105 Multimode Plate Reader (PerkinElmer) monitoring absorbance at 405 nm. “Reagent 1”, “Reagent 2”, and “Reagent 3” are components of a chromogenic assay (BIOPHEN™ FVIII:C) supplied by Aniara and used as recommended by the supplier.

HTP-Analysis of Supernatants (Clotting Assay)

FVIII variant activity was determined by measuring the clotting rate of Hemophilia A patient plasma (George King Bio-Medical, Inc.) using the activated partial thromboplastin time assay. For an unchallenged assay, 25 μL of Expi293F or HepG2 conditioned media produced as described above were warmed to 37° C. and mixed with 30 μL of Hemophilia A patient plasma (George King Bio-Medical, Inc.) and 50 μL of aPTT-XL reagent (Pacific Hemostasis) in a 96-well, black, opaque bottom microtiter plate. The reactions were mixed briefly and incubated at 37° C. for 3 minutes. 10 μL of 200 μM calcium chloride (Sigma Aldrich) was then added, and the reactions were mixed briefly. Clotting was analyzed using an EnVision® 2105 Multimode Plate Reader (PerkinElmer) monitoring absorbance at 405 nm over time for 5-10 minutes.

HTP-Analysis of Supernatants Pretreated with Plasma

FVIII variants were challenged with Hemophilia A patient plasma (George King Bio-Medical, Inc.) to simulate the conditions that the variants may encounter within the bloodstream. First, 50 μL of Expi293F or HepG2 conditioned media produced as described above and 50 μL of Hemophilia A patient plasma (George King Bio-Medical, Inc.) were added to the wells of a 96-well round bottom microtiter plate. The plates were sealed with a PlateLoc® thermal microplate sealer (Agilent), and incubated at 37-55° C. for 1-24 h. The samples were then analyzed by either the chromogenic or clotting assays described above.

Example 4
FVIII Variants Derived from SEQ ID NO: 2

In this Example, experiments conducted to determine FVIII variant activity are described. These variants were tested using the chromogenic assays, as described in Example 3. Results are shown in Tables 4.1 and 4.2 for variants expressed using Expi293F cells.

TABLE 4.1

Activity of FVIII Variants Relative to SEQ ID NO: 2

Amino Acid
Unchallenged Chromogenic

SEQ ID
Differences
Activity FIOPC

NO:
(Relative to
Relative to

(nt/aa)
SEQ ID NO: 2)
SEQ ID NO: 2

1/2

+

3/4
T1148N
+

5/6
P85Q/I708T/T1148N
++

7/8
K798T
+

9/10
L706G
+

11/12
L706A
+

13/14
L171Q
+

15/16
Q335E
+

17/18
V63R
++

19/20
K344A
++

21/22
S428F
++

23/24
E879Q
++

25/26
F59L
+

27/28
Q429S
+

29/30
K205R
++

31/32
I1264W
+

33/34
M797P
+

35/36
L1248S
+

37/38
I1264R
+

39/40
Y873T
+

41/42
T792Y
+

43/44
I708K
+

45/46
K344L
++

47/48
F57V
+

49/50
Q96L
+

51/52
A362W
+

53/54
V63G
++

55/56
Y873L
+

57/58
M1292Y
++

59/60
T1148Q
++

61/62
D346V
++

63/64
M797G
+

65/66
L706Y
+

67/68
L1248R
++

69/70
N433A
++

71/72
Y65A
+

73/74
F59W
+

75/76
Y873Q
+

77/78
V795C
+

79/80
E684K/K798R
++

81/82
F57L
+

83/84
Q353S
+

85/86
S332Q
+

87/88
I708A
+

89/90
G1259T
+

91/92
V63M
+

93/94
M356L
++

95/96
L1248G
++

97/98
L1248P
++

99/100
Q436G
+

101/102
F57A
+

103/104
F59S
++

105/106
T1245R
+

107/108
F59R
+

109/110
Q96W
+

111/112
M797Y
+

113/114
K1145R
+

115/116
Q436D
+

117/118
F716L
+

119/120
S56E
+

121/122
R773H
+

123/124
T734I
++

125/126
N358S
++

127/128
Q335P
+

129/130
F59Q
++

131/132
S729T
+

133/134
F716K
++

135/136
T665G
+

137/138
A362T
+

139/140
F57T
+

141/142
N359G
+

143/144
T61S
++

145/146
V1250A
+

147/148
A362V
+

149/150
N359E
+

151/152
F716W
+

153/154
S332W
++

155/156
I708V
+

157/158
V63H
++

159/160
M797C
+

161/162
T61Q
+

163/164
M356N
++

165/166
V1250S
+

167/168
K798C
+

169/170
N433G
++

171/172
V795N
++

173/174
Q96I
+

175/176
S56P
+

177/178
V63N
+

179/180
T792W
+

181/182
I1264L
+

183/184
V1250P
+

185/186
S332P
+

187/188
F59V
+

189/190
K726L
++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 2. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1

TABLE 4.2

Activity of FVIII Variants Relative to SEQ ID NO: 2

Unchallenged

Amino Acid
Chromogenic

SEQ ID
Differences
Activity FIOPC

NO:
(Relative to
Relative to

(nt/aa)
SEQ ID NO: 2)
SEQ ID NO: 2

1/2

+

191/192
Q37H/Q96R/L171P/Q429L/I708T/
++

K799R/T1148N

193/194
L171P
++

195/196
L171P/Q429L/K732R/T1148N
++

197/198
Q96R/L171P
++

199/200
L171P/E919G/G1151S
+++

201/202
L171P/T792Y/M797Q/K799R/T1148N
++

203/204
L171P/P569T/T1148N
++

205/206
Q96R/L171P/Q429L/T792Y/M797T/
++

K799R

207/208
L171P/Q429L/I708T/L1422I
+++

209/210
L171P/Q429L
++

211/212
D321Y/A362P/K732E/M797T/K799R
++

213/214
L171P/L1100I
++

215/216
I708T/T1148N/A1186E
+++++

217/218
Q429L/L724I/K732E/T1148N
++

219/220
L171P/A362E/V795T/M797Q/K799M
++

221/222
L171P/Q429L/K732E
++

223/224
Q96R/L171P/S643N/M797T/K799R
++

225/226
L171P/T792Y/M797T/K799M
++

227/228
R503S
++

229/230
L171P/K732E
++

231/232
T1148N/F1193L
++

233/234
Q96R/L171P/A362E/Q429L/K732R/
++

V795T/M797Q

235/236
K726A
+

237/238
F59M
+

239/240
T734R
++

241/242
A362N
+

243/244
H334G
++

245/246
K732G
++

247/248
V63Q
++

249/250
E351S
++

251/252
L1377D
+++++

253/254
V795S
++

255/256
N358K
++

257/258
Q999R
++

259/260
V1250T
++

261/262
E306G
++

263/264
P349G
++

265/266
L1377G
+++

267/268
S332F
++

269/270
L1377E
+++++

271/272
V257I
++

273/274
L646I
++

275/276
D346R
++

277/278
P435Q
++

279/280
Q96V
++

281/282
L1377R
++

283/284
L1377S
+++

285/286
F59G
++

287/288
L171M
++

289/290
T792H
+

291/292
L1248D
++

293/294
E306S
++

295/296
L706M
++

297/298
L171H
+++

299/300
L1377A
++

301/302
L1377V
++

303/304
T734S
++

305/306
T1148R
++

307/308
F57S
++

309/310
F59D
++

311/312
K344E
+

313/314
A362G
++

315/316
P58K
++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 2. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2, “++++” >3.5, “+++++” >5

Example 5
FVII Variants Derived from SEQ ID NO: 252

In this Example, experiments conducted to determine FVIII variant activity are described. These variants were tested using the chromogenic assays, as described in Example 3. Results are shown in Tables 5.1 and 5.2 for variants expressed using Expi293F cells.

TABLE 5.1

Activity of FVIII Variants Relative to SEQ ID NO: 252

Unchallenged

Amino Acid
Amino Acid
Chromogenic

SEQ ID
Differences
Differences
Activity FIOPC

NO:
(Relative to
(Relative to
Relative to

(nt/aa)
SEQ ID NO: 252)
SEQ ID NO: 2)
SEQ ID NO: 252

251/252

L1377D
+

317/318
M1380W
L1377D/M1380W
+++

319/320
I388A
I388A/L1377D
+

321/322
F1043E
F1043E/L1377D
+

323/324
F1043R
F1043R/L1377D
+

325/326
I610R
I610R/L1377D
+

327/328
I1274N
I1274N/L1377D
+

329/330
S1220T
S1220T/L1377D
+

331/332
I1442T
L1377D/I1442T
+

333/334
F1193L
F1193L/L1377D
+

335/336
Q621T
Q621T/L1377D
+

337/338
L1054C
L1054C/L1377D
+

339/340
A755Q
A755Q/L1377D
+

341/342
Y1041G
Y1041G/L1377D
+

343/344
Y748S
Y748S/L1377D
+

345/346
Y1049G
Y1049G/L1377D
+

347/348
L1221V
L1221V/L1377D
+

349/350
M1051E
M1051E/L1377D
+

351/352
R612G
R612G/L1377D
+

353/354
F1037H
F1037H/L1377D
+

355/356
N616E
N616E/L1377D
+

357/358
Y1041H
Y1041H/L1377D
+

359/360
F387S
F387S/L1377D
+

361/362
S489R
S489R/L1377D
+

363/364
Q621M
Q621M/L1377D
+

365/366
S489Q
S489Q/L1377D
+

367/368
L750G
L750G/L1377D
++

369/370
S1224P
S1224P/L1377D
+

371/372
A618C
A618C/L1377D
+

373/374
N616G
N616G/L1377D
+

375/376
R612T
R612T/L1377D
+

377/378
I1050Q
I1050Q/L1377D
+

379/380
T1053A
T1053A/L1377D
+

381/382
N616W
N616W/L1377D
+

383/384
I896V
I896V/L1377D
+

385/386
S489F
S489F/L1377D
+

387/388
I1050P
I1050P/L1377D
+

389/390
M897G
M897G/L1377D
+

391/392
L749T
L749T/L1377D
+

393/394
I1274V
I1274V/L1377D
+

395/396
S1220L
S1220L/L1377D
+

397/398
S260E
S260E/L1377D
+

399/400
L481V
L481V/L1377D
+++

401/402
F613S
F613S/L1377D
+

403/404
F387G
F387G/L1377D
+

405/406
Q621R
Q621R/L1377D
+

407/408
I756R
I756R/L1377D
+

409/410
V63R
V63R/L1377D
+

All activities were determined relative to the reference polypeptide of SEQ ID NO: 252. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

TABLE 5.2

Activity of FVIII Variants Relative to SEQ ID NO: 252

Amino Acid
Amino Acid
(Expi293F) Unchallenged

SEQ ID
Differences
Differences
Chromogenic Activity

NO:
(Relative to
(Relative to
FIOPC Relative to

(nt/aa)
SEQ ID NO: 252)
SEQ ID NO: 2)
SEQ ID NO: 252

251/252

L1377D

411/412
I265S
I265S/L1377D
+

413/414
L1221T
L1221T/L1377D
+

415/416
M1380G
L1377D/M1380G
++

417/418
K752G
K752G/L1377D
+

419/420
Y748K
Y748K/L1377D
++

421/422
A755L
A755L/L1377D
++

423/424
F1218A
F1218A/L1377D
++

425/426
Y748N
Y748N/L1377D
+

427/428
T1378E
L1377D/T1378E
++

429/430
L614I
L614I/L1377D
+

431/432
L749F
L749F/L1377D
++

433/434
F387V
F387V/L1377D
+

435/436
L750T
L750T/L1377D
+

437/438
F1193G
F1193G/L1377D
++

439/440
L749V
L749V/L1377D
++

441/442
T1378A
T1378A/L1377D
+

443/444
L750V
L750V/L1377D
++

445/446
F387T
F387T/L1377D
++

447/448
F387Q
F387Q/L1377D
++

449/450
L750A
L750A/L1377D
++

451/452
F1037N
F1037N/L1377D
++

453/454
H1440A
H1440A/L1377D
++

455/456
M1051S
M1051S/L1377D
+++

457/458
L1221G
L1221G/L1377D
++

459/460
T1378D
L1377D/T1378D
++

461/462
H1440S
L1377D/H1440S
+++

463/464
F387R
F387R/L1377D
+

465/466
F1193A
F1193A/L1377D
++

467/468
F387I
F387I/L1377D
++

469/470
F1218M
F1218M/L1377D
++

471/472
I265W
I265W/L1377D
++

473/474
H1440E
L1377D/H1440E
++

475/476
V1439L
L1377D/V1439L
+

477/478
H1440T
L1377D/H1440T
+++

479/480
R612S
R612S/L1377D
+

481/482
Y1049W
Y1049W/L1377D
+

483/484
L1221S
L1221S/L1377D
+++

485/486
S1220D
S1220D/L1377D
++

487/488
L749G
L749G/L1377D
++

489/490
L749S
L749S/L1377D
+

491/492
F1218G
F1218G/L1377D
++

493/494
I1205L
I1205L/L1377D
++

495/496
Y1222F
Y1222F/L1377D
++

497/498
F1193E
F1193E/L1377D
++

499/500
I1050S
I1050S/L1377D
++

501/502
S1389W
L1377D/S1389W
+

503/504
V1439E
L1377D/V1439E
+++

505/506
S260T
S260T/L1377D
++

507/508
F1037P
F1037P/L1377D
+

509/510
M1051K
M1051K/L1377D
+++

511/512
I1269F
I1269F/L1377D
+

513/514
I265R
I265R/L1377D
++

515/516
M1051R
M1051R/L1377D
++

517/518
F1218L
F1218L/L1377D
++

519/520
M897R
M897R/L1377D
++

521/522
I388P
I388P/L1377D
+

523/524
Y748G
Y748G/L1377D
++

525/526
I756P
I756P/L1377D
++

527/528
Y748F
Y748F/L1377D
+

529/530
I390A
I390A/L1377D
++

531/532
I610L
I610L/L1377D
+

533/534
A755G
A755G/L1377D
++

535/536
L749R
L749R/L1377D
++

537/538
Y748V
Y748V/L1377D
+

539/540
M1380A
L1377D/M1380A
+

541/542
H1440C
L1377D/H1440C
+

543/544
H397N/F1218M
H397N/F1218M/L1377D
++

545/546
F1037M
F1037M/L1377D
+

547/548
F1218P
F1218P/L1377D
+

549/550
E757K
E757K/L1377D
++

551/552
V1373S
V1373S/L1377D
++

553/554
H1440G
L1377D/H1440G
++

555/556
F1193I
F1193I/L1377D
++

557/558
V1439T
L1377D/V1439T
++

559/560
L750D
L750D/L1377D
++

561/562
I390G
I390G/L1377D
++

563/564
I756G
I756G/L1377D
+

565/566
S1220E
S1220E/L1377D
++

567/568
T1378C
L1377D/T1378C
+

569/570
A618G
A618G/L1377D
+

571/572
D346R
D346R/L1377D
++

573/574
V63Q/L1248D
V63Q/L1248D/L1377D
++

575/576
P349G
P349G/L1377D
++

577/578
E306G/R355C/L1422I
E306G/R355C/L1377D/L1422I
++

579/580
L1422I
L1377D/L1422I
++

581/582
E306G/D346R/I708T
E306G/D346R/I708T/L1377D
++

583/584
T1148N
T1148N/L1377D
+++

585/586
L171P/D346R/L1248D
L171P/D346R/L1248D/L1377D
+++

587/588
V63Q/M797T/K799R/L1248D/
V63Q/M797T/K799R/L1248D/
++

L1422I
L1377D/L1422I

589/590
L171P/K799R
L171P/K799R/L1377D
++

591/592
A362P
A362P/L1377D
++

593/594
L1248D
L1248D/L1377D
++

595/596
V63Q
V63Q/L1377D
++

597/598
L171P/E306G/D346R/Q999R/
L171P/E306G/D346R/Q999R/
+++

L1248D
L1248D/L1377D

599/600
L171P/T1148N
L171P/T1148N/L1377D
+++

601/602
T734S/K799R/L1248D/L1422I
T734S/K799R/L1248D/L1377D/
++

L1422I

603/604
V63Q/K732E
V63Q/K732E/L1377D
+++

605/606
V63Q/L171P/E306G/D346R/
V63Q/L171P/E306G/D346R/
++

K732G
K732G/L1377D

607/608
L171P
L171P/L1377D
++

609/610
L171P/L1422I
L171P/L1377D/L1422I
++

611/612
V63Q/A362P/K732G/K799R/
V63Q/A362P/K732G/K799R/
++

L1248D
L1248D/L1377D

613/614
Q999R/T1148N/L1422I
Q999R/T1148N/L1377D/L1422I
+

615/616
D346R/P349G
D346R/P349G/L1377D
+++

617/618
E306G/P349G
E306G/P349G/L1377D
++

619/620
V63Q/L171P/T1148N/G1151S
V63Q/L171P/T1148N/L1377D/
++

G1151S

621/622
E306G
E306G/L1377D
+

623/624
K344E/P349G/L354R/V1250T
K344E/P349G/L354R/V1250T/
++

L1377D

625/626
V63G
V63G/L1377D
+

627/628
V1250T
V1250T/L1377D
+

629/630
V63R/E306G/K344E/P349G/
V63R/E306G/K344E/P349G/
+++

L354R/R503S/L1248R
L354R/R503S/L1248R/L1377D

631/632
F59D/V63S/S332P/P349G/
F59D/V63S/S332P/P349G/R503S/
++

R503S/L706M/I708K
L706M/I708K/L1377D

633/634
V63S/E306G/K344A/P349G/
V63S/E306G/K344A/P349G/
++

L354R/L706M/L1248R
L354R/L706M/L1248R/L1377D

635/636
L1248R
L1248R/L1377D
++

637/638
F59D/V63G
F59D/V63G/L1377D
++

639/640
S332P/L706M/I708K/L1248R/
S332P/L706M/I708K/L1248R/
++

V1250T
V1250T/L1377D

641/642
R503S
R503S/L1377D
++

643/644
F59D/V63R/P349G/L354R
F59D/V63R/P349G/L354R/L1377D
+++

645/646
F59D/P349G/I708K
F59D/P349G/I708K/L1377D
+++

647/648
F59D/E306G/K344E/L1248R
F59D/E306G/K344E/L1248R/L1377D
+++

649/650
K344E/L1248R/V1250T
K344E/L1248R/V1250T/L1377D
++

651/652
I708K
I708K/L1377D
++

653/654
V63R/K344E/P349G
V63R/K344E/P349G/L1377D
++

655/656
F59D
F59D/L1377D
+

657/658
V63S/K344A/P349G/L354R
V63S/K344A/P349G/L354R/L1377D
++

659/660
L706M
L706M/L1377D
+

661/662
F59D/E306G/R770W
F59D/E306G/R770W/L1377D
+

663/664
V63G/S347N/L354R/L1248R
V63G/S347N/L354R/L1248R/L1377D
++

665/666
L354R/R503S
L354R/R503S/L1377D
+++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 252. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

Example 6
FVIII Variants Derived from SEQ ID NO: 504

In this Example, experiments conducted to determine FVIII variant activity are described. These variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Tables 6.1 and 6.2 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 6.1

Activity of FVIII Variants Relative to SEQ ID NO: 504

Amino Acid
Amino Acid
(Expi293F) Unchallenged

Differences
Differences
Chromogenic Activity

SEQ ID
(Relative to SEQ
(Relative to
FIOPC Relative to SEQ ID

NO: (nt/aa)
ID NO: 504)
SEQ ID NO: 2)
NO: 504

503/504

L1377D/V1439E
+

667/668
L750D
L750D/L1377D/V1439E
++

669/670
M1051K
M1051K/L1377D/V1439E
++

671/672
M1051S
M1051S/L1377D/V1439E
++

673/674
S260T
S260T/L1377D/V1439E
++

675/676
I756P
I756P/L1377D/V1439E
+

677/678
F1193A
F1193A/L1377D/V1439E
++

679/680
S332P
S332P/L1377D/V1439E
+

681/682
M897R
M897R/L1377D/V1439E
++

683/684
F387Q
F387Q/L1377D/V1439E
+

685/686
F1037N
F1037N/L1377D/V1439E
+

687/688
F387R
F387R/L1377D/V1439E
++

689/690
E306G/L749F
E306G/L749F/L1377D/V1439E
++

691/692
I1270V
I1270V/L1377D/V1439E
+

693/694
S489E
S489E/L1377D/V1439E
++

695/696
Q487N
Q487N/L1377D/V1439E
++

697/698
A488G
A488G/L1377D/V1439E
++

699/700
Q903E
Q903E/L1377D/V1439E
+

All activities were determined relative to the reference polypeptide of SEQ ID NO: 504. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1

TABLE 6.2

Activity of FVIII Variants Relative to SEQ ID NO: 504

(Expi293F)
(HepG2)
(Expi293F)

Unchallenged
Unchallenged
Unchallenged

Chromogenic
Chromogenic
Clotting

Amino Acid
Amino Acid
Activity
Activity
Activity

SEQ ID
Differences
Differences
FIOPC
FIOPC
FIOPC

NO:
(Relative to
(Relative to
Relative to
Relative to
Relative to

(nt/aa)
SEQ ID NO: 504)
SEQ ID NO: 2)
SEQ ID NO:504
SEQ ID NO: 504
SEQ ID NO: 504

503/504

L1377D/V1439E
+
+
+

701/702
L171P/L1248D/H1440T
L171P/L1248D/
+++
+++
++

L1377D/V1439E/H1440T

703/704
S260T/T1148N/T1378D
S260T/T1148N/L1377D/
+++
+
+

T1378D/V1439E

705/706
L171P/S260T/F387I/
L171P/S260T/F387I/
+++
+++
+++

L750D/L1248D
L750D/L1248D/L1377D/

V1439E

707/708
L171P/S260T
L171P/S260T/L1377D/
+++
+++
++

V1439E

709/710
L171P/S260T/F387I/
L171P/S260T/F387I/
+++
+++
+++

L750D/M1051S/T1148N
L750D/M1051S/T1148N/

L1377D/V1439E

711/712
L171P
L171P/L1377D/V1439E
+++
+++
++

713/714
L750D/M1051S/T1148N
L750D/M1051S/T1148N/
+++
++
++

L1377D/V1439E

715/716
L171P/S260T/I756P
L171P/S260T/I756P/
+++
+++
++

L1377D/V1439E

717/718
L171P/L1248D
L171P/L1248D/L1377D/
+++
+++
++

V1439E

719/720
L171P/T1148N
L171P/T1148N/L1377D/
++
+++
++

V1439E

721/722
F387I
F387I/L1377D/V1439E
++
+
+

723/724
F59D
F59D/L1377D/V1439E
++
+
++

725/726
H1440S
L1377D/V1439E/H1440S
++
+
+

727/728
S332P/L750D/L1248D
S332P/L750D/L1248D/
+++
++
+++

L1377D/V1439E

729/730
I756P/L1248D
I756P/L1248D/L1377D/
++
+
++

V1439E

731/732
L1248D
L1248D/L1377D/V1439E
++

++

733/734
L171P/F387I/L1248D/
L171P/F387I/L1248D/
+++
++
++

H1440S
L1377D/H1440S/V1439E

735/736
L750D/H1440S
L750D/L1377D/V1439E/
++
++
++

H1440S

737/738
F59D/L171P/L750D
F59D/L171P/L750D/
+++
+++
+++

L1377D/V1439E

739/740
L171P/F1193A/L1248D
L171P/F1193A/L1248D/
+++
+++
++

L1377D/V1439E

741/742
L750D/T1148N/
L750D/T1148N/F1193A/
+++
++
++

F1193A/H1440S
L1377D/V1439E/H1440S

743/744
T1148N
T1148N/L1377D/V1439E
++
++
++

745/746
L171P/F387I/M1051K
L171P/F387I/M1051K/
++
+++
++

L1377D/V1439E

747/748
Y748G
Y748G/L1377D/V1439E
++
+
+

749/750
E306G
E306G/L1377D/V1439E

++

751/752
E306G/F387R/I390A/
E306G/F387R/I390A/
+++
+++
++

L749F/S1220D
L749F/S1220D/L1377D/

V1439E

753/754
I390A
I390A/L1377D/V1439E
++
+++
++

755/756
E306G/I390A/F1037N
E306G/I390A/F1037N/
++
++
++

L1377D/V1439E

757/758
E306G/R612S/L749F
E306G/R612S/L749F/
++
+
++

L1377D/V1439E

759/760
F387V/I390A/L749F
F387V/I390A/L749F/
+++
++
++

L1377D/V1439E

761/762
V63S/I390A/R503S/
V63S/I390A/R503S/
++
++
++

L749F
L749F/L1377D/V1439E

763/764
F387R/I390A/L749R
F387R/I390A/L749R/
++
++
++

L1377D/V1439E

765/766
S1220D
S1220D/L1377D/V1439E
++

767/768
V63S/E306G
V63S/E306G/L1377D/
++
++
++

V1439E

769/770
L749F
L749F/L1377D/V1439E

++
++

771/772
E306G/I390A/L749F/
E306G/I390A/L749F/
++
++
++

F1037N
F1037N/L1377D/V1439E

773/774
F387V
F387V/L1377D/V1439E
++
++
+

775/776
E306G/L749F/S1220D
E306G/L749F/S1220D/
++
++
+

L1377D/V1439E

777/778
E306G/F387V/I390A/
E306G/F387V/I390A/
++
++
++

R612S/L749F/
R612S/L749F/F1037N/

F1037N
L1377D/V1439E

779/780
F328S
F328S/L1377D/V1439E
++
++
+

All activities were determined relative to the reference polypeptide of SEQ ID NO: 504. Level of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

Example 7
FVIII Variants Derived from SEQ ID NO: 706

In this Example, experiments conducted to determine FVIII variant activity are described. These variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Tables 7.1 and 7.2 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 7.1

Activity of FVIII Variants Relative to SEQ ID NO: 706

(Expi293F)

