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 IgG