An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE2, IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with “new method” lysate compared to DC pulsed with “classical method” lysate. Our results indicate that immunomagnetic isolation of CD14+ monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.

免疫疗法是一类极具前景的新型治疗手段，旨在激活机体免疫系统以特异性清除恶性肿瘤细胞。免疫治疗可诱导特异性抗肿瘤免疫应答，而树突状细胞（dendritic cells, DC）作为专职抗原呈递细胞，已被广泛应用于癌症免疫治疗的实验研究中。已有多项研究报道了可用于临床的成熟、抗原负载型DC的制备方法。为获得符合药品生产质量管理规范（Good Manufacturing Practice, GMP）的标准化制备流程，亟需进一步优化DC制备的质量与标准化水平。本研究中，我们以31例胶质母细胞瘤（Glioblastoma, GB）患者的外周血单核细胞为起始材料，通过CliniMACS®设备的免疫磁珠CD14分选技术分离得到CD14+单核细胞，并以此制备DC。将分离得到的CD14+单核细胞在白细胞介素4（interleukin-4, IL-4）与粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）的诱导下分化为未成熟DC，随后通过肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）、前列腺素E2（prostaglandin E2, PGE2）、白细胞介素1β（interleukin-1β, IL-1β）及白细胞介素6（interleukin-6, IL-6）诱导其成熟。本研究首次采用半自动解离仪GentleMACS®在封闭系统中制备全肿瘤裂解液，相较于手工解离法，该方法的蛋白质得率提升了130%。值得注意的是，经“新方法”制备的肿瘤裂解液负载的DC，其CD83分子的平均荧光强度显著高于经“经典方法”制备的肿瘤裂解液负载的DC。本研究结果表明，利用CliniMACS®设备通过免疫磁珠分选CD14+单核细胞，并以全肿瘤裂解液蛋白负载DC，是一种可在GMP级条件下实现临床规模制备高质量、功能成熟DC的可靠方法。