Appl Microbiol Biotechnol (2009) 84:1087–1094 DOI 10.1007/s00253-009-2029-1 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation M. M. Harmsen & C. B. van Solt & H. P. D. Fijten Received: 20 March 2009 / Revised: 29 April 2009 / Accepted: 29 April 2009 / Published online: 12 May 2009 # The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N- glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity. Keywords Nanobody . Recombinant antibody . Neutralization . N-glycosylation . Yeast M. M. Harmsen (*) : C. B. van Solt : H. P. D. Fijten Central Veterinary Institute of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands e-mail: michiel.harmsen@wur.nl Introduction Camelids produce antibodies that are naturally devoid of light chains (Hamers-Casterman et al. 1993). The single N- terminal domains of these heavy-chain antibodies (VHHs) are fully capable of antigen binding. They have several advantages for biotechnological purposes, such as a high physicochemical stability, a high production level in microorganisms, and the facile construction of genetic fusions of two VHH domains (Harmsen and De Haard 2007). As a result, there is currently much interest in various therapeutic applications of VHHs (Harmsen and De Haard 2007; Saerens et al. 2008). The mechanism of action of therapeutic antibody frag- ments is often based on blocking the interaction of their cognate antigen with a specific receptor. This is exemplified by antibodies neutralizing heat-labile toxin (LT), which is secreted by enterotoxigenic E. coli, and the closely related cholera toxin (CT), produced by Vibrio cholerae. Both toxins (reviewed by Spangler 1992) cause diarrheal diseases and belong to the AB5 toxin family. The five B- subunits are responsible for holotoxin binding to the monosialoganglioside (GM1) receptor on intestinal epithe- lial cells (Cuatrecasas 1973) after which the single A subunit is injected into the cells (Holmgren 1981). Neutralizing antibodies generally bind to the B-subunit and block receptor interaction (Apter et al. 1993). Similarly, virus neutralization by antibodies predominantly relies on the ability to block the interaction between the virion and a specific cellular receptor (Parren and Burton 2001). Such blocking may occur due to direct binding to a viral epitope involved with receptor interaction. Consistent with this we observed that most potent foot-and-mouth disease virus (FMDV) neutralizing VHHs recognized an epitope called the GH-loop (Harmsen et al. 2007), which binds to the