Enhanced Retention of NTSR1-Targeted Radionuclide Therapeutics via Covalent Inhibitors in Pancreatic, Colorectal, and Prostate Cancer Models

Neurotensin receptor subtype 1 (NTSR1) is overexpressed in numerous cancers. Our laboratory is exploring the utilization of covalent cysteine protease inhibitors (e.g., E-64) to increase tumor retention of targeted radionuclide therapeutics (TRTs) through protein adduct formation. Using this approach, we reported [177Lu]Lu-NA-ET1, an NTSR1-targeted construct. In this work, we continue the exploration of [177Lu]Lu-NA-ET1 in three different NTSR1-positive cancer models. [177Lu]Lu-3BP-227, a clinically investigated NTSR1-targeted construct, was utilized as a comparative benchmark. Both [177Lu]Lu-NA-ET1 and [177Lu]Lu-3BP-227 underwent in vitro investigation, including internalization and autoradiographic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies, in NTSR1-positive AsPC-1, HT-29, and PC-3 cell lines. Biodistribution, human radiation dosimetry, and in vivo autoradiographic SDS-PAGE studies were performed by using the same models. A dose escalation study using 585 MBq (15.8 mCi) of [177Lu]Lu-NA-ET1 was implemented in immunocompetent CF-1 mice. In all three cell lines, [177Lu]Lu-NA-ET1 demonstrated similar cellular uptake profiles relative to those of [177Lu]Lu-3BP-227. Biodistribution studies of [177Lu]Lu-NA-ET1 revealed increased (1.9–4.4-fold) tumor retention and radiation dose delivery relative to the control. Analysis of the in vitro and in vivo cellular and tissue lysates showed protein adducts that ranged from approximately 25–35 kDa, consistent with cysteine cathepsins, the speculative protein binding partner. A total of 585 MBq (15.8 mCi) of [177Lu]Lu-NA-ET1 was administered and found to be well-tolerated. Incorporating the covalent inhibitor in [177Lu]Lu-NA-ET1 resulted in an improved retention and radiation dose delivery profile compared to [177Lu]Lu-3BP-227. Examination of the therapeutic potential of [177Lu]Lu-NA-ET1 and further exploration of the chemical biology of this approach is underway.