BACKGROUND Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

中文翻译：

---

在封闭系统 G-Rex 生物反应器中通过激活、转导和扩增简化转基因 T 细胞的生产

背景 气体渗透性快速扩增 (G-Rex) 生物反应器已被证明可有效扩增用于治疗用途的免疫细胞，但并未解决许多工程化 T 细胞产品所需的病毒转导步骤的复杂性。在这里，我们展示了一种用 Vectofusin-1 试剂转导活化 T 细胞的新方法。转导是在 G-Rex 生物反应器中悬浮完成的。简化的转导步骤被整合到一个简化的过程中，该过程使用单个生物反应器，操作员干预有限。方法 来自健康供体的外周血单核细胞 (PBMC) 在细胞培养袋或 G-Rex 生物反应器中解冻、洗涤并用可溶性抗 CD3 和抗 CD28 抗体激活。细胞在含有白细胞介素 (IL)-7 和 IL-15 的 TexMACS GMP 培养基中培养，并用袋装 RetroNectin 或 G-Rex 中的 Vectorfusin-1 转导。比较两个过程之间的总活细胞数、倍数扩增、活力、转导效率、表型和功能。结果 简化的过程使用从激活到收获的单个容器，并在 11 天内实现了 56% 的转导和 29 倍的扩增。在简化的过程中产生的细胞在功能或表型上与传统的基于袋的过程中产生的细胞没有区别。讨论 本研究表明 T 细胞可以悬浮转导。此外，在用于临床使用的袋子中生成工程化 T 细胞的传统方法可以简化为更简单的方法，