Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia |
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| Institution: | 1. Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan;2. Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan;3. Department of Hematology, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Japan;4. Japanese Red Cross College of Nursing, Tokyo, Japan;5. First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan;6. Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi, Japan;7. Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University School of Medicine, Nashville, Tennessee, USA;8. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA;1. Peking University People’s Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, National Clinical Research Center for Hematologic Disease, No. 11 South Street of Xizhimen, Xicheng District, Beijing 100044, China;2. Peking-Tsinghua Center for Life Sciences, Beijing 100871, China;3. Lu Daopei Hospital, Langfang, Hebei 065201, China;4. Lu Daopei Institute of Hematology, Beijing 100176, China;5. Immunochina Pharmaceuticals Co., Ltd., Beijing 100089, China;6. Sartorius Stedim North America, Inc., 565 Johnson Avenue Bohemia, New York, NY 11716, USA;1. College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China;2. Shanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, China;3. Eastern Hepatobiliary Surgery Hospital, The Second Military University, Shanghai 201805, China;1. Department of Cardiovascular Research, Shinshu University Graduate School of Medicine, Matsumoto, Japan;2. Department of Regenerative Medicine, Research Institute of Biomedical Research and Innovation, Kobe, Japan;1. Cord & Marrow Transplant Facility, Kids Cancer Centre, Sydney Children''s Hospital, Sydney, Australia;2. Faculty of Medicine, University of New South Wales, Sydney, Australia;3. Children''s Cancer Institute Australia, Sydney, Australia;4. Westmead Millennium Institute, University of Sydney, Sydney, Australia;1. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;2. Immunology Program, Sloan Kettering Institute, New York, NY 10065, USA |
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| Abstract: | 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. 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.ResultsWe 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.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. 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. |
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| Keywords: | CAR tyrosine kinase inhibitor T315I |
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