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.