Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells
Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells
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DOI：
10.1016/j.jcyt.2014.02.017
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发表时间：
2014-07-01
期刊：
影响因子：
4.5
通讯作者：
Van Gool, Stefaan W.
中科院分区：
文献类型：
--
作者：
Eyrich, Matthias;Schreiber, Susanne C.;Van Gool, Stefaan W.
背景和目标。树突状细胞(DC)疫苗接种的主要挑战之一是建立协调的DC生产规程。在这里，我们报告了一种成功使用的开放式DC制造方法在封闭系统、良好制造规范(GMP)兼容协议中的转移和验证。方法：研究方法。所有生产步骤(裂解液生成、单核细胞选择、DC培养和冷冻保存)都经过标准化和验证。结果。肿瘤裂解液的组织学特征是机械匀浆和无机化。这种制剂每毫克肿瘤组织产生的蛋白质中位数为58+/-21微克。台盼蓝染色检测细胞活力，三磷酸腺苷释放法检测细胞活力。通过淋洗或CD14选择分离患者单核细胞，产生相同的结果。DC在含IL-4和粒-巨噬细胞集落刺激因子的CellGro培养液中诱导分化7d为最佳，经肿瘤裂解液冲击后在肿瘤坏死因子-α和IL-1β作用下成熟48h。这一方案导致DC成熟标志物如分化簇(CD)80、CD83、CD86、人类白细胞抗原DR和DC-SIGN的强健和可重复性的上调。这些DC的功能表现为定向向C-C基序趋化因子配体19/21迁移，阳性T细胞刺激能力和从初始CD8(+)T细胞激发抗原特异性T细胞的能力。冻存、解冻、洗涤后DC的表型稳定性、活性和功能均未见明显丧失。146名接受平板贴壁疫苗接种的患者的临床数据与GMP级DC的临床数据比较显示，后者并不逊色。结论。我们强大的、经过验证和批准的DC生产方案为生产癌症疫苗的协调程序奠定了基础，这为更大规模的多中心临床试验铺平了道路。
Background and aims. One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol. Methods. All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated. Results. Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 +/- 21 mu g protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-alpha and IL-1 beta after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter. Conclusions. Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.