A structural and functional study of anti-ganglioside antibodies

NeuGc GM3 is considered an attractive target for cancer immunotherapy since it is a tumor-associated antigen present in several types of cancer such as breast carcinoma, melanoma and non-small cell lung cancer (NSCLC) while being effectively absent in healthy human tissues. The monoclonal antibody (mAb) 14F7 is specific for NeuGc GM3 and does not cross-react with NeuAc GM3, a ganglioside found in healthy tissues, even though these two glycolipids have highly similar structures. 14F7 treatment has shown to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), but has in addition the ability to directly kill tumor cells without involving the complement system. We employed a mass spectrometry-based approach and SPR analysis for the characterization of the binding interactions between 14F7 and the NeuGc GM3 ganglioside or the anti-idiotypic antibody generated from 14F7, 4G9, respectively. In order to understand the mechanism by which the antibody kills the cells, we then performed stable isotope labeling with amino acids in cell culture (SILAC) combined with proteomics to identify and quantify proteins after antibody treatment. SILAC was also employed to investigate the protein expression in HeLa cells under hypoxic conditions. The combination of MS, SPR and SILAC analyses employed here have allowed us to gain a significantly improved understanding of the molecular interactions and functions of a highly promising antibody for cancer immunotherapies.