Figure 5.

Sequential combinatorial targeting mediated by AdCAR-T

AdCAR-T were incubated for 48 h with an equicellular mix of JeKo-1 ^WT, JeKo-1,^CD19KOJeKo1^CD20KO and Jeko-1^CD19/CD20KO at an E:T (AdCAR-T to JeKo-1 WT and KO variants) of 2:1 in the presence of either 10 ng/mL LLE-CD19 mAb (upper path) or 10 ng/mL LLE-CD20 mAb (lower path). After 48 h, distribution of AdCAR-T and JeKo-1 variants was determined by flow cytometry and either 10 ng/mL LLE-CD19 mAb or 10 ng/mL LLE-CD20 mAb was added to additional samples of each condition leading to the following four conditions: 1) LLE-CD19 mAb plus at 0 h plus LLE-CD20 mAb at 48 h (top plot, right panel), 2) LLE-CD19 mAb plus at 0 h plus LLE-CD19 mAb at 48 h (upper middle plot, right panel), 3) LLE-CD20 mAb plus at 0 h plus LLE-CD20 mAb at 48 h (lower middle plot, right panel) and 4) LLE-CD20 mAb plus at 0 h plus LLE-CD19 mAb at 48 h (bottom plot, right panel). After additional 24 h (total incubation time 72 h) distribution of AdCAR-T and JeKo-1 variants was determined by flow cytometry. The experiment demonstrates that AdCAR-T can be redirected to a second-target antigen sequentially even in the presence of the LLE-AM against the first-target antigen. Data display representative flow cytometric plots of (n = 2) independent experiments in triplicates of two different donors.