Yield improvement of a plant-made antibody against Enterohemorrhagic E. coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that harbors asymptomatically in a bovine intestinal reservoir and has been a consistent global health risk for the last thirty years. In this thesis, I have functionally characterized an anti-EHEC antibody and shown it to bind and neutralize four of the seven most prevalent EHEC strains. However, yield is arguably the greatest technical hurdle in advancing such an antibody from proof of principle to market. Although there have been many advances in the last thirty years in improving plant-based recombinant yields, little is still known about both the mutational potential in engineering the structure for better yield as well as the limitations of the antibody’s oxidative folding environment. This thesis explores two main strategies toward yield improvement: first, rational design of the antibody and second, targeting it to the thylakoid lumen. I have identified and characterised six rationally designed mutations that can be pyramided to improve the yield of the antibody by twenty-fold. I show that despite engineering its structure, the antibody retains its ability to assemble into a polymeric complex as well as its binding and neutralization efficacy against EHEC in in vitro assays. Because endoplasmic reticulum (ER)-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of antibodies in the ER, I sought to explore oxidative folding in an alternative subcompartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. I developed a set of in-house expression vectors targeting the antibody to the thylakoid lumen via either Sec or Tat import pathways. Compared to stromal, cytoplasm and Tat-imported pathways, the Sec-targeted antibody showed superior accumulation, but about one third less than its ER-targeted counterpart. Sec-targeted antibodies also retain binding and neutralization efficacy in in vitro assays. Additionally, the introduction of a rationally designed de novo disulfide enhances in vivo accumulation when introduced into the Sec-targeted antibody. These results collectively provide a proof of concept on the viability of rational design and thylakoid targeting as novel, broadly applicable strategies for yield improvement and potentially advancing an anti-EHEC antibody closer toward market adoption