Mu L J, Gaudernack G, Saebøe-Larssen S, Hammerstad H, Tierens A, Kvalheim G
Laboratory for Cellular Therapy, The Norwegian Radium Hospital, University of Oslo, Oslo, Norway.
Scand J Immunol. 2003 Nov;58(5):578-86. doi: 10.1046/j.1365-3083.2003.01333.x.
With the aim of producing large quantities of mRNA-transfected monocyte-derived dendritic cells (DCs) to be used as cancer vaccines, a new clinical grade procedure has been developed. Peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis were enriched for monocytes by immunomagnetic depletion of CD19+ B cells and CD2+ T cells employing the ISOLEX 300i device. After 5 days of culture of enriched monocytes in gas permeable Teflon bags, using serum-free medium supplemented with granulocyte/macrophage-colony stimulating factor and interleukin-4 (IL-4), immature DCs were generated. Following transfection with mRNA from three human prostate cancer cell lines (DU145, LNCaP and PC-3), employing a newly developed square wave electroporation procedure, the immature DCs were immediately transferred to Teflon bags and matured for 48 h, using serum-free medium supplemented with IL-1alpha, IL-6, tumour necrosis factor-alpha and PGE2. The electroporation procedure efficiently transferred mRNA into the DCs with minor effect on the viability of the cells. The generated matured transfected DCs show high expression of the antigens CD83, CD80, CD86 and human leucocyte antigen-DR. Freezing and thawing of the transfected matured DCs had minor effect on cell viability and the phenotype. From 4 x 109 PBMCs, about 1 x 108 transfected matured DCs are produced. The thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by enzyme-linked immunospot assay using mock-transfected DCs as control. Based on these results, clinical trials in cancer patients have been initiated.
为了大量生产用作癌症疫苗的mRNA转染单核细胞来源的树突状细胞（DCs），已开发出一种新的临床级程序。通过白细胞分离术获得的外周血单核细胞（PBMCs），使用ISOLEX 300i设备通过免疫磁珠法去除CD19+B细胞和CD2+T细胞来富集单核细胞。在透气的特氟龙袋中使用补充有粒细胞/巨噬细胞集落刺激因子和白细胞介素-4（IL-4）的无血清培养基培养富集的单核细胞5天后，产生未成熟DCs。采用新开发的方波电穿孔程序用来自三种人前列腺癌细胞系（DU145、LNCaP和PC-3）的mRNA转染后，未成熟DCs立即转移到特氟龙袋中，并使用补充有IL-1α、IL-6、肿瘤坏死因子-α和PGE2的无血清培养基成熟48小时。电穿孔程序有效地将mRNA转移到DCs中，对细胞活力影响较小。产生的成熟转染DCs显示出抗原CD83、CD80、CD86和人类白细胞抗原-DR的高表达。转染成熟DCs的冻融对细胞活力和表型影响较小。从4×109个PBMCs中可产生约1×108个转染成熟DCs。解冻的转染DCs能够在体外引发针对前列腺癌mRNA编码抗原的原发性T细胞反应，以mock转染DCs作为对照通过酶联免疫斑点试验显示。基于这些结果，已启动癌症患者的临床试验。
