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Virus neutralization: Sweet antibodies have more bite

Functional Glycomics (11 June 2009) | doi:10.1038/fg.2009.21

New production systems focusing on the defined glycosylation of antibodies are improving potency and therapeutic potential.

Schematic representation of 'classic' mammalian and camelid antibody formats.

Antibodies can block binding of virus particles to the cell surface receptors that they exploit, preventing infection. When produced in mammalian cells, antibodies have a heterogeneous N-glycan profile known to influence antibody efficacy; however, the importance of individual glycan structures is undetermined. Non-mammalian expression systems can produce antibodies more cheaply but these have different glycosylation capabilities. Two new studies highlight how understanding and modifying antibody glycosylation can increase anti-viral potency: Strasser et al. report in the Journal of Biological Chemistry that a plant-based system producing homogenously beta1,4-galactosylated anti-HIV antibodies improves neutralization of the virus; while in Applied Microbiology and Biotechnology Harmsen et al. show that glycosylation of camelid antibodies produced in yeast increases their anti-toxin and anti-viral capacities.

Mammalian antibodies generally consist of two light and two heavy chains, but half of the circulating antibodies found in camelids lack light chains. The single N-terminal variable domain of these heavy-chain antibodies is fully capable of antigen binding. These 'nanobodies' present interesting therapeutic possibilities owing to their small size, high stability, good production levels in microorganisms and ease of modification by genetic fusion. About a tenth of nanobodies are glycosylated. Harmsen et al. immunized llamas with a toxin and used phage display and DNA sequencing to select nanobody clones for expression in the yeast Saccharomyces cerevisiae. The most potent toxin-neutralizing clone identified was found to be glycosylated and its neutralization efficiency was reduced threefold by de-glycosylation. The study also found that by fusing non-glycosylated anti-virus nanobodies to another that is glycosylated, anti-viral efficacy is increased.

Microorganisms are not the only source of recombinant proteins: production in plants has been gradually improved to increase yields, reduce production time and improve cost-effectiveness. Strasser et al. recently generated a plant glycosylation mutant that does not add unwanted plant specific beta1,2-xylose and core alpha1,3-fucose residues to glycoproteins. Full-size anti-HIV antibodies produced by this mutant were comparable in potency to mammalian cell-derived antibodies. In their new study, the authors went a step further by engineering the plants to add terminal beta1,4-linked galactose residues, thus producing anti-HIV antibodies homogeneously displaying the major human serum antibody N-glycan species. Additionally, by targeting the beta1,4-galactosyltransferase to a late Golgi compartment they avoided the problem of incomplete glycan processing previously seen with over-expression of the enzyme. In in vitro binding and neutralization assays the beta1,4-galactosylated antibodies were over three times as effective as other plant-derived glycoforms, which were already slightly more potent than antibodies produced in mammalian cells.

The exact mechanisms of improved virus neutralization by the glycosylated antibodies in these studies are unknown and likely to be slightly different. The small size of nanobodies means that the addition of a bulky sugar chain could considerably increase steric hindrance of virus-receptor binding. Meanwhile, small changes to terminal sugar residues can subtly affect full-size antibody conformation, potentially influencing antigen binding, stability and effector functions. Although the proposed mechanism for increasing nanobody potency would not require a specific glycan, switching to the plant-based system described by Strasser et al. would produce human-like glycosylations that avoid the problem of rapid clearance in vivo. Both papers highlight the potential therapeutic benefits of correct glycosylation during antibody production.

Emma Leah

Original research paper:

  1. Harmsen, M. M., van Solt, C. B. & Fijten H. P. D. Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation. Appl. Microbiol. Biotechnol. (12 May 2009) doi: 10.1007/s00253-009-2029-1 | Article |
  2. Strasser, R. et al. Improved virus neutralization by plant-produced anti-HIV antibodies with a homogenous beta1,4-galactosylated N-glycan profile. J. Biol. Chem. (28 May 2009) doi: 10.1074/jbc.M109.014126 | Article |