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Affinity is an important determinant of the anti-trypanosome activity of nanobodies

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Organization
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. 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.
Keywords
HEAVY-CHAIN ANTIBODIES, VARIANT SURFACE GLYCOPROTEIN, SINGLE-DOMAIN ANTIBODIES, ANTIGENIC VARIATION, AFRICAN TRYPANOSOMES, IN-VITRO, BRUCEI, IDENTIFICATION, ENDOCYTOSIS, EXPRESSION

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Chicago
Caljon, Guy, Benoit Stijlemans, Dirk Saerens, Jan Van Den Abbeele, Serge Muyldermans, Stefan Magez, and Patrick De Baetselier. 2012. “Affinity Is an Important Determinant of the Anti-trypanosome Activity of Nanobodies.” Plos Neglected Tropical Diseases 6 (11).
APA
Caljon, Guy, Stijlemans, B., Saerens, D., Van Den Abbeele, J., Muyldermans, S., Magez, S., & De Baetselier, P. (2012). Affinity is an important determinant of the anti-trypanosome activity of nanobodies. PLOS NEGLECTED TROPICAL DISEASES, 6(11).
Vancouver
1.
Caljon G, Stijlemans B, Saerens D, Van Den Abbeele J, Muyldermans S, Magez S, et al. Affinity is an important determinant of the anti-trypanosome activity of nanobodies. PLOS NEGLECTED TROPICAL DISEASES. 2012;6(11).
MLA
Caljon, Guy et al. “Affinity Is an Important Determinant of the Anti-trypanosome Activity of Nanobodies.” PLOS NEGLECTED TROPICAL DISEASES 6.11 (2012): n. pag. Print.
@article{8131705,
  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. 
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 {\textrangle}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.},
  articleno    = {e1902},
  author       = {Caljon, Guy and Stijlemans, Benoit and Saerens, Dirk and Van Den Abbeele, Jan and Muyldermans, Serge and Magez, Stefan and De Baetselier, Patrick},
  issn         = {1935-2735},
  journal      = {PLOS NEGLECTED TROPICAL DISEASES},
  language     = {eng},
  number       = {11},
  pages        = {8},
  title        = {Affinity is an important determinant of the anti-trypanosome activity of nanobodies},
  url          = {http://dx.doi.org/10.1371/journal.pntd.0001902},
  volume       = {6},
  year         = {2012},
}

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