Rice endosperm produces an underglycosylated and potent form of the HIV‐neutralizing monoclonal antibody 2G12

Abstract

SummaryProtein microbicides against HIV can help to prevent infection but they are required in large, repetitive doses. This makes current fermenter-based production systems prohibitively expensive. Plants are advantageous as production platforms because they offer a safe, economical and scalable alternative, and cereals such as rice are particularly attractive because they could allow pharmaceutical proteins to be produced economically and on a large scale in developing countries. Pharmaceutical proteins can also be stored as unprocessed seed, circumventing the need for a cold chain. Here, we report the development of transgenic rice plants expressing the HIV-neutralizing antibody 2G12 in the endosperm. Surprisingly for an antibody expressed in plants, the heavy chain was predominantly aglycosylated. Nevertheless, the heavy and light chains assembled into functional antibodies with more potent HIV-neutralizing activity than other plant-derived forms of 2G12 bearing typical high-mannose or plant complex-type glycans. Immunolocalization experiments showed that the assembled antibody accumulated predominantly in protein storage vacuoles but also induced the formation of novel, spherical storage compartments surrounded by ribosomes indicating that they originated from the endoplasmic reticulum. The comparison of wild-type and transgenic plants at the transcriptomic and proteomic levels indicated that endogenous genes related to starch biosynthesis were downregulated in the endosperm of the transgenic plants, whereas genes encoding prolamin and glutaredoxin-C8 were up-regulated. Our data provide insight into factors that affect the functional efficacy of neutralizing antibodies in plants and the impact of recombinant proteins on endogenous gene expression.

Regulatory approval and a first‐in‐human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants

“…The production of 2G12 has previously been reported in plants although only thus far in the context of proof-of-principle studies that do not address the requirements of process-scale GMP manufacturing. The antibody has been expressed in transgenic maize seeds Ramessar et al, 2008), Arabidopsis seeds and leaves (Loos et al, 2011), tobacco leaves (Floss et al, 2009;Strasser et al, 2008), and most recently rice seeds (Vamvaka et al, 2015) with similar in vitro biding activity as C2G12 and variable HIV-neutralization efficacy. It has also been produced, along with a panel of other HIV-neutralizing antibodies, by transient expression (Rosenberg et al, 2013;Sainsbury et al, 2010).…”

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

“…Vamvaka et al [74] reported the development of transgenic rice plant expressing the HIV-neutralizing antibody 2G12 in the endosperm (42 µg/g dry seed weight) to evaluate the potential of rice seeds as a vehicle for inexpensive microbicide production. Production is higher than the initial achievement of maize-derived 2G12 (30 µg/g) by Rademacher et al [156].…”

Heritable Genomic Fragment Deletions and Small Indels in the Putative ENGase Gene Induced by CRISPR/Cas9 in Barley

“…Both activities have been detected in mature barley seeds, and there is a positive correlation between the removal of N-glycans from proteins and the mobilization of storage glycoproteins, reflecting the fact that both ENGase and PNGase activities increase during germination (Vuylsteker et al, 2000). Recombinant glycoproteins produced in cereal grains often carry a single GlcNAc linked to the asparagine residue, possibly reflecting endogenous ENGase activity (Rademacher et al, 2008; Hensel et al, 2015; Vamvaka et al, 2016). …”