Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans

Marcello Catellani,Mauro Cerasi,Silvia Massa,Chiara Lico,Cristina Capodicasa,Carla Bromuro,Eugenio Benvenuto,Antonella Torosantucci

doi:10.1186/s12896-020-00607-0

Abstract

BackgroundInfections 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.ResultsIn 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.ConclusionsProduction 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.

Highlights

Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy
We recently demonstrated that chimeric murinehuman antibodies directed against the fungal cell wall polysaccharide β1–3 glucan are able to confer a significant protection in vivo against systemic and mucosal infections caused by the fungal pathogen C. albicans [14]
Generation of N. benthamiana and S. lycopersicum hairy roots (HR) producing 2G8 antibody To generate HR producing the recombinant anti-βglucan Single-chain Fragment variable Fragment crystallisable (scFvFc) 2G8, N. benthamiana and S. lycopersicum leaf explants were infected with R. rhizogenes harbouring the pBIM2G8 vector containing the scFvFc 2G8 gene (Fig. 1a)

Summary

Introduction

Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. During the 2014 Ebola outbreak in Liberia, an experimental cocktail of three antibodies, named ZMapp, produced in plants [5] according to the cGMP and using RAMP platform (Rapid Antibody Manufacturing Platform), was made available on an emergency use basis for compassionate treatment of a patient population of seven individuals, five of which recovered [6]. This and other examples showcase the approach of producing biologicals in plants as a valuable low-cost alternative to synthesize pharmaceuticals to combat pathogenic agents. Rhizosecretion of foreign proteins has been already investigated for reporter proteins such as GFP [11], and for biopharmaceuticals, as LTB [12] or monoclonal antibodies [13]

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