Anti GPC3 CAR T lymphocytes effectively target NSCLC, including chemoresistant stem like cells

Purpose: The aim of our study was to explore a preclinical strategy of cell-based immunotherapy using anti-GPC3 CAR-lymphocytes in the context of lung cancer, with a particular focus on eradicating a chemoresistant tumor subset displaying stem-like features. Procedures : GPC3 expression was confirmed on a large series of surgical specimens from squamous non-small cell lung cancer (NSCLC) and on a panel of tumor cell lines. Functional assays were performed using CAR-T lymphocytes engineered from healthy donors with an anti-GPC3 2 nd generation CAR construct. To visualize the cancer stem cell (CSC) subset, tumor cell lines were transduced with a lentiviral CSC-detector vector in which GFP expression is driven by the stem-gene promoter OCT4, rendering green the cells with stem-like features. Results: GPC3 expression was confirmed on 21/50 squamous NSCLC samples and 5 tumor cell lines (EBC1; H226; NCI-H23; NCI-H596; A-427), by immunohistochemistry and flow cytometry respectively. Anti-GPC3 CAR-T lymphocytes (mean surface CAR expression 40%) effectively killed all tested tumor cell lines (n = 5), with mean specific cytotoxic activity ranging from 71% ±13 to 42%±21 at effector-to-target (E:T) ratios of 10:1 and 2:1, respectively (p<0.0001 vs unmodified T cell controls), while showing no activity against the GPC3-negative A-549 cell line. The cytotoxic activity fully included the putative cancer stem cell (CSC) compartment (GFP⁺), previously shown with increased resistance to cisplatin in vitro, but also displayed preferential killing toward this subset compared with GFP⁻ differentiated counterparts resulting in approximately twofold greater reduction (p<0.05 , E/T 5:1). Conclusions: GPC3 emerges as an effective and promising target for cell-based immunotherapy in lung cancer. Although preliminary, our data support the concept that anti-GPC3 CAR-T cells can effectively eliminate the CSC-enriched tumor compartment, which is believed to drive chemoresistance and recurrence, thus providing a rationale for future clinical development of cell-based immunotherapies in advanced or relapsed non-responsive lung cancer.