Design and Evaluation of Dual-Targeted Radionuclide Therapeutics for NTS1 with Improved Tumor Retention through Endolysosomal Trapping

This study investigates the development of neurotensin receptor subtype 1-(NTS1-) targeted constructs incorporated with an irreversible cysteine protease inhibitor (i.e., cysteine protease trapping agent (CPTA)) to improve tumor retention through adduct formation. Specifically, in this CPTA-incorporated NTS1-targeted agent (CPTA-NTS1TA) paradigm, we investigated the structure-activity impact of a series of alkyl, alkylamine, and ether linkers. The CPTA-NTS1TAs, [ ^<b>177</b> <b>Lu]-Lu-8a-e</b>, exhibited nanomolar binding affinities (9-38 nM) to NTS1 and were capable of rapid and irreversible inhibition (18,900 ± 1800-54,000 ± 7000 M^-1 s^-1) of a cysteine protease (i.e., cathepsin B). Linker modifications that included a positive charge ([ ^<b>177</b> <b>Lu]-Lu-8c</b>) or were less sterically restrained ([ ^<b>177</b> <b>Lu]-Lu-8a</b>) exhibited improved NTS1-targeting capabilities in an HT-29 colon cancer mouse model. Although, the level of tumor uptake (4.6 ± 0.7-16.5 ± 1.2%ID/g) of [ ^<b>177</b> <b>Lu]-Lu-8a-e</b> was often well below the benchmark control [ ^<b>177</b> <b>Lu]-Lu-3BP-227</b> (20.8 ± 1.1%ID/g), a NTS1-targeted agent that has reached clinical trials. However, all experimental constructs containing the irreversible inhibitor had higher tumor retention (17.8 ± 1.5-28 ± 7%) as a percentage of initial uptake compared to <b>[</b> ^<b>177</b> <b>Lu]-Lu-3BP-227</b> (13 ± 2%). In conclusion, this study provides valuable insights into the structure-activity relationships of these dual-targeted constructs for future development to improve tumor uptake and reduce the renal retention of NTS1-targeted radiotherapeutics.