Albumin variants

Abstract

The present invention relates to variants of a parent albumin, the variants having altered plasma half-life compared with the parent albumin. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

What is claimed is: 
     
         1 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having an altered binding affinity to FcRn compared with the binding affinity of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn, wherein the polypeptide comprises alterations at two or more positions selected from positions corresponding to positions (a) 492 and 580; (b) 492 and 574; (c) 492 and 550; (d) 550 and 573; (e) 550 and 574; (f) 550 and 580 in SEQ ID NO: 2. 
     
     
         2 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having an altered binding affinity to FcRn compared with the binding affinity of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn, wherein:
 (a) the polypeptide comprises alterations at two or more positions, in which at a position corresponding to position 492 of SEQ ID NO: 2 there is an alteration to generate at a position corresponding to position an amino acid from the group consisting of A, C, D, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, preferably D and at a position corresponding to position 573 of SEQ ID NO: 2 there is an alteration to generate at a position corresponding to position to an amino acid from the group consisting of C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or H, or   (b) at a position corresponding to position 492 of SEQ ID NO: 2 there is an alteration to generate G and at a position corresponding to position 573 there is an alteration to generate A or P and the polypeptide comprises an additional alteration at a position selected from the group consisting of 550, 574 and 580.   
     
     
         3 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having an altered binding affinity to FcRn compared with the binding affinity of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn, wherein:
 (a) the polypeptide comprises alterations at two or more positions, in which at a position corresponding to position 573 of SEQ ID NO: 2 there is an alteration to generate an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or H and at a position corresponding to position 574 of SEQ ID NO: 2 there is an alteration to generate an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S or Y, more preferably H, D, F or G, most preferably H, or   (b) at a position corresponding to position 573 of SEQ ID NO: 2 there is a P and at a position corresponding to position 574 of SEQ ID NO: 2 there is an N and the polypeptide comprises an additional alteration at a position selected from the group consisting of 492, 550, and 580.   
     
     
         4 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having an altered binding affinity to FcRn compared with the binding affinity of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn, wherein
 (a) the polypeptide comprises alterations at two or more positions, in which at a position corresponding to position 573 of SEQ ID NO: 2 there is an alteration to generate an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y, preferably Y, W or H and at a position corresponding to position 580 of SEQ ID NO: 2 there is an alteration to generate an amino acid from the group consisting of C, D, E, F, G, H, I, L, M, N, P, R, S, T, V, W, Y, or   (b) at a position corresponding to position 573 of SEQ ID NO: 2 there is an alteration to generate a P and at a position corresponding to position 580 of SEQ ID NO: 2 there is an alteration to generate a K and the polypeptide comprises an additional alteration at a position selected from the group consisting of 492, 550, and 574.   
     
     
         5 . A polypeptide which is a variant of albumin, fragment thereof or fusion polypeptide comprising said variant albumin or a fragment thereof having an altered binding affinity to FcRn compared with the binding affinity of a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn, wherein:
 (a) the polypeptide comprises alterations at two or more positions, in which at a position corresponding to position 574 of SEQ ID NO: 2 there is an alteration to generate an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y, H, D, F, G, N, S or Y, more preferably H, D, F or G, most preferably H and at a position corresponding to position 580 of SEQ ID NO: 2 there is an alteration to generate at a position corresponding to position to an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V, W, Y, or   (b) at a position corresponding to position 574 of SEQ ID NO: 2 there is an alteration to generate an N and at a position corresponding to position 580 of SEQ ID NO: 2 there is an alteration to generate a K and the polypeptide comprises an additional alteration at a position selected from the group consisting of 492, 550, and 573.   
     
     
         6 . The polypeptide according to  claim 1 , wherein the polypeptide comprises alterations at three or more positions selected from positions corresponding to positions 492, 550, 573, 574 and 580 in SEQ ID NO: 2. 
     
     
         7 . The polypeptide of  claim 1  wherein the reference albumin is HSA (SEQ ID No: 2) or a fragment thereof, or a fusion polypeptide comprising HSA or a fragment thereof, most preferably SEQ ID NO: 2. 
     
     
         8 . The polypeptide according to  claim 1  having a stronger binding affinity to FcRn and/or longer plasma half-life than a parent albumin, reference albumin, fragment thereof or fusion polypeptide comprising said parent albumin, reference albumin or fragment or fusion thereof. 
     
     
         9 . The polypeptide according  claim 1 , wherein the sequence identity of the polypeptide 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%. 
     
     
         10 . A fusion polypeptide comprising a polypeptide according to  claim 1  and a fusion partner polypeptide selected from a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         11 . 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, 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 comprising alterations at two or more positions corresponding to positions selected from 492, 550, 573, 574 and 580 in SEQ ID NO: 2, preferably as described in  claim 1 ;   (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.   
     
     
         12 . The method of  claim 11  wherein the reference albumin is HSA (SEQ ID No: 2) or a fragment thereof, or a fusion polypeptide comprising HSA or a fragment thereof, most preferably SEQ ID NO: 2. 
     
     
         13 . The method according to  claim 11 , wherein the sequence identity of the polypeptide 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%. 
     
     
         14 . A conjugate comprising a polypeptide according to  claim 1  and a conjugation partner. 
     
     
         15 . An associate comprising a polypeptide according to  claim 1  or obtainable by a method according to  claim 11  and a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         16 . A nanoparticle or microparticle comprising a polypeptide according to  claim 1 . 
     
     
         17 . A composition comprising a polypeptide according to  claim 1  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 corresponding parent albumin, reference albumin, fragment thereof or fusion polypeptide, conjugate, associate or nanoparticle or microparticle comprising said parent albumin, reference albumin or fragment or fusion thereof to FcRn. 
     
     
         18 . A composition according to  claim 17  where the binding affinity of the polypeptide, fusion polypeptide, conjugate, associate or nanoparticle or microparticle to FcRn is stronger than the binding affinity of HSA to FcRn. 
     
     
         19 . A composition according to  claim 17 , wherein the binding coefficient of the variant of the polypeptide, fusion polypeptide, conjugate, associate or nanoparticle or microparticle to FcRn is less than 0.9×KD of HSA to FcRn, more preferred less than 0.5×KD of HSA to FcRn, more preferred less than 0.1×KD of HSA to FcRn, even more preferred less than 0.05×KD of HSA to FcRn, even more preferred less than 0.02×KD of HSA to FcRn and most preferred less than 0.01×KD of HSA to FcRn. 
     
     
         20 . The composition according to  claim 17 , further comprising a compound comprising an antibody binding domain (ABD) and a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         21 . The composition according to  claim 17 , comprising a pharmaceutically acceptable carrier or excipient. 
     
     
         22 . 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;   (b) where the molecule is not a polypeptide, conjugating the molecule to a polypeptide according to claim.   
     
     
         23 . A method according to  claim 22  wherein the molecule is a therapeutic, prophylactic, diagnostic, imaging or other beneficial moiety. 
     
     
         24 . A nucleic acid encoding the polypeptide or fusion polypeptide of  claim 1 . 
     
     
         25 . A vector comprising a nucleic acid according to  claim 24 . 
     
     
         26 . A host cell comprising a nucleic acid according to  claim 24 . 
     
     
         27 . A method of prophylaxis, treatment or diagnosis comprising administering a polypeptide according to  claim 1  to a subject.