Peptide Nucleic Acid Probes for Pretargeted Affibody-Mediated Delivery of Cytotoxic Payloads to HER2-Positive Carcinoma

Abstract

This study investigates how the structural design of secondary PNA probes affects their ability to carry the cytotoxic agent DM1 in a novel affibody-PNA-based pretargeting approach directed at Human Epidermal growth factor Receptor 2 (HER2)-expressing cells. Six different DM1-conjugated secondary 8-mer PNA probes with strategic hydrophilic modifications were designed and evaluated. Surface plasmon resonance analysis confirmed that the modifications preserved hybridization to the HER2-targeting affibody-PNA primary probe, with all secondary probes showing similar binding kinetics (KD = 310–660 pM). Biodistribution studies in nontumor-bearing NMRI mice with lutetium-177 (177Lu)-labeled secondary probes revealed that probe modifications dramatically influenced organ distribution patterns. The secondary probes SP2, SP5, and SPc demonstrated favorable biodistribution profiles with reduced accumulation in critical organs, i.e., liver and kidneys. Receptor-mediated endocytosis was also affected by secondary probe design, showing an increase in cellular internalization for SP5 (20.4% after 24 h) compared to SP2 (12.1%). Our findings highlight how rational design can optimize in vivo pharmacokinetics while preserving binding properties essential for effective pretargeting, providing a foundation for developing novel targeted cytotoxic delivery systems with potentially improved therapeutic indexes.