Antibody–drug conjugates (ADCs) are a rapidly expanding class of anti-cancer agents comprised of a monoclonal antibody linked to a cytotoxic payload. By delivering the payload directly to cancerous cells, ADCs offer greater selectivity and cytotoxic potency than either sub-unit alone. Of the 14 FDA-approved ADCs, two utilise Topoisomerase I (Top1) inhibitors: Sacituzumab govitecan (SG) and Trastuzumab deruxctan (T-DXd). While T-DXd is considered the current gold standard for ADCs, Top1-targeting ADCs still possess several significant limitations, warranting further research. The development of resistance is one key factor, particularly through increased expression of efflux pumps such as P-glycoprotein (P-gp) and the Breast Cancer Resistance Protein (BCRP). These pumps can export the drug from the cell before it can elicit its therapeutic effect, increasing the likelihood of off-target effects, disease recurrence, and mortality. This project therefore seeks to develop novel efflux-resistant analogues of leading Top1 inhibitors and incorporate them into improved ADCs.

Following in silico modelling of a large compound set, the most promising candidates are synthesised and evaluated for their in vitro cytotoxicity across multiple human cancer cell lines. Lead compounds are further tested for their mechanism of action using flow cytometry and gel-based assays to ensure that structural modifications retain on-target activity and specificity. Finally, both direct and indirect measures of efflux liability are investigated through comparison of efflux ratios derived from cytotoxicity data and drug accumulation levels in wild-type and efflux-positive cell lines.

We have demonstrated that the types of structural modifications introduced are well tolerated, with on-target Top1 activity maintained in vitro. Initial in silico studies indicate that the leading analogues form increased hydrophobic interactions with P-gp and may interfere with the conformational change required for drug efflux, suggesting the potential for improved efflux resistance. Importantly, this hypothesis has been supported by early efflux studies, in which lead compounds demonstrated up to a 20-fold decrease in efflux ratio compared with current Top1 inhibitors. Based on these findings, a small panel of inhibitors has been selected as candidates for incorporation into ADCs, for which the conjugation and assay workflow has already been established in-house.

A series of efflux-resistant Top1 inhibitors has been developed, demonstrating that efflux liability can be reduced without compromising on-target activity. These compounds therefore hold promise as potent next-generation ADC payloads capable of overcoming current challenges associated with chemoresistance.

This research was co-funded by the Medical Research Council (project code 2888791) and Pheon Therapeutics.