Stroncek David F, Lee Daniel W, Ren Jiaqiang, Sabatino Marianna, Highfill Steven, Khuu Hanh, Shah Nirali N, Kaplan Rosandra N, Fry Terry J, Mackall Crystal L
Cell Processing Section, Department of Transfusion Medicine, NIH Clinical Center, NIH, 10 Center Drive-MSC-1184, Building 10, Room 3C720, Bethesda, MD, 20892-1184, USA.
Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Virginia, Charlottesville, USA.
J Transl Med. 2017 Mar 16;15(1):59. doi: 10.1186/s12967-017-1160-5.
Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes.
Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products.
When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells.
Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.
利用自体外周血单核细胞（PBMC）浓缩物制备嵌合抗原受体（CAR）T细胞用于治疗血液系统恶性肿瘤的临床试验前景良好，但CAR T细胞产量存在差异。这种差异部分归因于PBMC浓缩物被单核细胞和粒细胞污染。
逆流淘析可在封闭系统中将淋巴细胞与单核细胞和粒细胞分离。我们研究了使用淘析富集淋巴细胞的PBMC浓缩物来制备8种CD19 -和5种GD2 - CAR T细胞产品。
与PBMC浓缩物相比，富含淋巴细胞的淘析组分中CD3 +和CD56 +细胞比例更高，CD14 +和CD15 +细胞比例更低。使用淘析淋巴细胞制备的所有13种CAR T细胞产品均产生了足够数量的转导CAR T细胞以满足临床剂量标准。GD2 - CAR T细胞产品中的T细胞和转导T细胞明显多于CD19 - CAR T细胞产品。将淘析淋巴细胞产生的CAR T细胞产量与先前通过抗CD3/CD28磁珠选择或抗CD3/CD28磁珠选择加塑料贴壁从PBMC浓缩物中分离的细胞产生的CAR T细胞产量进行比较，发现淘析淋巴细胞产生的GD2 - CAR T细胞数量更多，但CD19 - CAR T细胞并非如此。
使用淘析法富集PBMC浓缩物中的淋巴细胞可增加GD2 - CAR T细胞的产量。这些结果进一步证明单核细胞和粒细胞会抑制CAR T细胞的扩增。
