Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV‐1 and contains predominantly single‐GlcNAc N‐glycans

Abstract

SummaryAntibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro , and the ability to prevent HIV-1 infection in animal models.It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N -glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

Manufacturing antibodies in the plant cell

“…In consequence, the development phase for recombinant mAb production is accelerated. Nevertheless, form the pionner work of Hiatt et al [13] to the most recent production of anti-HIV prophylactic IgG in corn [21,22], the lengthy process of establishing a homozygous transgenic plant line with stable and homogeneous IgG production has been the Achilles' heel of plant-made antibodies.…”

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication

“…Antibody concentrations were measured by surface plasmon resonance as described previously using a BIACORE 2000 (Biacore; GE Healthcare; Rademacher et al, 2008). 2G12 accumulation was measured from triplicate leaf extracts representing infiltrated tissue from six plants.…”

N-Glycopeptide Profiling in Arabidopsis Inflorescence

“…not cleave off a single GlcNAc); and 3) it is difficult to retain short glycan structures in released glycan analysis. It also has been observed that recombinant proteins expressed in plants can have glycans trimmed to a single sugar residue (32,33). The sample preparation for this study did not make a specific attempt to solubilize transmembrane proteins, so the results here may be biased toward more soluble proteins, with many Golgi-resident proteins identified.…”