Saito Shoji, Nakazawa Yozo, Sueki Akane, Matsuda Kazuyuki, Tanaka Miyuki, Yanagisawa Ryu, Maeda Yasuhiro, Sato Yuko, Okabe Seiichi, Inukai Takeshi, Sugita Kanji, Wilson Matthew H, Rooney Cliona M, Koike Kenichi
Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
Cytotherapy. 2014 Sep;16(9):1257-69. doi: 10.1016/j.jcyt.2014.05.022.
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).
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.
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.
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.
为开发一种针对对酪氨酸激酶抑制剂（TKIs）耐药的费城染色体阳性急性淋巴细胞白血病（Ph(+)ALL）的治疗方案，我们评估了经非病毒工程改造以表达CD19特异性嵌合抗原受体（CAR）的T细胞的抗白血病活性。
通过使用piggyBac转座子和4-D核转染系统，将CD19.CAR基因导入来自10 mL健康供者血液的单个核细胞中。用CD3/CD28抗体刺激经核转染的细胞，磁选CD19.CAR，并在含白细胞介素-15的无血清培养基中与自体饲养细胞共培养21天。为评估其细胞毒性效力，我们将CAR T细胞与7种Ph(+)ALL细胞系（包括3种TKI耐药（T315I突变）细胞系）以1:5或更低的效应细胞与靶细胞比例在无细胞因子的情况下共培养。
我们获得了约1.3×10⁸个CAR T细胞（CD4⁺，25.4%；CD8⁺，71.3%），共表达CD45RA和CCR7的比例高达约80%。共培养7天后，CAR T细胞在1:5和1:10的比例下根除了所有肿瘤细胞，在1:50的比例下显著减少了肿瘤细胞数量。动力学分析显示，在存在肿瘤细胞的情况下，CAR T细胞在20天的培养期内增殖高达37倍。暴露于肿瘤细胞时，CAR T细胞可瞬时且可重复地上调转基因以及肿瘤坏死因子相关凋亡诱导配体和白细胞介素-2的表达。
我们通过使用piggyBac转座子、4D核转染仪和无血清/无异种/无肿瘤细胞/无病毒培养系统，从10 mL血液中产生了临床相关数量的CAR T细胞。CAR T细胞对Ph(+)ALL表现出显著的细胞毒性，无论是否存在T315I突变。piggyBac介导的CD19特异性T细胞疗法可能为耐药Ph(+)ALL提供一种有效、廉价且安全的选择。
