HER2-Targeting and TRAIL-Presenting Protein Nanoparticles Induce a Concentration-Dependent Biphasic Response in HER2-Positive Breast Cancer Cells

Targeted therapy enables the selective delivery of therapeutics to specific cells, reducing off-target effects and improving efficacy. HER2-targeted approaches are particularly effective in HER2-positive breast cancer. Here, we engineered protein nanoparticles based on Aquifex aeolicus lumazine synthase (AaLS) to simultaneously display HER2-binding nanobodies (aHER2Nb; A10 or 2Rb17C) and/or TRAIL on their surface. Both single- and dual-ligand AaLS protein nanoparticles retained an intact cage architecture and showed strong binding to HER2-overexpressing breast cancer cells. Although SK-BR3 and MDA-MB-453 cells were resistant to soluble TRAIL, TRAIL-presenting AaLS (AaLS/TRAIL) markedly enhanced cytotoxicity by promoting death receptor clustering. Unexpectedly, dual-ligand AaLS protein nanoparticles (AaLS/TRAIL/A10 and AaLS/TRAIL/2Rb17C) exhibited biphasic cytotoxicity; low doses synergistically enhanced apoptosis in HER2-positive cells, whereas higher doses reduced efficacy, likely due to the activation of survival signaling. These results highlight the importance of dose optimization for maximizing the use of TRAIL-based targeted therapies.