Amino Acid

Unchallenged

Differences
Amino Acid
Chromogenic

(Relative to
Differences
Activity FIOPC

SEQ ID
SEQ ID NO:
(Relative to
Relative to

NO: (nt/aa)
706)
SEQ ID NO: 2)
SEQ ID NO: 706

705/706

L171P/S260T/F387I/L750D/L1248D/
+

L1377D/V1439E

781/782
M1051K
L171P/S260T/F387I/L750D/M1051K/
++

L1248D/L1377D/V1439E

783/784
L1221G
L171P/S260T/F387I/L750D/L1221G/
+

L1248D/L1377D/V1439E

785/786
F1218G
L171P/S260T/F387I/L750D/F1218G/
+

L1248D/L1377D/V1439E

787/788
I387R
L171P/S260T/F387R/L750D/L1248D/
+

L1377D/V1439E

789/790
M1051R
L171P/S260T/F387I/L750D/M1051R/
++

L1248D/L1377D/V1439E

791/792
L749F/
L171P/S260T/F387I/L749F/L750D/
++

F1218G
F1218G/L1248D/L1377D/V1439E

793/794
I387T
L171P/S260T/F387T/L750D/L1248D/
+

L1377D/V1439E

795/796
S332P
L171P/S260T/S332P/F387I/L750D/
+

L1248D/L1377D/V1439E

797/798
M897K
L171P/S260T/F387I/L750D/M897K/
+

L1248D/L1377D/V1439E

799/800
T696E
L171P/S260T/F387I/T696E/L750D/
+

L1248D/L1377D/V1439E

801/802
F816W
L171P/S260T/F387I/L750D/F816W/
+

L1248D/L1377D/V1439E

803/804
F824W
L171P/S260T/F387I/L750D/F824W/
+

L1248D/L1377D/V1439E

805/806
A826G
L171P/S260T/F387I/L750D/A826G/
+

L1248D/L1377D/V1439E

807/808
V393W
L171P/S260T/F387I/V393W/L750D/
+

L1248D/L1377D/V1439E

809/810
I825L
L171P/S260T/F387I/L750D/I825L/
+

L1248D/L1377D/V1439E

811/812
M897R
L171P/S260T/F387I/L750D/M897R/
+

L1248D/L1377D/V1439E

813/814
F816V
L171P/S260T/F387I/L750D/F816V/
++

L1248D/L1377D/V1439E

815/816
F698W
L171P/S260T/F387I/F698W/L750D/
++

L1248D/L1377D/V1439E

817/818
V393Q
L171P/S260T/F387I/V393Q/L750D/
+

L1248D/L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 706. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1

TABLE 7.2

Activity of FVIII Variants Relative to SEQ ID NO: 706

(Expi293F)
(HepG2)
(Expi293F)

Unchallenged
Unchallenged
Unchallenged

Chromogenic
Chromogenic
Clotting

Amino Acid
Amino Acid
Activity
Activity
Activity

SEQ ID
Differences
Differences
FIOPC
FIOPC
FIOPC

NO:
(Relative to
(Relative to
Relative to
Relative to
Relative to

(nt/aa)
SEQ ID NO: 706)
SEQ ID NO: 2)
SEQ ID NO: 706
SEQ ID NO: 706
SEQ ID NO: 706

705/706

L171P/S260T/F387I/L750D/
+
+
+

L1248D/L1377D/V1439E

819/820
S332P/I387R/
L171P/S260T/S332P/F387R/
+++
+++
+++

I390A/T1148N
I390A/L750D/T1148N/L1248D/

L1377D/V1439E

821/822
F328S/I387R/
L171P/S260T/F328S/F387R/
+
+
++

L749F
L749F/L750D/L1248D/L1377D/

V1439E

823/824
L749F
L171P/S260T/F387I/L749F/
+
+
+

L750D/L1248D/L1377D/V1439E

825/826
E306G/F328S/
L171P/S260T/E306G/F328S/
+++
+++
+

S332P/I390A/
S332P/F387I/I390A/L750D/

M1051R/F1193A/
M1051R/F1193A/F1218G/L1248D/

F1218G
L1377D/V1439E

827/828
V63Q/E306G/
V63Q/L171P/S260T/E306G/
+++
+++
+++

S332P/M1051R/
S332P/F387I/L750D/M1051R/

T1148N
T1148N/L1248D/L1377D/V1439E

829/830
V63Q/E306G/
V63Q/L171P/S260T/E306G/
++
+++
++

S332P/I387T/
S332P/F387T/L750D/M1051K/

M1051K
L1248D/L1377D/V1439E

831/832
I387T/I390A/
L171P/S260T/F387T/I390A/
++
+++
++

M1051R
L750D/M1051R/L1248D/L1377D/

V1439E

833/834
D186A/E306G/
L171P/D186A/S260T/E306G/
+++
++
+++

F328S/S332P/
F328S/S332P/F387I/I390A/

I390A/L749F/
L749F/L750D/M1051K/T1148N/

M1051K/T1148N/
F1193A/L1248D/L1377D/V1439E

F1193A

835/836
I387T/I390A/
L171P/S260T/F387T/I390A/
++
++
++

I756P/M1051R
L750D/I756P/M1051R/L1248D/

L1377D/V1439E

837/838
E306G/I390A/
L171P/S260T/E306G/F387I/
+++
++
+++

M1051R
I390A//L750D/M1051R/

L1248D/L1377D/V1439E

839/840
E306G/S332P/
L171P/S260T/E306G/S332P/
+++
+++
+++

I390A/L749F
F387I/I390A/L749F/L750D/

L1248D/L1377D/V1439E

841/842
E306G/I387R/
L171P/S260T/E306G/F387R/
++
++
+++

I390A/T1148N
I390A/L750D/T1148N/L1248D/

L1377D/V1439E

843/844
E306G/F328S/
L171P/S260T/E306G/F328S/
+++
+++
+++

S332P/I387T/
S332P/F387T/I390A/L749F/

I390A/L749F/
L750D/M1051R/T1148N/F1193A/

M1051R/T1148N/
L1248D/L1377D/V1439E

F1193A

845/846
S332P/I387R/
L171P/S260T/S332P/F387R/
+++
++
+++

I390A/L749F/
I390A/L749F/L750D/T1148N/

T1148N
L1248D/L1377D/V1439E

847/848
I387R/I390A/
L171P/S260T/F387R/I390A/
++
++
++

L749F
L749F/L750D/L1248D/L1377D/

V1439E

849/850
E306G/I387T/
L171P/S260T/E306G/F387T/
++
++
++

L749F/M1051K/
L749F/L750D/M1051K/T1148N/

T1148N
L1248D/L1377D/V1439E

851/852
I387R/L749F
L171P/S260T/F387R/L749F/
++
++
++

L750D/L1248D/L1377D/V1439E

853/854
E306G/Q945K/
L171P/S260T/E306G/F387I/
++
+
+

M1051R/F1193A/
L750D/Q945K/M1051R/F1193A/

L1221G
L1221G/L1248D/L1377D/V1439E

855/856
E306G/I390A/
L171P/S260T/E306G/F387I/
++
+
++

L749F/M1051K
I390A/L749F/L750D/M1051K/

L1248D/L1377D/V1439E

857/858
V63Q/E306G/
V63Q/L171P/S260T/E306G/
++
++
+++

S332P/I390A
S332P/F387I/I390A/L750D/

L1248D/L1377D/V1439E

859/860
E306G/I387T/
L171P/S260T/E306G/F387T/
+++
++
+

I390A/L749F/
I390A/L749F/L750D/T1148N/

T1148N/F1193A/
F1193A/L1221G/L1248D/L1377D/

L1221G
V1439E

861/862
E306G/S332P/
L171P/S260T/E306G/S332P/
+++
+++
++

I387R/I390A/
F387R//I390A/L749F/

L749F/F1218M
L750D/F1218M/L1248D/L1377D/

V1439E

863/864
V63Q/E306G/
V63Q/L171P/S260T/E306G/
+++
+++
+

S332P/I390A/
S332P/F387I/I390A/L750D/

M1051R/T1148N/
M1051R/T1148N/F1193A/F1218G/

F1193A/F1218G
L1248D/L1377D/V1439E

865/866
G924I
L171P/S260T/F387I/L750D/
+
++
+

G924I/L1248D/L1377D/V1439E

867/868
A755S
L171P/S260T/F387I/L750D/
+
+
+

A755S/L1248D/L1377D/V1439E

869/870
K752F
L171P/S260T/F387I/L750D/
+
+
++

K752F/L1248D/L1377D/V1439E

871/872
S751Y
L171P/S260T/F387I/L750D/
++
++
++

S751Y/L1248D/L1377D/V1439E

873/874
Q923M
L171P/S260T/F387I/L750D/
+
+
+

Q923M/L1248D/L1377D/V1439E

875/876
M797Y
L171P/S260T/F387I/L750D/
+
+
+

M797Y/L1248D/L1377D/V1439E

877/878
N754W
L171P/S260T/F387I/L750D/
+
+
+

N754W/L1248D/L1377D/V1439E

879/880
I793G
L171P/S260T/F387I/L750D/
+
+
+

1793G/L1248D/L1377D/V1439E

881/882
N753D
L171P/S260T/F387I/L750D/
++
+
+++

N753D/L1248D/L1377D/V1439E

883/884
R922W
L171P/S260T/F387I/L750D/
+
+
+

R922W/L1248D/L1377D/V1439E

885/886
I896L
L171P/S260T/F387I/L750D/
+
+
+

1896L/L1248D/L1377D/V1439E

887/888
K938R
L171P/S260T/F387I/L750D/
+
+
+

K938R/L1248D/L1377D/V1439E

889/890
I775G
L171P/S260T/F387I/L750D/
++
++
++

I775G/L1248D/L1377D/V1439E

891/892
S751E
L171P/S260T/F387I/L750D/
+
+
+

S751E/L1248D/L1377D/V1439E

893/894
K732W
L171P/S260T/F387I/L750D/
+
+
++

K732W/L1248D/L1377D/V1439E

895/896
K752G
L171P/S260T/F387I/L750D/
+
+
++

K752G/L1248D/L1377D/V1439E

897/898
N810V
L171P/S260T/F387I/L750D/
+
+
+

N810V/L1248D/L1377D/V1439E

899/900
K938V
L171P/S260T/F387I/L750D/
+
+
+

K938V/L1248D/L1377D/V1439E

901/902
A747D
L171P/S260T/F387I/L750D/
++
+
++

A747D/L1248D/L1377D/V1439E

903/904
A755G
L171P/S260T/F387I/L750D/
+
+
+

A755G/L1248D/L1377D/V1439E

905/906
V393G/A407V
L171P/S260T/F387I/V393G/
++
++
+

A407V/L750D/L1248D/L1377D/

V1439E

907/908
M797W
L171P/S260T/F387I/L750D/
+
+
++

M797W/L1248D/L1377D/V1439E

909/910
K819V
L171P/S260T/F387I/L750D/
+
+
+

K819V/L1248D/L1377D/V1439E

911/912
V393G
L171P/S260T/F387I/V393G/
++
+++
+

L750D/L1248D/L1377D/V1439E

913/914
I787W
L171P/S260T/F387I/L750D/
+
++
++

I787W/L1248D/L1377D/V1439E

915/916
V393R
L171P/S260T/F387I/V393R/
++
+++
+

L750D/L1248D/L1377D/V1439E

917/918
F802V
L171P/S260T/F387I/L750D/
+
++
+

F802V/L1248D/L1377D/V1439E

919/920
I775E
L171P/S260T/F387I/L750D/
++
+
+

I775E/L1248D/L1377D/V1439E

921/922
G924P
L171P/S260T/F387I/L750D/
+
+
+

G924P/L1248D/L1377D/V1439E

923/924
K732G
L171P/S260T/F387I/K732G/
+
+
+

L750D/L1248D/L1377D/V1439E

925/926
Q811F
L171P/S260T/F387I/L750D/
+
+
++

Q811F/L1248D/L1377D/V1439E

927/928
F802S
L171P/S260T/F387I/L750D/
+
++
+

F802S/L1248D/L1377D/V1439E

929/930
K752L
L171P/S260T/F387I/L750D/
+
+
+++

K752L/L1248D/L1377D/V1439E

931/932
S392A
L171P/S260T/F387I/S392A/
+
+
+

L750D/L1248D/L1377D/V1439E

933/934
H397G
L171P/S260T/F387I/H397G/
+
++
+

L750D/L1248D/L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 706. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

Example 8
FVIII Variants Derived from SEQ ID NO: 840

In this Example, experiments conducted to determine FVIII variant activity by assaying the protein activity after a challenge is described. These variants were tested for FVIII activity after no incubation and after incubation in Hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Tables 8.1 and 8.2 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 8.1

Activity of FVIII Variants Relative to SEQ ID NO: 840

(Expi293F)
(Expi293F)

Unchallenged
Activity after

Chromogenic
Plasma

Amino Acid

Activity
Challenge

Differences
Amino Acid
FIOPC
FIOPC

SEQ
(Relative to
Differences
Relative to
Relative to

ID NO:
SEQ ID NO:
(Relative to
SEQ ID NO:
SEQ ID NO:

(nt/aa)
840)
SEQ ID NO: 2)
840
840

839/840

L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/L1248D/L1377D/V1439E

935/936
V620N
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

V620N/L749F/L750D/L1248D/L1377D/V1439E

937/938
P491E
L171P/S260T/E306G/S332P/F387I/I390A/
++
+

P491E/L749F/L750D/L1248D/L1377D/V1439E

939/940
M1229D
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/M1229D/L1248D/L1377D/V1439E

941/942
P615N
L171P/S260T/E306G/S332P/F387I/I390A/
++

P615N/L749F/L750D/L1248D/L1377D/V1439E

943/944
I1283S
L171P/S260T/E306G/S332P/F387I/I390A/
++

L749F/L750D/L1248D/I1283S/L1377D/V1439E

945/946
H822S
L171P/S260T/E306G/S332P/F387I/I390A/
++

L749F/L750D/H822S/L1248D/L1377D/V1439E

947/948
F328G
L171P/S260T/E306G/F328G/S332P/F387I/
++
+

I390A/L749F/L750D/L1248D/L1377D/V1439E

949/950
H212L
L171P/H212L/S260T/E306G/S332P/F387I/
+++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

951/952
K818W
L171P/S260T/E306G/S332P/F387I/I390A/
++
+

L749F/L750D/K818W/L1248D/L1377D/V1439E

953/954
I1283L
L171P/S260T/E306G/S332P/F387I/I390A/
+
++

L749F/L750D/L1248D/I1283L/L1377D/V1439E

955/956
F555G/
L171P/S260T/E306G/S332P/F387I/I390A/
++
+

E684G
F555G/E684G/L749F/L750D/L1248D/L1377D/

V1439E

957/958
V828K
L171P/S260T/E306G/S332P/F387I/I390A/
++
++

L749F/L750D/V828K/L1248D/L1377D/V1439E

959/960
R550G
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

R550G/L749F/L750D/L1248D/L1377D/V1439E

961/962
R830M
L171P/S260T/E306G/S332P/F387I/I390A/
++
+

L749F/L750D/R830M/L1248D/L1377D/V1439E

963/964
S839A
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/S839A/L1248D/L1377D/V1439E

965/966
H822T
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/H822T/L1248D/L1377D/V1439E

967/968
L13Y
L13Y/L171P/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

969/970
L14G
L14G/L171P/S260T/E306G/S332P/F387I/
+
+

I390A/L749F/L750D/L1248D/L1377D/V1439E

971/972
C17V
C17V/L171P/S260T/E306G/S332P/F387I/
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

973/974
M836V
L171P/S260T/E306G/S332P/F387I/I390A/
+
++

L749F/L750D/M836V/L1248D/L1377D/V1439E

975/976
S839P
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/S839P/L1248D/L1377D/V1439E

977/978
K1210W
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/K1210W/L1248D/L1377D/V1439E

979/980
Y1281R
L171P/S260T/E306G/S332P/F387I/I390A/
++

L749F/L750D/L1248D/Y1281R/L1377D/V1439E

981/982
V64D
V64D/L171P/S260T/E306G/S332P/F387I/
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

983/984
H212G
L171P/H212G/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

985/986
V682R
L171P/S260T/E306G/S332P/F387I/I390A/
++

V682R/L749F/L750D/L1248D/L1377D/V1439E

987/988
L14M
L14M/L171P/S260T/E306G/S332P/F387I/
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

989/990
L318C
L171P/S260T/E306G/L318C/S332P/F387I/

++

I390A/L749F/L750D/L1248D/L1377D/V1439E

991/992
C17S
C17S/L171P/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

993/994
A20L
A20L/L171P/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

995/996
K818G
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/K818G/L1248D/L1377D/V1439E

997/998
M836G
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/M836G/L1248D/L1377D/V1439E

999/1000
T21M
T21M/L171P/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

1001/1002
M836R
L171P/S260T/E306G/S332P/F387I/I390A/
+
++

L749F/L750D/M836R/L1248D/L1377D/V1439E

1003/1004
F325Y
L171P/S260T/E306G/F325Y/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

1005/1006
T533V
L171P/S260T/E306G/S332P/F387I/I390A/
++

T533V/L749F/L750D/L1248D/L1377D/V1439E

1007/1008
S19G
S19G/L171P/S260T/E306G/S332P/F387I/
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

1009/1010
L14V
L14V/L171P/S260T/E306G/S332P/F387I/
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

1011/1012
Q817D
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/Q817D/L1248D/L1377D/V1439E

1013/1014
L831W
L171P/S260T/E306G/S332P/F387I/I390A/
+
+

L749F/L750D/L831W/L1248D/L1377D/V1439E

1015/1016
K225R
L171P/K225R/S260T/E306G/S332P/F387I/
++
++

I390A/L749F/L750D/L1248D/L1377D/V1439E

1017/1018
H1156R
L171P/S260T/E306G/S332P/F387I/I390A/
+
++

L749F/L750D/H1156R/L1248D/L1377D/V1439E

1019/1020
R830V
L171P/S260T/E306G/S332P/F387I/I390A/
++
++

L749F/L750D/R830V/L1248D/L1377D/V1439E

1021/1022
V393G/
L171P/S260T/E306G/S332P/F387I/I390A/
++
++

A747D
V393G/A747D/L749F/L750D/L1248D/L1377D/

V1439E

1023/1024
N753D
L171P/S260T/E306G/S332P/F387I/I390A/
++

L749F/L750D/N753D/L1248D/L1377D/V1439E

1025/1026
V393G/
L171P/S260T/E306G/S332P/F387I/I390A/
++
++

K732W/
V393G/K732W/L749F/L750D/S751Y/L1248D/

S751Y
L1377D/V1439E

1027/1028
V63Q/
V63Q/L171P/S260T/E306G/S332P/F387I/
++
+

M797W
I390A/L749F/L750D/M797W/L1248D/L1377D/

V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 840. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

TABLE 8.2

Activity of FVIII Variants Relative to SEQ ID NO: 840

(Expi293F)

(Expi293F)
Chromogenic
(HepG2)
(Expi293F)

Unchallenged
Activity
Unchallenged
Unchallenged

Chromogenic
after Plasma
Chromogenic
Clotting

Activity
Challenge
Activity
Activity

Amino Acid
Amino Acid
FIOPC
FIOPC
FIOPC
FIOPC

SEQ ID
Differences
Differences
Relative to
Relative to
Relative to
Relative to

NO:
(Relative to
(Relative to
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:

(nt/aa)
SEQ ID NO: 840)
SEQ ID NO: 2)
840
840
840
840

839/840

L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+
+
+

L1248D/L1377D/V1439E

1029/1030
L622D
L171P/S260T/E306G/S332P/F387I/I390A/L622D/L749F/
++
+
+
+

L750D/L1248D/L1377D/V1439E

1031/1032
P617E
L171P/S260T/E306G/S332P/F387I/I390A/P617E/L749F/
++
++
+
++

L750D/L1248D/L1377D/V1439E

1033/1034
P617N
L171P/S260T/E306G/S332P/F387I/I390A/P617N/L749F/
++

+
+

L750D/L1248D/L1377D/V1439E

1035/1036
P907D
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+

+

P907D/L1248D/L1377D/V1439E

1037/1038
N1040E
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+++

++

N1040E/L1248D/L1377D/V1439E

1039/1040
P617D
L171P/S260T/E306G/S332P/F387I/I390A/P617D/L749F/
++
++
+
+

L750D/L1248D/L1377D/V1439E

1041/1042
Q611E
L171P/S260T/E306G/S332P/F387I/I390A/Q611E/L749F/
++
++
++
+

L750D/L1248D/L1377D/V1439E

1043/1044
V620P
L171P/S260T/E306G/S332P/F387I/I390A/V620P/L749F/
++
++

+

L750D/L1248D/L1377D/V1439E

1045/1046
P617G
L171P/S260T/E306G/S332P/F387I/I390A/P617G/L749F/
++
++
+
++

L750D/L1248D/L1377D/V1439E

1047/1048
A488G
L171P/S260T/E306G/S332P/F387I/I390A/A488G/L749F/

+
+

L750D/L1248D/L1377D/V1439E

1049/1050
P1192D
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++

+

P1192D/L1248D/L1377D/V1439E

1051/1052
S1194P
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++

+

S1194P/L1248D/L1377D/V1439E

1053/1054
G619E
L171P/S260T/E306G/S332P/F387I/I390A/G619E/L749F/
++
++
++
+

L750D/L1248D/L1377D/V1439E

1055/1056
P615D
L171P/S260T/E306G/S332P/F387I/I390A/P615D/L749F/
+
+
+
+

L750D/L1248D/L1377D/V1439E

1057/1058
F16A
F16A/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
++
++
+

L750D/L1248D/L1377D/V1439E

1059/1060
F802S
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+
++
+

F802S/L1248D/L1377D/V1439E

1061/1062
K818V
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
++
+
+

K818V/L1248D/L1377D/V1439E

1063/1064
L211K
L171P/L211K/S260T/E306G/S332P/F387I/I390A/L749F/
+
++
+
++

L750D/L1248D/L1377D/V1439E

1065/1066
M836W
L171P/S260T/E306G/S332P/F387I/I390A/L749F/M836W/
++
++

++

L750D/L1248D/L1377D/V1439E

1067/1068
F555R
L171P/S260T/E306G/S332P/F387I/I390A/F555R/L749F/
++
++

++

L750D/L1248D/L1377D/V1439E

1069/1070
V536A
L171P/S260T/E306G/S332P/F387I/I390A/V536A/L749F/
+
++
+
+

L750D/L1248D/L1377D/V1439E

1071/1072
I527A
L171P/S260T/E306G/S332P/F387I/I390A/I527A/L749F/

+

+

L750D/L1248D/L1377D/V1439E

1073/1074
F555E
L171P/S260T/E306G/S332P/F387I/I390A/F555E/L749F/
++
++
+
++

L750D/L1248D/L1377D/V1439E

1075/1076
H841W
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
++

+

H841W/L1248D/L1377D/V1439E

1077/1078
M320Q
L171P/S260T/E306G/M320Q/S332P/F387I/I390A/L749F/
+
++
+
+

L750D/L1248D/L1377D/V1439E

1079/1080
S837G
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++

++
+

S837G/L1248D/L1377D/V1439E

1081/1082
M558R
L171P/S260T/E306G/S332P/F387I/I390A/M558R/L749F/
+
+

L750D/L1248D/L1377D/V1439E

1083/1084
F555G
L171P/S260T/E306G/S332P/F387I/I390A/F555G/L749F/
+
++
+
+

L750D/L1248D/L1377D/V1439E

1085/1086
L211R
L171P/L211R/S260T/E306G/S332P/F387I/I390A/L749F/
+
+
+
+

L750D/L1248D/L1377D/V1439E

1087/1088
V64G
V64G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
+
+

L750D/L1248D/L1377D/V1439E

1089/1090
M320R
L171P/S260T/E306G/M320R/S332P/F387I/I390A/L749F/
++
+++
++
++

L750D/L1248D/L1377D/V1439E

1091/1092
T533Y
L171P/S260T/E306G/S332P/F387I/I390A/T533Y/L749F/
+

++
++

L750D/L1248D/L1377D/V1439E

1093/1094
L914V
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+
++
++

L914V/L1248D/L1377D/V1439E

1095/1096
F16V
F16V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
++
++

L750D/L1248D/L1377D/V1439E

1097/1098
I1283E
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++

++
+

L1248D/I1283E/L1377D/V1439E

1099/1100
V536M
L171P/S260T/E306G/S332P/F387I/I390A/V536M/L749F/
+

++
+

L750D/L1248D/L1377D/V1439E

1101/1102
F312L
L171P/S260T/E306G/F312L/S332P/F387I/I390A/L749F/
++
++
++
++

L750D/L1248D/L1377D/V1439E

1103/1104
M681N
L171P/S260T/E306G/S332P/F387I/I390A/M681N/L749F/
++
+++
+
++

L750D/L1248D/L1377D/V1439E

1105/1106
S838T
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+
++
+

S838T/L1248D/L1377D/V1439E

1107/1108
V536R
L171P/S260T/E306G/S332P/F387I/I390A/V536R/L749F/
+
+
++
+

L750D/L1248D/L1377D/V1439E

1109/1110
R15G
R15G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
++
+

L750D/L1248D/L1377D/V1439E

1111/1112
V536P
L171P/S260T/E306G/S332P/F387I/I390A/V536P/L749F/
++
++

++

L750D/L1248D/L1377D/V1439E

1113/1114
M558W
L171P/S260T/E306G/S332P/F387I/I390A/M558W/L749F/
++
++
++
+

L750D/L1248D/L1377D/V1439E

1115/1116
M558E
L171P/S260T/E306G/S332P/F387I/I390A/M558E/L749F/
++

++
+

L750D/L1248D/L1377D/V1439E

1117/1118
L1427Y
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++
++
++

L1248D/L1377D/L1427Y/V1439E

1119/1120
S839G
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/

++
+

S839G/L1248D/L1377D/V1439E

1121/1122
F18L
F18L/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
+
++
+
+

