Spatiotemporal Quantification of HER2-targeting Antibody–Drug Conjugate Bystander Activity and Enhancement of Solid Tumor Penetration

Abstract Purpose: Antibody–drug conjugate (ADC) has had a transformative effect on the treatment of many solid tumors, yet it remains unclear how ADCs exert bystander activity in the tumor microenvironment. Experimental Design: Here, we directly visualized and spatiotemporally quantified the intratumor biodistribution and pharmacokinetics of different ADC components by developing dual-labeled fluorescent probes. Results: Mechanistically, we found that tumor penetration of ADCs is distinctly affected by their ability to breach the binding site barrier (BSB) in perivascular regions of tumor vasculature, and bystander activity of ADC can only partially breach BSB. Furthermore, bystander activity of ADCs can work in synergy with coadministration of their parental antibodies, leading to fully bypassing BSBs and enhancing tumor penetration via a two-step process. Conclusions: These promising preclinical data allowed us to initiate a phase I/II clinical study of coadministration of RC48 and trastuzumab in patients with malignant stomach cancer to further evaluate this treatment strategy in humans.