Figure 2.

(A) Schematic design of the lentiviral vector for expression of the second-generation CARs. The constructs contain CD19, EGFR or CD44v6 scFv, CD34 hinge domain, CD28 transmembrane and intracellular domain, CD3ζ cytosolic domain and blue fluorescent protein (BFP). (B) Enrichment of CAR-positive T-cells. Primary human T-cells were lentivirally transduced with constructs expressing CD19, EGFR, or CD44v6 CARs. Three days after transduction, CAR-positive T-cells were stained with CD34 microbeads (Miltenyi) and finally enriched by magnetic cell sorting. Different fractions (pre, flow-through, post MACS) were analyzed by flow cytometry using the QBEND-PE antibody ( Supplementary Table S1 ). CAR expression (x-axis) is plotted against the absolute cell count (y-axis). Illustrations were gated with the percentage of positive cells. Data are depicted as mean ± SEM for three different experiments.