L750D/L1248D/L1377D/V1439E

1123/1124
L13G
L13G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
++
+
+

L750D/L1248D/L1377D/V1439E

1125/1126
K1210M
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+

++
+

K1210M/L1248D/L1377D/V1439E

1127/1128
H1156G
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
++
++
+

H1156G/L1248D/L1377D/V1439E

1129/1130
M558L
L171P/S260T/E306G/S332P/F387I/I390A/M558L/L749F/
+
++

L750D/L1248D/L1377D/V1439E

1131/1132
S839K
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
++
++
++

S839K/L1248D/L1377D/V1439E

1133/1134
L211P
L171P/L211P/S260T/E306G/S332P/F387I/I390A/L749F/
+

+
+

L750D/L1248D/L1377D/V1439E

1135/1136
L211G
L171P/L211G/S260T/E306G/S332P/F387I/I390A/L749F/
+

+
+

L750D/L1248D/L1377D/V1439E

1137/1138
L211Q
L171P/L211Q/S260T/E306G/S332P/F387I/I390A/L749F/
++
++
+
++

L750D/L1248D/L1377D/V1439E

1139/1140
F16D
F16D/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
+
++

L750D/L1248D/L1377D/V1439E

1141/1142
M320G
L171P/S260T/E306G/M320G/S332P/F387I/I390A/L749F/
+
+

L750D/L1248D/L1377D/V1439E

1143/1144
Y683W
L171P/S260T/E306G/S332P/F387I/I390A/Y683W/L749F/
++
++

+

L750D/L1248D/L1377D/V1439E

1145/1146
Y24V
Y24V/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
++

+

L750D/L1248D/L1377D/V1439E

1147/1148
V534I
L171P/S260T/E306G/S332P/F387I/I390A/V534I/L749F/
++
++
+
++

L750D/L1248D/L1377D/V1439E

1149/1150
M681A
L171P/S260T/E306G/S332P/F387I/I390A/M681A/L749F/
++
++
+
+

L750D/L1248D/L1377D/V1439E

1151/1152
P840G
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+

+

P840G/L1248D/L1377D/V1439E

1153/1154
F1160R
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+

+

F1160R/L1248D/L1377D/V1439E

1155/1156
M681C
L171P/S260T/E306G/S332P/F387I/I390A/M681C/L749F/
+
++
++
+

L750D/L1248D/L1377D/V1439E

1157/1158
F328Q
L171P/S260T/E306G/F328Q/S332P/F387I/I390A/L749F/

+
+

L750D/L1248D/L1377D/V1439E

1159/1160
R15S
R15S/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
+
+
+
+

L750D/L1248D/L1377D/V1439E

1161/1162
V536K
L171P/S260T/E306G/S332P/F387I/I390A/V536K/L749F/
+
+
+
+

L750D/L1248D/L1377D/V1439E

1163/1164
V536G
L171P/S260T/E306G/S332P/F387I/I390A/V536G/L749F/
++
++

+

L750D/L1248D/L1377D/V1439E

1165/1166
T533R
L171P/S260T/E306G/S332P/F387I/I390A/T533R/L749F/
+
++
+
++

L750D/L1248D/L1377D/V1439E

1167/1168
M681E
L171P/S260T/E306G/S332P/F387I/I390A/M681E/L749F/
++

++
++

L750D/L1248D/L1377D/V1439E

1169/1170
V536W
L171P/S260T/E306G/S332P/F387I/I390A/V536W/L749F/
++
++

++

L750D/L1248D/L1377D/V1439E

1171/1172
H1156N
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+
++
++

H1156N/L1248D/L1377D/V1439E

1173/1174
F16P
F16P/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
+
+

L750D/L1248D/L1377D/V1439E

1175/1176
Q817A
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++

++

Q817A/L1248D/L1377D/V1439E

1177/1178
F18R
F18R/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
++

+

L750D/L1248D/L1377D/V1439E

1179/1180
Y551W
L171P/S260T/E306G/S332P/F387I/I390A/Y551W/L749F/
++
+
+
+

L750D/L1248D/L1377D/V1439E

1181/1182
F555T
L171P/S260T/E306G/S332P/F387I/I390A/F555T/L749F/
+
++

+

L750D/L1248D/L1377D/V1439E

1183/1184
M681G
L171P/S260T/E306G/S332P/F387I/I390A/M681G/L749F/
++
+++
++
+

L750D/L1248D/L1377D/V1439E

1185/1186
H212A
L171P/H212A/S260T/E306G/S332P/F387I/I390A/L749F/
+
++
+
+

L750D/L1248D/L1377D/V1439E

1187/1188
F16G
F16G/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+
++
+

L750D/L1248D/L1377D/V1439E

1189/1190
S838G
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+

+

S838G/L1248D/L1377D/V1439E

1191/1192
M681R
L171P/S260T/E306G/S332P/F387I/I390A/M681R/L749F/
+
+
+
+

L750D/L1248D/L1377D/V1439E

1193/1194
V393G
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
++
++
++

L750D/L1248D/L1377D/V1439E

1195/1196
V393G/A747D/K752G/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/
++
++
++
++

Q811F/K819V/
L749F/L750D/K752G/Q811F/K819V/Q923M/T1148N

Q923M/T1148N
L1248D/L1377D/V1439E

1197/1198
V393G/S751Y/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
++
+++
+
+

I775G
L750D/S751Y/I775G/L1248D/L1377D/V1439E

1199/1200
V63Q/V393G/K732W/
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/
++
+++
++
++

I775G/T1148N
K732W/L749F/L750D/I775G/T1148N/L1248D/L1377D/

V1439E

1201/1202
V393G/K732W/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/K732W/
++
++
++
++

T1148N
L749F/L750D/T1148N/L1248D/L1377D/V1439E

1203/1204
I775G/T1148N
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+++
++
++

I775G/T1148N/L1248D/L1377D/V1439E

1205/1206
V393G/K752L/N753D/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
++
+++
+
++

I775G/I793G/
L750D/K752L/N753D/I775G/I793G/M797W/L1248D/

M797W
L1377D/V1439E

1207/1208
V393G/Q923M
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
++
++
++
+

L750D/Q923M/L1248D/L1377D/V1439E

1209/1210
V393G/Q923M/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
++
+++
++
+

T1148N
L750D/Q923M/T1148N/L1248D/L1377D/V1439E

1211/1212
V393G/A747D/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/
++
++
++
++

S751Y
L749F/L750D/S751Y/L1248D/L1377D/V1439E

1213/1214
V393G/A747D/N753D/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/A747D/
++
+++
++
++

I775G/T1148N
L749F/L750D/N753D/I775G/T1148N/L1248D/L1377D/

V1439E

1215/1216
K752L/Q923M/
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++
++
+

M1051R
K752L/Q923M/M1051R/L1248D/L1377D/V1439E

1217/1218
V63Q/I775G
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+++
++
++

L750D/I775G/L1248D/L1377D/V1439E

1219/1220
V63Q/V393G/K752L/
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/V393G/
++
++
+
+

I775G/I793G/
L749F/L750D/K752L/I775G/I793G/M1051R/T1053R

M1051R/T1053R
L1248D/L1377D/V1439E

1221/1222
K752G/N753D/I775G/
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
++
++
++

I793G/K819V
K752G/N753D/I775G/I793G/K819V/L1248D/L1377D/

V1439E

1223/1224
V63Q/M1051R
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+++
++
++

L750D/M1051R/L1248D/L1377D/V1439E

1225/1226
V63Q/A747D/K752G/
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/A747D/
++
+++
++
++

N753D/I775G/Q811F/
L749F/L750D/K752G/N753D/I775G/Q811F/K819V/

K819V/Q923M/
Q923M/M1051R L1248D/L1377D/V1439E

M1051R

1227/1228
V393G/K752G/
L171P/S260T/E306G/S332P/F387I/I390A/V393G/L749F/
+
++
++
++

I787W
L750D/K752G/I787W/L1248D/L1377D/V1439E

1229/1230
S751Y
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+
+
++

S751Y/L1248D/L1377D/V1439E

1231/1232
V63Q/T1148N
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
++
++
++

L750D/T1148N/L1248D/L1377D/V1439E

1233/1234
V63H/A747D/K752G/
V63H/L171P/S260T/E306G/S332P/F387I/I390A/A747D/
++
+
++
++

N753D/K819V/
L749F/L750D/K752G/N753D/K819V/Q923M/T1148N

Q923M/T1148N
L1248D/L1377D/V1439E

1235/1236
T1148N
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
++
+
++

T1148N/L1248D/L1377D/V1439E

1237/1238
A747D/K752L/
L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/
++
++
+
++

N753D/I793G/
L750D/K752L/N753D/I793G/Q811F/L1248D/L1377

Q811F
V1439E

1239/1240
V63Q
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/L749F/
++
+++
++
++

L750D/L1248D/L1377D/V1439E

1241/1242
M797W/T1148N
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
+
+
++
+

M797W/T1148N/L1248D/L1377D/V1439E

1243/1244
S751Y/K752L/
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+++
++
++

N753D/M1051R
S751Y/K752L/N753D/M1051R/L1248D/L1377D/V1439E

1245/1246
V63Q/K732W/M1051R
V63Q/L171P/S260T/E306G/S332P/F387I/I390A/K732W/
++
++
++
+

L749F/L750D/M1051R/L1248D/L1377D/V1439E

1247/1248
K752G/M797W/S815I/
L171P/S260T/E306G/S332P/F387I/I390A/L749F/L750D/
++
+++
++
++

Q923M/M1051R/
K752G/M797W/S815I/Q923M/M1051R/T1148N

T1148N
L1248D/L1377D/V1439E

1249/1250
A747D/N753D
L171P/S260T/E306G/S332P/F387I/I390A/A747D/L749F/
++
++
++
++

L750D/N753D/L1248D/L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 840. Levels of increased activity are defined as follows:

“+” 0.9 to 1.1,

“++” >1.1,

“+++” >2

Example 9
FVIII Variants Derived from SEQ ID NO: 1200

In this Example, experiments conducted to determine FVIII variant activity by assaying the protein activity after a challenge is described. These variants were tested for FVIII activity after no incubation and after incubation in Hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Tables 9.1 and 9.2 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 9.1

Activity of FVII Variants Relative to SEQ ID NO: 1200

(Expi293F)

(Expi293F)
Chromogenic

Unchallenged
Activity after

Chromogenic
Plasma

Amino Acid

Activity
Challenge

Differences
Amino Acid
FIOPC
FIOPC

SEQ ID
(Relative to
Differences
Relative to
Relative to

NO:
SEQ ID NO:
(Relative to
SEQ ID NO:
SEQ ID NO:

(nt/aa)
1200)
SEQ ID NO: 2)
1200
1200

1199/1200

V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/T1148N/L1248D/L1377D/

V1439E

1251/1252
M681A
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/M681A/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1253/1254
L622D
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/L622D/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1255/1256
P617G
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/P617G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1257/1258
M558L
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/M558L/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1259/1260
V536K
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/V536K/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1261/1262
M681G
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1263/1264
L211Q
V63Q/L171P/L211Q/S260T/E306G/
+
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1265/1266
M320R
V63Q/L171P/S260T/E306G/M320R/
+++
+++

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1267/1268
M558W
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/M558W/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1269/1270
S839K
V63Q/L171P/S260T/E306G/S332P/
+++
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S839K/T1148N/L1248D/

L1377D/V1439E

1271/1272
L211K
V63Q/L171P/L211K/S260T/E306G/
++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1273/1274
M797W
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/M797W/T1148N/L1248D/

L1377D/V1439E

1275/1276
T533R
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/T533R/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1277/1278
S751A
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/S751A/I775G/T1148N/L1248D/

L1377D/V1439E

1279/1280
A303T
V63Q/L171P/S260T/A303T/E306G/
+++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1281/1282
T951V
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/T951V/I775G/T1148N/L1248D/

L1377D/V1439E

1283/1284
G164E
V63Q/G164E/L171P/S260T/E306G/
++
+++

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1285/1286
K818G
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K818G/T1148N/L1248D/

L1377D/V1439E

1287/1288
E807L
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E807L/T1148N/L1248D/

L1377D/V1439E

1289/1290
K799A
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K799A/T1148N/L1248D/

L1377D/V1439E

1291/1292
G735S
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/G735S/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1293/1294
S794R
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S794R/T1148N/L1248D/

L1377D/V1439E

1295/1296
E926V
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E926V/T1148N/L1248D/

L1377D/V1439E

1297/1298
A755P
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/K732W/L749F/

L750D/A755P/I775G/T1148N/L1248D/

L1377D/V1439E

1299/1300
Q921W
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/Q921W/T1148N/L1248D/

L1377D/V1439E

1301/1302
D801A
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D801A/T1148N/L1248D/

L1377D/V1439E

1303/1304
E1094S
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E1094S/T1148N/L1248D/

L1377D/V1439E

1305/1306
L968A
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/L968A/T1148N/L1248D/

L1377D/V1439E

1307/1308
K929Q
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K929Q/T1148N/L1248D/

L1377D/V1439E

1309/1310
G924W
V63Q/L171P/S260T/E306G/S332P/
++
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/G924W/T1148N/L1248D/

L1377D/V1439E

1311/1312
E807P
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E807P/T1148N/L1248D/

L1377D/V1439E

1313/1314
F698D
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/F698D/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1315/1316
S168G
V63Q/S168G/L171P/S260T/E306G/
+
++

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1317/1318
K929V
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K929V/T1148N/L1248D/

L1377D/V1439E

1319/1320
K818A
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K818A/T1148N/L1248D/

L1377D/V1439E

1321/1322
K818S
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K818S/T1148N/L1248D/

L1377D/V1439E

1323/1324
R581K
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/R581K/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1325/1326
K818P
V63Q/L171P/S260T/E306G/S332P/
+
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K818P/T1148N/L1248D/

L1377D/V1439E

1327/1328
S916K
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916K/T1148N/L1248D/

L1377D/V1439E

1329/1330
F698S
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/F698S/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1331/1332
A303G
V63Q/L171P/S260T/A303G/E306G/
+++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1333/1334
S916C
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916C/T1148N/L1248D/

L1377D/V1439E

1335/1336
T1428G
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/T1148N/L1248D/L1377D/

T1428G/V1439E

1337/1338
Y805L
V63Q/L171P/S260T/E306G/S332P/
+++
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/Y805L/T1148N/L1248D/

L1377D/V1439E

1339/1340
G924M
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/G924M/T1148N/L1248D/

L1377D/V1439E

1341/1342
L1114M
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/L1114M/T1148N/L1248D/

L1377D/V1439E

1343/1344
T311G
V63Q/L171P/S260T/E306G/T311G/
+++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1345/1346
Y737N
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/Y737N/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1347/1348
Y805V
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/Y805V/T1148N/L1248D/

L1377D/V1439E

1349/1350
D801I
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D801I/T1148N/L1248D/

L1377D/V1439E

1351/1352
S751D
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/S751D/I775G/T1148N/L1248D/

L1377D/V1439E

1353/1354
S693T
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/S693T/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1355/1356
K818E
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K818E/T1148N/L1248D/

L1377D/V1439E

1357/1358
K929A
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K929A/T1148N/L1248D/

L1377D/V1439E

1359/1360
T311E
V63Q/L171P/S260T/E306G/T311E/
+++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1361/1362
Y737L
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/Y737L/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1363/1364
E809W
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E809W/T1148N/L1248D/

L1377D/V1439E

1365/1366
E757A
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/E757A/I775G/T1148N/L1248D/

L1377D/V1439E

1367/1368
A1099G
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/A1099G/T1148N/L1248D/

L1377D/V1439E

1369/1370
K799G
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K799G/T1148N/L1248D/

L1377D/V1439E

1371/1372
S916I
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916I/T1148N/L1248D/

L1377D/V1439E

1373/1374
Y805G
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/Y805G/T1148N/L1248D/

L1377D/V1439E

1375/1376
Y737F
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/Y737F/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1377/1378
Y737M
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/Y737M/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1379/1380
K799P
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K799P/T1148N/L1248D/

L1377D/V1439E

1381/1382
E807C
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E807C/T1148N/L1248D/

L1377D/V1439E

1383/1384
S916E
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916E/T1148N/L1248D/

L1377D/V1439E

1385/1386
T676Q
V63Q/L171P/S260T/E306G/S332P/
++
+

F387I/I390A/V393G/T676Q/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1387/1388
R581V
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/R581V/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1389/1390
L968P
V63Q/L171P/S260T/E306G/S332P/
++
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/L968P/T1148N/L1248D/

L1377D/V1439E

1391/1392
A755Y
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/A755Y/I775G/T1148N/L1248D/

L1377D/V1439E

1393/1394
S916Q
V63Q/L171P/S260T/E306G/S332P/
+++
+++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916Q/T1148N/L1248D/

L1377D/V1439E

1395/1396
G735D
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/G735D/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1397/1398
D740G
V63Q/L171P/S260T/E306G/S332P/
+++
+++

F387I/I390A/V393G/K732W/D740G/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1399/1400
F698Y
V63Q/L171P/S260T/E306G/S332P/
+
+

F387I/I390A/V393G/K732W/F698Y/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1401/1402
E757G
V63Q/L171P/S260T/E306G/S332P/
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/E757G/I775G/T1148N/L1248D/

L1377D/V1439E

1403/1404
D791T
V63Q/L171P/S260T/E306G/S332P/
+++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D791T/T1148N/L1248D/

L1377D/V1439E

1405/1406
L1114A
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/L1114A/T1148N/L1248D/

L1377D/V1439E

1407/1408
G735V
V63Q/L171P/S260T/E306G/S332P/
+
++

F387I/I390A/V393G/K732W/G735V/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 1200. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >2

TABLE 9.2

Activity of FVIII Variants Relative to SEQ ID NO: 1200

(Expi293F)

(HepG2)

(Expi293F)
Chromogenic
(HepG2)
Chromogenic
(Expi293F)

Unchallenged
Activity
Unchallenged
Activity after
Unchallenged

Chromogenic
after Plasma
Chromogenic
Plasma
Clotting

Amino Acid

Activity
Challenge
Activity
Challenge
Activity

Differences
Amino Acid
FIOPC
FIOPC
FIOPC
FIOPC
FIOPC

SEQ ID
(Relative to
Differences
Relative to
Relative to
Relative to
Relative to
Relative to

NO:
SEQ ID NO:
(Relative to
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:

(nt/aa)
1200)
SEQ ID NO: 2)
1200
1200
1200
1200
1200

1199/1200

V63Q/L171P/S260T/E306G/S332P/
+
+
+
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1409/1410
V536P
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
+

F387I/I390A/V393G/V536P/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1411/1412
L211R/V536K/
V63Q/L171P/L211R/S260T/E306G/
++
++
+
++
+

L622D/M681A
S332P/F387I/I390A/V393G/V536K/

L622D/M681A/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1413/1414
V536K/M558L
V63Q/L171P/S260T/E306G/S332P/
++
+
++
+
+

F387I/I390A/V393G/V536K/M558L/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1415/1416
L211R/V536R/
V63Q/L171P/L211R/S260T/E306G/
++
++
++
++
+

F555G/P617G/
S332P/F387I/I390A/V393G/V536R/

W732K
F555G/P617G/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1417/1418
L211R/M681R/
V63Q/L171P/L211R/S260T/E306G/
++

++
+
+

W732K
S332P/F387I/I390A/V393G/M681R/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1419/1420
L211R/V536K/
V63Q/L171P/L211R/S260T/E306G/
++
++
+
+
+

F555E
S332P/F387I/I390A/V393G/V536K/

F555E/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1421/1422
V536R
V63Q/L171P/S260T/E306G/S332P/
++
++
+
+
+

F387I/I390A/V393G/V536R/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1423/1424
F555E
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
++

F387I/I390A/V393G/F555E/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1425/1426
L211R/V536P
V63Q/L171P/L211R/S260T/E306G/
++
++++
++
++
+

S332P/F387I/I390A/V393G/V536P/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1427/1428
L211R/H212P
V63Q/L171P/L211R/H212P/S260T/
++
+
++
++
+

E306G/S332P/F387I/I390A/V393G/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1429/1430
L211R/V536K
V63Q/L171P/L211R/S260T/E306G/
+
+
+
++
+

S332P/F387I/I390A/V393G/V536K/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1431/1432
L211R/V536K/
V63Q/L171P/L211R/S260T/E306G/
++
+
+
++
+

P617G/M681A/
S332P/F387I/I390A/V393G/

W732K
V536K/P617G/M681A/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1433/1434
L211R
V63Q/L171P/L211R/S260T/E306G/
++
++
++
++
++

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1435/1436
L211R/V536P/
V63Q/L171P/L211R/S260T/E306G/
++
+
++
++
++

M558L/P617G/
S332P/F387I/I390A/V393G/V536P/

M681A
M558L/P617G/M681A/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1437/1438
F555G/W732K
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
+

F3871/I390A/V393G/F555G/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1439/1440
V536P/F555E/
V63Q/L171P/S260T/E306G/S332P/
++
+++
++
++
+

M558L/P617G/
F387I/I390A/V393G/V536P/F555E/

W732K
M558L/P617G/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1441/1442
V536K/P617G/
V63Q/L171P/S260T/E306G/S332P/
++
++
+
++
+

M681A/W732K
F387I/I390A/V393G/V536K/P617G/

M681A/L749F/L750D/I775G/

NT1148/L1248D/L1377D/V1439E

1443/1444
V536K/F555G
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
+

F387I/I390A/V393G/V536K/F555G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1445/1446
V536P/L622D/
V63Q/L171P/S260T/E306G/S332P/
++
++

+
+

M681A
F3871/I390A/V393G/V536P/L622D/

M681A/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1447/1448
W732K
V63Q/L171P/S260T/E306G/S332P/
++

++
++
+

F387I/I390A/V393G/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1449/1450
H212A/M320Q/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+

T533R/P617E/
M320Q/S332P/F387I/I390A/V393G/

M681G
T533R/P617E/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1451/1452
L211K/F312L/
V63Q/L171P/L211K/S260T/E306G/
++
+
++
++
++

T533R/V534I/
F312L/S332P/F387I/I390A/V393G/

P617D/G619E/
T533R/V534I/P617D/G619E/M681N/

M681N/S839K
K732W/L749F/L750D/I775G/S839K/

T1148N/L1248D/L1377D/V1439E

1453/1454
H212A
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1455/1456
L211K/H212A/
V63Q/L171P/L211K/H212A/S260T/
+++
++
++
++
++

M320Q/G619E/
E306G/M320Q/S332P/F387I/I390A/

M681G/S839K
V393G/G619E/M681G/K732W/

L749F/L750D/I775G/S839K/T1148N/

L1248D/L1377D/V1439E

1457/1458
M797W/S839K
V63Q/L171P/S260T/E306G/S332P/
+
++
+
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/M797W/S839K/

T1148N/L1248D/L1377D/V1439E

1459/1460
H212A/F312L/
V63Q/L171P/H212A/S260T/E306G/
++
+++
++
++
+

M320Q/T533R/
F312L/M320Q/S332P/F387I/I390A/

V534I/P617E/
V393G/T533R/V534I/P617E/M681G/

M681G
K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1461/1462
M558W/G619E/
V63Q/L171P/S260T/E306G/S332P/
++
++
++
+++
++

M681G
F387I/I390A/V393G/M558W/G619E/

M681G/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1463/1464
M320Q/M558W/
V63Q/L171P/S260T/E306G/M320Q/
++
++
++
++
+

M681G
S332P/F387I/I390A/V393G/

M558W/M681G/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1465/1466
H212A/M320Q/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+

V534I/G619E/
M320Q/S332P/F387I/I390A/V393G/

M681G/M797W/
V534I/G619E/M681G/K732W/L749F/

S839K
L750D/I775G/M797W/S839K/T1148N/

L1248D/L1377D/V1439E

1467/1468
M320R/M558W/
V63Q/L171P/S260T/E306G/M320R/
++
++++
+
++
+

G619E/W732K/
S332P/F387I/I390A/V393G/M558W/

S839K
G619E/L749F/L750D/I775G/S839K/

T1148N/L1248D/L1377D/V1439E

1469/1470
L211K/H212A/
V63Q/L171P/L211K/H212A/S260T/
+++
++++
++
++
+

F312L/V534I/
E306G/F312L/S332P/F387I/I390A/

M558W/G619E/
V393G/V534I/M558W/G619E/M681N/

M681N/W732K/
L749F/L750D/I775G/

S839K
S839K/T1148N/L1248D/L1377D/

V1439E

1471/1472
M320R/V534I/
V63Q/L171P/S260T/E306G/M320R/
+++
++
++
++
++

M558R/G619E/
S332P/F387I/I390A/V393G/V534I/

W732K/M797W
M558R/G619E/L749F/L750D/I775G/

M797W/T1148N/L1248D/L1377D/

V1439E

1473/1474
M558W/P617D/
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
++

G619E/M681N
F387I/I390A/V393G/M558W/P617D/

G619E/M681N/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1475/1476
L211Q/V534I/
V63Q/L171P/L211Q/S260T/E306G/
++
++
++
++
++

M558W/Q611E/
S332P/F387I/I390A/V393G/V534I/

M681N
M558W/Q611E/M681N/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1477/1478
L211K/M320R/
V63Q/L171P/L211K/S260T/E306G/
+++
++
++
+++
++

P617E/G619E/
M320R/S332P/F387I/I390A/V393G/

M681G
P617E/G619E/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1479/1480
M558W/P617D/
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
++

M681G/M797W
F387I/I390A/V393G/M558W/P617D/

M681G/K732W/L749F/L750D/I775G/

M797W/T1148N/L1248D/L1377D/

V1439E

1481/1482
M320R/V534I/
V63Q/L171P/S260T/E306G/M320R/
+++
+++
++
++
++

G619E/W732K
S332P/F387I/I390A/V393G/

V534I/G619E//L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1483/1484
L211K/G619E/
V63Q/L171P/L211K/S260T/E306G/
+++
++
++
++
++

M681N
S332P/F387I/I390A/V393G/

G619E/M681N/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1485/1486
L211K/H212A/
V63Q/L171P/L211K/H212A/S260T/
++
+++
++
++
+

V534I/M558W/
E306G/S332P/F387I/I390A/V393G/

G619E/W732K/
V534I/M558W/G619E/L749F/L750D/

S839K
I775G/S839K/T1148N/L1248D/

L1377D/V1439E

1487/1488
H212A/M320R/
V63Q/L171P/H212A/S260T/E306G/
+++
+++++
+++
++
++

G619E/M681N/
M320R/S332P/F387I/I390A/V393G/

M797W/S839K
G619E/M681N/K732W/L749F/L750D/

I775G/M797W/S839K/T1148N/

L1248D/L1377D/V1439E

1489/1490
H212A/M320R/
V63Q/L171P/H212A/S260T/E306G/
++
+++
++
++
+

Q611E/S839K
M320R/S332P/F387I/I390A/V393G/

Q611E/K732W/L749F/L750D/I775G/

S839K/T1148N/L1248D/L1377D/

V1439E

1491/1492
M320Q/M558W/
V63Q/L171P/S260T/E306G/M320Q/
+++
+++
+++
+++
++

G619E/M681N
S332P/F387I/I390A/V393G/M558W/

G619E/M681N/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1493/1494
F312L/T533R/
V63Q/L171P/S260T/E306G/F312L/
++
+++
++
++
++

