Protein Nanoparticles Simultaneously Displaying TRAIL and EGFR-Binding Ligands Effectively Induce Apoptotic Cancer Cell Death and Overcome EGFR-TKI Resistance in Lung Cancer

Lung cancer remains one of the most lethal cancers globally, with nonsmall cell lung cancer (NSCLC) representing the predominant subtype. Despite significant advancements in targeted therapies, overcoming therapeutic resistance in NSCLC remains a significant challenge, particularly in cases resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Here, we developed target-specific, apoptosis-inducing protein nanoparticles using <i>Aquifex aeolicus</i> lumazine synthase (AaLS), which were engineered to simultaneously display multiple TRAIL molecules and EGFR-binding ligands, including EGFR affibody (Afb) or anti-EGFR nanobodies (7D12, 9G8, and EgB4). These nanoparticles utilize the EGFR-binding ligand to enhance selective targeting of EGFR-overexpressing lung adenocarcinoma (PC9, HCC827, A549) and squamous cell carcinoma (H226) cells, regardless of mutations within the intracellular kinase domain of EGFR, which are primarily driven by tyrosine kinase inhibitors commonly used as first-line treatments in lung cancer therapy. The codisplayed EGFR-binding ligands enhance the attachment of TRAIL-displaying protein nanoparticles to cancer cells by stabilizing interactions with EGFR, promoting cell surface clustering of TRAIL molecules and improving TRAIL engagement with death receptors (DRs). This sustained interaction significantly amplifies TRAIL-mediated apoptotic cancer cell death signaling, effectively overcoming both TRAIL and EGFR-TKI resistance in NSCLC cells. Our findings suggest that dual ligand-displaying protein nanoparticles targeting DRs and EGFR represent a promising therapeutic strategy to potentiate TRAIL efficacy and circumvent EGFR-TKI resistance in NSCLC.