A Multi-Targeting Chimeric Antigen Receptor-T Cell Platform to Overcome Antigen Heterogeneity in the Treatment of Non-Small Cell Lung Cancer.

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors is limited by antigen heterogeneity and T cell exhaustion. To address these limitations, we develop a novel multi-targeting "Bicephali" CAR-T platform featuring a dual-transmembrane protein with two distinct extracellular antigen-binding domains and a shared intracellular 4-1BB co-stimulatory/CD3ζ signaling domain. CD276 and NKG2D ligands (NKG2DLs) show high and heterogeneous expression in non-small cell lung cancer (NSCLC) and are undetectable in normal tissues. Bicephali CAR-T cells targeting CD276 and NKG2DLs demonstrate superior tumoricidal activity against NSCLC than conventional BB002 CAR-T cells in vitro and in vivo, together with improved immunological synapse formation and mitochondrial metabolic fitness. In homogeneous NSCLC models co-expressing CD276 and NKG2DLs, Bicephali CAR-T cells achieve prolonged survival outcomes compared to monospecific CAR-T cells. In antigenically heterogeneous NSCLC, Bicephali CAR-T cells more consistently control tumors and prolong survival, whereas monospecific CAR-T cells fail to eliminate tumors following antigen loss. Mechanistically, improved mitochondrial fitness and antioxidant capacity in Bicephali CAR-T cells are associated with sustained T cell function, preserved stem-like differentiation, and durable effector responses. These findings support a multi-targeting CAR-T approach to address antigen heterogeneity in NSCLC and potentially other solid tumors.