title: Affinity is an important determinant of the anti-trypanosome activity of nanobodies.
abstract: 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.
  
  
  RESULTS
  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 (K(D)), 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 K(D) remained unaffected.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biomedical Sciences, Unit of Veterinary Protozoology, Institute of Tropical Medicine Antwerp, Antwerp, Belgium. gcaljon@itg.be
authors.forename: Guy
authors.initials: G
authors.lastname: Caljon
authors.forename: Benoît
authors.initials: B
authors.lastname: Stijlemans
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Jan
authors.initials: J
authors.lastname: Van Den Abbeele
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Stefan
authors.initials: S
authors.lastname: Magez
authors.forename: Patrick
authors.initials: P
authors.lastname: De Baetselier
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity; D051379:Mice; D008810:Mice, Inbred C57BL; D050296:Microbial Viability / Q000187:drug effects; D061905:Single-Domain Antibodies / Q000276:immunology* / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D061905:Single-Domain Antibodies