Utilizing a dual action agent for targeted neuroendocrine tumor therapy

Neuroendocrine tumors (NETs) are rare, heterogeneous neoplasms with an incidence of 8 per 100,000 individuals, a rate that has increased significantly over the past four decades. These tumors frequently overexpress somatostatin receptor subtype 2 (SSTR2), enabling a form of NET-targeted radiotherapy called peptide-receptor radionuclide therapy (PRRT). Although PRRT improves progression-free survival (PFS), over half of patients in the NETTER-2 trial did not respond, and recurrence often occurs after the four FDA-approved therapy cycles. Strategies to improve outcomes for patients on PRRT have been explored, such as combining PRRT with the DNA alkylating agent temozolomide (TMZ). This combination has improved objective response rates but has not significantly extended overall or progression-free survival. To overcome these limitations, our lab developed tumor-targeted TMZ (ttTMZ), a drug conjugate that selectively delivers its chemotherapeutic payload directly to SSTR2-positive tumors and can chelate radioisotopes for imaging and therapy. Chelation of gallium-67 ( 67 Ga) by ttTMZ yields 67 Ga-ttTMZ, a dual-action agent that combines radiotherapy and chemotherapy in a single targeted construct. The Auger emitter 67 Ga has high linear energy transfer (4-4500 keV/μm) and a small radius of emission (2.4 μm), properties that lead to increased damage from emissions and decreased off-target effects, respectively. Radiolabeling ttTMZ with 67 Ga yields 67 Ga-ttTMZ with >95% efficiency. We evaluated 67 Ga-ttTMZ in IMR-32 cells using Western blotting for cleaved PARP and gamma-H2AX to assess apoptosis and DNA damage, respectively. Optimal dosing was identified as 0.5 MBq/mL 67 Ga and 100 μM ttTMZ. gamma-H2AX immunofluorescence confirmed a dose-dependent increase in DNA damage. In vivo PET imaging with 68 Ga-ttTMZ demonstrated selective localization to SSTR2-positive tumors and rapid clearance in dual-implant mouse models 1 h post-injection. These findings demonstrate that 67 Ga-ttTMZ induces significant DNA damage and apoptosis. Western blotting and gamma-H2AX immunofluorescence microscopy results both indicate that DNA damage is a primary mechanism of action for 67 Ga-ttTMZ. The selective tumor targeting and dual-action mechanism of 67 Ga-ttTMZ highlight its potential as a next-generation combination therapeutic for NETs, addressing key limitations of current PRRT and TMZ combination therapy.