Mutant interleukin-2 (IL-2) polypeptides
The present invention relates to IL-2 mutants with increased affinity for the IL-2 alpha-receptor subunit (IL-2Rα). The invention thus includes IL-2 mutants with improved biological potency. The invention also includes methods for directed evolution of IL-2α using yeast surface display to generate mutants with increased affinity for IL-2Rα.
1. A mutant interleukin-2 (IL-2) polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2 and further comprising one or more amino acid substitutions at positions 69, 74, and 128 of SEQ ID NO: 2, wherein the polypeptide binds an IL-2 receptor alpha subunit (IL-2Rα) with an affinity that is greater than the affinity with which the polypeptide represented by SEQ ID NO: 3 binds to IL-2Rα.
2. The mutant IL-2 polypeptide of claim 1 , wherein amino acid residue 69 has been substituted.
3. The mutant IL-2 polypeptide of claim 1 , wherein amino acid residue 74 has been substituted.
4. The mutant IL-2 polypeptide of claim 1 , wherein amino acid residue 128 has been substituted.
5. The mutant IL-2 polypeptide of claim 1 , wherein amino acid residues 69 and 74 have been substituted.
6. The mutant IL-2 polypeptide of claim 1 , wherein amino acid residues 69, 74 and 128 have been substituted.
7. The mutant IL-2 polypeptide of claim 1 , wherein aside from amino acid substitutions at positions 69, 74, and 128 of SEQ ID NO: 2, the amino acid sequence of the mutant IL-2 polypeptide is otherwise identical to SEQ ID NO: 2.
8. The mutant IL-2 polypeptide of claim 1 , further comprising an additional amino acid substitution.
9. The mutant IL-2 polypeptide of claim 1 , wherein alanine is substituted for valine at position 69, proline is substituted for glutamine at position 74, and threonine is substituted for isoleucine at position 128.
10. The mutant IL-2 polypeptide of claim 1 , wherein the mutant polypeptide binds to the IL-2Rα subunit with a K d of less than 28 nM.
11. The mutant IL-2 polypeptide of claim 10 , wherein the mutant IL-2 polypeptide binds to the IL-2α subunit with a K d of equal to or less than about 25 nM.
12. The mutant IL-2 polypeptide of claim 11 , wherein the mutant IL-2 polypeptide binds to the IL-2R.alpha, subunit with a K d of equal to or less than about 5 nM.
13. The mutant IL-2 polypeptide of claim 12 , wherein the mutant IL-2 polypeptide binds to the IL-2R.alpha, subunit with a K d of equal to or less than about 1 nM.
14. The mutant IL-2 polypeptide of claim 1 , further comprising a heterologous amino acid sequence.
15. The mutant IL-2 polypeptide of claim 14 , wherein the heterologous amino acid sequence increases the circulating half-life of the mutant IL-2 polypeptide, enhances expression of the mutant IL-2 polypeptide, directs cellular localization of the mutant IL-2 polypeptide, or serves as a marker or tag.
16. The mutant IL-2 polypeptide of claim 14 , wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin, a FLAG epitope, a c-myc epitope, albumin, or an Aga2p agglutinin polypeptide.
17. The mutant IL-2 polypeptide of claim 14 , wherein the heterologous amino acid sequence is the sequence of an antibody or antigen-binding fragment thereof.
18. The mutant IL-2 polypeptide of claim 14 , wherein the heterologous amino acid sequence is a toxin.
19. The mutant IL-2 polypeptide of claim 1 , wherein the polypeptide is glycosylated.
20. The mutant IL-2 polypeptide of claim 1 , wherein the polypeptide is not glycosylated.
21. A physiologically acceptable composition comprising the mutant IL-2 polypeptide of claim 1 in a physiologically acceptable carrier.