![]() An alternative, effective and safe option is needed for patients with TKI-resistant Ph +ALL.Īdoptive immunotherapy with the use of T cells expressing a chimeric antigen receptor (CAR) targeting CD19 (CD19.CAR) is a novel approach for the treatment of B-cell malignancies. Consequently, the outcome of patients with T315I mutations is very poor ( 3). In particular, T315I–mutated leukemic clones are resistant to all approved TKIs (imatinib, nilotinib and dasatinib). Although the introduction of BCR-ABL tyrosine kinase inhibitors (TKIs) into conventional chemotherapy has improved the outcomes ( 1), TKI resistance, mainly because of the emergence of mutations in the BCR-ABL kinase domain, remains a problem in a substantial proportion of patients with Ph +ALL ( 2). Philadelphia chromosome–positive (Ph +) acute lymphoblastic leukemia (ALL) characterized by the existence of a BCR-ABL fusion gene is observed in 20–30% of adult patients and in 2–3% of pediatric patients with ALL ( 1). PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph(+)ALL. CAR T cells exhibited marked cytotoxicity against Ph(+)ALL regardless of T315I mutation. 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.ConclusionsWe 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. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. 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. 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.ResultsWe obtained ∼1.3 × 10(8) CAR T cells (CD4(+), 25.4% CD8(+), 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. 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. Background aimsTo 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).MethodsA 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.
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