HER2-mediated internalization of a targeted prodrug cytotoxic conjugate is dependent on the valency of the targeting ligand

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, AHNPmonovalent and AHNPbivalent) with different valency and correlated their internalization-promoting ability to biological potency. Since AHNPbivalent (but not AHNPmonovalent) induces rapid receptor internalization, we exploited this feature to deliver cytotoxic conjugates coupling AHNPbivalent and Taxol (Taxol . AHNPbivalent). The prodrug conjugate releases Taxol after receptor-mediated internalization, and cytotoxicity can be used as a marker of internalization. Taxol . AHNPbivalent is significantly more cytotoxic than free Taxol + free AHNPbivalent. Hence, the Taxol x 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.