%0 Journal Article
%T Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies
%A Guy Caljon
%A Beno£¿t Stijlemans
%A Dirk Saerens
%A Jan Van Den Abbeele
%A Serge Muyldermans
%A Stefan Magez
%A Patrick De Baetselier
%J PLOS Neglected Tropical Diseases
%D 2012
%I Public Library of Science (PLoS)
%R 10.1371/journal.pntd.0001902
%X Background The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket. Methodology/Principal Findings Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (KD), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the KD remained unaffected. Conclusions/Significance This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.
%U http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001902