Albumin variants

Abstract

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

1 . A Method for preparing a variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof, comprising following steps:
 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 variant albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof having one or more substitutions corresponding to the substitutions in SEQ ID NO: 2 selected among:
 Q417A,C,D,E,F,G,H,I,K,L,M,N,P, R,S,T,V,W,Y; 
 H440A,C,D,E,F,G, I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 A490C,D,E, F,G,H,I,K,L,M,N,P, R,S,T,V,W,Y 
 E492A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 V493A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T.W,Y: 
 D494A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 E495A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 T496A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y; 
 P499A,C,D,E,F,G,H,I,K,L,M,N, Q,R,S,T,V,W,Y; 
 K500A,C,D,E,F,G,H,I, ,L,M,N, Q,R,S,T,V,W,Y; 
 E501A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 N503A,C,D,E,F,G,H,I,K,L,M, P,Q,R,S,T,V,W,Y; 
 A504C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 E505A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 T506A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S, V,W,Y; 
 H510A,C,D,F,G,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 H535A,C,D,F,G, I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 K536A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; 
 P537A,C,D,E,F,G,H,I,K,L,M,N, Q,R,S,T,V,W,Y; 
 K538A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; 
 T540A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S, ,V,W,Y; 
 K541A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; 
 E542A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 D550A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 K573A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; 
 K574A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; 
 Q580A,C,D,E,F,G,H,I, K, L,M,N,P,R,S,T,V,W,Y; 
 A581C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
 A582C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; or 
 G584A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 
   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 variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof; and   e. Recovering the variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof from the growth medium;   wherein the variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof, has an altered plasma half-life compared with the parent albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof.   
     
     
         2 . A variant of albumin, fragments thereof or fusion polypeptides comprising said variant albumin or a fragment thereof having altered plasma half-life compared with the parent albumin, fragment thereof or fusion polypeptide comprising said parent albumin or fragment thereof, comprising one or more substitutions in positions corresponding to the positions in SEQ ID NO:2 selected among: 573, 500, 550, 492, 580, 574, 417, 440, 464, 490, 493, 494, 495, 496, 499, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 575, 577, 578, 579, 581, 582 and 584, where the variant is not the variant consisting of SEQ ID NO: 2 with the substitution D494N, E501K, K541E, D550G,A, K573E or K574N. 
     
     
         3 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 2 , having a longer plasma half-life than the parent albumin. 
     
     
         4 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 3 , comprising one or more substitutions corresponding to the following substitutions in SEQ ID NO: 2: K573Y,W,P,H,F,V,I,T,N,S,G,M,C,A,E,Q,R,L,D. K573P+K574N+A577T+A578R+S579C+Q580K+A581D+G584A. K573P+A577E+A578S+Q580K+A582T, K573P+A578S+S579T+G584A, K573P+L575F+G584A, E492G, N503K, K500R N503H, D550E, K574N, Q580K and E492G+N503K, E492G+N503H. E492H+E501P+N503H+E505D+T506S+T540S+K541E, E492G+K573A, E492G+N503K+K573A, E492G+N503H, E492G+K573P, E492G+N503K+K573P, E492G+N503H+K573P. 
     
     
         5 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 2 , having a shorter plasma half-life than the parent albumin 
     
     
         6 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 5 , comprising one or more substitution(s) corresponding to the substitutions in SEQ ID NO: 2 selected among: K500E,G,D,A,S,C,P,H,F,N,W,T,M,Y,V,Q,L,I,R. D550N, H464Q, H510Q, H535Q, D494N,Q,A. E495Q,A, T496A, E492G+V493P, K541G,D, D494N+E495Q+T496A, E495Q+T496A, E492G+V493P+K538H+K541N+E542D, N503K,D P499A, K541A,N, D494E+Q417H, Q417A, E492T+N503D, A490D+E492T+V493L+E501P+N503D+A504E+E505K+T506F+K541D, K536A, P537A, E501A,Q, E492G+K538H+K541N+E542D. 
     
     
         7 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 1 , comprising one or more further alteration that generates thio group on the surface. 
     
     
         8 . The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to  claim 1 , wherein the sequence identity of the variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof to SEQ ID NO: 2 is more than 80%, preferably more than 90%, more preferred more than 95%, more preferred more than 96%, even more preferred more than 97%, more preferred more than 98% and most preferred more than 99%. The variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof according to the previous claims being a variant fragment of albumin or a fusion polypeptide comprising a variant fragment of albumin wherein the fragment is at least 20 amino acids, preferably at least 50 amino acids, preferably at least 100 amino acids, more preferred 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. 
     
     
         9 . A nucleic acid encoding the variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof of  claim 2 . 
     
     
         10 . A conjugate comprising the variant albumin or fragment thereof according to  claim 2  and a beneficial therapeutic moiety. 
     
     
         11 . An Associate comprising the variant albumin or fragment thereof according to  claim 2  and a beneficial therapeutic moiety. 
     
     
         12 . A fusion polypeptide comprising a variant albumin or fragment thereof according to  claim 2  and a fusion partner polypeptide. 
     
     
         13 . A composition comprising a variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof or a conjugate comprising said variant albumin, wherein the binding to FcRn is stronger than for the corresponding albumin or fragment thereof or fusion polypeptide comprising said albumin or fragment thereof or a conjugate comprising said albumin. 
     
     
         14 . The composition of  claim 13  where the binding to FcRn is stronger than the binding of HSA to FcRn. 
     
     
         15 . The composition of  claim 14 , wherein the binding coefficient of the variant of albumin, fragments thereof or fusion polypeptide comprising said variant albumin or fragment thereof or a conjugate comprising said variant albumin to FcRn is less than 0.9× KD for HSA, more preferred less than 0.5× KD for HSA, more preferred less than 0.1× KD for HSA, even more preferred less than 0.05× KD for HSA, even more preferred less than 0.02× KD for HSA and most preferred less than 0.01× KD for HSA. 
     
     
         16 . The composition according to  claim 12 , comprising a variant albumin or a fragment thereof according to  claim 1 , a conjugate of  claim 10 , an associate of  claim 11  or a fusion polypeptide of  claim 12 . 
     
     
         17 . The composition according to  claim 13 , comprising a variant albumin or a fragment thereof according to  claim 1 , further comprising a compound comprising a ABD and a pharmaceutically beneficial moiety. 
     
     
         18 . The composition according to  claim 13 , being a pharmaceutical composition.