Enhanced homing and efficacy of HER2-CAR T cells via CXCR5/CCR6 co-expression for HER2-positive NSCLC.

Chimeric antigen receptor T-cell (CAR T) therapy development represents a promising therapeutic strategy for HER2-positive non-small cell lung cancer (NSCLC), a subtype accounting for 1-5% of NSCLC cases. However, the clinical efficacy of CAR T cells remains limited by poor tumor infiltration. Here, we identify NSCLC-specific overexpression of the CXCL13 and CCL20 chemokines within the tumor microenvironment (TME) and develop a dual chemokine receptor strategy to overcome this barrier. Western blotting and qRT-PCR were used to quantify chemokine receptor expression (CXCR5, CCR6) in NSCLC. Cytotoxicity and antigen recognition sensitivity of CXCR5-CCR6-HER2-CAR T cells against target cells were assessed using in vitro co-culture assays. In vitro proliferation and migration capacities of these engineered T cells were also evaluated. Anti-tumor activity was determined through in vivo animal experiments. We demonstrate for the first time that HER2-targeted CAR T cells co-expressing the chemokine receptors CXCR5 and CCR6 selectively respond to CXCL13 and CCL20, which are highly expressed in the NSCLC TME. This dual chemokine receptor co-expression strategy has not been previously applied to solid tumors. The CXCR5/CCR6 pairing synergistically enhanced the antitumor activity of HER2-CAR T cells in both in vitro and in vivo models. Furthermore, CXCR5 and CCR6 co-expression significantly improved the in vitro cytotoxicity, antigen recognition sensitivity, proliferation, and migration of HER2-CAR T cells. In vivo, this modification enhanced HER2-CAR T cell survival, expansion, and tumor infiltration. CXCR5/CCR6 co-expression establishes a novel therapeutic paradigm for refractory HER2-positive NSCLC. Its modular design facilitates rapid clinical translation and adaptation to other chemokine-defined solid tumors.