<p>Terbium-161 (¹⁶¹Tb) is regarded as a highly promising radionuclide for therapeutic applications due to its emission of not only β⁻ particles but also conversion electrons and Auger electrons. This unique decay characteristic makes it particularly effective for eliminating micrometastases. To fully exploit the therapeutic potential of ¹⁶¹Tb while addressing issues such as severe off-target toxicity and low tumor-to-non-tumor uptake ratios caused by the large molecular weight and slow clearance of antibodies in traditional radioimmunotherapy (RIT), this study innovatively employs the pretargeting radioimmunotherapy (PRIT) strategy combined with ¹⁶¹Tb. By decoupling the targeting and radionuclide delivery processes, this approach effectively reduces the radiation background in the blood and normal organs. In this study, cetuximab was selected as the primary antibody targeting the epidermal growth factor receptor (EGFR), and peptide nucleic acid (PNA)—a DNA analog with excellent in vivo stability and high hybridization affinity—was employed for the first time as a ¹⁶¹Tb labelled pretargeting agent. We successfully designed and synthesized a ¹⁶¹Tb-labeled PNA-mediated pretargeting radiopharmaceutical, and subsequently conducted a comprehensive evaluation of its in vivo biodistribution, SPECT/CT imaging, and antitumor effects in the HCC827 xenograft mouse model. The experimental results showed that group A (Cetuximab-PNA-1 + [¹⁶¹Tb]Tb-PNA-2) exhibited significantly reduced radioactive accumulation in the blood and a significantly increased tumor-to-background ratio compared with group B ([¹⁶¹Tb]Tb-DOTA-Cetuximab). After 4&#xa0;weeks of treatment, compared to the control group, a&#xa0;significant tumor growth inhibition was observed in group A (Cetuximab-PNA-1 + [¹⁶¹Tb]Tb-PNA-2).</p> Graphical abstract <p>Construction of PNA mediated pretargeting strategy platform and in vivo mechanism.</p> <p></p>