Antibody-target binding quantification in living tumors using macroscopy fluorescence lifetime Forster resonance energy transfer imaging (MFLI FRET)

Abstract

Emergent breast tumor resistance and tumor microenvironment (TME) heterogeneity can lead to decreased drug delivery efficacy, resulting in therapeutic failure. Preclinical molecular imaging is a crucial tool in the advancement of targeted therapeutics for supporting the development of new drugs but also to elucidate factors hampering optimal drug delivery. However, noninvasive tumor imaging modalities that can quantify drug-target engagement, which is critical for therapeutic actuation, are still lacking. We have demonstrated the utility of macroscopic fluorescence lifetime Forster's Resonance Energy Transfer (MFLI FRET)-based optical imaging to measure labeled trastuzumab (TZM)-human epidermal growth factor receptor (HER2) binding in human HER2+ cell lines and breast tumor xenograft mice models. We have established a clinically relevant TZM antibody drug containing the Meditope (MDT) peptide conjugated to near-infrared (NIR) dyelabeled FRET pairs, that retain full HER2 binding capability. Herein, we demonstrate FRET measurements using MFLI in vivo imaging platform to measure the ability of MDT-TZM to bind HER2 in living breast tumor xenografts. HER2+ AU565 breast tumor xenografts bearing nude mice were injected retro-orbitally with TZM (NHS-conjugated) or MDT-TZM labeled with AlexaFluor700 (donor) and AlexaFluor750 (acceptor) and MFLIFRET imaging was performed 24 h and 48 h post-injection. Preliminary data suggest that MDT-TZM shows higher uniform and consistent FRET signal compared to TZM, suggesting increased efficacy of TZM-MDT-HER2 binding. Also staggered injections of donor and acceptor MDT-TZM may be optimal for quantifying MDT-TZM using MFLIFRET compared to single injections.