Key role of a structural water molecule for the specificity of 14F7—An antitumor antibody targeting the NeuGc GM3 ganglioside

Abstract

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody–antigen interactions.

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

“…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 ). In mouse models, 14F7 showed strong anti-tumor effects ( 20 , 33 ).…”

“…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.…”