Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland.
Center for Cellular Engineering, National Institutes of Health Clinical Center, Bethesda, Maryland.
Transfusion. 2019 May;59(5):1755-1764. doi: 10.1111/trf.15287. Epub 2019 Apr 11.
When manufacturing chimeric antigen receptor (CAR) T cells using anti-CD3/anti-CD28 beads, ex vivo T-cell expansion is dependent on the composition of leukocytes used in the manufacturing process. We investigated the effects of leukocyte composition on CAR T-cell expansion and characteristics using an alternative manufacturing method.
Anti-B-cell maturation antigen and CD19-CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T-cell culture initiation. T-cell expansion was stimulated with soluble anti-CD3 and interleukin-2.
Fifty-one CAR T-cell products were evaluated; 28 anti-B-cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T-cell expansion was reduced when greater quantities of monocytes were present in the post-density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T-cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post-density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells.
The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T-cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T-cell products.
在使用抗 CD3/抗 CD28 珠粒制造嵌合抗原受体 (CAR) T 细胞时，体外 T 细胞扩增依赖于制造过程中使用的白细胞组成。我们使用替代制造方法研究了白细胞组成对 CAR T 细胞扩增和特性的影响。
使用自体外周血单个核细胞 (PBMNC) 浓缩物制造抗 B 细胞成熟抗原和 CD19-CAR T 细胞。使用密度梯度分离法从 PBMNC 中富集淋巴细胞，用于 CAR T 细胞培养起始。用可溶性抗 CD3 和白细胞介素-2 刺激 T 细胞扩增。
评估了 51 个 CAR T 细胞产品；为 24 名患者生产了 28 种抗 B 细胞成熟抗原 (BCMA) CAR T 细胞和 27 种 CD19 CAR T 细胞。在后密度梯度分离 PBMNC 中存在更多单核细胞时，CAR T 细胞扩增减少。此外，培养 7 天后 CAR T 细胞产品中 CD4 与 CD8 细胞的比值取决于后密度梯度分离 PBMNC 中单核细胞、红细胞和中性粒细胞的数量。更多数量的单核细胞和红细胞与更多比例的 CD4+细胞相关，更多数量的中性粒细胞与更多比例的 CD8+细胞相关。
用于制造 CAR T 细胞的白细胞组成会影响细胞扩增和 CAR T 细胞产品的组成。更均匀或完全的 PBMNC 淋巴细胞富集可提高最终 CAR T 细胞产品的一致性。
