title: Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Saito
authors.affiliation: Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan. Electronic address: yxnakaza@shinshu-u.ac.jp.
authors.forename: Yozo
authors.initials: Y
authors.lastname: Nakazawa
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Sueki
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Ryu
authors.initials: R
authors.lastname: Yanagisawa
authors.affiliation: Department of Hematology, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Maeda
authors.affiliation: Japanese Red Cross College of Nursing, Tokyo, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Okabe
authors.affiliation: Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Inukai
authors.affiliation: Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi, Japan.
authors.forename: Kanji
authors.initials: K
authors.lastname: Sugita
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Wilson
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Cliona M
authors.initials: CM
authors.lastname: Rooney
authors.affiliation: Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Koike
mesh_terms: D018941:Antigens, CD19 / Q000235:genetics / Q000378:metabolism; D019496:Cancer Vaccines; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D016895:Culture Media, Serum-Free / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D019409:Interleukin-15 / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy*; D009154:Mutation / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000502:physiology* / Q000637:transplantation; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D018941:Antigens, CD19; D019496:Cancer Vaccines; D016895:Culture Media, Serum-Free; D004251:DNA Transposable Elements; D019409:Interleukin-15; D007376:Interleukin-2; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand
keywords: CAR; Ph(+)ALL; T315I; piggyBac-transposon; tyrosine kinase inhibitor
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