Tracking the Fate of Therapeutic Proteins Using Ratiometric Imaging of Responsive Shortwave Infrared Probes

Monoclonal antibodies (mAbs) are essential agents for cancer treatment and diagnosis. Advanced optical imaging strategies have the potential to address specific questions regarding their complex in vivo life cycle. This study presents responsive shortwave infrared (SWIR) probes and an associated imaging scheme to assess mAb biodistribution, cellular uptake, and proteolysis. Specifically, we identify a Pegylated benzo-fused norcyanine derivative (Benz-NorCy7) that is activated in acidic environments and can be appended to mAbs without significant changes in optical properties. As a mAb conjugate, this agent shows high tumor specificity in a longitudinal imaging study in a murine model. To enable independent tracking of mAb uptake and lysosomal uptake and retention, a two-color ratiometric imaging strategy was employed using an "always-ON" heptamethine cyanine dye (ex = 785 nm) and the pH-responsive Benz-NorCy7 (ex = 890 nm). To assess proteolytic catabolism, we append a cleavable carbamate to Benz-NorCy7 to create turn-ON probes. These agents facilitate the compari-son of two common peptide linkers and provide insights into their in vivo properties. Overall, these studies provide a strate-gy to assess the fate of protein-based therapeutics using optical imaging in the SWIR range.