TRAIL-based anti-cancer therapy in breast cancer utilizing multivalent protein cage nanoparticle with anti-HER2 nanobody

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent known for its ability to induce apoptosis selectively in cancer cells, sparing normal tissues. However, the application of TRAIL for clinical use faces some limitations due to the development of TRAIL resistance in cancer cells, reducing the efficacy of TRAIL standalone therapy. So, to address this challenge, we engineered protein cage nanoparticle AaLS to deliver a stronger apoptotic signal of TRAIL through its multivalent architecture, displaying multiple TRAIL molecules on the surface of a single AaLS, thus increasing the local concentration and inducing clustering and activation of death recreators, thereby overcoming TRAIL resistance. Our result demonstrates that AaLS/TRAIL effectively overcomes TRAIL resistance in SK-BR3 and MDA-MB453 by delivering stronger apoptotic signals. Also, to specifically target HER2 overexpressing breast cancer cells, anti-HER2 nanobodies A10 and 2Rb17C were used. A10 and 2Rb17C displayed high binding affinity showing KD values in lower nanomolar range, facilitating targeted delivery of TRAIL using AaLS/TRAIL/aHER2Nb dual functionalized nanoparticle system. In vitro studies demonstrate that dually displaying TRAIL and HER2Nb on the surface of AaLS resulted in enhanced cytotoxicity even in lower doses of TRAIL, due to the strong binding of AaLS containing TRAIL, allowing it to transmit stronger apoptotic signals, leading to enhanced caspase activations and cell death. However, in higher concentrations, due to intensified signal transmission, it induced the formation of secondary complex, leading to fractional survival of cancer cells. These findings suggest that displaying TRAIL on the surface of AaLS potentiates the therapeutic effect of TRAIL, overcoming TRAIL resistance by enhancing its ability to cluster death receptors on the surface of cancer cells. Additionally, by co-displaying TRAIL and aHER2Nb on the surface of AaLS, therapeutic efficacy is further enhanced due to the targeted delivery of TRAIL to HER2-positive breast cancer cells, maximizing its cytotoxic effect. However, when TRAIL is delivered with high affinity cancer-targeting molecules like A10, the therapeutic dose should be carefully adjusted to avoid the formation of a secondary complex, which induces fraction survival of partial cancer mass, and affects the therapeutic outcomes of TRAIL-base therapy.