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Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

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.

SUBMITTER: Harmsen MM

PROVIDER: S-EPMC2755796 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

Harmsen M M MM van Solt C B CB Fijten H P D HP

Applied microbiology and biotechnology 20090512 6

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 c ...[more]

PMID: 19455325

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Project description:Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.

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Dataset Information

Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

Publications

Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

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