Fig. 6

MAGE-A1-CAR-T cell construction and identification. a The lentiviral vector construct of MAGE-A1-CAR (mCAR), the TM transmembrane portion. The mCAR is composed of the MAGE-A1-scFv linked to a human CD8a leader, CD8a hinge, and the transmembrane domain fused to an intracellular signaling domain derived from human CD137 and CD3ζ. b CD3ζ was detected by western blotting in HEK-293T cells transfected with mCAR. HEK-293T cells transfected with an unrelated-CAR were used as a positive control. Untransfected 293T cells (blank) were employed as a negative control. c A sandwich ELISA was performed to evaluate the binding ability of mCAR to MAGE-A1. 293T cells transfected with mCAR and unrelated-CAR were enrolled. Untransfected 293T cells were employed as control (blank). d The transfection efficiencies of mCART and unrelated-CART by GFP (ZsGreen) were 77.0% and 74.3%, respectively. In comparison, the transfection efficiencies of mCART and unrelated-CART by MAGE-A1-PE staining were 65.2% and 1.22%, respectively. e Flow cytometry analysis showed that CD3-positive, CD4-positive, and CD8-positive T cells in mCART were obtained from PBMCs by magnetic bead separation, activated by CD3/CD28 co-stimulation and transfected by mCAR lentivirus. An unrelated-CART was used as a positive control