Next Article in Journal
New Autoantibody Specificities in Systemic Sclerosis and Very Early Systemic Sclerosis
Previous Article in Journal
Utilizing Immunocytokines for Cancer Therapy
Article

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

1
NMI, Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany
2
Medannex Ltd., 1 Lochrin Square, Fountainbridge, Edinburgh EH3 9QA, UK
3
Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Geschwister-Scholl-Platz, 72074 Tuebingen, Germany
4
Department of Life Sciences, University of Applied Sciences Albstadt-Sigmaringen, Anton-Guentherstr. 51, 72488 Sigmaringen, Germany
*
Author to whom correspondence should be addressed.
Antibodies 2021, 10(1), 11; https://doi.org/10.3390/antib10010011
Received: 24 December 2020 / Revised: 15 January 2021 / Accepted: 2 March 2021 / Published: 19 March 2021
Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-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 Ca2+-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 Ca2+-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. View Full-Text
Keywords: HDX-MS; hydrogen–deuterium exchange; mass spectrometry; proteolysis-resistant protein; ANXA1; annexin-A1; conformational epitope mapping HDX-MS; hydrogen–deuterium exchange; mass spectrometry; proteolysis-resistant protein; ANXA1; annexin-A1; conformational epitope mapping
Show Figures

Figure 1

MDPI and ACS Style

Gramlich, M.; Hays, H.C.W.; Crichton, S.; Kaiser, P.D.; Heine, A.; Schneiderhan-Marra, N.; Rothbauer, U.; Stoll, D.; Maier, S.; Zeck, A. HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1. Antibodies 2021, 10, 11. https://doi.org/10.3390/antib10010011

AMA Style

Gramlich M, Hays HCW, Crichton S, Kaiser PD, Heine A, Schneiderhan-Marra N, Rothbauer U, Stoll D, Maier S, Zeck A. HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1. Antibodies. 2021; 10(1):11. https://doi.org/10.3390/antib10010011

Chicago/Turabian Style

Gramlich, Marius, Henry C.W. Hays, Scott Crichton, Philipp D. Kaiser, Anne Heine, Nicole Schneiderhan-Marra, Ulrich Rothbauer, Dieter Stoll, Sandra Maier, and Anne Zeck. 2021. "HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1" Antibodies 10, no. 1: 11. https://doi.org/10.3390/antib10010011

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop