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

Marius Gramlich,Scott Crichton,Sandra Maier,Nicole Schneiderhan-Marra,Henry C W Hays,Dieter Stoll,Anne Heine,Anne Zeck,Ulrich Rothbauer,Philipp D Kaiser

doi:10.3390/antib10010011

Abstract

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.

Highlights

Annexin A1 (ANXA1) is a 38-kDa protein which belongs to the annexin family of calcium-dependent phospholipid-binding proteins [1]
We report two epitope-mapping approaches based on HDX followed by pepsin digestion in solution and immobilized on beads, which both led to the elucidation of the ANXA1 surface region affected by antibody binding
hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a well-established technology to study the structural dynamics of proteins or elucidate protein–protein interaction sites

Summary

Introduction

Annexin A1 (ANXA1) is a 38-kDa protein which belongs to the annexin family of calcium-dependent phospholipid-binding proteins [1] It contains a C-terminal core region, consisting of four homologous repeat domains (I–IV), of which each has five α-helices and a 41-amino-acid N-terminal region [2]. In addition to the well-documented role in neutrophil and monocyte function in the innate immune system, it has been shown to modulate the signaling strength of the T cell receptor and the T cell activation and differentiation [9,10] Because of these properties, ANXA1 has been proposed as a therapeutic target for the treatment of T cell activation dysregulation diseases such as rheumatoid arthritis or multiple sclerosis [11,12]

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