Wiesinger Manuel, März Johannes, Kummer Mirko, Schuler Gerold, Dörrie Jan, Schuler-Thurner Beatrice, Schaft Niels
Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany.
Cancers (Basel). 2019 Aug 16;11(8):1198. doi: 10.3390/cancers11081198.
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 × 10 CAR-expressing T cells, starting from 1.77x10 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.
嵌合抗原受体（CAR）-T细胞已在白血病和淋巴瘤的治疗中展现出令人瞩目的临床缓解效果。然而，针对实体瘤的CAR-T细胞研发却相对滞后。在此，我们展示了在完全符合药品生产质量管理规范（GMP）的条件下，用于治疗黑色素瘤的CAR-T细胞的临床规模生产。出于安全性考虑，在这种方法中，通过mRNA电穿孔技术使一种针对硫酸软骨素蛋白聚糖4（CSPG4）的CAR被短暂表达。临床规模方案针对以下方面进行了优化：（i）T细胞的扩增；（ii）电穿孔效率；（iii）活力；（iv）冷冻保存；（v）效力。四次一致性运行从1.77×10外周血单个核细胞（PBMC）开始，产生了临床数量充足的CAR-T细胞，即2.4×10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%，冷冻保存后活力为84%。纯度为98.7%的CD3细胞，其中78.1%为CD3/CD8 T细胞，NK细胞污染率为1.2%，B细胞污染率为 &emsp
