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 Å 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.

SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7

“…14F7 is a clinically promising monoclonal antibody raised against the ganglioside NeuGc GM3, which represents an attractive target for cancer immunotherapy since this glycolipid is absent from healthy adult human tissues ( 15 ), but present in several malignancies ( 16 – 26 ). 14F7 is an IgG1 antibody with high affinity for its antigen (in the low nanomolar range) ( 19 , 27 – 29 ). This interaction has been characterized structurally (complex with the carbohydrate part of the glycolipid) ( 30 , 31 ) and by mutation analysis ( 32 ).…”

“…We have recently solved the crystal structure of the complex between 14F7 (a single-chain version) and the NeuGc GM3 trisacharide ( 31 ) and investigated how 14F7 recognizes NeuGc GM3 in a membrane-like environment ( 29 ). Here we seek to understand the effects that 14F7 induces in the cell, to gain a deeper understanding of the novel oncosis-like cell death mechanism induced by 14F7.…”