Department of Sustainability, Laboratory Biotechnologies, ENEA, Casaccia Research Center, Via Anguillarese 301, 00123, Rome, Italy.
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
BMC Biotechnol. 2020 Mar 12;20(1):15. doi: 10.1186/s12896-020-00607-0.
Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions.
In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results.
Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.
由真菌引起的感染通常对常规疗法有抗性，因此迫切需要开发新的治疗方法，如免疫疗法。与哺乳动物细胞培养相比，植物表达平台是生产治疗性抗体的一种有吸引力的生物技术策略。除了整个植物之外，还可以使用发根（HR）培养物，这是一种易于构建的表达系统，在密闭条件下可以无限期生长。
本研究报告了在 HR 中生产一种重组抗体的情况，该抗体被证明是针对真菌感染的人免疫治疗的良好候选物。通过对烟草和番茄的 HR 进行工程化，以单链（scFvFc）形式表达和分泌该抗体，旨在直接使用衍生提取物或培养基来对抗致病性真菌。尽管两种茄科 HR 都显示出良好的表达水平（高达 68mg/kg），但仅对烟草 HR 实现了rhizosecretion 的优化。初步评估结果表明，不仅存在蛋白酶，而且生长培养基的化学特性也会影响抗体产量，这对 HR 中的重组蛋白生产有影响。最后，评估了在烟草 HR 中产生的 scFvFc 2G8 抗体对白色念珠菌生长抑制试验的抗真菌活性，结果令人鼓舞。
在烟草和番茄的 HR 中生产这种抗真菌抗体，阐明了影响该系统制药的因素，并获得了针对白色念珠菌的有前途的即用型免疫疗法。
