Abstract

Prodrug strategies for protein therapeutics can enable spatiotemporal control, minimize off-target effects and improve safety. However, a universal chemical platform that concurrently enables reversible activity switching and efficient delivery remains a challenge. Here, we introduce a dithiolane-based “Trojan tag” (DTL tag) as a simple, multifunctional tool for constructing modular protein prodrugs with traceless intracellular activation. The DTL tag promotes efficient cellular internalization of diverse proteins, independent of size and isoelectric point. When applied to the anti-PD-L1 (programmed cell death ligand 1) nanobody, the DTL tag enhanced internalization of the resulting complex, effectively trimming PD-L1 from tumor cell surfaces. Furthermore, the tag served as a reduction-triggered activity switch for enzymes like luciferase and RNase A, enabling their efficient delivery and subsequent functional reactivation. Notably, the DTL tag synergized with commercial transfection reagents to enhance cytosolic delivery of RNase A, leading to enhanced cancer cell killing. This straightforward DTL platform streamlines the creation of smart protein prodrugs, offering a new strategy for developing protein prodrugs.