Validation of epitopes and paratopes for antibodies and antigens is crucial for understanding antibody binding efficiency and future antibody development, and thus has raised grown interest in pharmaceutical field. Multiple structural techniques like Cryo-EM, NMR and HDX were readily applied to the antibody-antigen systems to confirm the interaction interface. Cross-linking and covalent labeling coupled with mass spectrometry have been known as useful tools to monitor protein structures as well as protein-protein interactions in their native states. As such, it is desirable to develop new strategies with these techniques to better identify the antigen binding interface.

Different from common proteins, antigen-antibody complexes raise new challenges for cross-linking and covalent labeling experiments due to the lack of lysine residues on CDR regions of antibodies.

Here, we propose using unconventional cross-linkers like EDC, which is reactive towards carboxylic acid residues, for EGFR and Cetuximab or Zalutumumab complexes. Results are compared with that of NHS ester cross-linkers. Similarly, DEPC, which is a labeling reagent reactive towards six residues, are used for covalent labeling experiments. The results are compared with that of sulfo-NHS acetate.

EGFR-Cetuximab complex showed higher percentage of inter-molecular cross-linking across all cross-linkers tested compared to EGFR-Zalutumumab complex based on SDS-PAGE. Both EDC and NHS ester cross-linkers were able to obtain many intra-molecular cross-links within expected distance based on cross-linkers. However, only NHS ester was able to obtain inter-molecular cross-links in EGFR-Cetuximab complex. For covalent labeling experiments, both DEPC and sulfo-NHS were able to obtain results that are consistent with cross-linking in the EGFR-Cetuximab system.

Yiran Ma

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