Studying the mechanism of action and bystander effect of the GSK5764227 antibody-drug conjugate in patient-derived cancer organoids

B7-H3, a member of the B7 family of proteins, is expressed in a variety of tumour types and has emerged as a novel target for cancer therapies due to its specific expression in tumour cells and minimal expression in normal tissues. One potential therapy route comprises of an antibody-drug conjugate (ADC) that specifically binds to B7-H3 and delivers a topoisomerase inhibitor (TOPOi) as its cytotoxic payload. The GSK clinical-stage B7-H3 ADC (GSK5764227) has shown promising results in cancer patients where other treatment options have been exhausted. However, the precise mechanism underlying its therapeutic efficacy remains unclear. We utilised patient-derived organoids (PDOs) generated from different tumour types to investigate the mechanism of action of GSK5764227, from target binding and internalisation to trafficking through endosomal and autophagy pathways to treatment response. By leveraging a partially knockout PDO model, containing a mixture of B7-H3 positive and negative cells, we explored the potential bystander effect of the compound using spatial multiplex and real time imaging at multiple timepoints. In parallel, we analysed the cargo of extracellular vesicles to assess their role in delivering the payload to neighbouring B7-H3 negative cells and enhancing the overall treatment response. Our findings shows that GSK5764227 after binding to B7-H3 within minutes of treatment initiation, is internalised, and trafficked from early endosomes to lysosomes within 24 hours, where the cytotoxic payload is likely cleaved and released. The cleaved payload induces DNA damage, cell cycle arrest and apoptosis through Topoisomerase I inhibition. Furthermore, we observed that payload is trafficking into neighbouring cells through extracellular vesicles derived from treated PDOs, inducing treatment effects, including DNA damage in B7-H3 negative cells, further supporting the drug's bystander effect. Altogether, this work characterises how GSK5764227 induces cell cycle arrest and tumour apoptosis directly in B7-H3 positive cells and indirectly in neighbouring B7-H3 negative cells via bystander effect. Combining clinical, molecular and imaging data, this study enhances our understanding of the drug's mechanism of action and provides a foundation for optimising its use in difficult-to-treat-cancers, ultimately guiding personalised therapeutic strategies for patients.