Fig. 3

Construction and identification of the second-generation CARs targeting CD19 and CD20 antigen. A Schematic illustration of the construction of mRNA encoding CD19 CARs and CD20 CARs utilized IVT. B Schematic diagrams of the CD19 CAR-mRNA and the CD20CAR-mRNA. FMC63(anti-CD19) scFv and LEU16 (anti-CD20) scFv were linked to the CD8α hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain, respectively. C Denatured agarose gel electrophoresis of the CAR-mRNA. Lane (1) represents the CD19 CAR-mRNA group, lane (3) represents the CD20 CAR-mRNA group, and lane (2) represents mRNA Marker (6000nt). D Detection of CAR expression on NK cells by flow cytometry. CAR expression was detected by using one or two of four staining reagents: PE-Labeled monoclonal anti-FMC63 Antibody (column 1), Biotinylated Human CD20/MS4A1 full length protein followed by Streptavidin Protein-Alexa Fluor 647 (column 2), or FITC-Labeled Monoclonal Anti-FMC63 Antibody (column 3). The expression percentage of CAR on NK cells is noted on the right of each histogram. EGFP-transduced cells served as an additional negative control. E CD19 CARs and CD20 CARs expression kinetics on dual-target CAR-NK over 4 days. IVT in vitro transcription, scFv single-chain fragment variable regions, Cap1 Cap1 structure, UTR untranslated region