Processing of recombinant human IgGs in agroinfiltrated leaves of Nicotiana benthamiana

Thesis

Plants offer a powerful platform for recombinant protein expression, and have been used for successful production of human immunoglobulins G (IgGs). This offers an enticing alternative to current strategies for production of pharmaceutical antibodies. However, degradation of recombinant proteins by endogenous proteases causes severe yield losses and this minimises plants’ potential as viable production platforms. To tackle this issue, we investigated the in vitro and in vivo processing of four human anti-viral IgGs by plant proteases of Nicotiana benthamiana. Specifically, we tested anti-HIV 2F5, anti-SARS-CoV2 COVA2-15, anti-EBOV 2G4 and anti-HIV VRC01. In vitro incubation of IgGs with apoplastic fluids demonstrated that subtilase SBT5.2 is the sole protease responsible for 2F5 cleavage. Moreover, we found that different antibodies exhibit varying levels of susceptibility to cleavage by subtilases. However, we also desired to ascertain if other proteases impact IgG processing intracellularly, along the secretory pathway. We also carried out in vivo experiments, examining the subtilases previously tested in vitro, along with cysteine proteases, and identified varying impact on IgG accumulation. To get a better understanding of IgG susceptibility to protease processing, we used a site-directed mutagenesis approach on 2F5 and COVA2-15 IgGs and provide preliminary evidence that formation of disulphide bonds enclosing the cleavage site can reduce processing. Finally, we followed a combined immunoblotting and confocal approach to investigate the subcellular localisation of 2F5 light and heavy chains. Furthermore, we assessed the effect of IgG targeting to the endoplasmic reticulum or the vacuole as opposed to the apoplast. Our results demonstrate that the ER can be a promising compartment to maximise IgG accumulation. Cumulatively, our results indicate that individual protease depletion does not uniformly enhance IgG accumulation in N. benthamiana and must be tested on a caseby- case basis. Additionally, this work reveals the need to better understand IgG subcellular localisation and their processing profile upon expression in N. benthamiana, in order to make this a competitive production platform.

Authors: Beritza, K

• DOI: 10.5287/ora-9ev0qaakz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Nicotiana benthamiana; protein degradation; proteases; antibody production; plant molecular farming
• Subjects: Biotechnology; Plant Sciences; Synthetic biology