Generation and characterization of CD70-CAR T cells. (A) Representative histograms of CD70-CAR expression levels after transduction of activated T cells. CD70scFv-CAR T cells were detected by staining with biotin-labeled protein L, followed by staining with APC-conjugated streptavidin. CD27z-CAR expression was confirmed by detection of a truncated CD19 using an APC-conjugated CD19 specific antibody. (B) The frequency of CD70-CAR expressing T cells of 4 different donors. (C) The frequency of viable cells for CD70-CAR and NT T cells of 4 different donors, as determined by forward vs side scatter gating. (D) Fold change in the number of CD70-CAR T cells in culture during the manufacturing process without antigen stimulation. CAR T cells from 4 different donors were counted in weekly intervals. (E) Phenotypical characterization of CD70-CAR T cells by flow cytometry. The frequency of CD70-CAR T cells with a less (naive/CM T cells; left) and a more (effector memory/terminally differentiated effector memory cells reexpressing CD45RA [EM/EMRA] T cells; right) differentiated phenotype, as determined by the expression of CD45RO and CCR7 in 4 different donors. The colored bars represent the mean of results from 4 different donors, and the error bars indicate the standard deviation (SD). *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant, by unpaired Student t test. (F-G) A chromium-51 release assay was used to determine antigen-dependent lysis of CD70-CAR T cells against CD70-positive AML cell lines (Molm-13, THP-1) (F) and KG-1a (CD70⁻) cells (G) as the control. The graph shows the mean results of 3 technical replicates from 4 different donors, and the error bars indicate the SD. P values were calculated by 2-way ANOVA and are shown in the table below the graphs. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.