P617D/M681N/
S332P/F387I/I390A/V393G/T533R/

M797W/S839K
P617D/M681N/K732W/L749F/L750D/

I775G/M797W/S839K/T1148N/

L1248D/L1377D/V1439E

1495/1496
M558W/Q611E/
V63Q/L171P/S260T/E306G/S332P/
++
+++
+
++
++

M681N
F387I/I390A/V393G/M558W/Q611E/

M681N/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1497/1498
F312L
V63Q/L171P/S260T/E306G/F312L/
++
++
++
++
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1499/1500
L211Q/M320R/
V63Q/L171P/L211Q/S260T/E306G/
++
++
++
++
+

M558W/G619E/
M320R/S332P/F387I/I390A/V393G/

M681G/W732K/
M558W/G619E/M681G/L749F/L750D/

M797W/S839K
I775G/M797W/S839K/T1148N/

L1248D/L1377D/V1439E

1501/1502
M320Q/M558W/
V63Q/L171P/S260T/E306G/M320Q/
++
+
++
++
+

W732K/M797W
S332P/F387I/I390A/V393G/M558W/

L749F/L750D/I775G/M797W/T1148N/

L1248D/L1377D/V1439E

1503/1504
T533R/M558W/
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
+

Q611E/G619E/
F387I/I390A/V393G/T533R/M558W/

F716L/W732K/
Q611E/G619E/F716L//L749F/L750D/

S839K
I775G/S839K/T1148N/L1248D/

L1377D/V1439E

1505/1506
F312L/M681G
V63Q/L171P/S260T/E306G/F312L/
+++
++
++
+++
++

S332P/F387I/I390A/V393G/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1507/1508
M320Q/V534I/
V63Q/L171P/S260T/E306G/M320Q/
++
+++
++
++
++

G619E/M681N
S332P/F387I/I390A/V393G/V534I/

G619E/M681N/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1509/1510
L211K/M320Q/
V63Q/L171P/L211K/S260T/E306G/
+
++
+
+
+

T533R/V536L/
M320Q/S332P/F387I/I390A/V393G/

G619E
T533R/V536L/G619E/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1511/1512
M558W/P617E/
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
++

M681N/S839K
F387I/I390A/V393G/M558W/P617E/

M681N/K732W/L749F/L750D/I775G/

S839K/T1148N/L1248D/L1377D/

V1439E

1513/1514
H212A/M320R/
V63Q/L171P/H212A/S260T/E306G/
+++
++++
++
++
+

V534I/M558W/
M320R/S332P/F387I/I390A/V393G/

M681G
V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1515/1516
T533R/V534I/
V63Q/L171P/S260T/E306G/S332P/
++
++
+
++
+

M681N/S839K
F387I/I390A/V393G/T533R/V534I/

M681N/K732W/L749F/L750D/I775G/

S839K/T1148N/L1248D/L1377D/

V1439E

1517/1518
H212A/V534I/
V63Q/L171P/H212A/S260T/E306G/
+++
+++
++
+++
++

G619E/M681G
S332P/F387I/I390A/V393G/V534I/

G619E/M681G/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1519/1520
P617E/G619E/
V63Q/L171P/S260T/E306G/S332P/
++
++
+
++
++

M681G
F387I/I390A/V393G/P617E/G619E/

M681G/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1521/1522
L211Q/H212A/
V63Q/L171P/L211Q/H212A/S260T/
+++
+++
++
++
++

M320R/M558W/
E306G/M320R/S332P/F387I/I390A/

P617E/M681N
V393G/M558W/P617E/M681N/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1523/1524
H212A/V534I/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++

M558W/M681N/
S332P/F387I/I390A/V393G/V534I/

M797W
M558W/M681N/K732W/L749F/L750D/

I775G/M797W/T1148N/L1248D/

L1377D/V1439E

1525/1526
H212A/M320R/
V63Q/L171P/H212A/S260T/E306G/
+++
+++
++
++
++

V534I
M320R/S332P/F387I/I390A/V393G/

V534I/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1527/1528
L211K/F312L/
V63Q/L171P/L211K/S260T/E306G/
++
++
+
+
++

G619E
F312L/S332P/F387I/I390A/V393G/

G619E/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1529/1530
H212A/F312L/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+

G619E/S839K
F312L/S332P/F387I/I390A/V393G/

G619E/K732W/L749F/L750D/I775G/

S839K/T1148N/L1248D/L1377D/

V1439E

1531/1532
T210P/H212A/
V63Q/L171P/T210P/H212A/S260T/
++
+++
++
++
+

M320R/M558W/
E306G/M320R/S332P/F387I/I390A/

M681G
V393G/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1533/1534
H212A/F312L/
V63Q/L171P/H212A/S260T/E306G/
+++
++
++
+++
++

V534I/M681N/
F312L/S332P/F387I/I390A/V393G/

M797W/S839K
V534I/M681N/K732W/L749F/L750D/

I775G/M797W/S839K/T1148N/

L1248D/L1377D/V1439E

1535/1536
L211K/M320R/
V63Q/L171P/L211K/S260T/E306G/
++
++
++
++
++

M558W/S839K
M320R/S332P/F387I/I390A/V393G/

M558W/K732W/L749F/L750D/I775G/

S839K/T1148N/L1248D/L1377D/

V1439E

1537/1538
G924Q
V63Q/L171P/S260T/E306G/S332P/
+
+
++
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/G924Q/T1148N/L1248D/

L1377D/V1439E

1539/1540
S916L
V63Q/L171P/S260T/E306G/S332P/
+
+
+
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916L/T1148N/L1248D/

L1377D/V1439E

1541/1542
E757T
V63Q/L171P/S260T/E306G/S332P/
+
++
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/E757T/I775G/T1148N/L1248D/

L1377D/V1439E

1543/1544
Y737K
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++

F3871/I390A/V393G/K732W/Y737K/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1545/1546
K818V
V63Q/L171P/S260T/E306G/S332P/
++

++
++
+

F3871/I390A/V393G/K732W/L749F/

L750D/I775G/K818V/T1148N/L1248D/

L1377D/V1439E

1547/1548
D801G
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D801G/T1148N/L1248D/

L1377D/V1439E

1549/1550
T294G
V63Q/L171P/S260T/T294G/E306G/
++
++

+
+

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1551/1552
K799S
V63Q/L171P/S260T/E306G/S332P/
++

++
++
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K799S/T1148N/L1248D/

L1377D/V1439E

1553/1554
K799E
V63Q/L171P/S260T/E306G/S332P/
+

+
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/K799E/T1148N/L1248D/

L1377D/V1439E

1555/1556
E809G
V63Q/L171P/S260T/E306G/S332P/
+
+
+
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E809G/T1148N/L1248D/

L1377D/V1439E

1557/1558
D791M
V63Q/L171P/S260T/E306G/S332P/
+
++
++
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D791M/T1148N/L1248D/

L1377D/V1439E

1559/1560
Y737V
V63Q/L171P/S260T/E306G/S332P/
++
++
++
++
+

F387I/I390A/V393G/K732W/Y737V/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1561/1562
Y805Q
V63Q/L171P/S260T/E306G/S332P/
++
+++
++
++
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/Y805Q/T1148N/L1248D/

L1377D/V1439E

1563/1564
K799Q
V63Q/L171P/S260T/E306G/S332P/
+
+
+
++
+

F3871/I390A/V393G/K732W/L749F/

L750D/I775G/K799Q/T1148N/L1248D/

L1377D/V1439E

1565/1566
Y737S
V63Q/L171P/S260T/E306G/S332P/
+
++
+
+
+

F3871/I390A/V393G/K732W/Y737S/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1567/1568
S916M
V63Q/L171P/S260T/E306G/S332P/
++
+
+
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916M/T1148N/L1248D/

L1377D/V1439E

1569/1570
R759S
V63Q/L171P/S260T/E306G/S332P/
+

+
++

F387I/I390A/V393G/K732W/L749F/

L750D/R759S/I775G/T1148N/L1248D/

L1377D/V1439E

1571/1572
K799V
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
++

F3871/I390A/V393G/K732W/L749F/

L750D/I775G/K799V/T1148N/L1248D/

L1377D/V1439E

1573/1574
D538V
V63Q/L171P/S260T/E306G/S332P/
++
+
+
++
+

F387I/I390A/V393G/D538V/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1575/1576
E1109G
V63Q/L171P/S260T/E306G/S332P/
++
++
+
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E1109G/T1148N/L1248D/

L1377D/V1439E

1577/1578
S168R
V63Q/S168R/L171P/S260T/E306G/
+
++

+
++

S332P/F387I/I390A/V393G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1579/1580
Q921R
V63Q/L171P/S260T/E306G/S332P/
++
+
++
++
+

F3871/I390A/V393G/K732W/L749F/

L750D/I775G/Q921R/T1148N/L1248D/

L1377D/V1439E

1581/1582
E926T
V63Q/L171P/S260T/E306G/S332P/
++

++
+
++

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/E926T/T1148N/L1248D/

L1377D/V1439E

1583/1584
S916T
V63Q/L171P/S260T/E306G/S332P/
++
+++
+
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/S916T/T1148N/L1248D/

L1377D/V1439E

1585/1586
Y737G
V63Q/L171P/S260T/E306G/S332P/
++
+
++
+
+

F387I/I390A/V393G/K732W/Y737G/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1587/1588
D801R
V63Q/L171P/S260T/E306G/S332P/
+
++
+
+
+

F387I/I390A/V393G/K732W/L749F/

L750D/I775G/D801R/T1148N/L1248D/

L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 1200. Levels of increased activity are defined as follows:

“+” 0.9 to 1.1,

“++” >1.1,

“+++” >2,

“++++” >3.5,

”+++++” >5

Example 10
FVIII Variants Derived from SEQ ID NO: 1514

In this Example, experiments conducted to determine FVIII variant activity by assaying the protein activity after a challenge is described. These variants were tested for FVIII activity after no incubation and after incubation in Hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Tables 10.1 and 10.2 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 10.1

Activity of FVIII Variants Relative to SEQ ID NO: 1514

(Expi293F)
(Expi293F)

Unchallenged
Chromogenic

Chromogenic
Activity after

Amino Acid

Activity
Plasma

Differences
Amino Acid
FIOPC
Challenge

SEQ ID
(Relative to
Differences
Relative to
FIOPC Relative

NO:
SEQ ID
(Relative to
SEQ ID NO:
to SEQ ID NO:

(nt/aa)
NO: 1514)
SEQ ID NO: 2)
1514
1514

1513/1514

V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/L1377D/

V1439E

1589/1590
R1042E
V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/R1042E/T1148N/L1248D/

L1377D/V1439E

1591/1592
Y1041N
V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/Y1041N/T1148N/L1248D/

L1377D/V1439E

1593/1594
M1051H
V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/M1051H/T1148N/L1248D/

L1377D/V1439E

1595/1596
I1283H
V63Q/L171P/H212A/S260T/E306G/

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/I1283H/

L1377D/V1439E

1597/1598
I1283T
V63Q/L171P/H212A/S260T/E306G/

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/I1283T/

L1377D/V1439E

1599/1600
Y1281Q
V63Q/L171P/H212A/S260T/E306G/

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/Y1281Q/

L1377D/V1439E

1601/1602
Y1281P
V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/Y1281P/

L1377D/V1439E

1603/1604
S916E
V63Q/L171P/H212A/S260T/E306G/
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/S916E/T1148N/L1248D/

L1377D/V1439E

1605/1606
M1051W
V63Q/L171P/H212A/S260T/E306G/
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/M1051W/T1148N/L1248D/

L1377D/V1439E

1607/1608
L187C
V63Q/L171P/L187C/H212A/S260T/
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

9L74F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1609/1610
I215E
V63Q/L171P/H212A/I215E/S260T/

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1611/1612
I215P
V63Q/L171P/H212A/I215P/S260T/
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1613/1614
I215S
V63Q/L171P/H212A/I215S/S260T/

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1615/1616
L178C
V63Q/L171P/L178C/H212A/S260T/
++
++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1617/1618
I1050T
V63Q/L171P/H212A/S260T/E306G/
++
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/I1050T/T1148N/L1248D/

L1377D/V1439E

1619/1620
K1210T
V63Q/L171P/H212A/S260T/E306G/
++
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/K1210T/L1248D/

L1377D/V1439E

1621/1622
Y1281K
V63Q/L171P/H212A/S260T/E306G/
++
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/T1148N/L1248D/Y1281K/

L1377D/V1439E

1623/1624
R1042A
V63Q/L171P/H212A/S260T/E306G/
+
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/L749F/

L750D/I775G/R1042A/T1148N/L1248D/

L1377D/V1439E

1625/1626
V104S
V63Q/V104S/L171P/H212A/S260T/
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1627/1628
P615D
V63Q/L171P/H212A/S260T/E306G/
+
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/P615D/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1629/1630
I610S
V63Q/L171P/H212A/S260T/E306G/
+
++

320R/S332P/F387I/I390A/V393G/

V534I/M558W/I610S/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1631/1632
I610K
V63Q/L171P/H212A/S260T/E306G/

+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/I610K/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1633/1634
I610T
V63Q/L171P/H212A/S260T/E306G/
+
++

320R/S332P/F387I/I390A/V393G/

V534I/M558W/I610T/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1635/1636
V536P
V63Q/L171P/H212A/S260T/E306G/
+
++

320R/S332P/F387I/I390A/V393G/

V534I/V536P/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1637/1638
A618Y
V63Q/L171P/H212A/S260T/E306G/
++
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/A618Y/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1639/1640
Q621W
V63Q/L171P/H212A/S260T/E306G/
+
+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q621W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1641/1642
A618P
V63Q/L171P/H212A/S260T/E306G/
++
++

320R/S332P/F387I/I390A/V393G/

V534I/M558W/A618P/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1643/1644
L614F
V63Q/L171P/H212A/S260T/E306G/

+

320R/S332P/F387I/I390A/V393G/

V534I/M558W/L614F/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1645/1646
M110H
V63Q/M110H/L171P/H212A/S260T/

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

1647/1648
R301K
V63Q/L171P/H212A/S260T/R301K/

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

All activities were determined relative to the reference polypoptide of SEQ ID NO: 1514. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1

TABLE 10.2

Activity of FVIII Variants Relative to SEQ ID NO: 1514

(HepG2)

(Expi293F)
(Expi293F)

Chromo
(HepG2)

Chromogenic
Clotting

genic
Clotting

Amino Acid

(Expi293F)
(Expi293F)
Activity
Activity
(HepG2)
(HepG2)
Activity
Activity

Differences
Amino Acid
Unchallenged
Unchallenged
after
after
Unchallenged
Unchallenged
after
after

SEQ ID
(Relative to
Differences
Chromogenic
Clotting
Plasma
Plasma
Chromogenic
Clotting
Plasma
Plasma

NO:
SEQ ID
(Relative to
Activity
Activity
Challenge
Challenge
Activity
Activity
Challenge
Challenge

(nt/aa)
NO: 1514)
SEQ ID NO: 2)
FIOPC
FIOPC
FIOPC
FIOPC
FIOPC
FIOPC
FIOPC
FIOPC

1513/1514

V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1649/1650
I1078S
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/I1078S/

T1148N/L1248D/L1377D/V1439E

1651/1652
S812G
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S812G/

T1148N/L1248D/L1377D/V1439E

1653/1654
E786C
V63Q/L171P/H212A/S260T/E306G/
++
+
+

++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E786C/

T1148N/L1248D/L1377D/V1439E

1655/1656
A127V
V63Q/A127V/L171P/H212A/S260T/
+
+
++
++
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1657/1658
Y175G
V63Q/L171P/Y175G/H212A/S260T/

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1659/1660
K1093W
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093W/

T1148N/L1248D/L1377D/V1439E

1661/1662
F1088G
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++

++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1088G/T1148N/

L1248D/L1377D/V1439E

1663/1664
K1093T
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093T/T1148N/

L1248D/L1377D/V1439E

1665/1666
L650M
V63Q/L171P/H212A/S260T/E306G/

+
+
++
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/L650M/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1667/1668
R301M
V63Q/L171P/H212A/S260T/R301M/
+
+
+

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1669/1670
T535N
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/T535N/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1671/1672
D806R
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
++
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D806R/T1148N/

L1248D/L1377D/V1439E

1673/1674
Y738E
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1675/1676
S700E
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/S700E/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1677/1678
E132R
V63Q/E132R/L171P/H212A/S260T/

+
+
++
+
++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1679/1680
E1076G
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
++
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E1076G/T1148N/

L1248D/L1377D/V1439E

1681/1682
S1084L
V63Q/L171P/H212A/S260T/E306G/

+
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1084L/T1148N/

L1248D/L1377D/V1439E

1683/1684
G498A
V63Q/L171P/H212A/S260T/E306G/
+
+
++

+

M320R/S332P/F387I/I390A/V393G/

G498A/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1685/1686
E786L
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E786L/T1148N/

L1248D/L1377D/V1439E

1687/1688
Y738F
V63Q/L171P/H212A/S260T/E306G/
+

+
+
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738F/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1689/1690
T535G
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
+

++

M320R/S332P/F387I/I390A/V393G/

V534I/T535G/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1691/1692
D806E
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D806E/T1148N/

L1248D/L1377D/V1439E

1693/1694
I756G
V63Q/L171P/H212A/S260T/E306G/
++
+
++

++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I756G/I775G/T1148N/

L1248D/L1377D/V1439E

1695/1696
F1108S
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1108S/T1148N/

L1248D/L1377D/V1439E

1697/1698
K1093V
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++

++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093V/

T1148N/L1248D/L1377D/V1439E

1699/1700
S746P
V63Q/L171P/H212A/S260T/E306G/
++

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

S746P/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1701/1702
L160E
V63Q/L160E/L171P/H212A/S260T/
++
+
+
+
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1703/1704
L160T
V63Q/L160T/L171P/H212A/S260T/
+
+
+
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1705/1706
L178V
V63Q/L171P/L178V/H212A/S260T/
+
+
+
++
+++
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1707/1708
S543L
V63Q/L171P/H212A/S260T/E306G/
+
+
++

+++

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/S543L/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1709/1710
I310G
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++

+

I310G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1711/1712
S700Q
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/S700Q/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1713/1714
K1093A
V63Q/L171P/H212A/S260T/E306G/
++
++
+++
++
+
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093A/

T1148N/L1248D/L1377D/V1439E

1715/1716
Q584A
V63Q/L171P/H212A/S260T/E306G/

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q584A/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1717/1718
E918S
V63Q/L171P/H212A/S260T/E306G/
++
++
+++

++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E918S/T1148N/

L1248D/L1377D/V1439E

1719/1720
E1452L
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E/E1452L

1721/1722
Y738P
V63Q/L171P/H212A/S260T/E306G/

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738P/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1723/1724
D790W
V63Q/L171P/H212A/S260T/E306G/
+
+
++

++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790W/T1148N/

L1248D/L1377D/V1439E

1725/1726
D790I
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790I/T1148N/

L1248D/L1377D/V1439E

1727/1728
G292D
V63Q/L171P/H212A/S260T/G292D/
+
+
++

++
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1729/1730
E800M
V63Q/L171P/H212A/S260T/E306G/
+
+
++

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E800M/T1148N/

L1248D/L1377D/V1439E

1731/1732
F1108M
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
++
+
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1108M/

T1148N/L1248D/L1377D/V1439E

1733/1734
F1088S
V63Q/L171P/H212A/S260T/E306G/

+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1088S/T1148N/

L1248D/L1377D/V1439E

1735/1736
A167G
V63Q/A167G/L171P/H212A/S260T/
+
+
+
+
++
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1737/1738
V892T
V63Q/L171P/H212A/S260T/E306G/

+
+
+

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/V892T/T1148N/

L1248D/L1377D/V1439E

1739/1740
D790P
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790P/T1148N/

L1248D/L1377D/V1439E

1741/1742
E800R
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E800R/T1148N/

L1248D/L1377D/V1439E

1743/1744
Y738K
V63Q/L171P/H212A/S260T/E306G/
++

+
+
+++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738K/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1745/1746
S304G
V63Q/L171P/H212A/S260T/S304G/
+
+
+
++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1747/1748
S1116G
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1116G/T1148N/

L1248D/L1377D/V1439E

1749/1750
L178Q
V63Q/L171P/L178Q/H212A/S260T/
++
+
+

++++
++
++
++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1751/1752
S1116A
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1116A/T1148N/

L1248D/L1377D/V1439E

1753/1754
K752G
V63Q/L171P/H212A/S260T/E306G/
+
++
++

++
++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/K752G/I775G/T1148N/

L1248D/L1377D/V1439E

1755/1756
F1108W
V63Q/L171P/H212A/S260T/E306G/
+
+

+
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1108W/T1148N/

L1248D/L1377D/V1439E

1757/1758
Y738D
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+
+
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738D/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1759/1760
T281A
V63Q/L171P/H212A/S260T/T281A/

+

++
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1761/1762
Y917G
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/Y917G/T1148N/

L1248D/L1377D/V1439E

1763/1764
H712M
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712M/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1765/1766
S1080V
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1080V/T1148N/

L1248D/L1377D/V1439E

1767/1768
D806G/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+

+

K1352E
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D806G/T1148N/

L1248D/K1352E/L1377D/

V1439E

1769/1770
E1095G
V63Q/L171P/H212A/S260T/E306G/
+
+

+
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E1095G/T1148N/

L1248D/L1377D/V1439E

1771/1772
K1093G
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093G/

T1148N/L1248D/L1377D/V1439E

1773/1774
D744A
V63Q/L171P/H212A/S260T/E306G/
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

D744A/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1775/1776
S543G
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/S543G/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1777/1778
M1098G
V63Q/L171P/H212A/S260T/E306G/
+
+

+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/M1098G/

T1148N/L1248D/L1377D/V1439E

1779/1780
T281S
V63Q/L171P/H212A/S260T/T281S/E306G/
++
+
+
+
+
+

M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1781/1782
I756T
V63Q/L171P/H212A/S260T/E306G/
++
+
++

++
+
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I756T/I775G/T1148N/

L1248D/L1377D/V1439E

1783/1784
M948S
V63Q/L171P/H212A/S260T/E306G/
+
+

++
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/M948S/T1148N/

L1248D/L1377D/V1439E

1785/1786
A119V
V63Q/A119V/L171P/H212A/S260T/

+

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1787/1788
S1116L
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1116L/T1148N/

L1248D/L1377D/V1439E

1789/1790
E785V
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E785V/T1148N/

L1248D/L1377D/V1439E

1791/1792
M1113F
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/M1113F/T1148N/

L1248D/L1377D/V1439E

1793/1794
S700A
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/S700A/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1795/1796
E786D
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E786D/T1148N/

L1248D/L1377D/V1439E

1797/1798
Y175A
V63Q/L171P/Y175A/H212A/S260T/

+

+

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1799/1800
D790M
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790M/T1148N/

L1248D/L1377D/V1439E

1801/1802
F667I
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/F667I/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1803/1804
Q158G
V63Q/Q158G/L171P/H212A/S260T/
+
+

++

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1805/1806
Q580S
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q580S/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1807/1808
W707H
V63Q/L171P/H212A/S260T/E306G/
++
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/W707H/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1809/1810
D731I
V63Q/L171P/H212A/S260T/E306G/
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/D731I/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1811/1812
E918M
V63Q/L171P/H212A/S260T/E306G/
++
+

+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E918M/T1148N/

L1248D/L1377D/V1439E

1813/1814
L1100S
V63Q/L171P/H212A/S260T/E306G/
++
+

+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/L1100S/T1148N/

L1248D/L1377D/V1439E

1815/1816
T1089S
V63Q/L171P/H212A/S260T/E306G/
+
+

+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1089S/T1148N/

L1248D/L1377D/V1439E

1817/1818
H712Y
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712Y/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1819/1820
Y175H
V63Q/L171P/Y175H/H212A/S260T/
+
+
+

+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1821/1822
K1092G
V63Q/L171P/H212A/S260T/E306G/

+
++

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1092G/T1148N/

L1248D/L1377D/V1439E

1823/1824
T939E
V63Q/L171P/H212A/S260T/E306G/

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T939E/T1148N/

L1248D/L1377D/V1439E

1825/1826
Y1104E
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/Y1104E/T1148N/

L1248D/L1377D/V1439E

1827/1828
E800G
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E800G/T1148N/

L1248D/L1377D/V1439E

1829/1830
D736F
V63Q/L171P/H212A/S260T/E306G/
+
+
++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

D736F/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1831/1832
S543W
V63Q/L171P/H212A/S260T/E306G/

+
+

++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/S543W/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1833/1834
N1297A
V63Q/L171P/H212A/S260T/E306G/
++
+
+

++
++
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

N1297A/L1377D/V1439E

1835/1836
H712L
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712L/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1837/1838
I1127V
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/I1127V/T1148N/

L1248D/L1377D/V1439E

1839/1840
T939S
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T939S/T1148N/

L1248D/L1377D/V1439E

1841/1842
L178M
V63Q/L171P/L178M/H212A/S260T/
+
+
++
+
++

++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1843/1844
D731R
V63Q/L171P/H212A/S260T/E306G/
++
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/D731R/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1845/1846
T686W
V63Q/L171P/H212A/S260T/E306G/
+
+

+
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/T686W/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1847/1848
S543P
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+++
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/S543P/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1849/1850
D736G
V63Q/L171P/H212A/S260T/E306G/
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

D736G/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1851/1852
E1129P
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/E1129P/

L1248D/L1377D/V1439E

1853/1854
L1138Q
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/L1138Q/T1148N/

L1248D/L1377D/V1439E

1855/1856
S812V
V63Q/L171P/H212A/S260T/E306G/
++
+

++

+
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S812V/T1148N/

L1248D/L1377D/V1439E

1857/1858
E786D/
V63Q/L171P/H212A/S260T/E306G/
+
+

+

+

+

R821Q
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E786D/R821Q/

T1148N/L1248D/L1377D/V1439E

1859/1860
T535C
V63Q/L171P/H212A/S260T/E306G/

+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/T535C/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1861/1862
K680R
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/K680R/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1863/1864
S1116I
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1116I/T1148N/

L1248D/L1377D/V1439E

1865/1866
T1089F
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1089F/T1148N/

L1248D/L1377D/V1439E

1867/1868
S304E
V63Q/L171P/H212A/S260T/S304E/
++
+
++
+

+
+
+

E306G/M320R/S332P/F3871/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1869/1870
L178W
V63Q/L171P/L178W/H212A/S260T/
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1871/1872
Y1104C
V63Q/L171P/H212A/S260T/E306G/

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/Y1104C/T1148N/

L1248D/L1377D/V1439E

1873/1874
D744R
V63Q/L171P/H212A/S260T/E306G/
+

++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

D744R/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1875/1876
Y738R
V63Q/L171P/H212A/S260T/E306G/

