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

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, while the interaction between IL-21 and γC, albeit essential for signalling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity towards the signalling 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 modelling 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, while undiminished IL-21Rα affinity were successfully identified. Functional studies confirmed that these two novel hIL-21 double-mutants do act as hIL21 antagonists.