Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside

Abstract

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is an anti-tumor antibody with high clinical potential, which has extraordinary specificity for NeuGc GM3, but does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 [A] crystal structure of the 14F7 binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Intriguingly, a water molecule appears to shape the specificity of 14F7. Using model membrane systems, we show that 14F7 recognizes NeuGc GM3 only above lipid concentrations that are likely to form glycolipid-rich domains. This "all-or-nothing" effect was exacerbated in giant unilamellar vesicles and multilamellar vesicles, whereas no binding was observed to 100 nm liposomes, emphasizing that the 14F7-NeuGc GM3 interaction is additionally modulated by membrane curvature. Unexpectedly, adding NeuAc GM3 strongly increased binding affinity to NeuGc GM3-containing liposomes. This effect may be important for tumor recognition, where the ubiquitous NeuAc GM3 may enhance 14F7 binding to NeuGc GM3-expressing cancer cells.