+

++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

Y738R/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1877/1878
K1093S
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093S/T1148N/

L1248D/L1377D/V1439E

1879/1880
H712S
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712S/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1881/1882
L187G
V63Q/L171P/L187G/H212A/S260T/

+
++

+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1883/1884
E132P
V63Q/L171P/H212A/S260T/E306G/
+

+
++

E132P/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1885/1886
S812I
V63Q/L171P/H212A/S260T/E306G/
+
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S812I/T1148N/

L1248D/L1377D/V1439E

1887/1888
S1080G
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1080G/T1148N/

L1248D/L1377D/V1439E

1889/1890
E800C
V63Q/L171P/H212A/S260T/E306G/
+
+

++

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E800C/T1148N/

L1248D/L1377D/V1439E

1891/1892
Y1101G
V63Q/L171P/H212A/S260T/E306G/
++
++
++

+
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/Y1101G/T1148N/

L1248D/L1377D/V1439E

1893/1894
D808K
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
+

++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D808K/T1148N/

L1248D/L1377D/V1439E

1895/1896
D808S
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D808S/T1148N/

L1248D/L1377D/V1439E

1897/1898
D790A
V63Q/L171P/H212A/S260T/E306G/
++
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790A/T1148N/

L1248D/L1377D/V1439E

1899/1900
E800V
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+++
++
++
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E800V/T1148N/

L1248D/L1377D/V1439E

1901/1902
K680L
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+

++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/K680L/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1903/1904
A167R
V63Q/A167R/L171P/H212A/S260T/
++
+
+
++
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1905/1906
K752E
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/K752E/I775G/T1148N/

L1248D/L1377D/V1439E

1907/1908
K1093N
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++

++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093N/T1148N/

L1248D/L1377D/V1439E

1909/1910
A119G
V63Q/A119G/L171P/H212A/S260T/
++
+
++

++
++
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1911/1912
E739G
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
++++
+
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

E739G/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1913/1914
S1116R
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1116R/T1148N/

L1248D/L1377D/V1439E

1915/1916
Q580Y
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q580Y/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1917/1918
S304A
V63Q/L171P/H212A/S260T/S304A/
+
+
+

++
++
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1919/1920
H712Q
V63Q/L171P/H212A/S260T/E306G/
++
+
+

++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712Q/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1921/1922
D806S
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D806S/T1148N/

L1248D/L1377D/V1439E

1923/1924
H712G
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1925/1926
D790G
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D790G/T1148N/

L1248D/L1377D/V1439E

1927/1928
F1108V
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++

+
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/F1108V/T1148N/

L1248D/L1377D/V1439E

1929/1930
D731L
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/D731L/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1931/1932
S1084N
V63Q/L171P/H212A/S260T/E306G/
++
+

++
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S1084N/T1148N/

L1248D/L1377D/V1439E

1933/1934
I1127L
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/I1127L/T1148N/

L1248D/L1377D/V1439E

1935/1936
S308P
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+

+
+
+

S308P/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1937/1938
E1076S
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
++
++
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/E1076S/T1148N/

L1248D/L1377D/V1439E

1939/1940
K1093P
V63Q/L171P/H212A/S260T/E306G/
+
++
++
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093P/T1148N/

L1248D/L1377D/V1439E

1941/1942
K1093Y
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
++
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093Y/T1148N/

L1248D/L1377D/V1439E

1943/1944
E739R
V63Q/L171P/H212A/S260T/E306G/

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

E739R/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1945/1946
E739P
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

E739P/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1947/1948
I310V
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+
+

I310V/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1949/1950
H712T
V63Q/L171P/H212A/S260T/E306G/

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/H712T/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1951/1952
A167H
V63Q/A167H/L171P/H212A/S260T/
+
+
+
+
+
+
+

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1953/1954
K1093R
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K1093R/T1148N/

L1248D/L1377D/V1439E

1955/1956
V536P/F555E/
V63Q/L171P/H212A/S260T/E306G/
++
+
++++

+
+
++

G619E/
M320R/S332P/F387I/I390A/V393G/

W732K/K799S/
V534I/V536P/F555E/M558W/G619E/

Y805Q
M681G/L749F/L750D/I775G/

K799S/Y805Q/T1148N/L1248D/

L1377D/V1439E

1957/1958
F555E/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
+

D801G
M320R/S332P/F387I/I390A/V393G/

V534I/F555E/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

D801G/T1148N/L1248D/L1377D/

V1439E

1959/1960
F555E/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++
++
+

K799S
M320R/S332P/F387I/I390A/V393G/

V534I/F555E/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

K799S/T1148N/L1248D/L1377D/

V1439E

1961/1962
G619E/K799S/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
+
++

D801G/
M320R/S332P/F387I/I390A/V393G/

Y805Q
V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/K799S/

D801G/Y805Q/T1148N/L1248D/

L1377D/V1439E

1963/1964
V536P/F555E/
V63Q/L171P/H212A/S260T/E306G/
++
+
++

++
+
+
+

G619E/
M320R/S332P/F387I/I390A/V393G/

W732K/K799S
V534I/V536P/F555E/M558W/G619E/

M681G/L749F/L750D/I775G/

K799S/T1148N/L1248D/L1377D/

V1439E

1965/1966
K799S/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
++
+
+

Y805Q
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K799S/Y805Q/

T1148N/L1248D/L1377D/V1439E

V63Q/

1967/1968
D801G
L171P/H212A/S260T/E306G/
++
+
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D801G/T1148N/

L1248D/L1377D/V1439E

1969/1970
A246D/G619E/
V63Q/L171P/H212A/A246D/S260T/
++
++
++
++

+

+

K799S/D801G
E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

K799S/D801G/T1148N/L1248D/

L1377D/V1439E

1971/1972
V536P/F555E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
+
+
+

G619E
M320R/S332P/F387I/I390A/V393G/

V534I/V536P/F555E/M558W/G619E/

M681G/K732W/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

1973/1974
G619E/S839K
V63Q/L171P/H212A/S260T/E306G/
++
+
++

+

++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/S839K/

T1148N/L1248D/L1377D/V1439E

1975/1976
G619E/K799S/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
+

Y805Q
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/K799S/

Y805Q/T1148N/L1248D/L1377D/

V1439E

1977/1978
V536P/D801G/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+
+
+
+

Y805Q
M320R/S332P/F387I/I390A/V393G/

V534I/V536P/M558W/M681G/

K732W/L749F/L750D/I775G/D801G/

Y805Q/T1148N/L1248D/L1377D/

V1439E

1979/1980
F555E/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
+
+
+

K799S/
M320R/S332P/F387I/I390A/V393G/

Y805Q
V534I/F555E/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

K799S/Y805Q/T1148N/L1248D/

L1377D/V1439E

1981/1982
G619E/D801G/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
+

+

Y805Q
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/D801G/

Y805Q/T1148N/L1248D/L1377D/

V1439E

1983/1984
F555E/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
+

+

G619E
M320R/S332P/F387I/I390A/V393G/

V534I/F555E/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

1985/1986
V536P/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
+
+
+

D801G
M320R/S332P/F387I/I390A/V393G/

V534I/V536P/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

D801G/T1148N/L1248D/L1377D/

V1439E

1987/1988
D801G/
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+
++
+
+
+

Y805Q
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/D801G/Y805Q/

T1148N/L1248D/L1377D/V1439E

1989/1990
V536P/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
+
+

K799S/
M320R/S332P/F387I/I390A/V393G/

Y805Q
V534I/V536P/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

K799S/Y805Q/T1148N/L1248D/

L1377D/V1439E

1991/1992
F555E/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
++
+
+

W732K/K799S/
M320R/S332P/F387I/I390A/V393G/

D801G/
V534I/F555E/M558W/G619E/M681G/

Y805Q
L749F/L750D/I775G/K799S/

D801G/Y805Q/T1148N/L1248D/

L1377D/V1439E

1993/1994
Y1281H
V63Q/L171P/H212A/S260T/E306G/

+

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

Y1281H/L1377D/V1439E

1995/1996
F749L
V63Q/L171P/H212A/S260T/E306G/
+

++

+

++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L750D/I775G/T1148N/L1248D/L1377D/

V1439E

1997/1998
L614H
V63Q/L171P/H212A/S260T/E306G/

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/L614H/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

1999/2000
N616D
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/N616D/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2001/2002
Q621K
V63Q/L171P/H212A/S260T/E306G/
+
+

+
++
++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q621K/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2003/2004
I1050E
V63Q/L171P/H212A/S260T/E306G/
++
+
+

+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/I1050E/T1148N/

L1248D/L1377D/V1439E

2005/2006
I610H
V63Q/L171P/H212A/S260T/E306G/
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/I610H/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2007/2008
Q611D
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/Q611D/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2009/2010
R1042T
V63Q/L171P/H212A/S260T/E306G/
+

+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/R1042T/T1148N/

L1248D/L1377D/V1439E

2011/2012
N616Q
V63Q/L171P/H212A/S260T/E306G/
++
+
+

++
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/N616Q/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2013/2014
L614N
V63Q/L171P/H212A/S260T/E306G/
+
+

++
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/L614N/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2015/2016
T370A/
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
+
+

L614A
M320R/S332P/T370A/F387I/I390A/

V393G/V534I/M558W/L614A/

M681G/K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

2017/2018
H1156D
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/H1156D/

L1248D/L1377D/V1439E

2019/2020
K1210V
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
++
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/K1210V/

L1248D/L1377D/V1439E

2021/2022
L614T
V63Q/L171P/H212A/S260T/E306G/
++
+
+

+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/L614T/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2023/2024
R1158N
V63Q/L171P/H212A/S260T/E306G/
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/R1158N/

L1248D/L1377D/V1439E

2025/2026
Y1281S
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/T1148N/L1248D/

Y1281S/L1377D/V1439E

2027/2028
A618E
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/A618E/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2029/2030
F555E/G681N/
V63Q/L171P/H212A/S260T/E306G/
+

+
++
+
+

Y737K/
M320R/S332P/F387I/I390A/V393G/

K799Q
V534I/F555E/M558W/M681N/

K732W/Y737K/L749F/L750D/I775G/

K799Q/T1148N/L1248D/L1377D/

V1439E

2031/2032
V536P/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++
+

E809G/
M320R/S332P/F387I/I390A/V393G/

E1109G
V534I/V536P/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/

E809G/E1109G/T1148N/L1248D/

L1377D/V1439E

2033/2034
V536K/F555G/
V63Q/L171P/H212A/S260T/E306G/
++

++

G619E/Y737K/
M320R/S332P/F387I/I390A/V393G/

K799Q/
V534I/V536K/F555G/M558W/

E809G/S839K/
G619E/M681G/K732W/Y737K/L749F/

S916T/Q921R
L750D/I775G/K799Q/E809G/

S839K/S916T/Q921R/T1148N/L1248D/

L1377D/V1439E

2035/2036
S916T/
V63Q/L171P/H212A/S260T/E306G/
+
+
++
++
+
+
+

E1109G
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S916T/E1109G/

T1148N/L1248D/L1377D/V1439E

2037/2038
V536R/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++

+

G681N/K799Q/
M320R/S332P/F387I/I390A/V393G/

S916T/
V534I/V536R/M558W/G619E/

E1109G
M681N/K732W/L749F/L750D/I775G/

K799Q/S916T/E1109G/T1148N/

L1248D/L1377D/V1439E

2039/2040
F555G/G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
+
++

G681N/E807R/
M320R/S332P/F387I/I390A/V393G/

E1109G
V534I/F555G/M558W/G619E/M681N/

K732W/L749F/L750D/I775G/

E807R/E1109G/T1148N/L1248D/

L1377D/V1439E

2041/2042
F555E/G619E/
V63Q/L171P/H212A/S260T/E306G/
++

++

+

+

G681N/Y737K/
M320R/S332P/F387I/I390A/V393G/

K799Q
V534I/F555E/M558W/G619E/M681N/

L749F/L750D/I775G/K799Q/

T1148N/L1248D/L1377D/V1439E

2043/2044
G619E/G681N/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++
+
++

K799S/S916T/
M320R/S332P/F387I/I390A/V393G/

E1109G
V534I/M558W/G619E/M681N/

K732W/L749F/L750D/I775G/K799S/

S916T/E1109G/T1148N/L1248D/

L1377D/V1439E

2045/2046
G619E/G681N
V63Q/L171P/H212A/S260T/E306G/
++
++
++
+++
+
++
+
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681N/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2047/2048
F555G/Q921R
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/F555G/M558W/M681G/

K732W/L749F/L750D/I775G/Q921R/

T1148N/L1248D/L1377D/V1439E

V63Q/

2049/2050
G619E/G681N/
L171P/H212A/S260T/E306G/
++
++
++
++
+
++

+

S916T
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681N/

K732W/L749F/L750D/I775G/S916T/

T1148N/L1248D/L1377D/V1439E

V63Q/

2051/2052
F555G/G619E/
L171P/H212A/S260T/E306G/
+
++
++
++
+
++
+
+

G681N/K799S
M320R/S332P/F387I/I390A/V393G/

V534I/F555G/M558W/G619E/M681N/

K732W/L749F/L750D/I775G/

K799S/T1148N/L1248D/L1377D/

V1439E

2053/2054
F555E
V63Q/L171P/H212A/S260T/E306G/
+
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/F555E/M558W/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2055/2056
W732K
V63Q/L171P/H212A/S260T/E306G/

+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/L749F/

L750D/I775G/T1148N/L1248D/L1377D/

V1439E

2057/2058
G619E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++

++
+
+
+

K799S
M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/K799S/

T1148N/L1248D/L1377D/V1439E

2059/2060
G619E
V63Q/L171P/H212A/S260T/E306G/
+
+

+
++
++

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/G619E/M681G/

K732W/L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2061/2062
G681N
V63Q/L171P/H212A/S260T/E306G/
+
++
++
++
+
+
+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681N/K732W/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2063/2064
F555G/S916T
V63Q/L171P/H212A/S260T/E306G/
+
+
++
+

+
+

M320R/S332P/F387I/I390A/V393G/

V534I/F555G/M558W/M681G/

K732W/L749F/L750D/I775G/S916T/

T1148N/L1248D/L1377D/V1439E

2065/2066
S839K
V63Q/L171P/H212A/S260T/E306G/
+
+
+

++

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/S839K/T1148N/

L1248D/L1377D/V1439E

2067/2068
K799Q
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
+
+

+

M320R/S332P/F387I/I390A/V393G/

V534I/M558W/M681G/K732W/

L749F/L750D/I775G/K799Q/T1148N/

L1248D/L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 1514. Levels of increased activity are defined as follows:

“+” 0.9 to 1.1,

“++” >1.1,

“+++” >2,

“++++” >2.5

Example 11
FVIII Variants Derived from SEQ ID NO: 2072

In this Example, experiments conducted to determine FVIII variant activity by assaying the protein activity after a challenge is described. These variants were tested for FVIII activity after no incubation and after incubation in Hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. Variants were tested using the chromogenic and clotting assays, as described in Example 3. Results are shown in Table 11.1 for variants expressed using Expi293F cells or HepG2 cells.

TABLE 11.1

Activity of FVIII Variants Relative to SEQ ID NO: 2072

FIOP
FIOP inv
FIOP
FIOP
FIOP inv
FIOP

Unchallenged
Unchallenged
Challenged
Unchallenged
Unchallenged
Challenged

Amino Acid

Biophen
APTT
Biophen
Biophen
APTT
Biophen

Differences
Amino Acid
(Expi)
(Expi)
(Expi)
(HepG2)
(HepG2)
(HepG2)

(Relative to
Differences
Relative to
Relative to
Relative to
Relative to
Relative to
Relative to

SEQ ID
SEQ ID NO:
(Relative to
SEQ ID NO
SEQ ID NO
SEQ ID NO
SEQ ID NO
SEQ ID NO
SEQ ID NO

NO: (nt/aa)
2072)
SEQ ID NO: 2)
2072
2072
2072
2072
2072
2072

2073/2074
A167G
V63Q/A167G/L171P/H212A/S260T/
+++
++
+
+
+

E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2075/2076
A167G/E738Y/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
+
+
++

E1076S/K1093G
E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/K732W/L749F/L750D/

I775G/E1076S/K1093G/T1148N/

L1248D/L1377D/V1439E

2077/2078
A167G/E738Y/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
+
+
++

K752E/E800V/Y805Q/
E306G/L318G/M320R/S332P/

D808S/E809G/
F387I/I390A/V393G/V534I/M558W/

S1116I
M681G/K732W/L749F/L750D/

K752E/I775G/E800V/Y805Q/

D808S/E809G/S1116I/T1148N/L1248D/

L1377D/V1439E

2079/2080
A167G/E800V/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
+
+
+

E809G/E1076S
E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/E800V/E809G/E1076S/

T1148N/L1248D/L1377D/

V1439E

2081/2082
A167G/G318L
V63Q/A167G/L171P/H212A/S260T/
+++
+
++
++
++
++

E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

2083/2084
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
+++
++
+
++
++
++

E800V/Y805Q/
E306G/M320R/S332P/F387I/I390A/

D808S/E809G
V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E800V/Y805Q/D808S/E809G/

T1148N/L1248D/L1377D/

V1439E

2085/2086
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
++
+
+
++
++
++

G681N
E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

2087/2088
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
++
++
++

G681N/S700E/
E306G/M320R/S332P/F387I/I390A/

Y805Q/D808S/
V393G/V534I/M558W/M681N/

E809G/E1076S/
S700E/K732W/Y738E/L749F/

S1116I
L750D/I775G/Y805Q/D808S/E809G/

E1076S/S1116I/T1148N/L1248D/

L1377D/V1439E

2089/2090
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
++
++
++

S700E
E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2091/2092
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
++
+
++
++
++
++

S700E/E1076S
E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/E1076S/T1148N/

L1248D/L1377D/V1439E

2093/2094
A167G/G318L/
V63Q/A167G/L171P/H212A/S260T/
++
+
+
++
++
++

S700E/E738Y/
E306G/M320R/S332P/F387I/I390A/

E739G/E1076S
V393G/V534I/M558W/M681G/

S700E/K732W/E739G/L749F/

L750D/I775G/E1076S/T1148N/

L1248D/L1377D/V1439E

2095/2096
A167G/G681N/
V63Q/A167G/L171P/H212A/S260T/
++
++
+
+
+
++

E738Y/E800V/
E306G/L318G/M320R/S332P/

D808S/E809G
F387I/I390A/V393G/V534I/M558W/

M681N/K732W/L749F/L750D/

I775G/E800V/D808S/E809G/T1148N/

L1248D/L1377D/V1439E

2097/2098
A167G/L178Q/
V63Q/A167G/L171P/L178Q/H212A/
++
++
+
++
++
++

S700E/K752E/
S260T/E306G/L318G/M320R/

K1093G
S332P/F387I/I390A/V393G/V534I/

M558W/M681G/S700E/K732W/

Y738E/L749F/L750D/K752E/I775G/

K1093G/T1148N/L1248D/

L1377D/V1439E

2099/2100
A167G/S700E
V63Q/A167G/L171P/H212A/S260T/
+++
+
+
+
+
++

E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/I775G/T1148N/

L1248D/L1377D/V1439E

2101/2102
A167G/S700E/
V63Q/A167G/L171P/H212A/S260T/
+
++
+
+
+
++

E738Y/E739G/
E306G/L318G/M320R/S332P/

K1093G
F387I/I390A/V393G/V534I/M558W/

M681G/S700E/K732W/E739G/

L749F/L750D/I775G/K1093G/

T1148N/L1248D/L1377D/V1439E

2103/2104
A167G/S700E/
V63Q/A167G/L171P/H212A/S260T/
+
++
+
+
++
+++

E738Y/K752E/
E306G/L318G/M320R/S332P/

K1093G
F387I/I390A/V393G/V534I/M558W/

M681G/S700E/K732W/L749F/

L750D/K752E/I775G/K1093G/

T1148N/L1248D/L1377D/V1439E

2105/2106
A167G/S700E/
V63Q/A167G/L171P/H212A/S260T/
+
++
+
+
+
++

E738Y/K752E/
E306G/L318G/M320R/S332P/

Y805Q/D808S/
F387I/I390A/V393G/V534I/M558W/

E809G/K1093A/
M681G/S700E/K732W/L749F/

S1116I
L750D/K752E/I775G/Y805Q/D808S/

E809G/K1093A/S1116I/T1148N/

L1248D/L1377D/V1439E

2107/2108
D808S/E809G
V63Q/L171P/H212A/S260T/E306G/
++
++
++
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/D808S/E809G/T1148N/

L1248D/L1377D/V1439E

2109/2110
E1076S
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E1076S/T1148N/L1248D/

L1377D/V1439E

2111/2112
E738Y
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

2113/2114
E738Y/D808S/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+
++

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

D808S/E809G/T1148N/L1248D/

L1377D/V1439E

2115/2116
E738Y/E1076S
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

E1076S/T1148N/L1248D/L1377D/

V1439E

2117/2118
E738Y/E739G
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

2119/2120
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
+

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/E809G/T1148N/L1248D/

L1377D/V1439E

2121/2122
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
++

E809G/K1093G/
L318G/M320R/S332P/F387I/I390A/

S1116I
V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/E809G/K1093G/S1116I/

T1148N/L1248D/L1377D/V1439E

2123/2124
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
+
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/K1093G/T1148N/L1248D/

L1377D/V1439E

2125/2126
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++

K752E/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

K752E/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2127/2128
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++

K799S/E800V/
L318G/M320R/S332P/F387I/I390A/

K1093G
V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/K799S/E800V/K1093G/

T1148N/L1248D/L1377D/V1439E

2129/2130
E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/E739G/L749F/L750D/

I775G/S1116I/T1148N/L1248D/

L1377D/V1439E

2131/2132
E738Y/E800V
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

E800V/T1148N/L1248D/L1377D/

V1439E

2133/2134
E738Y/K1093G
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

K1093G/T1148N/L1248D/L1377D/

V1439E

2135/2136
E738Y/K752E
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/K752E/

I775G/T1148N/L1248D/L1377D/

V1439E

2137/2138
E738Y/K752E/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/K752E/

I775G/K1093G/T1148N/L1248D/

L1377D/V1439E

2139/2140
E738Y/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

K799S
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/K752E/

I775G/K799S/T1148N/L1248D/

L1377D/V1439E

2141/2142
E738Y/K799S/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
++

E809G/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

K799S/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2143/2144
E738Y/K799S/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/L749F/L750D/I775G/

K799S/K1093G/T1148N/L1248D/

L1377D/V1439E

2145/2146
E739G
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/E739G/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2147/2148
E739G/E800V/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/E739G/L749F/

L750D/I775G/E800V/E809G/T1148N/

L1248D/L1377D/V1439E

2149/2150
E800V
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E800V/T1148N/L1248D/

L1377D/V1439E

2151/2152
E800V/Y805Q/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
++
++

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E800V/Y805Q/E809G/

T1148N/L1248D/L1377D/V1439E

2153/2154
E809G
V63Q/L171P/H212A/S260T/E306G/
++++
+
+
++
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E809G/T1148N/L1248D/

L1377D/V1439E

2155/2156
E809G/K1093G
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2157/2158
E809G/K1093G/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
+
++

S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E809G/K1093G/S1116I/

T1148N/L1248D/L1377D/V1439E

2159/2160
E809G/S1116I
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E809G/S1116I/T1148N/

L1248D/L1377D/V1439E

2161/2162
G318L
V63Q/L171P/H212A/S260T/E306G/
+++
+
++
++
++
++

M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

2163/2164
G318L/E1076S
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++
++

M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/

E1076S/T1148N/L1248D/L1377D/

V1439E

2165/2166
G318L/E1076S/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

S1116I
M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/

E1076S/S1116I/T1148N/L1248D/

L1377D/V1439E

2167/2168
G318L/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++

Y805Q/D808S/
M320R/S332P/F387I/I390A/

E809G/E1076S
V393G/V534I/M558W/M681G/K732W/

Y738E/E739G/L749F/L750D/

I775G/Y805Q/D808S/E809G/

E1076S/T1148N/L1248D/L1377D/

V1439E

2169/2170
G318L/I499L/S700E/
V63Q/L171P/H212A/S260T/E306G/
++++
++
+
++
++
+++

E738Y/E809G
M320R/S332P/F387I/I390A/

V393G/I499L/V534I/M558W/M681G/

S700E/K732W/L749F/L750D/

I775G/E809G/T1148N/L1248D/

L1377D/V1439E

2171/2172
G318L/K1093A
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
++

M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/

K1093A/T1148N/L1248D/L1377D/

V1439E

2173/2174
G318L/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
++
+++

K1093A
M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/K752E/

I775G/K1093A/T1148N/L1248D/

L1377D/V1439E

2175/2176
G318L/S1116I
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/K732W/

Y738E/L749F/L750D/I775G/

S1116I/T1148N/L1248D/L1377D/

V1439E

2177/2178
G318L/S543P/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

E739G
M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/M681G/

K732W/Y738E/E739G/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2179/2180
G318L/S543P/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
+++

G68IN/K752E/
M320R/S332P/F387I/I390A/

S1116I
V393G/V534I/S543P/M558W/M681N/

K732W/Y738E/L749F/L750D/

K752E/I775G/S1116I/T1148N/

L1248D/L1377D/V1439E

2181/2182
G318L/S543P/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

K1093A
M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/K1093A/T1148N/L1248D/

L1377D/V1439E

2183/2184
G318L/S700E/E738Y/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++
++++

K1093A/S1414N
M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/S700E/

K732W/L749F/L750D/I775G/

K1093A/T1148N/L1248D/L1377D/

S1414N/V1439E

2185/2186
G681N
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

I775G/T1148N/L1248D/L1377D/

V1439E

2187/2188
G681N/E738Y
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/L749F/L750D/I775G/

T1148N/L1248D/L1377D/V1439E

2189/2190
G681N/E738Y/
V63Q/L171P/H212A/S260T/E306G/
++
+
++
+
++
++

E739G/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/E739G/L749F/L750D/

I775G/K1093G/T1148N/L1248D/

L1377D/V1439E

2191/2192
G681N/E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+

K752E/K799S/
L318G/M320R/S332P/F387I/I390A/

E800V/E809G/
V393G/V534I/M558W/M681N/

S1116I/G1339C
K732W/E739G/L749F/L750D/

K752E/I775G/K799S/E800V/E809G/

S1116I/T1148N/L1248D/G1339C/

L1377D/V1439E

2193/2194
G681N/E738Y/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
+
++

E739G/S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/E739G/L749F/L750D/

I775G/S1116I/T1148N/L1248D/

L1377D/V1439E

2195/2196
G681N/E738Y/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+

E800V
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/L749F/L750D/I775G/

E800V/T1148N/L1248D/L1377D/

V1439E

2197/2198
G681N/E800V/
V63Q/L171P/H212A/S260T/E306G/
++++
++
+
++
++
++

Y805Q/E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

I775G/E800V/Y805Q/E809G/

T1148N/L1248D/L1377D/V1439E

2199/2200
G681N/E809G/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
+
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

