Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Abstract

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.

Keywords: epitope mapping; hydrogen/deuterium exchange mass spectrometry; mAb/antigen complexes; monoclonal antibody; native mass spectrometry.

Figure 1.

Fab:JAM-A binding stoichiometries as deduced from native mass spectrometry. Relative proportions of each species were deduced from native MS experiments in large excess of JAM-A (≥ 4 molar equivalents).

Figure 2.

Label-free interaction analysis monitored by surface plasmon resonance. Workflow of indirect and sequential competition experiments designed for the SPR study (a). Parts 2b-c shows the superposition of three sensorgrams starting with the capture of JAM-A by an anti-6His mouse monoclonal antibody chemically grafted on a CM5 sensor chip followed by the injection of a 50 µg/ml solution of antibody either hz6F4-2 (b), F11 (c) and J10.4 (d). A second solution of antibody (50 µg/ml) is then injected at each cycle either hz6F4-2 (red), F11 (green) or J10.4 (blue). The regeneration step at the end of each cycle is not shown.

Figure 3.

HDX difference plots and heat maps for free JAM-A compared to hz6F4-2:JAM-A (a), F11:JAM-A (b) and J10.4:JAM-A (c) complexes. The difference plots illustrate differential deuterium uptake for each identified peptide at different exposure times 0.5 (orange), 2 (grey), 10 (yellow), 30 (blue) and 60 min (green). Differences are summed into striped histograms which highlight localized variations. Significant differences in deuterium uptake are highlighted by red horizontal dotted line. Heat maps are colored from −3% (blue) to 9% (red) difference of deuterium uptake after 60 min. Color coding for relative deuterium uptake difference is shown at the bottom right (cold colors correspond to areas that incorporate more deuterium in the complexed state, and so become more exposed to deuterated buffer; while hot colors are associated with areas that incorporate less deuterium in the complexed state, and so are protected from labelling buffer).

Figure 4.

HDX-MS epitope mapping results plotted on JAM-A crystal structure (PDB: 1NBQ). Relative deuterium uptake differences were plotted on JAM-A crystal structure (PDB: 1NBQ) for hz6F4-2:JAM-A (a), F11:JAM-A (b) and J10.4:JAM-A (c). Deuterium uptake graphs of peptides showing significant differences in deuterium uptake are provided in panels (b), (d) and (f). Red lines stand for JAM-A alone while mAb:JAM-A complex are represented in blue curves.

Figure 5.

JAM-A epitopes superimposition for hz6F4-2, F11 and J10.4 binding. JAM-A epitopes were superimposed on monomeric (a) and dimeric (b) structures of JAM-A (PDB: 1NBQ). Blue: common area to hz6F4-2 and F11 epitope; Cyan: specific hz6F4-2 epitope; Magenta: specific F11 epitope; Red: J10.4 epitope.