Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance
Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance
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DOI：
10.3390/cancers11081198
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发表时间：
2019-08-01
期刊：
影响因子：
5.2
通讯作者：
Schaft, Niels
中科院分区：
文献类型：
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作者：
Wiesinger, Manuel;Maerz, Johannes;Schaft, Niels
嵌合抗原受体(CAR)-T细胞在白血病和淋巴瘤中已经显示出令人印象深刻的临床回归。然而，针对实体瘤的CAR-T细胞的发展相对滞后。在这里，我们提出了临床规模生产的CAR-T细胞治疗黑色素瘤完全符合GMP。在这种方法中，出于安全原因，通过mRNA电穿孔有意瞬时表达了一种针对硫酸软骨素蛋白多糖4(CSPG4)的CAR。临床规模方案被优化为：(I)T细胞的扩增，(Ii)电穿孔效率，(Iii)活力，(Iv)冷冻保存，和(V)效力。4次连续培养后，CAR-T细胞数量达到临床所需水平，即从1.77×10(8)个外周血单核细胞开始，获得2.4×10(9)个表达CAR的T细胞，平均扩增倍数为13.6倍，CAR阳性细胞的电穿孔效率为88.0%，电穿孔后存活率为74.1%，冻存后存活率为84%。纯度为98.7%的CD3(+)细胞、78.1%的CD3(+)/CD8(+)T细胞、1.2%的NK细胞和0.6%的B细胞。得到的CAR-T细胞在冷冻保存后进行了细胞溶解活性测试，显示出抗原特异性和非常有效的肿瘤细胞裂解能力。虽然我们的工作是描述性的，而不是研究性的，但我们预计，提供这一临床适用的方案来产生足够数量的mRNA转基因CAR-T细胞将有助于推动癌症采用细胞疗法的领域向前发展。
Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 x 10(9) CAR-expressing T cells, starting from 1.77x10(8)PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3(+) cells, with 78.1% CD3(+)/CD8(+) T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.