ProTide-enabled antibody-drug conjugates: A novel platform for the targeted delivery of phosphorylated drugs

Herein, we report the first integration of ProTide technology with antibody-drug conjugates (ADCs) to create a novel platform that enables the targeted delivery of active, phosphorylated drugs. Our ADCs contain a specially designed ProTide linker that exploits the ProTide enzymatic activation pathway to release monophosphorylated nucleoside analogues directly into target cells. Using gemcitabine as a model drug conjugated to trastuzumab lysines, we demonstrated good antiproliferative activity in HER2-positive cancer cell lines (SKBR3 and MIA PaCa-2) compared to the unconjugated antibody and existing ProTide formulations. The lead compound achieved IC₅₀ values of 0.25 μM (SKBR3) and 0.19 μM (MIA PaCa-2), which represent a significant improvement over current therapies. Mechanistic studies confirmed the successful enzymatic release of the drug and the maintenance of the internalization properties of the antibody. This platform fulfils the requirement to deliver phosphorylated drugs while minimising resistance and systemic toxicity. Beyond nucleoside analogues, this approach opens up new possibilities for the targeted delivery of other phosphate-containing therapeutics, potentially expanding the scope of ADC technology. • The manuscript reports the first example of the combination of ProTide technology with ADCs for the targeted release of phosphorylated drugs • The ADCs showed improved antiproliferative activity compared to unconjugated trastuzumab and gemcitabine ProTide NUC-1031, demonstrating successful internalization and enzymatic drug release in HER2-positive cancer cell lines • The approach can potentially be applied to other nucleoside analogues and non-nucleoside phosphate or phosphonate drugs, expanding the capabilities of ADC technology beyond traditional cytotoxic agents • This work represents an advance in targeted drug delivery as it provides a new mechanism for the delivery of phosphorylated drugs that could circumvent cellular resistance mechanisms