Figure 2.
Kinetics, potency and durability of target cell lysis activity of in vitro expanded CCR4 CAR T cells. The cell line ATL41214 has been derived from a patient with HTLV-1 associated chronic adult T cell leukemia and the BJAB cell line that has been derived from a patient with Burkitt lymphoma. BJAB/CCR4 cell line is a stable transfectant of parental BJAB that expresses human CCR4. All three cell lines are tagged with luciferase and the surface expression of CCR4 on these cell lines were determined by flow cytometry using an anti-human CCR4-APC antibody (clone D8SEE from eBioscence) depicted in red or an isotype matched control antibody depicted in blue (panel A). CAR T cells that were expanded in vitro for 12 days following lentiviral transduction in the presence of IL-2, and CD3/CD28 microbeads were co-cultured with ATL41214 cells in triplicate at indicated effector:target ratios for indicated durations of time. The percentage of cell lysis was calculated as indicated in the Methods section after the addition of D-luciferin and measuring bioluminescence using a microplate counter (panel B). Long term retention of lytic activity was determined by in vitro expansion of CAR T cells for a period of 8 weeks as described in the Methods section and then co-culturing with ATL41214 target cells for a period of 20 hours at indicated effector:target ratios shown on the x-axis (panel C). In Panel D, BJAB and BJAB/CCR4 cells were co-cultured with CCR4 CAR T cells for a period of 24 hrs and the cytolytic activity was measured by biophotonic cytotoxicity assay.