Albumin variants

Abstract

The invention relates to variants of a parent albumin having altered plasma half-life compared with the parent albumin. The invention also relates to fusion polypeptides and conjugates comprising said variant albumin.

What is claimed is: 
     
         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. 
   
     
     
         2 . The method of  claim 1 , wherein the alteration is a substitution. 
     
     
         3 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having at least 90% sequence identity to human serum albumin (SEQ ID NO: 2) and an altered binding affinity to FcRn compared with the binding affinity of human serum albumin (SEQ ID No: 2), fragment thereof or fusion polypeptide comprising human serum albumin (SEQ ID No: 2), or fragment or fusion thereof to FcRn, the polypeptide comprising one or more alterations at one or more position corresponding to positions in SEQ ID NO: 2 selected from: (a) 75 to 91 (preferably 83); (b) 492 to 538; (c) 505, 531, 524, 472, 108, 190, 197 and 425; (d) 186 to 201; (e) 457 to 472; (f) 414 to 426; (g) 104 to 120; (h) 144 to 150; (i) 30 to 41; (j) 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) the group consisting of positions D63N, E82K, E84K, D87N, L90P, K106E, R114G, E119K, V146E, H464A, H464N, C201F, D494N, E501K, E503K, E505K, H510A, I513N, 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. 
     
     
         4 . The polypeptide according to  claim 3  wherein the alteration is a substitution. 
     
     
         5 . The polypeptide according to  claim 4 , having a stronger binding affinity to FcRn and/or longer plasma half-life than SEQ ID NO: 2, fragment thereof or fusion polypeptide comprising SEQ ID NO: 2. 
     
     
         6 . The polypeptide according to  claim 3 , comprising one or more further alterations that provides a conjugatable thiol group on the polypeptide. 
     
     
         7 . The polypeptide according to  claim 5 , wherein the fragment is at least 200 amino acids, more preferred at least 300 amino acids, more preferred at least 400 amino acids and most preferred at least 500 amino acids. 
     
     
         8 . A fusion polypeptide comprising a polypeptide according to  claim 3  and a fusion partner polypeptide. 
     
     
         9 . A conjugate comprising a polypeptide according to  claim 3  and a conjugation partner. 
     
     
         10 . An associate comprising a polypeptide according to  claim 3  and a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         11 . A nanoparticle or microparticle comprising a polypeptide according to  claim 3 , a fusion polypeptide according to  claim 8 , a conjugate according to  claim 9  or an associate according to  claim 10 . 
     
     
         12 . A composition comprising a polypeptide, fusion polypeptide, conjugate, associate or nanoparticle or microparticle according to  claim 3 , wherein the binding affinity of the polypeptide, fusion polypeptide, conjugate, associate or nanoparticle or microparticle to FcRn is stronger than the binding affinity of a composition comprising the human serum albumin (SEQ ID NO: 2), fragment thereof or fusion polypeptide, conjugate, associate or nanoparticle or microparticle comprising human serum albumin (SEQ ID NO: 2) or fragment or fusion thereof to FcRn. 
     
     
         13 . The composition according to  claim 12 , comprising a polypeptide or fusion polypeptide according to  claim 3 , a conjugate according to  claim 9 , an associate according to  claim 10  or a nanoparticle or microparticle according to  claim 11 , further comprising a compound comprising an antibody binding domain (ABD) and a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         14 . The composition according to  claim 13 , comprising a pharmaceutically acceptable carrier. 
     
     
         15 . A method of using of a polypeptide or fusion polypeptide according to  claim 3 , a conjugate according to  claim 9 , an associate according to  claim 10  or a nanoparticle or microparticle according to  claim 11  or a composition according to  claim 12  to increase the binding affinity to FcRn or half-life, preferably in plasma, of a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety relative to the binding affinity of a reference comprising or consisting of HSA (SEQ ID NO: 2) or a fragment, fusion, conjugate, associate, nanoparticle or microparticle thereof to FcRn. 
     
     
         16 . A method for altering the binding affinity to FcRn or half-life preferably in plasma, of a molecule comprising:
 (a) where the molecule is a polypeptide, fusing or conjugating the molecule to a polypeptide according to  claim 3  or to a conjugate according to  claim 9 ; associating the molecule to a polypeptide according to  claim 3  or to a conjugate according to  claim 9 ; incorporating the molecule in an associate according to  claim 10 , in nanoparticle or microparticle according to  claim 11  or a composition according to  claim 12 ;   (b) where the molecule is not a polypeptide, conjugating the molecule to a polypeptide according to  claim 3  or to a conjugate according to  claim 9 ; associating the molecule to a polypeptide according to  claim 3  or to a conjugate according to  claim 9 ; incorporating the molecule in an associate according to  claim 10 , in nanoparticle or microparticle according to  claim 11  or a composition according to  claim 12 .   
     
     
         17 . A nucleic acid encoding the polypeptide or fusion polypeptide of  claim 3 . 
     
     
         18 . A vector comprising a nucleic acid according to  claim 17 . 
     
     
         19 . A host cell comprising a nucleic acid according to  claim 17 . 
     
     
         20 . A host cell according to  claim 19  wherein the host cell is a eukaryote, preferably a yeast (such as  Saccharomyces cerevisiae ) or a mammalian cell (such as CHO or HEK) or a plant cell (such as rice). 
     
     
         21 . A method of prophylaxis, treatment or diagnosis comprising administering a polypeptide, fusion polypeptide, conjugate, composition, associate, nanoparticle or microparticle or polynucleotide according to  claim 3  to a subject.