Albumin variants and conjugates
Based on the three-dimensional structure of albumin, the inventors have designed variant polypeptides (muteins) which have one or more cysteine residues with a free thiol group (hereinafter referred to as “thio-albumin”). The variant polypeptide may be conjugated through the sulphur atom of the cysteine residue to a conjugation partner such as a bioactive compound.
1. A conjugation competent polypeptide comprising up to and including five conjugation competent cysteine residues,
a) wherein said polypeptide comprises an albumin sequence having at least 96% sequence identity to SEQ ID NO: 1; and
b) wherein said polypeptide is a mutant comprising a Cysteine at or immediately following an amino acid residue in the albumin sequence which corresponds to the second, third, fourth or fifth residue relative to the N- or C-terminus of the albumin sequence; and/or
c) wherein the polypeptide is a mutant comprising a Cysteine at or adjacent to an amino acid residue in the albumin sequence which:
i) has a solvent accessibility of at least 80%, is not located within secondary structure, has a B-factor of a C-alpha atom of at least 50 as defined by x-ray crystallography and has no polymorphism at this position that is known to cause thermal instability; or
ii) has a solvent accessibility of at least 80%, is located within secondary structure, has a B-factor of a C-alpha atom of at least 30 as defined by x-ray crystallography and has no polymorphism at this position is known to cause thermal instability; and
d) optionally, wherein the polypeptide comprises additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, or insertion;
with the proviso that, where there are two or fewer conjugation competent cysteine residues within the polypeptide, such polypeptide does not include a cysteine at the position corresponding to position 1 of SEQ ID NO: 1.
2. A polypeptide according to claim 1 comprising conjugation competent cysteine residues at positions equivalent to positions 2 and 585 of SEQ ID NO: 1.
3. A polypeptide according to claim 1 comprising four or more conjugation competent cysteine residues.
4. A polypeptide according to claim 3 comprising:
conjugation competent cysteine residues at positions equivalent to positions 2 and 585 of SEQ ID NO: 1; and
a conjugation competent cysteine residue at one or both of positions equivalent to positions 364 and 562 of SEQ ID NO: 1.
5. A polypeptide according to claim 1 in which the polypeptide comprises one or more of:
a) substitution of an amino acid, other than cysteine, with a cysteine at a position corresponding to a position equivalent to any of residues L585, A2, D562, A364, A504, E505, E86, D129, D549, A581, D121, E82, Q397and A578 of SEQ ID No. 1;
b) insertion of a cysteine at a position adjacent the N- or C- side of an amino acid corresponding to a position equivalent to any of residues L585, A2, D562, A364, A504, E505, E86, D129, D549, A581, D121, E82, Q397 and A578 of SEQ ID No. 1; and
c) addition of a cysteine to the N- side of the N-terminal residue of an albumin sequence or to the C- side of the C-terminal residue of an albumin sequence; such that the net result of the substitution, deletion, addition or insertion events of a), b) and c) is that the number of conjugation competent cysteine residues of the polypeptide sequence is increased relative to the polypeptide prior to the substitution, insertion, deletion and addition events.
6. The polypeptide according to claim 1 , wherein at least one cysteine residue has been substituted with a different amino acid residue.
7. A polypeptide according to claim 1 in which one or more of the conjugation competent cysteines is conjugated to a bioactive molecule.
8. A polypeptide according to claim 1 in which the polypeptide has at least 98% sequence identity to SEQ ID NO: 1.
9. A polypeptide according to claim 1 in which the polypeptide further comprises a further linker to which a bioactive compound may be linked.
10. A polypeptide according to claim 1 in which the polypeptide has at least 95% 96% sequence identity to SEQ ID NO: 1 and comprises conjugation competent cysteine residues located at: (a) A2+L585, (b) A2+A364+D562+L585C, (c) A2 and adjacent the C-side of the C-terminus of the albumin (d) T79+A364; (f) T79+D562+A364; (g) D562+A364+D1; (h) T79+D562+A364+A504; (i) T79+D562+A364+L585; (j) T79+D562+A364+D1; (k) T79+D562+A364+L585+D1; (1) E86+D562+A364+A504+A2; (m) $270+A581; (n) $270+D129; (o) $270+A581+E82; (p) $270+A581+D129; (q) $270+A581+E82+D129; (r) $270+A581+E82 +D129+Q397; (s) C369+C177; (t) A364+A581; (u) T79+A364+A581; (v) A364+A581+D129; (w) A364+C177; (x) D562+C369; (y) D129+C369; (z) A581+C369; or (ai) D562+D129+C369.
11. The polypeptide according to claim 1 in which the polypeptide has at least 97% sequence identity to SEQ ID NO: 1.
12. The polypeptide according to claim 1 in which the polypeptide has at least 98% sequence identity to SEQ ID NO: 1.
13. The polypeptide according to claim 1 in which the polypeptide has at least 99% sequence identity to SEQ ID NO: 1.
14. A conjugate which comprises a bioactive compound and a polypeptide according to claim 1 , wherein the bioactive compound is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide.
15. A conjugate which comprises a bioactive compound and a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to residues 1 to 585 of SEQ ID NO: 1 in which:
a) at a position equivalent to position 34 of SEQ ID NO: 1, there is a conjugation competent cysteine residue; and
b) within those residues corresponding to positions 1-585 of SEQ ID NO: 1 there are two or more conjugation competent cysteine residues, wherein the bioactive compound is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide.
16. A conjugate which comprises a bioactive compound and a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to residues 1 to 585 of SEQ ID NO: 1 in which:
a) at a position equivalent to position 34 of SEQ ID NO: 1, there is not a conjugation competent cysteine residue; and
b) elsewhere in the polypeptide there are three or more conjugation competent cysteine residues, wherein the bioactive compound is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide.
17. A conjugation competent polypeptide comprising an amino acid sequence which has at least 98% sequence identity to residues 1 to 585 of SEQ ID NO: 1 in which:
a) at a position equivalent to position 34 of SEQ ID NO: 1, there is a conjugation competent cysteine residue; and
b) within those residues corresponding to positions 1-585 of SEQ ID NO: 1 there are two or more conjugation competent cysteine residues.
18. A conjugation competent polypeptide comprising an amino acid sequence which is at least 98% identical to residues 1 to 585 of SEQ ID NO: 1in which:
a) at a position equivalent to position 34 of SEQ ID NO: 1, there is not a conjugation competent cysteine residue; and
b) elsewhere in the polypeptide there are three or more conjugation competent cysteine residues.
19. A polynucleotide which encodes the polypeptide of claim 1 .
20. A plasmid comprising the polynucleotide of claim 19 .
21. A host cell comprising a polynucleotide of claim 19 .
22. The host cell of claim 21 , which is a yeast cell.
23. A method of producing a polypeptide of claim 1 , comprising:
culturing the host cell of claim 21 under conditions that allow expression of the polypeptide; and
recovering the polypeptide from the host cell and/or from host cell growth medium.