Preclinical activity of Mesothelin-specific CAR.CIK lymphocytes against Cholangiocarcinoma

Advanced and unresectable cholangiocarcinoma (CCA) is currently an unmet clinical need, with no effective therapies available. Here we explored the preclinical anti-CCA activity of a cellular immunotherapy approach with cytokine-induced killer lymphocytes (CIK) redirected with a chimeric antigen receptor (CAR) against mesothelin (MSLN), recently emerging as a relevant CCA target. MSLN-CAR.CIK were successfully generated from patients’ PBMC and engineered with a second-generation MSLN-CAR. As tumor targets, we employed and characterized a panel of CCA cell lines. CAR expression and phenotype of CAR.CIK were analyzed by flow cytometry. MSLN-CAR.CIK killing ability in 2D models was evaluated at different effector:target ratio (E:T) by flow cytometry. MSLN-CAR.CIK displayed significantly superior in vitro cytolytic activity against CCA as compared with NTD.CIK, even at low E:T ratios. In order to recapitulate the complexity of CCA, 3D tumor spheroids were developed from CCA cells bearing a reporter gene (RFP) and co-incubated with effector cells. Images were acquired at different time-points using fluorescence microscopy. The tumor recruitment of MSLN-CAR.CIK and infiltration in CCA spheroids were analyzed using confocal microscopy. The intense tumor killing activity, along with tumor localization and infiltration, by MSLN-CAR.CIK was confirmed also against CCA 3D spheroids resulting significantly superior as compared to unmodified NTD.CIK. This study demonstrated that MSLN-CAR.CIK are effective against CCA in both 2D models and 3D structures, supporting MSLN as a valuable target for advanced CCA. Our findings provide reliable translational rationale to explore cellular immunotherapy with MSLN-CAR.CIK in clinical studies within the field of advanced CCA.