Fig. 1

MET-CAR design, expression, and characterization. (A, B) Schematic drawing of second-generation MET-CARs. #948: MET-CAR with DO24 scFv as binding domain; #949: MET-CAR with DO24 scFAb as binding domain. VL: Variable Light domain; VH: Variable Heavy domain; CL: constant region from human Igκ chain; CH1, CH2, CH3: constant regions 1, 2, 3 from human IgG1 chain; CD28: transmembrane and juxtamembrane regions from human CD28; CD3ζ: zeta region from human CD3. Black lines represent the plasma membrane. (C) Schematic drawing of the lentiviral vector (LV) expressing MET-CARs. In grey vector backbone in color the bidirectional expression cassette. P-PGK: human phosphoglycerate kinase promoter; MET-CAR: cDNA encoding for #948 or #949 MET-CAR; Pmin-CMV: minimal promoter from the cytomegalovirus; eGFP: cDNA encoding for the enhanced Green Fluorescent Protein. Pmin-CMV and eGFP are in antisense with respect to the P-PGK and MET-CAR. Arrows indicate the divergent RNA transcripts originating from the bidirectional internal promoter. (D) Representative flow-cytometry analysis of MET-CAR and eGFP expression in PBMC transduced with #948-LV or #949-LV. Numbers in the plots indicate the percentage of events for each quadrant. (E) Representative flow-cytometry analysis of immune-phenotype markers in PBMC transduced with MET-CAR LVs. The table reports the percentage of expression for each marker in the total cell population. NTD: Not-transduced PBMC