Development and Evaluation of the Cystiene Cathepsin Trapping Approach Incorporated in NTSR1- and FAP-Targeted Radiotherapeutics

Author

Katherine Brake, University of Nebraska Medical CenterFollow

ORCID ID

0009-0001-3115-7882

Graduation Date

Spring 5-10-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Jered C. Garrison

Abstract

Fibroblast activation protein (FAP) and neurotensin receptor subtype 1 (NTSR1) are highly expressed in multiple malignancies, making them promising targets for radiopharmaceutical therapy. FAP is a membrane-bound serine protease predominantly expressed in cancer-associated fibroblasts within the tumor stroma of over 90% of epithelial cancers. Its restricted expression in normal adult tissues makes it an ideal candidate for targeted radionuclide therapy. However, existing FAP-targeted agents, such as ¹⁷⁷Lu-FAPI-46, exhibit rapid tumor washout, limiting their potential for sustained radiation dose delivery. Similarly, NTSR1, a G-protein-coupled receptor overexpressed in pancreatic, colorectal, and prostate cancers, has been explored as a radiopharmaceutical target. Although peptide-based ¹⁷⁷Lu-3BP-227 has shown clinical feasibility, its limited tumor retention has hindered therapeutic efficacy.

To address these challenges, we developed ¹⁷⁷Lu-FAPI-ET1 and ¹⁷⁷Lu-NA-ET1, two novel radiopharmaceuticals incorporating a cysteine cathepsin trapping agent designed to prolong tumor retention via endolysosomal trapping. This strategy utilizes an epoxysuccinyl peptide inhibitor that selectively and irreversibly binds to the catalytic cysteine residue of endolysosomal cysteine cathepsins, forming stable high-molecular-weight adducts. Since cysteine cathepsins are highly expressed in the tumor microenvironment and play a role in extracellular matrix remodeling and cancer progression, leveraging their proteolytic activity for intracellular trapping enhances the retention of radiopharmaceutical constructs within tumor cells.

In vitro studies confirmed that both constructs maintained high binding affinity to their respective targets and efficiently formed covalent adducts with cysteine cathepsins. Biodistribution studies in glioblastoma cancer xenograft models demonstrated significantly prolonged tumor retention, with ¹⁷⁷Lu-FAPI-ET1 showing a five-fold increase in tumor residence at 168 hours post-injection compared to ¹⁷⁷Lu-FAPI-46. Similarly, ¹⁷⁷Lu-NA-ET1 displayed 1.9- to 4.4-fold enhanced tumor retention relative to ¹⁷⁷Lu-3BP-227 across multiple cancer models. Human radiation dosimetry estimates indicated that these modifications improve the therapeutic index while maintaining tolerability.

These findings validate the endolysosomal trapping approach as an effective strategy to overcome rapid clearance in small-molecule targeted radiopharmaceuticals. By leveraging cysteine cathepsins to facilitate intracellular trapping, this approach significantly enhances tumor retention and radiation dose delivery. Future work will focus on optimizing specificity to minimize off-target effects, but this strategy holds substantial promise for advancing precision radiopharmaceutical therapy in FAP- and NTSR1-expressing malignancies.

Comments

2025 Copyright, the authors

Recommended Citation

Brake, Katherine, "Development and Evaluation of the Cystiene Cathepsin Trapping Approach Incorporated in NTSR1- and FAP-Targeted Radiotherapeutics" (2025). Theses & Dissertations. 935.
https://digitalcommons.unmc.edu/etd/935