Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies

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. © 2012 caljon et al.