Conditional Activation of Protein Therapeutics by Templated Removal of Peptide Nucleic Acid Masking Groups

Abstract 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.