ALBUMIN VARIANTS
The present invention relates to variants of a parent albumin; the variants comprise one or more mutations in Domain II of albumin which lead to a change in binding affinity to FcRn and/or a change in half-life compared to the “parent” albumin. The invention allows tailoring of binding affinity and/or half-life of an albumin to the requirements and desires of a user or application. The invention also relates to fragments and fusion polypeptide of the variant albumins, as well as to polynucleotides encoding the variants, nucleic acid constructs, vectors, host cells comprising the polynucleotides and methods of using said variants.
1 . A polypeptide which is a variant of a parent 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 one or more (several) alterations in Domain II of albumin selected from the group consisting of positions corresponding to positions 349, 342, 381, 345, 384, 198, 206, 340, 341, 343, 344, 352, 382, 348, and/or 383 in SEQ ID NO: 2, wherein the polypeptide does not consist of SEQ ID NO: 2 with alteration E382K.
2 . The polypeptide according to claim 1 wherein the alteration at the position corresponding to position 349, 342, 381, 345, 384, 198, 206, 340, 341, 343, 344, 352, 382, 348, and/or 383 is a substitution.
3 . The polypeptide according to claim 2 wherein the substitution at the position corresponding to position 349 is to F, W Y H P, K or Q.
4 . The polypeptide according to claim 2 wherein the substitution at the position corresponding to position 342 is to Y, W, F, H, T, N, Q, A, C, I, L, P, V.
5 . The polypeptide according to claim 2 wherein the substitution at the position corresponding to position 381 is to G or A.
6 . The polypeptide according to claim 2 wherein the substitution at the position corresponding to position 345 is to E, H, I or Q.
7 . The polypeptide according to claim 1 further comprising a substitution at the position corresponding to position 573.
8 . The polypeptide according to claim 7 wherein the substitution at the position corresponding to position 573 is to P, Y or W.
9 . The polypeptide according to claim 1 further comprising a substitution at the position corresponding to position 83.
10 . The polypeptide according to claim 9 wherein the substitution at the position corresponding to position 83 is to N, K or S.
11 . The polypeptide according to claim 1 comprising substitutions at positions corresponding to positions 83, 342 and 573 (or equivalent position of other albumins or variants or fragments thereof), preferably T83N, K or S+S342Y, W, F, H, T, N, or Q, A, C, I, L, P, V or Y+K573P, Y or W, more preferably T83N+S342Y+K573P (SEQ ID NO: 103).
12 . The polypeptide according to claim 1 comprising substitutions at positions corresponding to positions 83, 349 and 573 of SEQ ID NO: 2 (or equivalent position of other albumins or variants or fragments thereof), preferably T83N, K or S+L349F, W, Y, H, P, K or Q+K573P, Y or W, more preferably T83N+L349F+K573P (SEQ ID NO: 104).
13 . The polypeptide according to claim 1 comprising substitutions at positions corresponding to positions 83, 381, and 573 of SEQ ID NO: 2 (or equivalent position of other albumins or variants or fragments thereof), preferably T83N, K or S+V381, G or A+K573P, Y or W, more preferably T83N+V381G+K573P (SEQ ID NO: 105).
14 . The polypeptide according to claim 1 wherein the parent albumin or 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.
15 . 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.
16 . The polypeptide according to claim 1 , wherein the sequence identity of the polypeptide to SEQ ID NO: 2 is more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%.
17 . A fusion polypeptide comprising a polypeptide of claim 1 and a fusion partner polypeptide selected from a therapeutic, prophylactic, diagnostic, imaging or other beneficial polypeptide.
18 . 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 one or more (several) alterations in Domain II of albumin;
(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.
19 . The method according to claim 18 wherein the one or more (several) alterations in Domain II of albumin are selected from the positions selected from the group consisting of positions corresponding to 349, 342, 381, 345, 384, 198, 206, 340, 341, 343, 344, 352, 382, 348, and/or 383 in SEQ ID NO: 2, wherein the polypeptide does not consist of SEQ ID NO: 2 with alteration E382K.
20 . The method according to claim 19 wherein the alteration at the position corresponding to position 349, 342, 381, 345, 384, 198, 206, 340, 341, 343, 344, 352, 382, 348, and/or 383 is a substitution.
21 . The method according to claim 20 wherein the substitution at the position corresponding to position 349 is to F, W Y H P, K or Q.
22 . The method according to claim 20 , wherein the substitution at the position corresponding to position 342 is to Y, W, F, H, T, N, Q, A, C, I, L, P, V, preferably Y.
23 . The method according to claim 20 wherein the substitution at the position corresponding to position 381 is to G or A.
24 . The method according to claim 20 wherein the substitution at the position corresponding to position 345 is to E, H, I or Q.
25 . The method according to claim 19 further comprising a substitution at the position corresponding to position 573.
26 . The method according to claim 25 wherein the substitution at the position corresponding to position 573 is to P, Y or W.
27 - 50 . (canceled)