HER2-Mediated Internalization of a Targeted Prodrug Cytotoxic Conjugate Is Dependent on the Valency of the Targeting Ligand

Abstract

HER2 is a validated therapeutic target for cancer. There are no natural ligands, but monoclonal antibodies and peptides that bind HER2 act as artificial ligands, selectively affecting HER2-overexpressing tumors. One reported mechanism for this effect is receptor downregulation, but the expected correlation of ligand-dependent HER2 internalization and tumor inhibition remain poorly characterized. Moreover, HER2 ligands have limited therapeutic efficacy and often they require adjuvant treatment with the chemotherapeutic Taxol. Here, we generated a series of HER2 ligands (Anti-HER2/neu peptide ligands, AHNP_monovalent and AHNP_bivalent) with different valency and correlated their internalization-promoting ability to biological potency. Since AHNP_bivalent(but not AHNP_monovalent) induces rapid receptor internalization, we exploited this feature to deliver cytotoxic conjugates coupling AHNP_bivalent and Taxol (Taxol · AHN_Pbivalent). The prodrug conjugate releases Taxol after receptor-mediated internalization, and cytotoxicity can be used as a marker of internalization. Taxol · AHN_Pbivalent is significantly more cytotoxic than free Taxol + free AHNP_bivalent. Hence, the Taxol · AHNP_bivalent prodrug binds to HER2, induces receptor internalization and downregulation, and the subsequent release of free Taxol inside the targeted cell results in synergistic toxicity, The effect is selective towards HER2- expressing cells. This work links HER2 receptor internalization and growth arrest, and the chemical conjugation strategy may yield improved and HER2 selective therapeutics.