A dual screening strategy identifies delivery-dependent payload candidates for antibody-drug conjugates

<jats:p>The discovery of Antibody–Drug Conjugate (ADC) payloads relies predominantly on conventional cell-based cytotoxicity screening, an approach that inherently favors membrane-permeable compounds while potentially overlooking molecules possessing potent intracellular activities but limited cellular uptake. We hypothesized that such compounds represent a previously unexplored class of delivery-dependent payloads whose biological activity is revealed only following antibody-mediated internalization. To test this concept, we developed a dual screening strategy combining a cell viability assay with a cell-free GFP expression assay and applied it to a structurally diverse library of 51 pesticides, a largely unexplored source of bioactive molecules for ADC payload discovery. Multiple compounds displaying dissociated cellular and cell-free activity profiles were identified. From the subset combining low cellular toxicity with strong inhibition of cell-free GFP expression, fipronil was selected as a proof of concept molecule, derivatized with a cathepsin-cleavable Mc-Val-Cit-PAB linker, and conjugated to trastuzumab and rituximab through cysteine-based chemistry. As expected, free fipronil remained inactive in conventional cellular assays, whereas the trastuzumab–fipronil ADC significantly inhibited clonogenic growth of HER2-positive cells in a target-dependent manner. Neither the unconjugated components nor the nontargeting rituximab ADC reproduced this effect, demonstrating that intracellular delivery by the antibody was required to reveal payload activity. These results establish dual screening as a general strategy for identifying delivery-dependent payloads overlooked by conventional cytotoxicity screening. By expanding the chemical space accessible for ADC payload discovery, this approach provides a general framework for identifying unconventional payloads with novel mechanisms of action and the potential to improve the biological selectivity of next-generation ADCs.</jats:p>