I775G/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2201/2202
G681N/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
++
+++

K1093A
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

K752E/I775G/K1093A/T1148N/

L1248D/L1377D/V1439E

2203/2204
G681N/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
+++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

K752E/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2205/2206
G681N/S700E
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2207/2208
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+
++

E738Y/E739G/
L318G/M320R/S332P/F387I/I390A/

K1093G/S1116I
V393G/V534I/M558W/M681N/

S700E/K732W/E739G/L749F/

L750D/I775G/K1093G/S1116I/

T1148N/L1248D/L1377D/V1439E

2209/2210
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
+
++

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/I775G/E809G/T1148N/

L1248D/L1377D/V1439E

2211/2212
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2213/2214
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
++
+
++

K752E/D808S/
L318G/M320R/S332P/F387I/I390A/

E809G/K1093G/
V393G/V534I/M558W/M681N/

S1116I
S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/D808S/E809G/

K1093G/S1116I/T1148N/L1248D/

L1377D/V1439E

2215/2216
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+++
++
++
++
++

K752E/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/K1093G/

T1148N/L1248D/L1377D/V1439E

2217/2218
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++

R844H
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/I775G/R844H/T1148N/

L1248D/L1377D/V1439E

2219/2220
G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++

Y805Q/E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681N/

S700E/K732W/Y738E/L749F/

L750D/I775G/Y805Q/E809G/T1148N/

L1248D/L1377D/V1439E

2221/2222
K1093A
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/K1093A/T1148N/L1248D/

L1377D/V1439E

2223/2224
K1093G
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/K1093G/T1148N/L1248D/

L1377D/V1439E

2225/2226
K1093G/S1116I
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/K1093G/S1116I/T1148N/

L1248D/L1377D/V1439E

2227/2228
K752E
V63Q/L171P/H212A/S260T/E306G/
++
+
++
++
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/T1148N/L1248D/

L1377D/V1439E

2229/2230
K752E/E1076S
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/E1076S/T1148N/

L1248D/L1377D/V1439E

2231/2232
K752E/E800V/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+
++

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/E800V/E809G/T1148N/

L1248D/L1377D/V1439E

2233/2234
K752E/E800V/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/E800V/K1093G/

T1148N/L1248D/L1377D/V1439E

2235/2236
K752E/E809G
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/E809G/T1148N/

L1248D/L1377D/V1439E

2237/2238
K752E/G866A
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/G866A/T1148N/

L1248D/L1377D/V1439E

2239/2240
K752E/K1093G
V63Q/L171P/H212A/S260T/E306G/
+
++
++
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2241/2242
K752E/S1116I
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

K752E/I775G/S1116I/T1148N/

L1248D/L1377D/V1439E

2243/2244
L178Q/G318L/E739G/
V63Q/L171P/L178Q/H212A/S260T/
++
++
+
++
++
++

K752E/E809G/
E306G/M320R/S332P/F387I/I390A/

E1076S/S1116I
V393G/V534I/M558W/M681G/

K732W/Y738E/E739G/L749F/

L750D/K752E/I775G/E809G/E1076S/

S1116I/T1148N/L1248D/L1377D/

V1439E

2245/2246
L178Q/G318L/
V63Q/L171P/L178Q/H212A/S260T/
++
+
+
++
++
++

E800V/E809G
E306G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/E800V/E809G/T1148N/

L1248D/L1377D/V1439E

2247/2248
L178Q/G318L/G681N/
V63Q/L171P/L178Q/H212A/S260T/
++
++
+
++
++
++

E800V/D808S/
E306G/M320R/S332P/F387I/I390A/

E809G/E1076S
V393G/V534I/M558W/M681N/

K732W/Y738E/L749F/L750D/

I775G/E800V/D808S/E809G/E1076S/

T1148N/L1248D/L1377D/

V1439E

2249/2250
L178Q/G681N
V63Q/L171P/L178Q/H212A/S260T/
++
++
+
++
++
++

E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681N/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2251/2252
L178Q/G681N/
V63Q/L171P/L178Q/H212A/S260T/
++
++
+
++
++
++

E738Y/S1116I
E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681N/K732W/L749F/L750D/

I775G/S1116I/T1148N/L1248D/

L1377D/V1439E

2253/2254
L178Q/K752E/
V63Q/L171P/L178Q/H212A/S260T/
+
++
+
++
+
++

E809G
E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/K732W/Y738E/L749F/

L750D/K752E/I775G/E809G/T1148N/

L1248D/L1377D/V1439E

2255/2256
L178Q/S700E/E800V/
V63Q/L171P/L178Q/H212A/S260T/
++
++
+
++
+
++

Y805Q/D808S/
E306G/L318G/M320R/S332P/

E809G
F387I/I390A/V393G/V534I/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/I775G/E800V/Y805Q/

D808S/E809G/T1148N/L1248D/

L1377D/V1439E

2257/2258
L178Q/Y805Q/
V63Q/L171P/L178Q/H212A/S260T/
++
+
+
++
++
+

E809G/G866A
E306G/L318G/M320R/S332P/

F387I/I390A/V393G/V534I/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/Y805Q/E809G/G866A/

T1148N/L1248D/L1377D/

V1439E

2259/2260
S1116I
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/S1116I/T1148N/L1248D/

L1377D/V1439E

2261/2262
S543P
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2263/2264
S543P/E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/E739G/L749F/

L750D/I775G/E809G/T1148N/L1248D/

L1377D/V1439E

2265/2266
S543P/E738Y/E809G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/L749F/L750D/

I775G/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2267/2268
S543P/E739G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
++
+

K1093A
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/E739G/

L749F/L750D/I775G/K1093A/

T1148N/L1248D/L1377D/V1439E

2269/2270
S543P/E800V/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+

K1093G/S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/E800V/K1093G/S1116I/

T1148N/L1248D/L1377D/

V1439E

2271/2272
S543P/E809G/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2273/2274
S543P/E809G/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+

K1093G/S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/E809G/K1093G/S1116I/

T1148N/L1248D/L1377D/

V1439E

2275/2276
S543P/G681N/E738Y/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
++

E739G/K752E/
L318G/M320R/S332P/F387I/I390A/

E809G/K1093G
V393G/V534I/S543P/M558W/

M681N/K732W/E739G/L749F/

L750D/K752E/I775G/E809G/K1093G/

T1148N/L1248D/L1377D/

V1439E

2277/2278
S543P/G681N/E738Y/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++

E739G/K752E/
L318G/M320R/S332P/F387I/I390A/

K1093G
V393G/V534I/S543P/M558W/

M681N/K732W/E739G/L749F/

L750D/K752E/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2279/2280
S543P/G681N/E739G/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++

K752E/E800V/
L318G/M320R/S332P/F387I/I390A/

Y805Q/E809G/
V393G/V534I/S543P/M558W/

K1093G
M681N/K732W/Y738E/E739G/

L749F/L750D/K752E/I775G/E800V/

Y805Q/E809G/K1093G/T1148N/

L1248D/L1377D/V1439E

2281/2282
S543P/G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
+
+
+

E738Y/E739G/
L318G/M320R/S332P/F387I/I390A/

E800V/K1093G/
V393G/V534I/S543P/M558W/

S1116I
M681N/S700E/K732W/E739G/

L749F/L750D/I775G/E800V/K1093G/

S1116I/T1148N/L1248D/L1377D/

V1439E

2283/2284
S543P/G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
+
+
+

E738Y/E739G/
L318G/M320R/S332P/F387I/I390A/

Y805Q/D808S/
V393G/V534I/S543P/M558W/

E809G
M681N/S700E/K732W/E739G/

L749F/L750D/I775G/Y805Q/D808S/

E809G/T1148N/L1248D/L1377D/

V1439E

2285/2286
S543P/G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
+
+
++

K752E
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681N/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/T1148N/

L1248D/L1377D/V1439E

2287/2288
S543P/G681N/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++

K752E/E809G/
L318G/M320R/S332P/F387I/I390A/

K1093G
V393G/V534I/S543P/M558W/

M681N/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/E809G/

K1093G/T1148N/L1248D/L1377D/

V1439E

2289/2290
S543P/K1093G
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/K732W/Y738E/L749F/

L750D/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2291/2292
S543P/S700E
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2293/2294
S543P/S700E/E738Y
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+
++

E739G/K752E/
L318G/M320R/S332P/F387I/I390A/

E800V/K1093G/
V393G/V534I/S543P/M558W/

S1116I
M681G/S700E/K732W/E739G/

L749F/L750D/K752E/I775G/E800V/

K1093G/S1116I/T1148N/L1248D/

L1377D/V1439E

2295/2296
S543P/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
++

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/I775G/E809G/T1148N/

L1248D/L1377D/V1439E

2297/2298
S543P/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2299/2300
S543P/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
+
+
++

K752E
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/T1148N/

L1248D/L1377D/V1439E

2301/2302
S543P/S700E/K752E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
+
+
++

E800V/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/E800V/

K1093G/T1148N/L1248D/L1377D/

V1439E

2303/2304
S543P/S700E/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+
++

E809G/S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/E809G/

S1116I/T1148N/L1248D/L1377D/

V1439E

2305/2306
S543P/S700E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
+++

K752E/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/S543P/M558W/

M681G/S700E/K732W/Y738E/

L749F/L750D/K752E/I775G/K1093G/

T1148N/L1248D/L1377D/

V1439E

2307/2308
S700E
V63Q/L171P/H212A/S260T/E306G/
+++
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/T1148N/L1248D/

L1377D/V1439E

2309/2310
S700E/E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++

E800V/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/E739G/L749F/

L750D/I775G/E800V/K1093G/

T1148N/L1248D/L1377D/V1439E

2311/2312
S700E/E738Y/E739G/
V63Q/L171P/H212A/S260T/E306G/
+++
+
+
+
+

K799S/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/E739G/L749F/

L750D/I775G/K799S/K1093G/

T1148N/L1248D/L1377D/V1439E

2313/2314
S700E/E738Y/E800V/
V63Q/L171P/H212A/S260T/E306G/
+++
++
+
++
++
+++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/L749F/L750D/

I775G/E800V/K1093G/T1148N/

L1248D/L1377D/V1439E

2315/2316
S700E/E738Y/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
+
+
++

S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/L749F/L750D/

I775G/S1116I/T1148N/L1248D/

L1377D/V1439E

2317/2318
S700E/E809G
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/E809G/T1148N/

L1248D/L1377D/V1439E

2319/2320
S700E/E809G/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/E809G/K1093G/

T1148N/L1248D/L1377D/V1439E

2321/2322
S700E/K1093G
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
++
++

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/K1093G/T1148N/

L1248D/L1377D/V1439E

2323/2324
S700E/K1093G/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
++
++
++

S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/K1093G/S1116I/

T1148N/L1248D/L1377D/V1439E

2325/2326
S700E/K752E
V63Q/L171P/H212A/S260T/E306G/
++
++
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/T1148N/

L1248D/L1377D/V1439E

2327/2328
S700E/K752E/D808S/
V63Q/L171P/H212A/S260T/E306G/
++
++
+
++
+
++

E809G/K1093G/
L318G/M320R/S332P/F387I/I390A/

S1116I
V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/D808S/E809G/

K1093G/S1116I/T1148N/L1248D/

L1377D/V1439E

2329/2330
S700E/K752E/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
+
+

E809G/K1093G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/E809G/K1093G/

T1148N/L1248D/L1377D/

V1439E

2331/2332
S700E/K752E/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
+
+
++

K799S/S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/K799S/S1116I/

T1148N/L1248D/L1377D/

V1439E

2333/2334
S700E/K752E/
V63Q/L171P/H212A/S260T/E306G/
+
++
++
+
+
++

Y805Q/E809G/
L318G/M320R/S332P/F387I/I390A/

K1093A
V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/K752E/I775G/Y805Q/E809G/

K1093A/T1148N/L1248D/

L1377D/V1439E

2335/2336
S700E/K799S/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+
++

E800V
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/K799S/E800V/T1148N/

L1248D/L1377D/V1439E

2337/2338
S700E/S1116I
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+

L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/S1116I/T1148N/

L1248D/L1377D/V1439E

2339/2340
S700E/Y805Q/D808S/
V63Q/L171P/H212A/S260T/E306G/
++
++
++
++
+
++

E809G/E1076S
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

S700E/K732W/Y738E/L749F/

L750D/I775G/Y805Q/D808S/E809G/

E1076S/T1148N/L1248D/L1377D/

V1439E

2341/2342
V514L/S543P/G681N/
V63Q/L171P/H212A/S260T/E306G/
+
+
+
++
+
++

Y805Q/D808S/
L318G/M320R/S332P/F387I/I390A/

E809G
V393G/V514L/V534I/S543P/

M558W/M681N/K732W/Y738E/

L749F/L750D/I775G/Y805Q/D808S/

E809G/T1148N/L1248D/L1377D/

V1439E

2343/2344
Y805Q/D808S/
V63Q/L171P/H212A/S260T/E306G/
++
+
+
+
+
+

E809G
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/Y805Q/D808S/E809G/T1148N/

L1248D/L1377D/V1439E

2345/2346
Y805Q/E809G/
V63Q/L171P/H212A/S260T/E306G/
+
++
+
++
+

S1116I
L318G/M320R/S332P/F387I/I390A/

V393G/V534I/M558W/M681G/

K732W/Y738E/L749F/L750D/

I775G/Y805Q/E809G/S1116I/

T1148N/L1248D/L1377D/V1439E

All activities were determined relative to the reference polypeptide of SEQ ID NO: 2072. Levels of increased activity are defined as follows:

“+” 0.9 to 1.1,

“++” >1.1,

“+++” >2,

“++++” >2.5

Example 12
Identification of Active FVIII Variants with Reduced Immunogenicity

Putative T-cell epitopes in a B-domain deleted version of wild type human FVIII were identified using the Immune Epitope Database (IEDB; Immune Epitope Database and Analysis Resource website) tools, as known in the art and proprietary statistical analysis tools (See e.g., iedb.org and Vita et al., Nucl. Acids Res., 2010, 38 (Database issue): D854-62, Epub 2009 Nov. 11). The B-domain deleted version of wild type human FVIII (SEQ ID NO: 2) was parsed into all possible 15-mer analysis frames, with each frame overlapping the last by 14 amino acids. The 15-mer analysis frames were evaluated for immunogenic potential by scoring their predicted binding to eight common class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501), using methods recommended on the IEDB website. Potential T-cell epitope clusters contained within the polypeptide (i.e., sub-regions contained within human FVIII which have an unusually high potential for immunogenicity) were identified using statistical analysis tools, as known in the art. The identified T-cell epitope clusters were screened against the IEDB database of known epitopes.

Active FVIII variants identified in screens from Example 3 for their levels of predicted immunogenicity by evaluating their binding to the eight common Class II HLA-DR alleles. The total immunogenicity score and immunogenic hit count were calculated for each variant. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenic “hit count” (IHC) indicates the number of 15-mer analysis frames with an unusually high potential for immunogenicity (i.e., a higher score indicates a higher potential for immunogenicity). Mutations resulting in a reduced total immunogenicity score and/or an immunogenic hit count compared to the reference sequence were considered to be potential “deimmunizing mutations” and are shown in Tables 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, and 15.2. A collection of the most deimmunizing mutations were recombined to generate a number of variants that were active and predicted to be significantly less immunogenic than B-domain deleted version of WT human FVIII (SEQ ID NO: 2).

TABLE 12.1

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 2

SEQ ID

TIS reduction
IHC reduction

NO:
Amino Acid Differences
Relative to
Relative to

(nt/aa)
(Relative to SEQ ID NO: 2)
SEQ ID NO: 2
SEQ ID NO: 2

1/2

191/192
Q37H/Q96R/L171P/Q429L/I708T/K799R/T1148N
++
+

193/194
L171P
+
+

195/196
L171P/Q429L/K732R/T1148N
+
+

197/198
Q96R/L171P
++
+

199/200
L171P/E919G/G1151S
+

201/202
L171P/T792Y/M797Q/K799R/T1148N
+++
+

203/204
L171P/P569T/T1148N
+++
+

205/206
Q96R/L171P/Q429L/T792Y/M797T/K799R

+

207/208
L171P/Q429L/I708T/L1422I
++
+

209/210
L171P/Q429L

+

3/4
T1148N
+
+

211/212
D321Y/A362P/K732E/M797T/K799R
+

213/214
L171P/L1100I
++
+

215/216
I708T/T1148N/A1186E
+++
+

217/218
Q429L/L724I/K732E/T1148N

+

219/220
L171P/A362E/V795T/M797Q/K799M
++
+

221/222
L171P/Q429L/K732E

+

223/224
Q96R/L171P/S643N/M797T/K799R
++
+

225/226
L171P/T792Y/M797T/K799M
+
+

227/228
R503S
++
+

229/230
L171P/K732E
+
+

231/232
T1148N/F1193L
+
+

5/6
P85Q/1708T/T1148N
++
+

233/234
Q96R/L171P/A362E/Q429L/K732R/V795T/M797Q
+
+

7/8
K798T
+

9/10
L706G
++
++

11/12
L706A
+
+

13/14
L171Q
+

15/16
Q335E
+

17/18
V63R
++

235/236
K726A
+

19/20
K344A
+

237/238
F59M
+

241/242
A362N
+

243/244
H334G
+

25/26
F59L
+

247/248
V63Q
+++
++

251/252
L1377D
+++
+

27/28
Q429S
+

31/32
I1264W
+

259/260
V1250T
++
+

261/262
E306G
+

265/266
L1377G
++
+

269/270
L1377E
+++
+

273/274
L646I
+

33/34
M797P
+

35/36
L1248S
+
+

37/38
I1264R
+

39/40
Y873T
+

41/42
T792Y
+

43/44
I708K
+
+

45/46
K344L
+

47/48
F57V
+

49/50
Q96L
+

277/278
P435Q
+

51/52
A362W
++

53/54
V63G
+++
++

55/56
Y873L
+

59/60
T1148Q
+

63/64
M797G
+

65/66
L706Y
++
++

67/68
L1248R
++
+

71/72
Y65A
+
+

73/74
F59W
+

75/76
Y873Q
+

77/78
V795C
+

279/280
Q96V
+

81/82
F57L
+

83/84
Q353S
+

85/86
S332Q
+
+

87/88
I708A
+

89/90
G1259T
+
+

91/92
V63M
+

93/94
M356L
++

95/96
L1248G
+
+

97/98
L1248P
++
+

99/100
Q436G
+

101/102
F57A
+

103/104
F59S
+
+

105/106
T1245R
+

107/108
F59R
+

109/110
Q96W
+

111/112
M797Y
+

113/114
K1145R
+

281/282
L1377R
+

115/116
Q436D
+

117/118
F716L
+

119/120
S56E
+

283/284
L1377S
+
+

285/286
F59G
+

287/288
L171M
+

121/122
R773H
+

289/290
T792H
+

129/130
F59Q
+
+

131/132
S729T
+

133/134
F716K
+

135/136
T665G
+
+

137/138
A362T
++

139/140
F57T
+

141/142
N359G
+

291/292
L1248D
+
+

145/146
V1250A
++
+

147/148
A362V
+

149/150
N359E
++

151/152
F716W
+

153/154
S332W
+
++

155/156
I708V
+

157/158
V63H
++
++

159/160
M797C
+

295/296
L706M
+
++

161/162
T61Q
+
+

163/164
M356N
++

297/298
L171H
+

165/166
V1250S
+
+

167/168
K798C
+

169/170
N433G
+

299/300
L1377A
++

301/302
L1377V
+

171/172
V795N
+

173/174
Q96I
+

175/176
S56P
+

177/178
V63N
++
++

179/180
T792W
++

181/182
I1264L
+

183/184
V1250P
+
+

185/186
S332P
+
+

187/188
F59V
+

309/310
F59D
+
+

189/190
K726L
+

311/312
K344E
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2 and are defined as follows: “+” 1-10, “++” >10, “+++” >20.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2 and are defined as follows: “+” 1-3, “++” >3, “+++” >5

TABLE 12.2

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 252

SEQ ID

TIS reduction
IHC reduction

NO:
Amino Acid Differences
Relative to
Relative to

(nt/aa)
(Relative to SEQ ID NO: 252)
SEQ ID NO: 252
SEQ ID NO: 252

251/252

411/412
I265S
++
+

413/414
L1221T
+
++

415/416
M1380G
+

417/418
K752G
++
+

419/420
Y748K
+++
+++

423/424
F1218A
++
++

425/426
Y748N
+++
+++

427/428
T1378E
+

429/430
L614I
+
+

431/432
L749F
+
+

433/434
F387V
++
+

435/436
L750T
+
+

437/438
F1193G
++
+

441/442
T1378A
+

443/444
L750V

+

445/446
F387T
+++
+++

447/448
F387Q
+++
+++

451/452
F1037N
++
+

453/454
H1440A
+

455/456
M1051S
+
+

457/458
L1221G
++
++

459/460
T1378D
+

461/462
H1440S
+
+

463/464
F387R
++
+++

465/466
F1193A
+
+

467/468
F387I
+
+

469/470
F1218M
++
++

319/320
I388A
++
++

471/472
I265W
+++
+

473/474
H1440E
+++
+++

475/476
V1439L
+
+

477/478
H1440T
++
+++

479/480
R612S

+

481/482
Y1049W
+
++

321/322
F1043E
+++
+++

323/324
F1043R
++
++

483/484
L1221S
+
++

325/326
I610R
++
+

327/328
I1274N
+++
+++

329/330
S1220T
+
+

331/332
I1442T
+
+

485/486
S1220D
++
++

333/334
F1193L

+

335/336
Q621T
+
+

337/338
L1054C
+
+

339/340
A755Q
+
+

487/488
L749G
+++
+++

341/342
Y1041G
++
++

343/344
Y748S
+++
+++

345/346
Y1049G
+++
+++

347/348
L1221V
+
++

489/490
L749S
++
++

491/492
F1218G
++
++

497/498
F1193E
++
+

499/500
I1050S
++
++

501/502
S1389W
+

349/350
M1051E
+++
+++

503/504
V1439E
+++
+++

505/506
S260T
+
+

507/508
F1037P
++
+

351/352
R612G
+
+

353/354
F1037H
++
+

355/356
N616E
++
+

509/510
M1051K
++
++

511/512
I1269F
+
+

513/514
I265R
+
+

357/358
Y1041H
++
+++

515/516
M1051R
++
++

517/518
F1218L
+
++

359/360
F387S
+++
+++

361/362
S489R
++
+

363/364
Q621M
+
+

365/366
S489Q
++
+

367/368
L750G
+
+

519/520
M897R
+
+

521/522
I388P
+++
+++

369/370
S1224P
+
+

523/524
Y748G
+++
+++

371/372
A618C
+
+

525/526
I756P
++
+++

373/374
N616G
++
+

375/376
R612T

+

529/530
I390A
++
+

377/378
I1050Q
++
++

531/532
I610L

+

533/534
A755G
+
+

535/536
L749R
+
+

379/380
T1053A
+
+

381/382
N616W
+
+

537/538
Y748V
+
+

383/384
I896V
+
+

539/540
M1380A
+

541/542
H1440C
++
+++

385/386
S489F
+
+

543/544
H397N/F1218M
++
++

387/388
I1050P
++
++

389/390
M897G
++
+

545/546
F1037M
+
+

547/548
F1218P
++
++

549/550
E757K
+
+

391/392
L749T
++
+

551/552
V1373S
+

553/554
H1440G
++
+++

555/556
F1193I
+
+

557/558
V1439T
++
+++

393/394
I1274V
+
+

395/396
S1220L
+
+

559/560
L750D
+++
+++

397/398
S260E
++
+

561/562
I390G
+++
+

563/564
I756G
++
++

399/400
L481V
+

565/566
S1220E
++
++

401/402
F613S
+
+

403/404
F387G
+++
+++

405/406
Q621R
+
+

567/568
T1378C
+

407/408
I756R
++
+

569/570
A618G
++
+

573/574
V63Q/L1248D
+++
+++

577/578
E306G/R355C/L1422I
+++

579/580
L1422I
+

581/582
E306G/D346R/I708T
++

583/584
T1148N
+
+

585/586
L171P/D346R/L1248D
++
+

587/588
V63Q/M797T/K799R/L1248D/L1422I
+++
+++

589/590
L171P/K799R
+
+

591/592
A362P
+

593/594
L1248D
+
+

595/596
V63Q
+++
++

597/598
L171P/E306G/D346R/Q999R/L1248D
++
+

599/600
L171P/T1148N
++
+

601/602
T734S/K799R/L1248D/L1422I
++
+

603/604
V63Q/K732E
+++
++

605/606
V63Q/L171P/E306G/D346R/K732G
+++
+++

607/608
L171P
+
+

609/610
L171P/L1422I
++
+

611/612
V63Q/A362P/K732G/K799R/L1248D
+++
+++

613/614
Q999R/T1148N/L1422I
++
+

617/618
E306G/P349G
+

619/620
V63Q/L171P/T1148N/G1151S
+++
+++

621/622
E306G
+

623/624
K344E/P349G/L354R/V1250T
+++
+

625/626
V63G
+++
++

627/628
V1250T
++
+

629/630
V63R/E306G/K344E/P349G/L354R/R503S/L1248R
++++
++++

631/632
F59D/V63S/S332P/P349G/R503S/L706M/I708K
++++
++++

633/634
V63S/E306G/K344A/P349G/L354R/L706M/L1248R
++++
++++

635/636
L1248R
++
+

637/638
F59D/V63G
+++
++

639/640
S332P/L706M/I708K/L1248R/V1250T
+++
++++

641/642
R503S
++
+

643/644
F59D/V63R/P349G/L354R
+++
++

645/646
F59D/P349G/I708K
++
+

647/648
F59D/E306G/K344E/L1248R
+++
+

409/410
V63R
++
++

649/650
K344E/L1248R/V1250T
+++
+

651/652
I708K
+
+

653/654
V63R/K344E/P349G
+++
++

655/656
F59D
+
+

657/658
V63S/K344A/P349G/L354R
+++
++

659/660
L706M
+
++

661/662
F59D/E306G/R770W
++
+

663/664
V63G/S347N/L354R/L1248R
++++
+++

665/666
L354R/R503S
+++
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 252 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >50, “+++++”.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 252 and are defined as follows: “+” 1-3, “++” >3, “+++” >5, “++++” >8

