Optimizing peptide nucleic acid-based pretargeting for enhanced targeted radionuclide therapy

Radiolabeled targeting agents have emerged as valuable tools for the treatment of disseminated cancer. Monoclonal antibodies (mAbs) are widely employed as carriers for diagnostic and therapeutic radionuclides due to their exceptional specificity and affinity. However, their prolonged circulatory half-life can diminish diagnostic efficacy and increase radiation exposure to non-target tissues in therapeutic applications, resulting in dose-limiting toxicities. To overcome this limitation, pretargeting technologies emerge as promising strategies to enhance tumor-to-background ratio and reduce radiation exposure of healthy tissues. Our previous work introduced a pretargeting concept leveraging the specific interaction between two peptide nucleic acid (PNA) probes, HP1 and HP2, as the recognition mechanism. This early iteration of the PNA-based concept showed limited efficacy when used with mAb-based vectors. To improve its performance, we re-engineered the primary and secondary targeting agents by incorporating newly designed PNA-probes. As the primary targeting agent, we functionalized trastuzumab (T), a well-characterized human epidermal growth factor receptor 2 (HER2)-targeting IgG₁ mAb, with a 9-mer PNA probe (HP9). Both FcIII-based covalent UV-light crosslinking and enzyme-mediated glyco-engineering click-chemistry methods were applied to generate trastuzumab-PNA conjugates T-FcIII-HP9 and T-gly-HP9, respectively. As a radionuclide-carrying secondary agent, we utilized a 9-mer complementary PNA probe, HP16, which forms a stable duplex with HP9 as well as displaying favorable in vivo kinetics. Biacore and flow cytometry assessment of the HP9-conjugated trastuzumab agents demonstrated retained HER2-binding properties. The secondary HP16 probe, labeled with either a dye or a radionuclide, showed cell surface accumulation contingent on the presence of HP9 on the primary HER2-targeting agents. In vivo, T-gly-HP9 exhibited significantly longer blood circulation half-life and superior tumor uptake compared to T-FcIII-HP9. Further, therapeutic dosing with [¹⁷⁷Lu]-HP16 of trastuzumab-HP9 pretargeted HER2+ tumor models resulted in significantly delayed disease progression and extended survival compared to untreated subjects. Furthermore, pretargeted [¹⁷⁷Lu]-HP16 exhibited comparable efficacy to [¹⁷⁷Lu]-trastuzumab in both delaying disease progression and prolonging survival. In conclusion, the optimization of our PNA-based pretargeting system has resulted in exceptional in vivo targeting characteristics and therapeutic efficacy, validating the potential of this novel approach.