Natural products as payloads for antibody-drug conjugates: Cyclopamine linked to cetuximab for hedgehog pathway inhibition

Cyclopamine, a natural product that enabled the identification of Smoothened as a central regulator of the Hedgehog (HH) signalling pathway, has never advanced to clinical testing due to unfavourable pharmacokinetics and multiple off-target effects. In this study, we describe two novel antibody-drug conjugates (ADCs) in which Cetuximab is covalently linked to cyclopamine, to selectively deliver this natural product into melanoma cells exhibiting HH pathway activation and epidermal growth factor receptor (EGFR) expression. The ADC, which contains an acid-cleavable linker, effectively inhibited GLI1 and GLI2 expression and demonstrated antiproliferative activity in melanoma cell lines A375 and SK-MEL-5, highlighting the efficacy of cyclopamine and monoclonal antibody (mAb) conjugation. This activity was associated with a pro-apoptotic effect directly linked to HH pathway inhibition. The linker-payload system was stable in plasma yet released gradually cyclopamine at pH 5.5. These results suggest the feasibility of using natural products as payloads for the construction of ADCs to inhibit HH signalling in cancer cells. • Efficient conjugation of cyclopamine to an antibody with the aim of overcoming pharmacokinetic problems and off-target side effects. • Simultaneous inhibition of Hedgehog and EGFR signalling pathways in melanoma by targeted delivery. • The inhibition mechanism is based on apoptosis cell death following intracellular release of the drug.