Gulen Dumrul, Abe Fuminori, Maas Sarah, Reed Elizabeth, Cowan Kenneth, Pirruccello Samuel, Wisecarver James, Warkentin Phyllis, Northam Matt, Turken Orhan, Coskun Ugur, Senesac Joe, Talmadge James E
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, United States.
Int Immunopharmacol. 2008 Dec 20;8(13-14):1728-36. doi: 10.1016/j.intimp.2008.08.010. Epub 2008 Sep 13.
Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20,000:1, and a 2 or 3 hour co-culture, followed by a 1:10 dilution with media and an additional 16-22 hour incubation. Following incubation, the DCs were washed twice and the supernatants removed using a plasma extractor. The average viability after infection with Ad5CMV-p53 was 87.9+/-2.6% with an average of 20.3+5.4% of the DCs expressing p53. The calculated yield of DCs following Ad5CMV-p53 transduction, based on the number of monocytes in the apheresis products, averaged 12.4+3.8%. We conclude that it is possible to efficiently manufacture Adv transduced DCs using a functionally closed system.
使用基于树突状细胞（DC）的疫苗进行抗癌免疫治疗提供了一种辅助治疗策略，该策略与传统疗法无交叉反应。然而，DC疫苗的生产需要严格遵守药品生产质量管理规范（GMP）方法并进行严格的标准化。理想情况下，这包括一个用于单核细胞分离的封闭系统，以及封闭的培养和洗涤系统，还有有效的载体转导策略。在本研究中，我们使用Gambro Elutra从健康供体采集的未动员白细胞单采产品中富集单核细胞。这种方法将单核细胞的平均频率从13.6 + 3.2%（平均值 + 标准误）提高到富集后的平均频率79.5 + 4.3%，产率为79%至100%。然后，将单核细胞在封闭系统中使用透气的Vuelife氟乙烯丙烯（FEP）袋和含有10 ng/ml粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）和5 ng/ml白细胞介素（IL）4的X - vivo - 15培养基进行培养。在第2天和第4天，用25%的培养基体积和细胞因子对培养物进行再投喂。培养7天后，使用Cobe - 2991收获细胞，并使用改装有用于离心72 ml袋子的模块的台式离心机进行浓缩，然后使用血浆提取器去除上清液。这种方法将培养基体积平均减少到17.4 ml，DC平均浓度为6.3 + 1.0×10⁷个细胞/ml，活力为93.8 + 2.2%，纯度为88.9 + 3.3%，DC总产量为8.5 + 1.4×10⁸个。基于将DR⁺细胞鉴定为DC，我们根据单采产品中的单核细胞数量以及从单核细胞分化产生的DC数量进行计算，平均产率为46 + 8%。将DC用作疫苗时，需要用携带肿瘤抑制基因p53转基因（Adv5CMV - p53）作为抗原的腺病毒（Adv）载体进行转导，DC浓度为9×10⁶个DC/ml，Ad5CMV - p53与DC的比例为20,000:1，并进行2或3小时的共培养，然后用培养基1:10稀释并再孵育16 - 22小时。孵育后，将DC洗涤两次，并使用血浆提取器去除上清液。用Ad5CMV - p53感染后的平均活力为87.9 ± 2.6%，平均有20.3 + 5.4%的DC表达p53。基于单采产品中的单核细胞数量计算，Ad5CMV - p53转导后DC的计算产率平均为12.4 + 3.8%。我们得出结论，使用功能封闭系统可以有效地生产Adv转导的DC。
