Tissues, Cells and Advanced Therapeutics, Scottish National Blood Transfusion Service, Jack Copland Centre, Edinburgh, UK.
Blood Transfusion Centre, Scottish National Blood Transfusion Service, Aberdeen, UK.
Clin Exp Immunol. 2021 Oct;206(1):68-81. doi: 10.1111/cei.13640. Epub 2021 Jul 14.
Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.
采用 Epstein-Barr 病毒 (EBV)-特异性 T 细胞过继免疫疗法是一种潜在的治愈性治疗方法，适用于其他临床治疗方法无效的 EBV 相关恶性肿瘤患者。我们描述了改进的符合良好生产规范 (GMP) 的培养和分析工艺，用于常规淋巴母细胞系 (LCL) 驱动的 EBV 特异性 T 细胞制造，并描述了一种改进的用于分析 T 细胞产品的表型分析方法。我们优化了当前的 LCL 介导的 EBV 特异性 T 细胞临床制造工艺，建立了一种使用无异种蛋白的整个 GMP 合规试剂的改进工艺，并将得到的产品与我们之前的银行 T 细胞临床治疗进行了比较。我们评估了 T 细胞扩增中 LCL:T 细胞比例变化的影响，并开发了一个强大的流式细胞术标记面板，涵盖 T 细胞记忆、激活、分化和细胞内细胞因子释放，以更有效地对 T 细胞进行特征分析。这些数据使用 t-随机邻域嵌入 (t-SNE) 算法进行分析。优化的 GMP 合规工艺导致细胞处理时间缩短，中央记忆 T 细胞的保留和扩增得到改善。多参数流式细胞术确定了 LCL 刺激和 T 细胞扩增的最佳方案，并证明使用白细胞介素 (IL)-2、肿瘤坏死因子 (TNF)-α 和干扰素 (IFN)-γ 的细胞因子分析能够确定整个培养过程和最终产品中 T 细胞的分化状态。我们表明，完全符合 GMP 的 LCL 介导的 EBV 特异性 T 细胞封闭过程培养是可行的，通过细胞因子表达对 T 细胞进行分析可更好地描述起始材料、中间培养条件和最终产品。使用 t-SNE 分析可以简化复杂的多参数流式细胞术数据的可视化。