TABLE 12.3

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 504

SEQ ID

TIS reduction
IHC reduction

NO:
Amino Acid Differences
Relative to
Relative to

(nt/aa)
(Relative to SEQ ID NO: 504)
SEQ ID NO: 504
SEQ ID NO: 504

503/504

701/702
L171P/L1248D/H1440T
++
+

703/704
S260T/T1148N/T1378D
++
++

705/706
L171P/S260T/F387I/L750D/L1248D
++++
++++

707/708
L171P/S260T
+
++

709/710
L171P/S260T/F387I/L750D/M1051S/T1148N
++++
+++++

711/712
L171P
+
+

667/668
L750D
+++
+++

713/714
L750D/M1051S/T1148N
+++
++++

715/716
L171P/S260T/I756P
+++
++++

669/670
M1051K
++
++

717/718
L171P/L1248D
++
+

671/672
M1051S
+
+

719/720
L171P/T1148N
++
+

721/722
F387I
+
+

723/724
F59D
+
+

727/728
S332P/L750D/L1248D
+++
++++

729/730
I756P/L1248D
+++
+++

673/674
S260T
+
+

731/732
L1248D
+
+

675/676
I756P
++
+++

733/734
L171P/F387I/L1248D/H1440S
+++
+

677/678
F1193A
+
+

735/736
L750D/H1440S
+++
+++

737/738
F59D/L171P/L750D
+++
++++

739/740
L171P/F1193A/L1248D
++
++

679/680
S332P
+
+

741/742
L750D/T1148N/F1193A/H1440S
+++
++++

743/744
T1148N
+
+

745/746
L171P/F387I/M1051K
+++
+++

747/748
Y748G
+++
+++

681/682
M897R
+
+

683/684
F387Q
+++
+++

749/750
E306G
+

751/752
E306G/F387R/I390A/L749F/S1220D
++++
++++

685/686
F1037N
++
+

753/754
I390A
++
+

755/756
E306G/I390A/F1037N
+++
+

757/758
E306G/R612S/L749F
+
+

759/760
F387V/1390A/L749F
+++
+++

761/762
V63S/I390A/R503S/L749F
++++
++++

763/764
F387R/I390A/L749R
+++
+++

765/766
S1220D
++
++

687/688
F387R
++
+++

767/768
V63S/E306G
+++
++

689/690
E306G/L749F
+
+

769/770
L749F
+
+

771/772
E306G/I390A/L749F/F1037N
+++
+

773/774
F387V
++
+

775/776
E306G/L749F/S1220D
+++
++

777/778
E306G/F387V/I390A/R612S/L749F/F1037N
++++
++++

691/692
I1270V
+
+

693/694
S489E
+++
+

779/780
F328S
++
+++

695/696
Q487N
++
+

697/698
A488G
++
+

699/700
Q903E
++
+++

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 504 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >50, “+++++” >75.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 504 and are defined as follows: “+” 1-3, “++” >3, “+++” >5, “++++” >8, “+++++” >15

TABLE 12.4

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 706

SEQ ID

TIS reduction
IHC reduction

NO:
Amino Acid Differences
Relative to
Relative to

(nt/aa)
(Relative to SEQ ID NO: 706)
SEQ ID NO: 706
SEQ ID NO: 706

705/706

819/820
S332P/I387R/I390A/T1148N
+++
+++

821/822
F328S/I387R/L749F
+++
++++

781/782
M1051K
++
++

823/824
L749F
+

783/784
L1221G
++
++

825/826
E306G/F328S/S332P/I390A/M1051R/F1193A/F1218G
++++
++++

827/828
V63Q/E306G/S332P/M1051R/T1148N
++++
++++

829/830
V63Q/E306G/S332P/I387T/M1051K
++++
+++++

831/832
I387T/I390A/M1051R
+++
++++

833/834
D186A/E306G/F328S/S332P/I390A/L749F/M1051K/T1148N/
++++
++++

F1193A

835/836
I387T/I390A/I756P/M1051R
++++
++++

837/838
E306G/I390A/M1051R
+++
+++

839/840
E306G/S332P/I390A/L749F
+++
++

785/786
F1218G
++
++

841/842
E306G/I387R/I390A/T1148N
+++
+++

843/844
E306G/F328S/S332P/I387T/I390A/L749F/M1051R/T1148N/
+++++
++++

F1193A

845/846
S332P/I387R/I390A/L749F/T1148N
+++
+++

847/848
I387R/I390A/L749F
+++
++

787/788
I387R
++
++

849/850
E306G/I387T/L749F/M1051K/T1148N
+++
++++

789/790
M1051R
++
++

851/852
I387R/L749F
++
++

853/854
E306G/Q945K/M1051R/F1193A/L1221G
+++
++

855/856
E306G/I390A/L749F/M1051K
+++
+++

857/858
V63Q/E306G/S332P/I390A
+++
++++

791/792
L749F/F1218G
++
++

859/860
E306G/I387T/I390A/L749F/T1148N/F1193A/L1221G
++++
++++

861/862
E306G/S332P/I387R/I390A/L749F/F1218M
++++
++++

793/794
I387T
++
++

863/864
V63Q/E306G/S332P/I390A/M1051R/T1148N/F1193A/
+++++
+++++

F1218G

795/796
S332P
+
+

867/868
A755S
+

875/876
M797Y
+

797/798
M897K
+
+

877/878
N754W
+

879/880
I793G
+

881/882
N753D
+

883/884
R922W
+

885/886
I896L
+

799/800
T696E
+
+

889/890
I775G
+

801/802
F816W
+
+

803/804
F824W
++
+++

805/806
A826G
++
+++

891/892
S751E
+

807/808
V393W
++
+

809/810
I825L
+
+

811/812
M897R
+
+

895/896
K752G
+

899/900
K938V
+

903/904
A755G
+

905/906
V393G/A407V
+++
++

813/814
F816V
+
+

907/908
M797W
+

909/910
K819V
+
+

911/912
V393G
+++
++

913/914
I787W
+

921/922
G924P
+

815/816
F698W
+
++

931/932
S392A
+
+

933/934
H397G
+

817/818
V393Q
+
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 706 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >50, “+++++” >75.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 706 and are defined as follows: “+” 1-3, “++” >3, “+++” >5, “++++” >8, “+++++” >15

TABLE 12.5

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 840

Amino Acid

Differences
TIS reduction
IHC reduction

SEQ ID
(Relative to
Relative to
Relative to

NO: (nt/aa)
SEQ ID NO: 840)
SEQ ID NO: 840
SEQ ID NO: 840

839/840

1029/1030
L622D
+
+

1031/1032
P617E
+
+

1033/1034
P617N
+
+

935/936
V620N
+
+

1035/1036
P907D
+
+

1037/1038
N1040E
+
+

1039/1040
P617D
+
+

1041/1042
Q611E
+
+

937/938
P491E
+
+

1043/1044
V620P
+
+

1045/1046
P617G
+
+

1047/1048
A488G
++
+

1049/1050
P1192D
+
+

1051/1052
S1194P
+
+

1053/1054
G619E
+
+

939/940
M1229D
+++
++

1055/1056
P615D
++
+

941/942
P615N
+
+

1057/1058
F16A
+
+

943/944
I1283S
+++
++

945/946
H822S
+

1063/1064
L211K
++
+

1065/1066
M836W
+
+

947/948
F328G
+++
++

1067/1068
F555R
+
+

951/952
K818W
+
+

1069/1070
V536A
+
+

955/956
F555G/E684G
++
+

1071/1072
I527A
+
+

1073/1074
F555E
++
+

957/958
V828K

+

1075/1076
H841W

+

959/960
R550G
+
+

1077/1078
M320Q
+
+

1079/1080
S837G
++
+

1081/1082
M558R
++
++

1083/1084
F555G
++
++

1085/1086
L211R
++
+

961/962
R830M

+

963/964
S839A
+
+

965/966
H822T
+
+

1087/1088
V64G
+
++

1089/1090
M320R

+

1091/1092
T533Y
++
++

1095/1096
F16V
+

1097/1098
I1283E
+++
+++

1099/1100
V536M
+
+

969/970
L14G
++
++

1101/1102
F312L

+

1103/1104
M681N
+++
++

1107/1108
V536R
+
+

1109/1110
R15G
+
+

1111/1112
V536P
++
++

973/974
M836V
+
+

1113/1114
M558W
++
++

1115/1116
M558E
+++
++

975/976
S839P
+
+

977/978
K1210W
+++
++

979/980
Y1281R
++
++

981/982
V64D
+++
++

985/986
V682R
+
+

987/988
L14M
+

989/990
L318C
+++
+++

1119/1120
S839G
++
+

1123/1124
L13G
++
+

1125/1126
K1210M
+
+

1127/1128
H1156G
+
+

1129/1130
M558L
+
+

1131/1132
S839K
++
+

1133/1134
L211P
++
+

1135/1136
L211G
++
+

1137/1138
L211Q
++
+

1139/1140
F16D
+++
++++

995/996
K818G
+
+

1141/1142
M320G
++
+

997/998
M836G
++
+

1143/1144
Y683W
+
+

1145/1146
Y24V
+

1147/1148
V534I
+
+

1149/1150
M681A
+++
++

1001/1002
M836R
+
+

1151/1152
P840G

+

1153/1154
F1160R
+++
+

1155/1156
M681C
+++
++

1157/1158
F328Q
++
++

1159/1160
R15S
++
+

1005/1006
T533V
+
+

1007/1008
S19G
++
+++

1161/1162
V536K
+
+

1163/1164
V536G
+
++

1165/1166
T533R
+
++

1167/1168
M681E
+++
++

1011/1012
Q817D
++
+

1013/1014
L831W
+
+++

1169/1170
V536W
++
++

1171/1172
H1156N
+
+

1173/1174
F16P
++
+++

1175/1176
Q817A
+
+

1177/1178
F18R
+
+

1179/1180
Y551W
+

1181/1182
F555T
++
++

1017/1018
H1156R

+

1183/1184
M681G
+++
++

1185/1186
H212A
+
+

1187/1188
F16G
++
++

1189/1190
S838G
+
+

1019/1020
R830V

++

1191/1192
M681R
+
+

1021/1022
V393G/A747D
++
+

1193/1194
V393G
++
+

1195/1196
V393G/A747D/K752G/Q811F/K819V/Q923M/
+++
+++

T1148N
++
+

1197/1198
V393G/S751Y/I775G

1023/1024
N753D
++

1199/1200
V63Q/V393G/K732W/I775G/T1148N
+++
++++

1201/1202
V393G/K732W/T1148N
+++
++

1203/1204
I775G/T1148N
+
+

1205/1206
V393G/K752L/N753D/I775G/I793G/M797W
+++
+

1207/1208
V393G/Q923M
++
+

1209/1210
V393G/Q923M/T1148N
+++
++

1211/1212
V393G/A747D/S751Y
++
+

1025/1026
V393G/K732W/S751Y
++
+

1213/1214
V393G/A747D/N753D/I775G/T1148N
+++
++

1215/1216
K752L/Q923M/M1051R
++
++

1217/1218
V63Q/I775G
+++
++

1219/1220
V63Q/V393G/K752L/I775G/I793G/M1051R/T1053R
++++
++++

1221/1222
K752G/N753D/I775G/I793G/K819V
+++
+

1027/1028
V63Q/M797W
+++
++

1223/1224
V63Q/M1051R
+++
++++

1225/1226
V63Q/A747D/K752G/N753D/I775G/Q811F/K819V/
++++
++++

Q923M/M1051R

1227/1228
V393G/K752G/I787W
+++
+

1231/1232
V63Q/T1148N
+++
+++

1233/1234
V63H/A747D/K752G/N753D/K819V/Q923M/
+++
++++

T1148N

1235/1236
T1148N
+
+

1237/1238
A747D/K752L/N753D/I793G/Q811F
++

1239/1240
V63Q
+++
++

1241/1242
M797W/T1148N
++
+

1243/1244
S751Y/K752L/N753D/M1051R
+++
++

1245/1246
V63Q/K732W/M1051R
+++
++++

1247/1248
K752G/M797W/S815I/Q923M/M1051R/T1148N
+++
++

1249/1250
A747D/N753D
++

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 840 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >50.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 840 and are defined as follows: “+” 1-3, “++” >3, “+++” >5, “++++” >8

TABLE 12.6

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 1200

SEQ ID

TIS reduction
IHC reduction

NO:
Amino Acid Differences
Relative to
Relative to

(nt/aa)
(Relative to SEQ ID NO: 1200)
SEQ ID NO: 1200
SEQ ID NO: 1200

1199/1200

1409/1410
V536P
++
++

1411/1412
L211R/V536K/L622D/M681A
+++
++++

1413/1414
V536K/M558L
++
++

1415/1416
L211R/V536R/F555G/P617G/W732K
+++
++++

1417/1418
L211R/M681R/W732K
+++
++

1419/1420
L211R/V536K/F555E
+++
+++

1421/1422
V536R
+
+

1423/1424
F555E
++
++

1425/1426
L211R/V536P
+++
++

1427/1428
L211R/H212P
++
+

1251/1252
M681A
+++
++

1429/1430
L211R/V536K
+++
++

1431/1432
L211R/V536K/P617G/M681A/W732K
+++
++++

1433/1434
L211R
++
+

1435/1436
L211R/V536P/M558L/P617G/M681A
++++
++++

1437/1438
F555G/W732K
++
++

1439/1440
V536P/F555E/M558L/P617G/W732K
+++
++++

1253/1254
L622D
+
+

1255/1256
P617G
+
+

1441/1442
V536K/P617G/M681A/W732K
+++
+++

1443/1444
V536K/F555G
+++
+++

1257/1258
M558L
+
+

1445/1446
V536P/L622D/M681A
+++
++++

1447/1448
W732K
+

1259/1260
V536K
+
+

1449/1450
H212A/M320Q/T533R/P617E/M681G
+++
++++

1451/1452
L211K/F312L/T533R/V534I/P617D/G619E/
++++
+++++

M681N/S839K

1453/1454
H212A
+
+

1455/1456
L211K/H212A/M320Q/G619E/M681G/S839K
+++
++++

1457/1458
M797W/S839K
++
+

1459/1460
H212A/F312L/M320Q/T533R/V534I/P617E/
+++
+++++

M681G

1461/1462
M558W/G619E/M681G
+++
++++

1261/1262
M681G
+++
++

1463/1464
M320Q/M558W/M681G
+++
++++

1465/1466
H212A/M320Q/V534I/G619E/M681G/M797W/
++++
++++

S839K

1467/1468
M320R/M558W/G619E/W732K/S839K
+++
++++

1469/1470
L211K/H212A/F312L/V534I/M558W/G619E/
++++
++++

M681N/W732K/S839K

1471/1472
M320R/V534I/M558R/G619E/W732K/M797W
+++
+++

1473/1474
M558W/P617D/G619E/M681N
+++
++++

1475/1476
L211Q/V534I/M558W/Q611E/M681N
++++
++++

1477/1478
L211K/M320R/P617E/G619E/M681G
+++
++++

1479/1480
M558W/P617D/M681G/M797W
+++
++++

1481/1482
M320R/V534I/G619E/W732K
++
++

1483/1484
L211K/G619E/M681N
+++
+++

1263/1264
L211Q
++
+

1485/1486
L211K/H212A/V534I/M558W/G619E/W732K/
++++
++++

S839K

1487/1488
H212A/M320R/G619E/M681N/M797W/S839K
+++
++++

1489/1490
H212A/M320R/Q611E/S839K
++
++++

1265/1266
M320R

+

1491/1492
M320Q/M558W/G619E/M681N
+++
++++

1493/1494
F312L/T533R/P617D/M681N/M797W/S839K
+++
++++

1495/1496
M558W/Q611E/M681N
+++
++++

1497/1498
F312L

+

1499/1500
L211Q/M320R/M558W/G619E/M681G/W732K/
++++
++++

M797W/S839K

1501/1502
M320Q/M558W/W732K/M797W
+++
++

1503/1504
T533R/M558W/Q611E/G619E/F716L/W732K/
+++
++++

S839K

1505/1506
F312L/M681G
++
++

1267/1268
M558W
++
++

1507/1508
M320Q/V534I/G619E/M681N
+++
+++

1509/1510
L211K/M320Q/T533R/V536L/G619E
+++
+++

1269/1270
S839K
++
+

1511/1512
M558W/P617E/M681N/S839K
++++
++++

1271/1272
L211K
++
+

1513/1514
H212A/M320R/V534I/M558W/M681G
+++
++++

1515/1516
T533R/V534I/M681N/S839K
+++
++++

1517/1518
H212A/V534I/G619E/M681G
+++
+++

1519/1520
P617E/G619E/M681G
+++
+++

1521/1522
L211Q/H212A/M320R/M558W/P617E/M681N
++++
++++

1523/1524
H212A/V534I/M558W/M681N/M797W
+++
++++

1525/1526
H212A/M320R/V534I
+
++

1527/1528
L211K/F312L/G619E
++
+

1529/1530
H212A/F312L/G619E/S839K
++
+++

1531/1532
T210P/H212A/M320R/M558W/M681G
++++
++++

1273/1274
M797W
+

1275/1276
T533R
+
++

1533/1534
H212A/F312L/V534I/M681N/M797W/S839K
+++
++++

1535/1536
L211K/M320R/M558W/S839K
+++
++++

1277/1278
S751A
+

1279/1280
A303T
+
+

1539/1540
S916L
+

1549/1550
T294G
+
+

1285/1286
K818G
+
+

1551/1552
K799S
+

1553/1554
K799E
+

1299/1300
Q921W
+

1563/1564
K799Q
+

1567/1568
S916M
+

1305/1306
L968A
+

1309/1310
G924W
+

1569/1570
R759S
+

1313/1314
F698D
+++
++

1315/1316
S168G
+

1319/1320
K818A
+
+

1321/1322
K818S
+
+

1323/1324
R581K
+

1325/1326
K818P
+
+

1327/1328
S916K
+
+

1329/1330
F698S
+++
++

1331/1332
A303G
+
+

1333/1334
S916C
++
++

1335/1336
T1428G
+
+

1343/1344
T311G
+

1351/1352
S751D
+

1355/1356
K818E
++
+

1367/1368
A1099G
+

1369/1370
K799G
+

1371/1372
S916I
+
+

1379/1380
K799P
+

1383/1384
S916E
+++
++

1583/1584
S916T
+

1385/1386
T676Q
+
+

1387/1388
R581V
+

1389/1390
L968P
++

1391/1392
A755Y
+

1393/1394
S916Q
++
+

1399/1400
F698Y
+
++

1405/1406
L1114A
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 1200 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >50.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 1200 and are defined as follows: “+” 1-3, “++” >3, “+++” >5, “++++” >8, “+++++” >15

TABLE 12.7

Reduction of Total Immunogenicity Score (TIS) and Immunogenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 1514

Amino Acid

SEQ ID
Differences
TIS reduction
IHC reduction

NO:
(Relative to
Relative to
Relative to

(nt/aa)
SEQ ID NO: 1514)
SEQ ID NO: 1514
SEQ ID NO: 1514

1513/1514

1609/1610
I215E
++++
+

2069/2070
L318G
++++
+

1611/1612
I215P
++++
+

2033/2034
V536K/F555G/G619E/Y737K/K799Q/E809G/
+++
+

S839K/S916T/Q921R

2003/2004
I1050E
+++
+

1593/1594
M1051H
+++
+

1595/1596
I1283H
+++
+

1597/1598
I1283T
+++
+

1613/1614
I215S
+++
+

1599/1600
Y1281Q
+++
+

1601/1602
Y1281P
+++
+

1603/1604
S916E
+++
+

1857/1858
E786D/R821Q
+++
+

1589/1590
R1042E
+++
+

1615/1616
L178C
+++
+

1869/1870
L178W
+++
+

1955/1956
V536P/F555E/G619E/W732K/K799S/Y805Q
+++
+

1963/1964
V536P/F555E/G619E/W732K/K799S
+++
+

1617/1618
I1050T
++
+

1619/1620
K1210T
++
+

1999/2000
N616D
++
+

2005/2006
I610H
++
+

2025/2026
Y1281S
++
+

1591/1592
Y1041N
++
+

1657/1658
Y175G
++
+

1971/1972
V536P/F555E/G619E
++
+

2007/2008
Q611D
++
+

2027/2028
A618E
++
+

1605/1606
M1051W
++
+

1627/1628
P615D
++
+

1825/1826
Y1104E
++

1989/1990
V536P/G619E/K799S/Y805Q
++
+

1621/1622
Y1281K
++
+

1853/1854
L1138Q
++

1973/1974
G619E/S839K
++
+

1985/1986
V536P/G619E/D801G
++
+

1993/1994
Y1281H
++
+

1623/1624
R1042A
++
+

1749/1750
L178Q
++
+

1797/1798
Y175A
++
+

1939/1940
K1093P
++

1629/1630
I610S
++
+

1659/1660
K1093W
++

1661/1662
F1088G
++

1663/1664
K1093T
++

2009/2010
R1042T
++
+

1607/1608
L187C
++
+

1631/1632
I610K
++
+

1633/1634
I610T
++
+

1675/1676
S700E
++
+

1697/1698
K1093V
++

1907/1908
K1093N
++

2023/2024
R1158N
++
+

1625/1626
V104S
++
+

1739/1740
D790P
++

1881/1882
L187G
++
+

1635/1636
V536P
++
+

1637/1638
A618Y
++
+

1645/1646
M110H
++
+

1733/1734
F1088S
++

1871/1872
Y1104C
++

1925/1926
D790G
++

1977/1978
V536P/D801G/Y805Q
++
+

2015/2016
T370A/L614A
++
+

2019/2020
K1210V
++
+

2021/2022
L614T
++
+

2065/2066
S839K
++
+

1639/1640
Q621W
+
+

1819/1820
Y175H
+
+

1877/1878
K1093S
+

1991/1992
F555E/G619E/W732K/K799S/D801G/Y805Q
+
+

2031/2032
V536P/G619E/E809G/E1109G
+
+

2037/2038
V536R/G619E/G681N/K799Q/S916T/E1109G
+
+

2049/2050
G619E/G681N/S916T
+
+

1693/1694
I756G
+

1777/1778
M1098G
+

2001/2002
Q621K
+
+

1669/1670
T535N
+
+

1723/1724
D790W
+

1771/1772
K1093G
+

1781/1782
I756T
+

1959/1960
F555E/G619E/K799S
+
+

1969/1970
A246D/G619E/K799S/D801G
+
+

1979/1980
F555E/G619E/K799S/Y805Q
+
+

2011/2012
N616Q
+
+

2041/2042
F555E/G619E/G681N/Y737K/K799Q
+
+

2051/2052
F555G/G619E/G681N/K799S
+
+

1653/1654
E786C
+

1805/1806
Q580S
+

1909/1910
A119G
+

1919/1920
H712Q
+

1923/1924
H712G
+

1957/1958
F555E/G619E/D801G
+
+

1983/1984
F555E/G619E
+
+

2063/2064
F555G/S916T
+

1705/1706
L178V
+
+

1713/1714
K1093A
+

1719/1720
E1452L
+

1745/1746
S304G
+
+

1761/1762
Y917G
+

1793/1794
S700A
+
+

1961/1962
G619E/K799S/D801G/Y805Q
+
+

1975/1976
G619E/K799S/Y805Q
+
+

1997/1998
L614H
+
+

2017/2018
H1156D
+
+

2057/2058
G619E/K799S
+
+

1641/1642
A618P
+
+

1689/1690
T535G
+
+

1753/1754
K752G
+

1755/1756
F1108W
+

1783/1784
M948S
+

1785/1786
A119V
+

1791/1792
M1113F
+

1795/1796
E786D
+

1931/1932
S1084N
+

1981/1982
G619E/D801G/Y805Q
+
+

2043/2044
G619E/G681N/K799S/S916T/E1109G
+
+

2045/2046
G619E/G681N
+
+

2059/2060
G619E
+
+

1643/1644
L614F
+
+

1727/1728
G292D
+
+

1747/1748
S1116G
+

1787/1788
S1116L
+

1813/1814
L1100S
+

1905/1906
K752E
+

2013/2014
L614N
+
+

1647/1648
R301K
+
+

1867/1868
S304E
+
+

1941/1942
K1093Y
+

2029/2030
F555E/G681N/Y737K/K799Q
+

1709/1710
I310G
+

1711/1712
S700Q
+
+

1759/1760
T281A
+

1809/1810
D731I
+

1879/1880
H712S
+

1929/1930
D731L
+

1949/1950
H712T
+

2053/2054
F555E
+

2055/2056
W732K
+

1649/1650
I1078S
+

1665/1666
L650M
+

1725/1726
D790I
+

1731/1732
F1108M
+

1751/1752
S1116A
+

1845/1846
T686W
+

1859/1860
T535C
+
+

1897/1898
D790A
+

1953/1954
K1093R
+

1965/1966
K799S/Y805Q
+

2039/2040
F555G/G619E/G681N/E807R/E1109G
+
+

2067/2068
K799Q
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 1514 and are defined as follows: “+” 1-10, “++” >10, “+++” >20, “++++” >30.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 1514 and are defined as follows: “+” 1-3, “++” >3, “+++” >5

TABLE 12.8

Reduction of Total Immunogenicity Score (TIS) and Immungenic Hit

Count (IHC) for FVIII Variants Relative to SEQ ID NO: 2072

Amino Acid

IHC

SEQ ID
Differences
TIS reduction (rel
reduction (Rel

NO:
(Relative to
to SEQ ID NO
to SEQ ID

(nt/aa)
SEQ ID NO: 2072)
2072 = 1998)
NO 2072)

