Lysosome-Targeting Chimeras as Clearing Agents for Pretargeted ImmunoSPECT

Abstract In vivo pretargeting based on the inverse electron-demand Diels−Alder reaction between a trans-cyclooctene (TCO)-modified monoclonal antibody (mAb) and a radiolabeled tetrazine (Tz) is a highly effective approach for reducing radiation burden in nuclear imaging and therapy. However, the large size and slow pharmacokinetics of full-length immunoglobulins greatly hinder their bloodstream clearance. To circumvent this limitation of pretargeting, much effort has been put towards developing masking or clearing agents: molecules designed to be injected prior to the radioligand to rid the bloodstream of excess circulating TCO-mAbs via clearance organs. Yet even with the use of previously established clearing agents, high levels of TCO-mAb still remain in circulation. Moreover, some of these agents could lead to aggregate formation in the lungs or clogs in the bloodstream. Recently, a novel class of cell−surface protein degraders called lysosome-targeting chimeras (LYTACs) have been reported to simultaneously bind hepatocytic-expressed asialoglycoprotein receptor (ASGPR) and a harmful, disease-relevant protein, internalizing the latter for lysosomal degradation. Herein, we present a novel ASGPR-targeted tetrazine-bearing LYTAC  TriGalNAc-Tz (LYTAC-Tz)  as a clearing agent for pretargeting. First, the essential components of the pretargeted system were synthesized: LYTAC-Tz, a TCO-modified variant of the mAb CC49 (TCO-CC49), and an 111In-labeled DOTA-bearing Tz ([111In]In-DOTA-Tz). Then, LYTAC-Tz was evaluated in vivo for its efficacy as a clearing agent for pretargeting in a murine model of colorectal cancer. SPECT-CT images and ex vivo biodistribution data revealed that LYTAC-Tz resulted in lower blood activity levels than control cohorts along with higher tumor-to-blood ratios. Healthy organs boasted low activity levels (excluding the kidneys, which showed uptake due to their rapid excretion of excess radioligand). Ultimately, this work illustrates that LYTAC-Tzs are effective clearing agents and could be beneficial for reducing radiotoxicity in pretargeted systems.