Fig. 1

Validation of CD5-IL15/IL15sushi CAR construct. a Schematic representation of recombinant lentiviral vector encoding CD5 CAR linked with the P2A self-cleaving sequence to the IL-15/IL-15sushi domain of the IL-15 alpha receptor. Expression is driven by the spleen focus-forming virus (SFFV) promoter. The IL-15/IL-15sushi portion is composed of IL-2 signal peptide fused to IL-15 and linked to sushi domain via a 26-amino acid poly-proline linker. b Activated T cells from human peripheral blood buffy coat were transduced with either control (left) or CD5-IL15/IL15sushi CAR (right) viral supernatant from transfected HEK-293FT cells. 48 hours after transduction, cells were harvested, washed, and moved to tissue culture plates with fresh media and IL-2. After 2 days incubation, cells were harvested and stained with goat-anti-mouse F(Ab’), mouse anti-human CD3 and CD5, and against the RTX-binding epitope and analyzed by flow cytometry). Staining with F(Ab’) showed ~40% of T cells expressed CAR following transduction. T cells are displayed in red and transduced population is circled and colored green. c Transduced cells demonstrate normal CD3 expression and downregulation of CD5. T cells are colored red and circled. d Transduced cells show expression of the rituximab-binding epitopes on the cell surface. T cells are displayed in red and transduced population is circled and colored purple. e A co-culture experiment using CD5-positive MOLT4 cells was performed at E:T ratio of 2:1 for 24 hours. Cells were analyzed by flow cytometry using mouse anti-human CD5 and CD3 labeling. During 24-hour co-culture experiments, CD5IL-15/IL-15sushi CAR T cells showed profound killing (92%) of MOLT cells compared to control T cells. MOLT4 cells are blue, T cells are red, and target population is circled