Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
J Immunother Cancer. 2022 Sep;10(9). doi: 10.1136/jitc-2022-005189.
There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.
We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.
We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.
We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.
患者和护理人员对嵌合抗原受体 (CAR) T 细胞产品的需求不断增加。在这里，我们在 CliniMACS Prodigy 平台上建立了一种自动化的 CAR T 细胞制造工艺，该工艺可规模化提供治疗剂量，并通过无病毒 Sleeping Beauty (SB) 转位实现基因转移。
我们使用了连接到电穿孔单元的先进的 CliniMACS Prodigy，并使用原代人 T 细胞进行了一系列小规模开发和大规模确认运行。转位是使用 minicircle (MC) DNA 编码的 SB100X 转座酶和编码 CD19 CAR 的 pT2 转座子完成的。
我们定义了一个双脉冲电穿孔冲击，分别为双向和单向电场，允许高效的 MC 插入并保持高活力 T 细胞的频率。在三个大规模运行中，从白细胞分离产品中富集了 2E8 T 细胞，激活、基因工程和扩增，在 12 天内产生高达 3.5E9 个总 T 细胞/1.4E9 个 CAR 修饰的 T 细胞（CAR 修饰的 T 细胞：28.8%±12.3%）。所得细胞产物含有高度纯的 T 细胞（97.3±1.6%），具有平衡的 CD4/CD8 比值和高比例的中央记忆表型 T 细胞（87.5%±10.4%）。转座子拷贝数分别为#1-3 中的 7.0、9.4 和 6.8，基因分析显示激活/耗竭标志物表达平衡。CD19 CAR T 细胞产物在临床前模型中赋予了强大的抗淋巴瘤反应性。值得注意的是，与传统的非自动化 CAR T 细胞制造活动相比，操作人员的实际操作时间大大减少。
我们报告了首个基于转座子的自动化 CAR T 细胞制造工艺，该工艺已准备好进行正式验证，并可用于临床制造活动。该工艺和平台有可能使患者更容易获得 CAR T 细胞治疗，并加速学术和工业环境中的规模化、多重制造。
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