WBP-3425

Acquired resistance to third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge in non-small cell lung cancer (NSCLC), with ∼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 anti-tumor activity.

scRNA-seq and multicolor fluorescence staining were performed on NSCLC patient samples to analyze TME changes post-EGFR-TKI resistance. A triple therapy combining BC3448 (EGFR/CD3 BiTE), Tucidinostat (HDAC inhibitor), and WBP3425 (4-1BB agonist) was tested using in vitro co-culture assays, syngeneic cell-derived xenograft (CDX) models in humanized NOG-EXL mice, and multi-omics analyses.

scRNA-seq revealed reduced T-cell infiltration/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 due to 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/M2 macrophages. CD40/CD40L axis activation between T cells and monocyte-derived macrophages was critical for TME remodeling, with spatial profiling showing 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 NSCLC patient (>2 years of stable disease), presenting a translational potential of this approach.

This study establishes a novel triple therapy that overcomes the limitations of BiTEs in cold and immunosuppressive TMEs and provides an immunomodulatory approach to addressing third-generation EGFR-TKI resistance.