Figure 1.
Chemical structure of GD2 ganglioside.
Docking studies were performed with GD2-head, where the ceramide moiety was replaced by a methyl group.
Table 1.
Summary of crystallographic analysis.
Figure 2.
Crystal structure of MoAb 3F8 Fab fragment and docked model with GD2 pentasaccharide head group.
A. Space filling model of the antigen binding domain of MoAb 3F8. Heavy Chain CDR loops colored in dark blue; light chain CDR loops colored in light blue. B. Space filling model of 3F8 with docked GD2 pentasaccharide head group. C. Backbone ribbon diagram of 3F8 antigen binding domain. D. Key interacting residues in 3F8:GD2 docked complex.
Table 2.
Comparison of docking algorithms GLIDE versus CDOCKER in predicting known protein:ganglioside complexes.
Table 3.
Results of in silico scanning mutagenesis of CDR residues that directly interact with docked GD2 antigen. Energies are shown in units of kcal/mol.
Figure 3.
Hydrophobic surface map of MoAb 3F8 and MoAb 3F8-Ile (H:Gly54Ile).
Surfaces are rendered using Spatial Aggregation Propensity algorithm [34]. Highly hydrophobic patches are rendered in red, whereas hydrophilic surfaces are rendered in blue. A. MoAb 3F8 contains a hydrophobic patch that centers around H:Ile56. B. MoAb3F8-Ile has a binding pocket that creates additional surface contact with GD2 and contains a larger exposed hydrophobic surface area.
Figure 4.
Hu3F8 with H:Gly54Ile has enhanced binding to GD2.
A. ELISA assay of hu3F8 and hu3F8-Ile binding to ganglioside GD2 adhered to an ELISA plate. B. Retention of MoAbs hu3F8 and hu3F8-Ile to melanoma M14 cells after successive washes with PBS-EDTA. Decay rate constants and t1/2 are shown based on exponential decay curve fits.
Table 4.
Binding to GD2-positive and GD2-negative neuroblastoma cell lines by flow cytometry.
Figure 5.
Hu3F8 with H:Gly54Ile has enhanced ADCC.
A. ADCC on neuroblastoma LAN-1 cells. B. ADCC on melanoma M14 cells. C. ADCC on melanoma OCM-1 cells. IC50 values shown are based on sigmoidal curve fits.