Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.
Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.
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DOI：
10.1016/j.jcyt.2014.05.022
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发表时间：
2014-09
期刊：
影响因子：
4.5
通讯作者：
Koike K
中科院分区：
文献类型：
--
作者：
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
为了开发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，我们评估了表达cd19特异性嵌合抗原受体（CAR）的非病毒工程T细胞的抗白血病活性。CD19。利用piggybac -转座子和4-D核因子系统将CAR基因从健康供者的10ml血液中递送到单个核细胞中。用CD3/CD28抗体刺激核感染细胞，磁选择CD19。CAR，在含白细胞介素-15的无血清培养基中与自体饲养细胞培养21天。为了评估它们的细胞毒性，我们将CAR - T细胞与7种Ph+ALL细胞系共培养，其中包括3种tki抗性（t315i突变）细胞系，效应靶比为1:5或更低，不含细胞因子。我们获得约1.3 × 108个CART细胞（CD4+, 25.4%; CD8+, 71.3%），共表达CD45RA和CCR7高达约80%。共培养7天后，CAR - T细胞以1:5和1:10的比例杀灭了所有肿瘤细胞，并以1:50的比例显著减少了肿瘤细胞数量。动力学分析显示，在肿瘤细胞存在的情况下，在20天的培养期间，CART细胞的增殖率高达37倍。暴露于肿瘤细胞后，CAR - T细胞短暂且可重复地上调转基因以及肿瘤坏死因子相关的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-Nulcleofector和血清/xeno/肿瘤细胞/无病毒培养系统，从10ml血液中产生了临床相关数量的CAR - T细胞。无论T315I突变与否，CAR - T细胞对Ph+ALL均表现出明显的细胞毒性。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、廉价和安全的选择。
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 × 108 CART 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 CART 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.