HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

• Published in: Antibodies (Basel) 2021-03, Vol.10 (1), p.11
• Main Authors: Gramlich, Marius, Hays, Henry C W, Crichton, Scott, Kaiser, Philipp D, Heine, Anne, Schneiderhan-Marra, Nicole, Rothbauer, Ulrich, Stoll, Dieter, Maier, Sandra, Zeck, Anne
• Format: Article
• Language: English
• Subjects: Amino acids; annexin-A1; Antibodies; Antigens; ANXA1; Apoptosis; Beads; Binding sites; Calcium; Calcium ions; Calcium-binding protein; Cell activation; Cell proliferation; Cytotoxicity; Deuterium; Domains; Drug development; Epitopes; HDX-MS; Homology; Hydrogen-deuterium exchange; Hydrophobicity; Immune system; Inflammatory response; Lymphocytes; Lymphocytes T; Mass spectrometry; Mass spectroscopy; Metastases; Mode of action; Pepsin; Peptides; Phospholipids; Protein structure; Proteins; proteolysis-resistant protein; T cell receptors
• ISSN: 2073-4468; 2073-4468
• DOI: 10.3390/antib10010011

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca -dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca -dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen-deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca -bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.