Laboratory of Biotechnology, ENEA Casaccia Research Center, 00123 Rome, Italy.
Laboratory of Biomedical Technologies, ENEA Casaccia Research Center, 00123 Rome, Italy.
Biotechnol J. 2019 Mar;14(3):e1800081. doi: 10.1002/biot.201800081. Epub 2018 Jul 18.
Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.
发根（HR）培养物因其能够在培养基中分泌感兴趣的分子而成为生产异源蛋白的有吸引力的平台。主要限制是异源蛋白的积累产量低。本研究的目的是提高具有与人相容糖基化谱的肿瘤靶向抗体在 HR 培养基中的积累。为此，作者生产表达红色荧光蛋白（RFP）的菉麻 HR 培养物，以轻松筛选不同的激素，这些激素能够诱导培养基中异源蛋白的分泌。发现 2,4-二氯苯氧基乙酸（2,4-D）能够诱导 RFP 在培养基中高水平积累（334mg/L）。相同的方案用于提高糖基工程化的 ΔXTFT 菉麻 HR 培养物中肿瘤靶向、CD20 特异性 2B8-FcΔXF 重组抗体的分泌。添加 2,4-D 可使完全功能性 2B8-FcΔXF 在培养基中的积累增加 28 倍，达到约 16mg/L。抗体 N-糖基化分析表明 GnGn 结构的大量出现和含有木糖和岩藻糖的结构的低水平。这是首次以 HR 中高水平表达 scFv-Fc 格式的工程化抗 CD20 抗体的例子。
