Rational design of interleukin-21 antagonist through selective elimination of the γC binding epitope

Abstract

The cytokine interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Rα) and the common γ-chain (γC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Rα, whereas the interaction between IL-21 and γC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling γC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues, which compose the γC binding epitope using homology modeling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants, which have significantly impaired γC affinity with undiminished IL-21Rα affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.