The Characterization of anti body-antigen interactions provider Crucial information on the structural basis of the specificity of binding of two antibodies Their target anti-gens. In this project, an attempt was made two characterize the binding interactions Between a monoclonal anti-body (Mab) Designated as 14F7 and NeuGc-GM3 ganglioside antigen. The 14F7 strongly recognize the ganglioside antigen Fri human melanoma and breast tumors. The crystal structure of the Fab fragment 14F7 has Been solved by X-ray crystallography and bonding the two ganglioside Predicted by docking and MD Simulations. However, despite Significant efforts, so far no experimental structural analysis of the 14F7-NeuGc-GM3 complex is available. Such information Would ask extremely valuable for developing new and improved antibodies for cancer immunotherapy.There Parents, in this thesis, an alternative approach was overtaken two get experimental data. The binding interactions Within the complex were examined Using a newly Developed MS-based oxidative footprinting technique. Briefly, the anti-body sample, with and without the ligand, was overexposed two an excimer laser source in order two oxidize solvent accessible amino acid side chains. The oxidized samples were digested with trypsin simply. Finally, the tryptic fragments were analyzed by MALDI-TOF MS and ESI-LTQ-FT MS.
Sequencing of the MS / MS spectra and database searching identified two residue, Trp325 of the VL-CDR3 and Met112 of the VH-CDR3, That were oxidized in Both the free and ligand-bound 14F7. The Percentage of oxidation Of The peptides containing the residue were quantified based on the areas under the selected ion chromatograms of the oxidized and non-oxidized forms of the peptides. The Data Suggest That the Extent of oxidation of Both peptides is significantly lower in the ligand-bound 14F7. More over, an analysis of the spectral intensities and side chain solvent accessibilities confirmed That residue Met112 and Trp325 are protected from radical oxidation upon ligand binding the two anti-body. A new docking model of the complex is presented That is consis tent with all experimental data. The combined result, in contrast two the previous model, Suggest That the ligand volume in close proximity the two light chain, in Addition two bonding the two heavy chain CDR3 of the anti body. This information Can now ask unusually for Further Developmentof anti-tumor anti-body with improved potency and Affinity for the target tumor anti-gens.