| 主权项 |
1. A method for preparing a polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or fragment thereof having a binding affinity to FcRn which is altered compared to the binding affinity of a reference albumin comprising human serum albumin (SEQ ID No: 2), fragment or fusion thereof to FcRn, comprising:
(a) providing a nucleic acid encoding a parent albumin having at least 80% sequence identity to SEQ ID NO: 2; (b) modifying the sequence of step (a), to encode a polypeptide which is a variant albumin, fragment thereof or fusion polypeptide comprising said variant albumin or fragment thereof having one or more alterations at one or more position corresponding to positions in SEQ ID NO: 2 selected from: (a) any of 75 to 91 (preferably 83); (b) any of 492 to 538; (c) 505, 531, 524, 472, 108, 190, 197 and 425; (d) any of 186 to 201; (e) any of 457 to 472; (f) any of 414 to 426; (g) any of 104 to 120; (h) any of 144 to 150; (i) any of 30 to 41, (j) any of 550 to 585, (k) any of 276, 410 and 414 with one or more of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; wherein when the polypeptide comprises one or more alterations selected from: (v) the group consisting of positions 417, 464, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 550, 573, 574, 575, 577, 578, 579, 580, 581, 582 and 584; (vi) insertion of or substitution with a Cys at one or more (several) of the group consisting of positions 34, 38, 40, 75, 76, 80, 82, 83, 86, 90, 104, 113, 115, 116, 471, 496, 498, 501, 503, 504, 505, 506, 512, 538, 550, 560, 562, 564, 565, 573, 574, 577, 578, 579, 580, 581, 582, 584, 585; (vii) alteration at one or more (several) of positions 91, 200, 461, 514, 558, 559, 567 to break a disulphide bond, and/or (viii) D63N, E82K, E84K, D87N, L90P, K106E, R114G, E119K, V146E, H464A, H464N, C201F, D494N, E501K, E503K, E505K, H510A, 1513N, D518N, K525E, E529K, V533M, H535A, K536E, I537N, D550G, D550A, V557M, K560E, D563N, E565K, E570K, K573E, K574N, K574E, K584E;
the polypeptide also comprises one or more alterations at a position selected from group consisting of positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 144, 145, 146, 147, 148, 149, 150, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 276, 410, 411, 414, 415, 416, 418, 419, 420, 421, 422, 423, 424, 425, 426, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 469, 470, 471, 472, 497, 498, 502, 507, 508, 509, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 567, 568, 569, 570, 571, 572, 576, 583, 585 and/or a stop codon inserted or substituted at a position selected from 497 to 585; (c) introducing the modified sequence of step (b) in a suitable host cell; (d) growing the cells in a suitable growth medium under condition leading to expression of the polypeptide; and (e) recovering the polypeptide from the growth medium;
wherein the polypeptide has an altered binding affinity to FcRn and/or an altered plasma half-life compared with the half-life of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof. |
