Figure 4
Functional T cell bridging and target cell engagement by anti-mesothelin scFvs isolated from dCI selection experiments. (a) Induction of NFAT-driven expression of luciferase in Jurkat reporter cells transduced with anti-mesothelin CARs employing different scFv clones. Luciferase activity was measured after 24 h of co-culture with mesothelin expressing target cells (H-226, AsPC-1) or meso-negative Raji B cells. CD19 and P4 CARs are included as positive controls for CD19 and mesothelin respectively. NT, non-transformed control. (b) Induction of NFAT-driven expression of luciferase in Jurkat reporter cells in the presence of H-226 (left), and AsPC-1 (right) target cells and soluble T cell engagers. Dashed lines represent baseline stimulation in the presence of engager molecule and HEK293-6E mesothelin-negative cells. (c) Mesothelin specific T cell engager constructed from clone HS201 redirects primary human CD8+ T cells to kill H-226 tumor target cells. Image-based acquisition of Cytotox Red fluorescence reports the extent and real-time kinetics of killing. Baseline killing was determined against irrelevant meso-negative A673 cells using 750 pM engager. (d) Time-dependent killing of H-226 cells in the presence of 2 nM HS201 engager by primary human T cells (Pan-T purified; 14 days post isolation). Dead cell clusters emit red fluorescence.
