Journal
CYTOTHERAPY
Volume 16, Issue 9, Pages 1257-1269Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.jcyt.2014.05.022
Keywords
piggyBac-transposon; CAR; Ph(+)ALL; tyrosine kinase inhibitor; T315I
Categories
Funding
- JSPS KAKENHI [23591533, 24791050, 26461574]
- Takeda Science Foundation
- Japan Leukemia Research Fund
- Grants-in-Aid for Scientific Research [23591533, 24791050, 26461574] Funding Source: KAKEN
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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 CART cells with seven Ph(+)ALL cell lines including three TM-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines. Results. We obtained similar to 1.3 x 10(8) CART cells (CD4(+), 25.4%; CD8(+), 71.3%), co-expressing CD45RA and CCR7 up to similar to 80%. After 7-day co-culture, CART 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 CART 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. CART 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.
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