H/D-Exchange Applications –
Applying H/D-Exchange technology to advance the process of discovery and development of biological therapeutics through epitope mapping
Epitope mapping is an essential aspect of the discovery and development of diagnostic and therapeutic antibodies. Epitope mapping can streamline the selection of lead candidate molecules, particularly where epitope similarity or dissimilarity issues are involved. Intellectual property considerations of patentability, with freedom to operate consequences, can hinge upon the epitope itself. Furthermore, regulatory agencies recommend that prior to use in humans and, whenever possible, the protein bearing the reactive epitope, should be biochemically defined and the antigenic epitope itself determined (PTC/FDA, 94D-0259).
Crystal structure of neutralizing antibody / IL-17 complex with H/D-Exchange identified IL-17 epitope red highlighted
While an X-ray co-crystal of the antigen:antibody complex remains the gold standard of epitope determination, it is technically challenging, tedious and not always feasible due to the difficulty of obtaining high-quality, well-diffracting crystals. In the absence of a crystal complex, hydrogen/deuterium exchange (e.g., DXMS, H/D-Ex, etc.) is the next best available option. ExSAR employs hydrogen/deuterium exchange mass spectrometry as a tool to identify binding interfaces. Differences in the rate of exchange serve to highlight the location of an epitope.
Illustrated in the figure above, hydrogen/deuterium exchange (H/D-Ex) was utilized to identify the epitope of a potent neutralizing antibody to IL-17, a homodimeric cytokine involved in a number of pro-inflammatory signaling pathways. Illustrated in red are areas where exchange was significantly reduced on complex formation. Pink highlighted regions indicate areas that were moderately affected upon complex formation. The epitope was subsequently validated by the X-ray crystal structure of the complex.
Raymond F. Salemme, PhD, on H/D-Exchange vs. Crystallography for Antibody Epitope Mapping:
“The question often comes up, ‘What are the relative advantages or disadvantages of using H/D-Exchange to map antibody binding epitopes vs. direct X-ray crystal structure determination of an antibody-epitope complex.’
“There are numerous biophysical considerations that go into an informed response to this question (For example, is the low energy X-ray structure, impacted by lattice packing effects, the only, or even the most relevant structure defining the antibody-immunoglobulin complex?).
“Biophysical complexities aside, the short answer is simple.
“If epitope mapping is to be used as a component part of an antibody discovery or intellectual property strategy, the speed and reliability of the method used are the clear determinants governing the best approach. To be part of a discovery process that directs research decisions, the method needs to work quickly and reliably (essentially) every time.
“H/D-Exchange is the clear winner here, since in the vast majority of cases, it is relatively straightforward to do the required MS exchange measurements and get the answer. In contrast, crystallization of a immunoglobulin-epitope complex is a far more difficult, unreliable, and consequently time-consuming, experimental process.”