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

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HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1 antibodies Article HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1 Marius Gramlich 1, Henry C. W. Hays 2, Scott Crichton 2, Philipp D. Kaiser 1, Anne Heine 1, Nicole Schneiderhan-Marra 1, Ulrich Rothbauer 1,3, Dieter Stoll 1,4, Sandra Maier 1 and Anne Zeck 1,* 1 NMI, Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany; [email protected] (M.G.); [email protected] (P.D.K.); [email protected] (A.H.); [email protected] (N.S.-M.); [email protected] (U.R.); [email protected] (D.S.); [email protected] (S.M.) 2 Medannex Ltd., 1 Lochrin Square, Fountainbridge, Edinburgh EH3 9QA, UK; [email protected] (H.C.W.H.); [email protected] (S.C.) 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 * Correspondence: [email protected]; Tel.: +49-7121-51530-0; Fax: +49-7121-51530-816 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 ar- ranged 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, Citation: Gramlich, M.; Hays, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking H.C.W.; Crichton, S.; Kaiser, P.D.; selected regions on ANXA1 holds great potential for the development of novel therapeutics treating Heine, A.; Schneiderhan-Marra, N.; inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific Rothbauer, U.; Stoll, D.; Maier, S.; antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using Zeck, A. HDX-MS for Epitope amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, Characterization of a Therapeutic we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized ANTIBODY Candidate on the 2+ Calcium-Binding Protein Annexin-A1. on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca -bound Antibodies 2021, 10, 11. https:// conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the doi.org/10.3390/antib10010011 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 Received: 24 December 2020 binding sites of antibodies which will help to define a mode of action profile for their use in therapy. Accepted: 2 March 2021 Published: 19 March 2021 Keywords: HDX-MS; hydrogen–deuterium exchange; mass spectrometry; proteolysis-resistant protein; ANXA1; annexin-A1; conformational epitope mapping Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. 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 Copyright: © 2021 by the authors. a 41-amino-acid N-terminal region [2]. The C-terminal region is tightly compressed into Licensee MDPI, Basel, Switzerland. a slightly curved disk, which renders the protein resistant to enzymatic hydrolysis. The This article is an open access article convex face of the protein contains 12 calcium-binding sites [3] which, when occupied, distributed under the terms and lead to a conformational change that exposes the N-terminal domain III. Subsequently, two conditions of the Creative Commons ANXA1 molecules can form a dimer or interact with a second bilayer [2]. The N-terminal Attribution (CC BY) license (https:// domain is highly variable between the different members of the annexin family [4]. ANXA1 creativecommons.org/licenses/by/ function is mediated through binding to the formyl peptide receptor (FPR) and/or the 4.0/). Antibodies 2021, 10, 11. https://doi.org/10.3390/antib10010011 https://www.mdpi.com/journal/antibodies Antibodies 2021, 10, 11 2 of 12 phospholipid bilayer of the cell membrane [5]. The role of ANXA1 has been investigated in a variety of different diseases, including cardiology, immunology, neurology, endocrinology, and oncology [5–7]. One of ANXA1’s most important properties is its ability to alter the innate and adaptive immune system [5,8]. 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 consequently 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]. In this study, we characterize the binding region of a therapeutic antibody candidate binding to ANXA1 in complex with calcium [11]. The humanized antibody was generated from a murine antibody that has been shown to specifically inhibit T cell activation without any adverse cytotoxic effects [12]. Knowing the interaction region between the antibody and ANXA1 will help to define a mode of action profile for its therapeutic use. Epitopes of therapeutic monoclonal antibodies (mAbs) are often discontinuous and are only recognized by the mAb in their native conformational state [13]. A detailed map- ping of such epitopes is challenging and there is only a limited number of technologies available. Of these technologies, hydrogen–deuterium exchange coupled to mass spectrom- etry (HDX-MS) does not introduce changes to the binding partners, such as the removal or addition of modifications (e.g., glycosylation, mutations, cross-linkers). In compari- son to other structural high-resolution techniques, such as X-ray crystallography [14,15], NMR spectroscopy [16,17], or cryo-electron microscopy [18,19], HDX-MS is an effective method in terms of time, sample requirement, and throughput to supply coarse to highly resolved epitope structure information [13,20]. The method relies on differences in the solvent accessibility of epitope and non-epitope regions at the surface of an antigen. The exchange rate of backbone amide hydrogen atoms against deuterium slows down when the surface area is protected by the binding partner [21]. Following complex formation and deuteration, the antigen is proteolyzed by pepsin and the mass increase in the peptides is determined as a function of time to assess the level of deuteration [22]. Here, 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. Both approaches were adapted during the feasibility stage to the compact three-dimensional conformation of ANXA1 and to the calcium dependency of the antibody binding. 2. Results 2.1. Antigen and Antibody Characterization The antigen and the humanized murine anti-ANXA1 antibody (IgG1) were produced recombinant in E. coli and Chinese hamster ovary (CHO) cells, respectively. Their identity and purity were confirmed by HPLC-MS (Figure S1). Binding affinities of the anti-ANXA1- antibody for the two conformational states of ANXA1 (in the presence (holo) or absence (apo) of 1 mM Ca2+) were measured by surface plasmon resonance (SPR). For the Ca2+- bound holo-state of ANXA1, an equilibrium dissociation constant (KD) of 2.66 ± 0.02 nM was determined (Figure1a), whereas no binding of the anti-ANXA1 antibody to the apo- state of ANXA1 was detectable (Figure1b). This strongly suggests that the antibody exclusively binds to the Ca2+-bound holo-state of ANXA1. As a result, epitope characteri- zation of the antibody–antigen complex formation was only performed in the presence of 1 mM Ca2+. To ensure a high degree of complex formation (>89%) and thereby avoid a false EX1 signature, the KD value was used to calculate the antibody and antigen concentrations. Antibodies 2021, 10, 11 3 of 12 Antibodies 2021, 10, x FOR PEER REVIEW 3 of 12 FigureFigure 1.1.Binding Binding affinitiesaffinities ofof anti-ANXA1anti-ANXA1 antibody.antibody. ForFor surfacesurface plasmonplasmon resonanceresonance spectroscopyspectroscopy (SPR)-based(SPR)-based affinityaffinity measurements,measurements, anti-ANXA1anti-ANXA1 antibodyantibody waswas immobilized immobilized via via a a covalently covalently coupled coupled anti-human anti-human IgG IgG on on a a CM5 CM5 chip. chip. KineticKinetic measurementsmeasurements were were performed performed by by injecting injecting five five concentrations concentratio ofns ANXA1of ANXA1 in presence in presence and and absence absence of calcium of calcium (holo (holo (a), and (a), apoand (apob), respectively) (b), respectively) ranging ranging from from 1.6 to1.6 25 to nM. 25 nM. The The obtained obtained datasets datasets were were evaluated evaluated using using the the 1:1 1:1 Langmuir Langmuir binding binding model.model. AllAll measurementsmeasurements werewere performedperformed inin triplicate.triplicate.
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