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

Abstract

ABSTRACT 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.