Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Cytotherapy. 2013 Nov;15(11):1406-15. doi: 10.1016/j.jcyt.2013.06.003. Epub 2013 Aug 28.
Adoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells.
T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days.
The method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution.
We developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR-transduced T cells to support an ongoing clinical trial.
嵌合抗原受体 (CAR)-修饰的针对 CD19 的 T 细胞过继免疫疗法已显示出治疗 B 细胞淋巴瘤和白血病的良好效果。该疗法涉及用病毒载体转导自体 T 细胞，然后进行细胞扩增。我们描述了一种生产抗 CD19-CAR T 细胞的新的简化方法。
用抗 CD3/抗 CD28 顺磁珠从外周血单个核细胞 (PBMC) 中分离 T 细胞。第 2 天，将 T 细胞加入经 RetroNectin 预处理的培养袋中，并加载逆转录病毒抗 CD19 CAR 载体。将细胞、珠粒和载体孵育 24 小时，然后进行第二次转导。不进行旋转接种。然后再扩增细胞 9 天。
通过使用来自 B 细胞慢性淋巴细胞白血病患者的两个 PBMC 产品和来自健康供体的一个 PBMC 产品验证了该方法。来自 B 细胞慢性淋巴细胞白血病患者的两个 PBMC 产品中 T 细胞含量分别为 11.4%和 12.9%。该制造过程导致最终产品高度富集 T 细胞，平均 CD3+细胞含量为 98%，平均扩增 10.6 倍，平均转导效率为 68%。来自我们机构治疗的前 4 例急性淋巴细胞白血病患者的 PBMC 也获得了类似的结果。
我们开发了一种简化的半封闭系统，用于使用抗 CD3/抗 CD28 珠粒和袋子对 T 细胞进行初始选择、激活、转导和扩增，以生产用于支持正在进行的临床试验的自体抗 CD19 CAR 转导的 T 细胞。