2097/2098
A167G/L178Q/S700E/K752E/K1093G
+++
+++

2101/2102
A167G/S700E/E738Y/E739G/K1093G
++
++

2103/2104
A167G/S700E/E738Y/K752E/K1093G
+++
++

2105/2106
A167G/S700E/E738Y/K752E/Y805Q/D808S/E809G/
+++
++

K1093A/S1116I

2121/2122
E738Y/E739G/E809G/K1093G/S1116I
++

2137/2138
E738Y/K752E/K1093G
++

2141/2142
E738Y/K799S/E809G/K1093G
++

2189/2190
G681N/E738Y/E739G/K1093G
++

2203/2204
G681N/K752E/K1093G
++

2205/2206
G681N/S700E
++
++

2209/2210
G681N/S700E/E809G
++
++

2211/2212
G681N/S700E/K1093G
+++
++

2213/2214
G681N/S700E/K752E/D808S/E809G/K1093G/S1116I
+++
++

2215/2216
G681N/S700E/K752E/K1093G
+++
++

2219/2220
G681N/S700E/Y805Q/E809G
++
++

2225/2226
K1093G/S1116I
++

2227/2228
K752E
+

2233/2234
K752E/E800V/K1093G
+

2235/2236
K752E/E809G
+

2241/2242
K752E/S1116I
+

2249/2250
L178Q/G681N
++
+

2251/2252
L178Q/G681N/E738Y/S1116I
++
+

2253/2254
L178Q/K752E/E809G
++
+

2265/2266
S543P/E738Y/E809G/K1093G
++

2267/2268
S543P/E739G/K1093A
++

2271/2272
S543P/E809G/K1093G
++

2275/2276
S543P/G681N/E738Y/E739G/K752E/E809G/K1093G
++

2277/2278
S543P/G681N/E738Y/E739G/K752E/K1093G
++

2279/2280
S543P/G681N/E739G/K752E/E800V/Y805Q/E809G/
+

K1093G

2281/2282
S543P/G681N/S700E/E738Y/E739G/E800V/K1093G/
++
++

S1116I

2283/2284
S543P/G681N/S700E/E738Y/E739G/Y805Q/D808S/
++
++

E809G

2287/2288
S543P/G681N/S700E/K752E/E809G/K1093G
+++
++

2289/2290
S543P/K1093G
++

2301/2302
S543P/S700E/K752E/E800V/K1093G
++
++

2305/2306
S543P/S700E/K752E/K1093G
+++
++

2307/2308
S700E
++
++

2309/2310
S700E/E738Y/E739G/E800V/K1093G
++
++

2313/2314
S700E/E738Y/E800V/K1093G
++
++

2315/2316
S700E/E738Y/S1116I
++
++

2317/2318
S700E/E809G
++
++

2319/2320
S700E/E809G/K1093G
+++
++

2321/2322
S700E/K1093G
+++
++

2323/2324
S700E/K1093G/S1116I
+++
++

2325/2326
S700E/K752E
++
++

2329/2330
S700E/K752E/E809G/K1093G
+++
++

2333/2334
S700E/K752E/Y805Q/E809G/K1093A
+++
++

2335/2336
S700E/K799S/E800V
++
++

2337/2338
S700E/S1116I
++
++

2339/2340
S700E/Y805Q/D808S/E809G/E1076S
++
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2072 and are defined as follows: “+” 1-4, “++” 5-20, “+++” >20.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2072 and are defined as follows: “+” 4, “++” 5, “+++” >5

Example 13
Peptide T Cell Assay

In this Example, peptides were designed based on the wild-type B-domain deleted human factor VIII with or without mutations thought to alter the immunogenicity of the protein. Specifically, two polypeptides (SEQ ID NO: 2347 and SEQ ID NO: 2348) were prepared by Abzena Ltd. (Cambridge, United Kingdom). The sequence of the peptide of SEQ ID NO: 2347 has no amino acid changes compared to the sequence in SEQ ID NO: 2 while the sequence of the peptide of SEQ ID NO: 2348 contains a L→G substitution at amino acid position 12 of the peptide, which is equivalent to a L318G substitution relative to SEQ ID NO: 2. These peptides were analyzed for the presence of CD4⁺ T cell epitopes using EpiScreen™ T cell epitope matching technology, using peripheral blood mononuclear cells (PBMCs) from 50 healthy human donors. PBMCs were adjusted to a cell density of 2.5-3.0E6 cells/mL using AIM V© culture medium (Invitrogen, Paisley, UK). 5 μM of the peptides, or control medium, was added to the PBMCs in a flat bottomed 96-well plate. For each peptide-donor pair, 6 reactions were performed. Cultures were incubated for 6 days before adding 0.75 μCi of tritium-labeled thymidine (Perkin Elmer©, Beaconsfield, UK) to each well. Cultures were incubated another 18 hours before harvesting the cells onto filter mats using a TomTec Mach III cell harvester (Receptor Tech. Ltd., Warwick, UK). Counts per minute (CPM) for each well were determined Meltilex™ (Perkin Elmer®, Beaconsfield, UK) scintillation counting on a Microplate Beta Counter (Perkin Elmer®, Beaconsfield, UK) in paralux, low background counting mode. A Student's t-test comparing the response of the wells receiving peptide to the wells receiving medium was performed to determine the significance of the response (p<0.05). Positive responses were those whose mean CPM value of an individual peptide-donor pair divided by the average CPM value of the medium controls was above 1.90. The results for this experiment are shown in FIG. 1.

Similar to Example 1, directed evolution techniques were used to generate a gene variant containing the mutation tested using the Abzena, Ltd. Peptide T Cell Assay (SEQ ID NO: 2069 (nucleotide sequence); SEQ ID NO: 2070 (amino acid sequence)) derived from SEQ ID NO: 1514 within the same plasmid construct (See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103). This variant (SEQ ID NO: 2070) was produced using the methods described in Example 2 and tested for FVIII activity after no incubation and after incubation in Hemophilia A patient plasma (George King Bio-Medical, Inc.), as described in Example 3. The variant was tested using the chromogenic assay, as described in Example 3. The results are shown in FIG. 2.

Example 14
Activity of Truncated FVIII Variants Derived from SEQ ID NO: 2

Similar to Example 1, directed evolution techniques generally known by those skilled in the art were used to generate gene variants derived from SEQ ID NO: 2 containing truncated versions of factor VIII within the same plasmid construct (See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103). These variants were produced using the methods described in Example 2 and tested using the chromogenic assays, as described in Example 3. The results are shown in FIG. 3.

Example 15
Truncated FVIII Variants Derived from SEQ ID NO: 2

In this Example, experiments conducted to determine FVIII variant activity by assaying the protein activity are described. These variants were tested for FVIII activity using the chromogenic assay, as described in Example 3. Results are shown in Table 15.1 for variants expressed using Expi293F cells.

TABLE 15.1

Activity of FVIII Variants Relative to SEQ ID NO: 2

Amino Acid

SEQ ID
Differences
Expi293F Biophen

NO:
(Relative to
FIOP (Primary)

(nt/aa)
SEQ ID NO: 2)
Relative to SEQ ID NO: 2

3/4
T1148N
+

13/14
L171Q
+++

17/18
V63R
++

35/36
L1248S
++

67/68
L1248R
++

85/86
S332Q
++

95/96
L1248G
+++

153/154
S332W
+

157/158
V63H
++

185/186
S332P
++

193/194
L171P
+++

245/246
K732G
++

251/252
L1377D
++++

261/262
E306G
+++

265/266
L1377G
+++

267/268
S332F
+

269/270
L1377E
++++

281/282
L1377R
+++

283/284
L1377S
++++

287/288
L171M
+++

291/292
L1248D
++

293/294
E306S
+

297/298
L171H
+++

299/300
L1377A
+++

301/302
L1377V
+++

305/306
T1148R
++

2359/2360
E306T
+

2361/2362
E809L
+

2363/2364
E800L
+

2365/2366
F387A
+

2367/2368
Y738A
+

2369/2370
I499C
+

2371/2372
M681R
+

2373/2374
K1093E
+

2375/2376
T1148A
+

2377/2378
V63W
+

2379/2380
K752Q
+

2381/2382
I390E
+

2383/2384
V534M
+

2385/2386
L749P
+

2387/2388
K1093M
+

2389/2390
V1439S
+

2391/2392
S260K
+

2393/2394
M558V
+

2395/2396
S332N
+

2397/2398
I390T
+

2399/2400
Y738D
+

2401/2402
S700V
+

2403/2404
S1414P
+

2405/2406
K1093P
+

2407/2408
L749D
+

2409/2410
H212Y
+

2411/2412
M681C
+

2413/2414
Y805R
+

2415/2416
T1148S
+

2417/2418
E800R
+

2419/2420
S332V
+

2421/2422
L171K
+

2423/2424
E809Q
+

2425/2426
M681L
+

2427/2428
E800G
+

2429/2430
Y738T
+

2431/2432
M681E
+

2433/2434
K732C
+

2435/2436
L749C
+

2437/2438
S1414T
+

2439/2440
V63C
+

2441/2442
L171F
+

2443/2444
L318M
+

2445/2446
M681T
+

2447/2448
V534F
+

2449/2450
L750K
+

2451/2452
Y738N
+

2453/2454
K1093V
+

2455/2456
L749Q
+

2457/2458
H212F
+

2459/2460
H212M
+

2461/2462
I775S
+

2463/2464
F387W
+

2465/2466
I499L
+

2467/2468
F387R
+

2469/2470
T1148L
+

2471/2472
V1439T
+

2473/2474
E800M
+

2475/2476
L750R
+

2477/2478
V1439A
+

2479/2480
V63I
+

2481/2482
V393H
+

2483/2484
M558R
+

2485/2486
V63S
+

2487/2488
M558T
+

2489/2490
S332A
+

2491/2492
Y805C
+

2493/2494
L749F
+

2495/2496
H212A
+

2497/2498
L749A
+

2499/2500
K752L
+

2501/2502
L1248I
+

2503/2504
M681A
+

2505/2506
L171R
+

2507/2508
I775N
+

2509/2510
F387L
+

2511/2512
E809A
+

2513/2514
Y805G
+

2515/2516
K752R
+

2517/2518
M320I
+

2519/2520
V63E
+

2521/2522
Y805H
+

2523/2524
L749Y
+

2525/2526
S332G
+

2527/2528
Y805K
+

2529/2530
M558S
+

2531/2532
E800V
+

2533/2534
L1377M
+

2535/2536
E800W
+

2537/2538
E809C
+

2539/2540
Y738E
+

2541/2542
V1439H
+

2543/2544
L318V
+

2545/2546
E800Y
+

2547/2548
H212V
+

2549/2550
H212N
+

2551/2552
E800Q
+

2553/2554
H212K
+

2555/2556
L749M
+

2557/2558
S1414H
+

2559/2560
L1248C
++

2561/2562
L749K
++

2563/2564
M681Q
++

2565/2566
V393Y
++

2567/2568
Y738H
++

2569/2570
L750S
++

2571/2572
K732L
++

2573/2574
K752F
++

2575/2576
K1093S
++

2577/2578
L750T
++

2579/2580
E800I
++

2581/2582
E809R
++

2583/2584
S1414W
++

2585/2586
M558L
++

2587/2588
L318Q
++

2589/2590
V393Q
++

2591/2592
T1148E
++

2593/2594
L1248F
++

2595/2596
H212R
++

2597/2598
H212P
++

2599/2600
M681N
++

2601/2602
L749T
++

2603/2604
L171N
++

2605/2606
E800C
++

2607/2608
E809K
++

2609/2610
S260T
++

2611/2612
S332R
++

2613/2614
L750N
++

2615/2616
V534L
++

2617/2618
I390P
++

2619/2620
Y805S
++

2621/2622
M320Q
++

2623/2624
K732T
++

2625/2626
Y738S
++

2627/2628
V63K
++

2629/2630
E800N
++

2631/2632
L1248M
++

2633/2634
Y805L
++

2635/2636
E306A
++

2637/2638
S1414C
++

2639/2640
Y805P
++

2641/2642
H212L
++

2643/2644
L750G
++

2645/2646
K752M
++

2647/2648
S700E
++

2649/2650
S332Y
++

2651/2652
I390V
++

2653/2654
M681G
++

2655/2656
M558Q
++

2657/2658
V63F
++

2659/2660
E809W
++

2661/2662
M558E
++

2663/2664
M558G
++

2665/2666
L749I
++

2667/2668
K732Q
++

2669/2670
Y805M
++

2671/2672
I775K
++

2673/2674
F387T
++

2675/2676
L750Q
++

2677/2678
I390L
++

2679/2680
K732V
++

2681/2682
K752D
++

2683/2684
K752S
++

2685/2686
K732D
++

2687/2688
T1148C
++

2689/2690
M558P
++

2691/2692
E809I
++

2693/2694
L750W
++

2695/2696
S332H
++

2697/2698
E800P
++

2699/2700
K732R
++

2701/2702
L1248W
++

2703/2704
K752E
++

2705/2706
V63Y
++

2707/2708
V63T
++

2709/2710
L749G
++

2711/2712
I775G
++

2713/2714
K1093T
++

2715/2716
E306P
++

2717/2718
V393R
++

2719/2720
E809N
++

2721/2722
E809S
++

2723/2724
M681F
++

2725/2726
K732S
++

2727/2728
L750V
++

2729/2730
S1414A
++

2731/2732
E809T
++

2733/2734
K1093Q
++

2735/2736
L1248E
++

2737/2738
L171W
++

2739/2740
K732Y
++

2741/2742
L171T
++

2743/2744
Y805W
++

2745/2746
E809G
++

2747/2748
F387V
++

2749/2750
H212S
++

2751/2752
T1148M
++

2753/2754
I775P
++

2755/2756
K732M
++

2757/2758
V63L
++

2759/2760
L750P
++

2761/2762
V393F
++

2763/2764
M320P
++

2765/2766
V393G
++

2767/2768
S700T
++

2769/2770
K752V
++

2771/2772
E809V
++

2773/2774
K1093A
++

2775/2776
M320G
++

2777/2778
I390M
++

2779/2780
L1377I
++

2781/2782
M681W
++

2783/2784
S1414G
++

2785/2786
L750M
++

2787/2788
E800T
++

2789/2790
L318T
++

2791/2792
L171Y
++

2793/2794
M558W
++

2795/2796
Y805Q
++

2797/2798
M558A
++

2799/2800
M320S
++

2801/2802
L750D
++

2803/2804
I775V
++

2805/2806
S700A
++

2807/2808
L1377Y
++

2809/2810
M320L
+++

2811/2812
L318R
+++

2813/2814
V393A
+++

2815/2816
L171V
+++

2817/2818
V1439E
+++

2819/2820
L1248Y
+++

2821/2822
S332T
+++

2823/2824
L1248Q
+++

2825/2826
L1377C
+++

2827/2828
L1248H
+++

2829/2830
M681S
+++

2831/2832
I390G
+++

2833/2834
L171A
+++

2835/2836
L1248T
+++

2837/2838
V393M
+++

2839/2840
L171S
+++

2841/2842
E306D
+++

2843/2844
L1248A
+++

2845/2846
S1414E
+++

2847/2848
S1414D
+++

2849/2850
L1248K
+++

2851/2852
I390S
+++

2853/2854
L318K
+++

2855/2856
M320T
+++

2857/2858
L171D
+++

2859/2860
L171E
+++

2861/2862
M320V
+++

2863/2864
L1377P
+++

2865/2866
M320R
+++

2867/2868
L1377T
+++

2869/2870
L1377H
+++

All activities were determined relative to the reference polypeptide of SEQ ID NO: 2. Levels of increased activity are defined as follows: “+” 0.9 to 1.1, “++” >1.1, “+++” >1.5, “++++” >3.5

Active FVIII variants identified in screens from this Example were assessed for their levels of predicted immunogenicity by evaluating their binding to the eight common Class 11 HILA-DR alleles, as in Example 12. The total immunogenicity score and immunogenic hit count were calculated for each variant. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenic “hit count” (IHC) indicates the number of 15-mer analysis frames with an unusually high potential for immunogenicity (i.e., a higher score indicates a higher potential for immunogenicity). Mutations resulting in a reduced total immunogenicity score and/or an immunogenic hit count compared to the reference sequence were considered to be potential “deimmunizing mutations” and are shown in Table 15.2.

TABLE 15.2

Reduction of Total Immunogenicity Score (TIS) and Immungenic

Hit Count (IHC) for FVIII Variants Relative to SEQ ID NO: 2

Amino Acid

Differences
TIS reduction
IHC reduction

SEQ ID NO:
(Relative to
Relative to
Relative to

(nt/aa)
SEQ ID NO: 2)
SEQ ID NO: 2
SEQ ID NO: 2

2365/2366
F387A
+++
+

2369/2370
I499C
++

2371/2372
M681R
+
+

2373/2374
K1093E
++

2379/2380
K752Q
++
+

2381/2382
I390E
+++
+

2385/2386
L749P
++
+

2389/2390
V1439S
++
+

2391/2392
S260K
+
+

2393/2394
M558V
++
+

2395/2396
S332N
+
+

2397/2398
I390T
++
+

2401/2402
S700V

+

2405/2406
K1093P
++

2407/2408
L749D
+++
+

2411/2412
M681C
+++
+

2419/2420
S332V

+

2421/2422
L171K
+

2431/2432
M681E
+++
+

2435/2436
L749C
+++
+

2439/2440
V63C
+++
+

2445/2446
M681T
+++
+

2447/2448
V534F
+
+

2449/2450
L750K
+
+

2453/2454
K1093V
++

2455/2456
L749Q
++
+

2459/2460
H212M
+

2463/2464
F387W
+
+

2465/2466
1499L
+

2467/2468
F387R
++
+

2471/2472
V1439T
++
+

2475/2476
L750R
+

2477/2478
V1439A
+
+

2483/2484
M558R
++
+

2485/2486
V63S
+++
+

2487/2488
M558T
+++
+

2493/2494
L749F
+
+

2495/2496
H212A
+
+

2497/2498
L749A
+
+

2499/2500
K752L
+
+

2503/2504
M681A
+++
+

2505/2506
L171R
+

2509/2510
F387L
+
+

2517/2518
M320I
+
+

2519/2520
V63E
+++
+

2525/2526
S332G
+
+

2529/2530
M558S
++
+

2533/2534
L1377M
+
+

2541/2542
V1439H
+
+

2543/2544
L318V
++
+

2553/2554
H212K
+

2555/2556
L749M

+

2559/2560
L1248C
+
+

2561/2562
L749K
+
+

2563/2564
M681Q
+++
+

2569/2570
L750S
++
+

2573/2574
K752F
+
+

2575/2576
K1093S
+

2577/2578
L750T
+
+

2585/2586
M558L
+
+

2587/2588
L318Q
+++
+

2589/2590
V393Q
+

2591/2592
T1148E
+
+

2593/2594
L1248F
+
+

2597/2598
H212P
+
+

2599/2600
M681N
+++
+

2601/2602
L749T
++
+

2603/2604
L171N
++
+

2609/2610
S260T
+
+

2613/2614
L750N
++
+

2617/2618
I390P
+++
+

2621/2622
M320Q
+
+

2627/2628
V63K
++
+

2631/2632
L1248M
+
+

2637/2638
S1414C
+

2643/2644
L750G
+
+

2645/2646
K752M
+

2647/2648
S700E
++
+

2649/2650
S332Y
+
+

2651/2652
I390V
+
+

2653/2654
M681G
+++
+

2655/2656
M558Q
+++
+

2661/2662
M558E
+++
+

2663/2664
M558G
+++
+

2671/2672
I775K
+

2673/2674
F387T
+++
+

2675/2676
L750Q
+
+

2681/2682
K752D
++
+

2683/2684
K752S
+
+

2687/2688
T1148C
+
+

2689/2690
M558P
+++
+

2693/2694
L750W
+
+

2701/2702
L1248W

+

2703/2704
K752E
++
+

2707/2708
V63T
+++
+

2709/2710
L749G
+++
+

2711/2712
I775G
+

2713/2714
K1093T
++

2727/2728
L750V

+

2733/2734
K1093Q
++

2735/2736
L1248E
+
+

2737/2738
L171W
+

2741/2742
L171T
+

2747/2748
F387V
++
+

2749/2750
H212S
+
+

2753/2754
I775P
+

2759/2760
L750P
++
+

2761/2762
V393F
+

2763/2764
M320P
++
+

2765/2766
V393G
+++
+

2767/2768
S700T
+
+

2769/2770
K752V
++
+

2773/2774
K1093A
+

2775/2776
M320G
++
+

2777/2778
I390M
+

2779/2780
L1377I
+
+

2781/2782
M681W
+
+

2783/2784
S1414G
+

2789/2790
L318T
++

2793/2794
M558W
++
+

2797/2798
M558A
+++
+

2799/2800
M320S
+

2801/2802
L750D
+++
+

2803/2804
I775V
+

2805/2806
S700A
+
+

2807/2808
L1377Y
++
+

2809/2810
M320L
+
+

2817/2818
V1439E
+++
+

2819/2820
L1248Y
+
+

2823/2824
L1248Q
+
+

2825/2826
L1377C
++
+

2827/2828
L1248H
+
+

2829/2830
M681S
+++
+

2831/2832
I390G
+++
+

2833/2834
L171A
+
+

2835/2836
L1248T
+
+

2839/2840
L171S
+
+

2841/2842
E306D
+

2843/2844
L1248A
+
+

2847/2848
S1414D
+

2849/2850
L1248K
++
+

2851/2852
I390S
+
+

2853/2854
L318K
++
+

2855/2856
M320T
++
+

2857/2858
L171D
++
+

2859/2860
L171E
++
+

2861/2862
M320V
+
+

2863/2864
L1377P
++
+

2865/2866
M320R

+

2867/2868
L1377T
++
+

2869/2870
L1377H
++
+

TIS reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2 and are defined as follows: “+” 1-10, “++” >10, “+++” >20.

IHC reduction was measured as a reduction in the number of counts compared to the reference polypeptide of SEQ ID NO: 2 and are defined as follows: “+” 1-3, “++” >3, “+++” >5

Example 16
In Vitro Characterization of FVIII Variants

In this Example, experiments conducted to characterize FVIII variants produced as indicated herein are described.

Production of Factor VIII in Expi293F™ Cells or HepG2 cells

Expi293F™ (ThermoFisher Scientific) cells or HepG2 cells were transfected with pcDNA 3.1(+) or a CMV promoter and BGH-pA containing vectors encoding wild type FVIII with B-domain deleted, or variants, using the lipofection method with ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) or LIPOFECTAMINE® 3000 Reagent (ThermoFisher Scientific). Expi293F™ (ThermoFisher Scientific) cells were cultured in growth medium (Expi293™ Expression Medium; ThermoFisher Scientific). HepG2 cells were cultured in growth medium (EMEM with 10% fetal bovine serum, both from Corning). Expi293F™ cells or HepG2 cells were transfected with plasmid DNA and grown in suspension for 3-7 days. Conditioned media was then harvested, clarified by centrifugation and filtration, and stored at −80° C. until characterization.

Analysis of FVIII Variant Expression from Expi293F™ Cells or HepG2 Cells (ELISA)

FVIII variant expression in conditioned media was analyzed using human factor VIII ELISA kit (Ab272771, Abcam) according to the company's standard operating protocol. The colorimetric assay measures total human FVIII in the 0.0002 IU/ml-0.84 IU/ml range. FVIII variants above 0.84 IU/ml were diluted in tris-BSA buffer (A221406-RUO, Aniara Diagnostica) in a 96-well microtiter plate. The absorbance of all wells was measured at 450 nm using the EnVision Multimode Plate Reader. The results from this analysis are presented in FIG. 4 & FIG. 5, plotting mean±standard deviation of triplicates.

Analysis of FVIII Variant Activity (Chromogenic Assay)

FVIII variant activity was determined by measuring the release of paranitroaniline (pNa) from a synthetic peptide substrate. For an unchallenged assay, 25 μL of diluted or undiluted Expi293F or HepG2 conditioned media produced as described above were warmed to 37° C. and mixed with 25 μL of “Reagent 1” (BIOPHEN™ FVIII:C, Aniara) and 25 μL of “Reagent 2” (BIOPHEN™ FVIII:C, Aniara) in a 96-well, black, opaque bottom microtiter plate. The reactions were mixed briefly and incubated at 37° C. for 5 minutes. 25 μL of “Reagent 3” (BIOPHEN™ FVIII:C, Aniara) was then added, and the reactions were mixed briefly and incubated at 37° C. for 5 minutes prior to quenching with 25 μL pf 2% citric acid. Release of pNA was analyzed using an EnVision® 2105 Multimode Plate Reader (PerkinElmer) monitoring absorbance at 405 nm. “Reagent 1”, “Reagent 2”, and “Reagent 3” are components of a chromogenic assay (BIOPHEN™ FVIII:C) supplied by Aniara and used as recommended by the supplier. The results from this analysis are presented in FIG. 6 & FIG. 7 (before normalization) and FIG. 8 & FIG. 9 (after normalization using the protein concentration determined by ELISA above), plotting mean±standard deviation of triplicates.

Analysis of FVIII Variant Activity (Clotting Assay)

FVIII variant activity was determined by measuring the clotting rate of Hemophilia A patient plasma (George King Bio-Medical, Inc.) using the activated partial thromboplastin time assay. For an unchallenged assay, 25 μL of diluted or undiluted Expi293F or HepG2 conditioned media produced as described above were warmed to 37° C. and mixed with 30 μL of Hemophilia A patient plasma (George King Bio-Medical, Inc.) and 50 μL of aPTT-XL reagent (Pacific Hemostasis) in a 96-well, black, opaque bottom microtiter plate. The reactions were mixed briefly and incubated at 37° C. for 3 minutes. 10 μL of 200 μM calcium chloride (Sigma Aldrich) was then added, and the reactions were mixed briefly. Clotting was analyzed using an EnVision® 2105 Multimode Plate Reader (PerkinElmer) monitoring absorbance at 405 nm over time for 5-10 minutes. Analyses were performed using an R script to extrapolate the clot initiation time from the collected data points. The results from this analysis are presented in FIG. 10, plotting mean±standard deviation of triplicates.

Analysis of FVIII Variant Stability in Hemophilia A Patient Plasma

FVIII variants were challenged with Hemophilia A patient plasma (George King Bio-Medical, Inc.) to simulate the conditions that the variants may encounter within the bloodstream. First, 50 μL of Expi293F or HepG2 conditioned media produced as described above and 50 μL of Hemophilia A patient plasma (George King Bio-Medical, Inc.) were added to the wells of a 96-well round bottom microtiter plate. The plates were sealed with a PlateLoc® thermal microplate sealer (Agilent), and incubated at 37-55° C. for 1-96 h. The samples were then analyzed by the chromogenic assay described above. The results from this analysis are presented in FIG. 11, plotting mean±standard deviation of triplicates.

While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

	Number	Date	Country
	63297690	Jan 2022	US
	63478454	Jan 2023	US

RECOMBINANT FACTOR VIII VARIANTS

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