Olsen Cathrine Elisabeth, Cheung Lawrence H, Weyergang Anette, Berg Kristian, Vallera Daniel A, Rosenblum Michael G, Selbo Pål Kristian
Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, N-0310 Oslo, Norway.
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
J Clin Med. 2019 Dec 26;9(1):68. doi: 10.3390/jcm9010068.
The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133 and CD133 cell lines and a CD133 cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133 cells compared to CD133 cells. No cytotoxic responses were detected in either CD133 or CD133 cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133 cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133 and CD133 cancer cells.
本研究的目的是开发并探索一种新型的靶向CD133的免疫毒素（IT），用于与内体逃逸方法光化学内化（PCI）联合使用。通过将编码靶向人和鼠CD133/促红细胞生成素-1非糖基化和糖基化形式的单链抗体片段（scFv）的基因（scFvCD133）与编码核糖体失活蛋白（RIP）去甲基金鸡纳碱（rGelonin）的基因融合，重组构建了scFvCD133/rGelonin。在无细胞翻译试验中评估RIP活性。通过流式细胞术和荧光显微镜评估scFvCD133/rGelonin的选择性结合和细胞内积累。在CD133和CD133细胞系以及CD133细胞系中探索scFvCD133/rGelonin的PCI，通过MTT试验评估细胞毒性。与CD133细胞相比，scFvCD133/rGelonin在CD133细胞中表现出更强的结合能力和更高的积累。用<100 nM scFvCD133/rGelonin孵育72小时后，在CD133或CD133细胞中均未检测到细胞毒性反应。尽管与游离rGelonin相比，scFvCD133/rGelonin的RIP活性严重丧失，但与rGelonin的PCI相比，scFvCD133/rGelonin的PCI诱导细胞活力对数倍降低。令人惊讶的是，在CD133细胞中，scFvCD133/rGelonin的PCI也超过了rGelonin的PCI的细胞毒性。总之，PCI在CD133和CD133癌细胞中均促进了本身无毒的scFvCD133/rGelonin的强大细胞毒性活性。
