Fig. 1

Construction of effector cells and target cells. a Schematic representation of dCAR-engineered T cells. Engineered T cells endowed with the dCAR structure are activated in the presence of CEA and MSLN and eliminate target cells. b Structure of the plasmids used to construct modified T cells. After lentivirus transfection, effector cells yield expression of the novel dCAR structure consisting of anti-MSLN scFv, cytoplasmic domain of 4/1BB, anti-CEA scFv, cytoplasmic domain of CD3ζ, and green fluorescence protein (GFP) sequences. Simultaneously, we also constructed two negative controls, Cζ-CAR and MBB-CAR, and two positive controls, CEA-CAR and MSLN-CAR. c CAR-engineered T cells were successfully constructed by lentivirus transfection. With endogenous GFP expression, we measured the transduction efficiency by flow cytometry assays, quantifying fractions of CAR positive-CD4⁺ T and CAR positive-CD8⁺ T cells in different CAR groups. d Antigen expression on the cognate and non-cognate tumor cells. By flow cytometry assays, AsPC-1 cells highly expressed both CEA and MSLN while PANC-1 cells did not express the above antigens. In addition, HT29 cells could only highly express CEA and U87 cells only express MSLN. e Detection of the transfection efficacy of modified tumor cells. After puromycin screening, the transduction efficiencies were determined to be 93.51%, 92.17%, 83.20%, and 81.23% by flow cytometry assays for AsPC-1-RFP cells, PANC-1-RFP cells, HT29-RFP, and U87-RFP, respectively