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piggyBac构建的CD19-CAR-T治疗难治性费城染色体阳性急性淋巴细胞白血病

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发表于 2015-9-6 10:28:35 | 显示全部楼层 |阅读模式
Anti-leukemic potency of piggyBac-mediatedCD19-specific T cells against refractory Philadelphia chromosome positive acute lymphoblasticleukemia
piggyBac构建的CD19-CAR-T治疗难治性费城染色体阳性急性淋巴细胞白血病
Background aims. To develop a treatment option forPhiladelphia chromosome-positive acute lymphoblastic leukemia (PhtALL)resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemicactivity of T cells non-virally engineered to express a CD19-specific chimericantigen receptor (CAR). Methods. A CD19.CAR gene was delivered into mononuclearcells from 10 mL of blood of healthy donors through the use ofpiggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells werestimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR,and cultured in interleukin-15econtaining serum-free medium with autologousfeeder cells for 21 days. To evaluate their cytotoxic potency, we co-culturedCAR T cells with seven PhtALL cell lines including three TKI-resistant(T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower withoutcytokines. Results. We obtained w1.3 108 CAR T cells (CD4t, 25.4%;CD8t, 71.3%),co-expressing CD45RA and CCR7 up to w80%. After 7-day co-culture, CAR T cellseradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reducedtumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-foldproliferation of CAR T cells during a 20-day culture period in the presence oftumor cells. On exposure to tumor cells,CAR T cells transiently and reproduciblyupregulated the expression of transgene as well as tumor necrosisfactorerelated apoptosis-inducing ligand and interleukin-2. Conclusions. Wegenerated a clinically relevant number of CAR T cells from 10 mL of bloodthrough the use of piggyBac-transposons, a 4D-Nulcleofector, andserum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited markedcytotoxicity against PhtALL regardless of T315I mutation. PiggyBac-mediatedCD19-specific T-cell therapy may provide an effective, inexpensive and safeoption for drug-resistant PhtALL.
爱康得生物编译:
背景   为开发一种耐酪氨酸激酶抑制剂(TKI)费城染色体阳性急性淋巴细胞白血病(PhtALL)的替选治疗方案,我们评估了以非病毒载体构建的对CD19特异性的CAR-T的抗白血病活性。
方法    我们利用piggyBac转座子和4D细胞核转染系统将CD19CAR编码基因转染至10毫升来源于健康供体的单核细胞中。
我们用CD3 / CD28抗体去刺激核转染细胞,用于磁性选择CD19.CAR,并在在含有白介素15和自体滋养层细胞的无血清培养基中培养21天。为了检测这些CAR-T的细胞毒性,我们将其与7PhtALL细胞系共同培养;这些细胞系包括3株对TKI耐受的T3151突变型细胞株(效靶比1:5或更低,同时不添加任何细胞因子)
结果    我们获得约1.3* 108CART细胞(CD4+25.4;CD8 +71.3%),其中共表达CD45RACCR7的细胞占比高达80%。后7天的共同培养,效靶比1:51:10CAR-T完全杀灭肿瘤细胞,而效靶比1:50CAR-T基本清除肿瘤细胞。动力学分析表明,在肿瘤细胞的存在下,CART细胞数量在20天的培养期内增殖高达37倍。暴露于肿瘤细胞时,CART细胞可瞬时地重复的上调转染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。
结论    我们通过使用piggyBac4D细胞转染系统、无血清、无异种物质、无肿瘤细胞、无病毒培养系统从10毫升的血液中生产满足临床需求的CAR-T细胞。 这些CART细胞表现出对PhtALL明显的细胞毒作用。 piggyBac转导的CD19-T细胞疗法可能会为耐药的PhtALL提供有效,廉价和安全的治疗选择。

Anti-leukemic potency of piggyBac-mediated CD19-specic T cells against refractor.pdf

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