ANTIBODY-TO-IMAGING PIPELINE TO MONITOR DRUG-TARGET ENGAGEMENT IN BREAST TUMORS

Monoclonal antibody-based therapies have become standard treatments for both hematological and solid malignancies. Trastuzumab (TZM), a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2), is a key therapy for HER2+ breast cancer. However, therapeutic efficacy is often compromised by tumor microenvironment (TME) features, such as dense extracellular matrix and poor vascularization, that limit antibody access and binding. Thus, there is a critical need for imaging approaches that can measure whether an antibody drug actually reaches and binds its target within heterogeneous tumors. We have developed a near-infrared (NIR) fluorescence lifetime (FLI) Förster Resonance Energy Transfer (FRET) optical imaging platform to address this challenge. This method directly reports drug-target engagement by measuring the fraction of donor labeled antibodies undergoing FRET when in proximity to acceptor-labeled antibodies upon receptor binding, using in vitro microscopy and in vivo macroscopy. We applied NIR-FLI-FRET to quantify HER2 binding in HER2+ breast cancer cells, tumor spheroids, and xenografts. To assess the influence of TME, we examined tumors with differing levels of collagen and vascularity. NIR-FLI-FRET imaging revealed that tumors with elevated collagen content and reduced vascularity showed decreased TZM- HER2 binding, despite robust HER2 expression, suggesting physical barriers to antibody penetration and reduced therapeutic efficacy. We further integrated site-specific labeling and antibody engineering via meditope- enabled TZM (MDT-TZM) to improve imaging precision and antibody performance. MDT-TZM exhibited deeper tumor penetration and more uniform binding in vivo compared to traditional NHS-labeled TZM. FRET signal maps correlated strongly with immunohistochemistry for HER2 and TZM, validating imaging results. Together, this antibody-to-imaging pipeline offers a powerful, non-invasive method to quantify drug-target engagement and understand how TME impacts antibody therapy. It represents a critical step toward precision imaging to guide antibody design and optimize treatment strategies in breast cancer.