De novo design of protease-activatable cytokine prodrugs

Cytokines can elicit potent antitumor immunity, but their clinical use is often limited by systemic immune activation and dose-limiting toxicity. Protease-responsive masking offers a way to improve selectivity by linking cytokine activity to disease-associated protease cleavage, but current approaches have faced several challenges. Here we describe a de novo protein design strategy for generating protease-activatable cytokine prodrugs that overcomes these challenges. We use this strategy to design and characterize cytokine prodrugs and well-behaved AND-gated split systems that require both target-dependent colocalization and proteolytic unmasking to generate active cytokines. In challenging syngeneic tumor models, our IL-21 prodrugs reduced treatment-associated toxicity while maintaining antitumor efficacy.