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

Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, Maeda Y, Sato Y, Okabe S, Inukai T, Sugita K, Wilson MH, Rooney CM, Koike K
Cytotherapy. 2014 16 (9): 1257-69

PMID: 25108652 · PMCID: PMC4948190 · DOI:10.1016/j.jcyt.2014.05.022

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 × 10(8) 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.

Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

MeSH Terms (22)

Antigens, CD19 Cancer Vaccines Cell Line, Tumor Cell Proliferation Culture Media, Serum-Free Cytotoxicity, Immunologic DNA Transposable Elements Drug Resistance, Neoplasm Genetic Engineering Genetic Vectors Humans Immunotherapy, Adoptive Interleukin-2 Interleukin-15 Leukemia, Myelogenous, Chronic, BCR-ABL Positive Mutation Protein Kinase Inhibitors Receptors, Antigen, T-Cell Recombinant Fusion Proteins T-Lymphocytes TNF-Related Apoptosis-Inducing Ligand Up-Regulation

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