Sundarasetty B S, Chan L, Darling D, Giunti G, Farzaneh F, Schenck F, Naundorf S, Kuehlcke K, Ruggiero E, Schmidt M, von Kalle C, Rothe M, Hoon D S B, Gerasch L, Figueiredo C, Koehl U, Blasczyk R, Gutzmer R, Stripecke R
Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
Department of Hematological Medicine, Cell and Gene Therapy at King's, The Rayne Institute, King's College London, London, UK.
Gene Ther. 2015 Sep;22(9):707-20. doi: 10.1038/gt.2015.43. Epub 2015 May 28.
Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.
负载黑色素瘤抗原的单核细胞衍生常规树突状细胞（ConvDCs）在临床试验中显示出适度的反应。ConvDCs制造困难和效力低下阻碍了疗效研究。为克服这些问题，我们证明了慢病毒载体（LV）编程的树突状细胞具有更高的效力。在用编码细胞因子（粒细胞巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4））以及黑色素瘤抗原（酪氨酸酶相关蛋白2（TRP2））的三顺反子LV转导后，单核细胞被直接诱导自分化为树突状细胞（SmartDC-TRP2）。在此，对从五名晚期黑色素瘤患者的单核细胞生成的SmartDC-TRP2进行了自体树突状细胞：T细胞刺激试验，验证了所有患者功能性TRP2特异性细胞毒性T淋巴细胞（CTLs）的激活。我们描述了符合良好生产规范（GMP）的生产LV和SmartDC-TRP2的方法。实现了在袋系统中单核细胞转导的可行性以及遵循24小时标准操作程序后的冷冻保存。解冻后，回收了50%的初始单核细胞输入量，并且SmartDC-TRP2在体外自分化，显示出树突状细胞标志物的均匀表达、可检测的LV拷贝以及不偏向致癌位点的多克隆LV整合模式。GMP级SmartDC-TRP2在体外扩增了TRP2特异性自体CTLs。这些结果证明了一种更简单的符合GMP的有效个体化树突状细胞疫苗生产方法。这种树突状细胞疫苗与检查点抑制疗法联合使用时，可能对黑色素瘤具有更高的特异性。
