The aim of this research is to use hydrogen/deuterium (H/D) exchange coupled with proteolysis and mass spectrometry to map antigen-antibody interactions. While the epitope mapping of therapeutic monoclonal antibody is a critical step for the characterization of the antibody, approaches used to characterize antigen-antibody interactions, such as X-ray crystallography and site directed mutagenesis are target limited and labor intensive; not all proteins can be crystallized and site-directed mutagenesis requires the production of a significant number of mutants. Furthermore, other techniques, such as binding assay of overlapping peptides, may miss conformational epitopes. H/D exchange is not subject to these limitations. We propose to measure the H/D exchange of an antigen in the presence and absence of its antibody and compare the deuteration levels of each peptide fragment generated under these two conditions. The binding of an antibody to its antigen should retard the exchange of amide hydrogen through solvent exclusion, and/or restriction of conformational fluctuations at the antigen-antibody interface. The comparison of the antigen H/D exchange patterns in the presence and absence of its antibody should generate a """"""""footprint"""""""" of the antibody binding site - the antigen epitope. H/D exchange is less disruptive and less resource intensive than traditional epitope mapping technologies. During exchange, the analyte protein retains its native solution state. On halting the reaction, the partially deuterated protein is then fragmented proteolytically to analyze deuterium incorporation of each peptide fragment by mass spectrometry. ExSAR has recently developed fully automated instrumentation to measure H/D exchange for protein dynamics and protein-ligand interactions. ExSAR would like to expand this capability to study protein- protein interactions and more specifically, antigen-antibody interactions. Our strategy requires antibody immobilization, creation of an antibody column, and execution of the antigen H/D exchange in the antibody column. The confirmation of the crystallographic contact residues by the H/D exchange determined epitope will validate this strategy. Our long-term objective is to develop a fully automated instrument capable of measuring H/D exchange of an antigen in the presence or absence of antibody. ? ?

Public Health Relevance

- PROJECT NARRATIVE: The epitope mapping of therapeutic monoclonal antibody is a critical step for the characterization of the antibody. ExSAR's developing technologies could be applicable to the investigation of therapeutic antibodies. A detailed knowledge of the dynamic interaction of an antibody and its antigen may contribute towards an understanding of mechanisms underlying antibody cross-reactivity, an important factor in therapeutic efficacy. Establishing a fast, cheap and reliable methodology for the epitope mapping should have a direct impact on the monoclonal antibody therapeutic business. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IMM-K (13))
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Edmonds, Charles G
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Exsar Corporation
Monmouth Junction
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Pandit, Deepangi; Tuske, Steven J; Coales, Stephen J et al. (2012) Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking. J Mol Recognit 25:114-24
Coales, Stephen J; Tuske, Steven J; Tomasso, Justine C et al. (2009) Epitope mapping by amide hydrogen/deuterium exchange coupled with immobilization of antibody, on-line proteolysis, liquid chromatography and mass spectrometry. Rapid Commun Mass Spectrom 23:639-47