The radionuclide-labeled bevacizumab (BV) is a potential therapeutic approach for vascular endothelial growth factor overexpressed tumors. Because of its large molecular weight, BV is cleared slowly in vivo, which caused damage to healthy tissues and organs. On account of this situation, using the pretargeting strategy with DNA/RNA analogs, such as peptide nucleic acid (PNA), is an effective way of treating solid tumors.The BV-PNA conjugate (BV-PNA-1) was injected intravenously as the pretargeted probe, which was specifically accumulated in a solid tumor and gradually metabolically cleared. Then the [177Lu]Lu-labeled complementary PNA strand ([177Lu]Lu-PNA-2) as the second probe was injected, and bound with BV-PNA-1 by the base complementary pairing. In this study, the BV-based PNA-mediated pretargeting strategy was systematically studied, including stability of probes, specific binding ability, biodistribution in animal model, evaluation of single photon emission computed tomography/computed tomography imaging, and therapeutic effect.Compared with group A ([177Lu]Lu-BV), the group B (BV-PNA-1 + [177Lu]Lu-PNA-2) showed lower blood radiotoxicity (22.55 ±1.62 vs. 5.18 ± 0.40%, %ID/g, P < 0.05), and similar accumulation of radioactivity in tumor (5.32 ± 0.66 vs. 6.68 ± 0.79%, %ID/g, P > 0.05). Correspondingly, there was no significant difference in therapeutic effect between groups A and B.The PNA-mediated pretargeting strategy could increase the tumor-to-blood ratio, thereby reducing the damage to normal tissues, while having a similar therapeutic effect to solid tumor. All the experiments in this study showed the potential and effectiveness of pretargeting radioimmunotherapy.Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.