Exploration of the cysteine protease trapping approach to prolong tumor retention of antifolate targeted radionuclide therapeutics.

BACKGROUND: Aberrant overexpression of the folate receptor subtype 1 (FOLR1, FRα) in metastatic cancers has long been studied and therapeutically exploited via successful chemotherapeutics targeting folate metabolism. However, small-molecule FOLR1-targeted radionuclide therapeutics have typically exhibited poor radiation dose delivery profiles, particularly in terms of tumor-to-kidney ratios. Our laboratory has aimed to improve tumor retention and dose delivery profiles through a cysteine protease trapping approach (CPTA), designed to enhance tumor residence time, radiation dose delivery and cell death. This work presents our first investigation into the efficacy of CPTA in the context of FOLR1-targeted radionuclide therapeutics. Specifically, we incorporated an irreversible cysteine protease inhibitor (i.e., CPTA) into a FOLR1-targeted construct ([177Lu]Lu-FOLR1-CPTA) to examine the potential of this approach to form irreversible macromolecular adducts and increase tumor residence times.

RESULTS: We synthesized [177Lu]Lu-FOLR1-CPTA and a structurally matched control, [177Lu]Lu-FOLR1-SHAM, which lacks the epoxysuccinyl warhead required for cysteine protease inhibition. Both constructs exhibited high hydrophilicity (LogD7.4 ≈ -2.5) and low nanomolar FOLR1 binding affinity. FOLR1-CPTA irreversibly inhibited cysteine cathepsin B with a second-order rate constant (kinact/KI) of (6.6 ± 0.4) × 103 M-1 s-1 and, unlike SHAM, formed macromolecular-weight adducts in vitro and ex vivo as visualized by autoradiographic SDS-PAGE. In IGROV-1 xenograft mice, both radioconstructs demonstrated rapid blood clearance and high renal uptake. Tumor uptake at 4 h was 5.1 ± 0.3%ID/g for [177Lu]Lu-FOLR1-CPTA and 6.0 ± 0.4%ID/g for [177Lu]Lu-FOLR1-SHAM. Between construct differences in tumor uptake at individual timepoints were not statistically significant; however, longitudinal analysis revealed stable tumor retention for [177Lu]Lu-FOLR1-CPTA across 4-168 h, whereas [177Lu]Lu-FOLR1-SHAM exhibited significant washout over the same interval.

CONCLUSIONS: Incorporation of CPTA into a FOLR1-targeted TRT enabled robust intracellular adduct formation but did not yield a clear improvement in in vivo tumor targeting relative to a matched sham control. However, this initial work provides insight into the biological consequences of structural modifications (i.e., CPTA and zwitterionic peptide incorporation) in FOLR1-targeted radionuclide therapeutics, informing future optimization strategies to better isolate and leverage CPTA-driven retention.