Figure 8.

Differential target cell lysis by AdCAR-T

A) Target-antigen expression profile of CD19, CD10, and CD138 on freshly isolated peripheral B cells and BCP-ALL NALM6 tumor cells determined by flow cytometry. B–C) AdCAR-T were incubated for 24 h with NALM6 and freshly isolated peripheral B-cells at an E:T ratio of 2:1:1 (AdCAR-T: NALM6: B cells) in the presence or absence of 25 pg/mL LLE-CD19 mAb, 50 pg/mL LLE-CD10 mAb, or 200 pg/mL CD138-LLE mAb or the combination thereof or 10 ng/mL LLE-CD19 mAb and analyzed by flow cytometry. B) Schematic illustration of specific lysis based on differential expression profiles (upper panel). As illustrated, the combination of below threshold concentrations of AMs against the antigens CD19, CD10, and CD138, all expressed on NALM6, causes AdCAR-T activation and lysis of NALM6 while sparing B-cells only expressing CD19 (middle upper panel). In contrast, above threshold concentration of CD19 targeted AM mediates complete lysis of both NALM6 and B-cells. Representative flow cytometry plots of (n = 3) independent experiments in triplicates from three different donors are shown (lower panel). AdCAR-T are identified by CD3 expression, NALM6 by mCherry expression, B-cells by being negative for CD3 and mCherry. C) Viability of NALM6 and B cells after incubation at indicated conditions was quantified. Data shown represent mean ± SEM of (n = 4) independent experiments from four different donors in triplicates. In C) significance was determined by unpaired, two-tailed Mann-Whitney test (n = 4, p < .0001). (n.s.) not significant.