Bispecific T-cell Engager (CD3 × EGFR)-Based Triplet Therapy Unlocks CD40/CD40L Crosstalk to Revert Immunosuppression in Third-Generation EGFR-Tyrosine Kinase Inhibitor–Refractory NSCLC

INTRODUCTION: Acquired resistance to third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge in NSCLC, with approximately 50% of cases lacking precise resistance mechanisms. This study investigates the immunosuppressive tumor microenvironment (TME) driving resistance and develops a novel triple-combination therapy to restore T-cell antitumor activity. METHODS: Single-cell RNA sequencing and multicolor fluorescence staining were performed on NSCLC patient samples to analyze TME changes post-EGFR-TKI resistance. A triple therapy combining BC3448 (EGFR and CD3 bispecific T-cell engager), tucidinostat (histone deacetylase inhibitor), and WBP3425 (4-1BB agonist) was tested using in vitro co-culture assays, syngeneic cell-derived xenograft models in humanized NOG-EXL mice, and multi-omics analyses. RESULTS: Single-cell RNA sequencing revealed reduced T-cell infiltration or activation and increased immunosuppressive myeloid cells in resistant NSCLC. BC3448 monotherapy activated T cells and induced tumor cell apoptosis in vitro but was limited in vivo because of myeloid-driven immunosuppression. The triple therapy significantly enhanced tumor regression in osimertinib-resistant models (tumor growth inhibition >70%, p < 0.001), promoted CD8+ effector T-cell differentiation, and suppressed Tregs and M2 macrophages. CD40-CD40L axis activation between T cells and monocyte-derived macrophages was critical for TME remodeling, with spatial profiling revealing increased CD40L+ T-cell and CD40+ macrophage proximity, correlating with higher IFN-γ and reduced angiogenesis. A durable response to BC3448 monotherapy was observed in an immunotherapy-resistant patient with NSCLC (>2 y of stable disease), presenting a translational potential of this approach. CONCLUSIONS: This study establishes a novel triple therapy that overcomes the limitations of bispecific T-cell engagers in cold and immunosuppressive TMEs and provides an immunomodulatory approach to addressing third-generation EGFR-TKI resistance.