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. 2015 Nov 17;16(11):27497–27507. doi: 10.3390/ijms161126037

Figure 3.

Figure 3

Figure 3

TriFab mediated targeted delivery of a large molecule. The applied bsAb formats are schematically depicted on the right (cell targeting entities in blue, Dig-binding entities in red and Bio-binding entities in green colour). (a) TriFab specific for Dig and GPC3 or CD33 or LeY combined with Dig-Saporin or Bio and GPC3 or CD33 or LeY combined with Bio-Saporin were applied for targeted delivery of saporin. TriFab-Saporin complexes were generated by a simple and robust charging procedure as previously described for hapten-coupled payloads [11,13,14,15]: Dig-Saporin and TriFabs are incubated in a 1:1 molar ratio in cell culture medium for at least 15 min, followed by subsequent dilution to the concentrations indicated. BrdU incorporation and ATP-content (Cell Titer Glo, CTG) assays were applied to measure the viability of cells 48 h after exposure to TriFab and Saporin; (b) Targeted delivery of Dig-Saporin with IgG-derived (two antigen binding sites + two Dig-binding sites) or Fab-derived (one antigen binding site + two Dig-binding sites) bsAbs of the same targeting specificity indicates that TriFabs have at least the same specificity and delivery potential as other bsAb Formats (monovalent cell surface targeting with LeY specificity is less potent than bivalent (avidity-enhanced) targeting).