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.

中文翻译：

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优化茄科毛状根中抗真菌抗体的生产，以开发针对白色念珠菌的新配方


由真菌引起的感染通常难以用常规疗法治疗，迫切需要开发新的选择，例如免疫疗法。为了生产治疗性抗体，与哺乳动物细胞培养物相比，基于植物的表达平台是一种有吸引力的生物技术策略。除了整株植物外，还可以使用毛状根（HR）培养物，这代表了一种易于构建的表达系统，在封闭条件下处理时可以无限期生长。在这项研究中，报道了在 HR 中生产的重组抗体，该抗体被证明是针对真菌感染的人类免疫疗法的良好候选者。该抗体以工程化单链 (scFvFc) 形式的表达和分泌，由 HR 从本塞姆氏烟草和番茄中进行了评估，目的是直接使用衍生提取物或培养基来对抗病原真菌。尽管两种 Solanaceae HR 均表现出良好的表达水平（高达 68 mg/kg），但仅 N. Benthamiana HR 获得了根部分泌的优化。解释这一结果的初步评估强调了这样一个事实：不仅蛋白酶的存在，而且生长培养基的化学特性都会影响抗体产量，从而影响 HR 中重组蛋白的产生。最后，在白色念珠菌生长抑制测定中评估了本塞姆氏烟草 HR 中产生的 scFvFc 2G8 抗体的抗真菌活性，证明了令人鼓舞的结果。在本塞姆氏烟草和番茄的 HR 中产生这种抗真菌抗体，阐明了影响该系统中药用的因素，并允许获得有前景的针对白色念珠菌的即用型免疫治疗药物。