Anti-leukemic potency of piggyBac -mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

Abstract Background aims To develop a treatment option for Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR). Methods A CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac -transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15–containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph+ ALL cell lines including three TKI-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines. Results We obtained ∼1.3 × 108 CAR T cells (CD4+ , 25.4%; CD8+ , 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor–related apoptosis-inducing ligand and interleukin-2. Conclusions We generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac -transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph+ ALL regardless of T315I mutation. PiggyBac -mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph+ ALL.