Interleukin-2 (IL-2)-based therapeutics are emerging as treatments for immunotherapy
however, systemic activation of immune cells hampers their success. Chemically controlling the activity of potent cytokines could mitigate unwanted T cell stimulation and widen their therapeutic window. In this study, we developed a strategy for the conditional activation of proteins utilizing removable peptide nucleic acid (PNA) masking groups. Site-specific installation of "Lock"-PNAs containing a cleavage thioester linkage enabled steric blockage of receptor binding sites. Rapid unmasking and activation were performed by the addition of a complementary "Key"-PNA containing a cysteine (Cys) residue, which forms a PNA-PNA duplex leading to a proximity-accelerated cleavage step and release of the active protein. We exemplified the versatility of this methodology on de novo cytokine neoleukin-2/15 (Neo-2/15) through the preparation of PNA conjugates including homodimers, PNA-stapled conjugates, and dual PNA-bridged dimers. All constructs were effectively unmasked at low micromolar concentrations. Further, we demonstrated the conditional activation of a masked conjugate of Neo-2/15 in binding studies to the IL-2 receptors and in an ex vivo T cell signaling assay displaying a 480-fold potency increase upon activation. Finally, we extended the strategy to a designed ankyrin repeat protein (DARPin) activating the human CD40 receptor demonstrating successful masking and unmasking.