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 that is at least 80% identical to SEQ ID NO:2 and that binds an IL-2 receptor α subunit (IL-2Rα) with an affinity that is greater than the affinity with which the polypeptide represented by SEQ ID NO:3 binds the IL-2Rα,wherein the amino acid sequence of the mutant IL-2 polypeptide differs from SEQ ID NO:2 by virtue of containing alanine at position 69 rather than valine.
2. The mutant IL-2 polypeptide of claim 1 , wherein the amino acid sequence varies from SEQ ID NO:2 only by virtue of containing one or more amino acid substitutions.
3. The mutant IL-2 polypeptide of claim 1 , wherein the amino acid sequence varies from SEQ ID NO:2 by virtue of containing one or more amino acid substitutions and one or more additional amino acid residues.
4. The mutant IL-2 polypeptide of claim 1 , wherein the amino acid sequence varies from SEQ ID NO:2 by virtue of containing one or more amino acid substitutions and one or more amino acid deletions.
5. 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.
6. The mutant IL-2 polypeptide of claim 5 , wherein the mutant IL-2 polypeptide binds to the IL-2Rα subunit with a K d of equal to or less than about 25 nM.
7. The mutant IL-2 polypeptide of claim 6 , wherein the mutant IL-2 polypeptide binds to the IL-2Rα subunit with a K d of equal to or less than about 5 nM.
8. The mutant IL-2 polypeptide of claim 7 , wherein the mutant IL-2 polypeptide binds to the IL-2Rα subunit with a K d of equal to or less than about 1 nM.
9. The mutant IL-2 polypeptide of claim 1 , wherein the amino acid sequence is at least 90% identical to SEQ ID NO:2.
10. The mutant IL-2 polypeptide of claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO:2.
11. The mutant IL-2 polypeptide of claim 1 , further comprising a heterologous amino acid sequence.
12. The mutant IL-2 polypeptide of claim 11 , 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.
13. The mutant IL-2 polypeptide of claim 11 , 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.
14. The mutant IL-2 polypeptide of claim 11 , wherein the heterologous amino acid sequence is the sequence of an antibody or antigen-binding fragment thereof.
15. The mutant IL-2 polypeptide of claim 11 , wherein the heterologous amino acid sequence is a toxin.
16. The mutant IL-2 polypeptide of claim 1 , wherein the polypeptide is glycosylated.
17. The mutant IL-2 polypeptide of claim 1 , wherein the polypeptide is not glycosylated.
18. A physiologically acceptable composition comprising the mutant IL-2 polypeptide of claim 1 in a physiologically acceptable carrier.