Immunology Letters 133 (2010) 78–84 Contents lists available at ScienceDirect Immunology Letters journal homepage: www.elsevier.com/locate/ DARPins against a functional IgE epitope Michael J. Baumann a,b , Alexander Eggel a , Patrick Amstutz b , Beda M. Stadler a , Monique Vogel a,∗ a Institute of Immunology, University of Bern, Inselspital, Sahlihaus 2, 3010 Bern, Switzerland b Molecular Partners AG, Wagistrasse 14, 8952 Zürich-Schlieren, Switzerland article info Article history: Received 30 May 2010 Received in revised form 13 July 2010 Accepted 22 July 2010 Available online 29 July 2010 Keywords: DARPins Designed ankyrin repeat proteins Anti-IgE Allergic diseases Functional epitope Omalizumab abstract The monoclonal anti-IgE antibody omalizumab (Xolair ® ) is mostly used for the treatment of severe allergic asthma. However, the requirement of high doses and suboptimal cost-effectiveness limits the use of the treatment. Here we propose to use a new drug format based on non-immunoglobulin structures, poten- tially offering increased clinical efficacy while being more cost-effective. For this purpose, DARPins TM (designed ankyrin repeat proteins) against the constant heavy chain region of IgE have been isolated. DARPins were binding to IgE with high specificity and affinities in the low nanomolar range. Selected DARPins antagonized the interaction between IgE and its high-affinity receptor in inhibition assays. Fur- thermore, anti-IgE DARPins were shown to inhibit proinflammatory mediator release from rat basophilic leukemia cells expressing human high-affinity IgE receptors with higher efficacy than the monoclonal anti-IgE antibody omalizumab. DARPins may thus represent promising future drug candidates for the treatment of allergy. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Since the identification of immunoglobulin E (IgE) as key medi- ator of immediate hypersensitivity in asthma and allergic disorders [1–4], controlling of the IgE response has become one of the main therapeutic objectives. Direct targeting of IgE interaction with its high-affinity receptor (FcRI) [5–7] using monoclonal antibodies has been shown to be promising for the therapy of allergy [8,9]. Inhibition studies using -chain peptides [10,11] and the deter- mination of the crystal structure of the IgE–FcRI complex [12] mapped the two binding sites of FcRI to the N-terminal region of the C3 domains of both IgE heavy chains. Consistently with this requirement, omalizumab as well as other non-anaphylactogenic anti-IgE antibodies have been shown to recognize epitopes over- lapping with the binding site of FcRI [13]. Indeed, binding of these epitopes is thought to be of key importance for any bivalent IgE inhibitor, as the binding of anti-IgE antibodies to other epitopes allows the cross-link of IgE molecules already bound by its recep- tors, leading to the activation and degranulation of the underlying effector cells [14]. Murine anti-IgE antibodies of such epitope specificity are capa- ble to neutralize IgE in vitro [7,13,15]. One such antibody binding serum-free IgE is omalizumab (Xolair ® , Novartis Pharmaceuti- cals, Germany; Genentech Inc., USA). It has been humanized [16] and was shown to efficiently decrease serum IgE levels in vivo ∗ Corresponding author. Tel.: +41 31 632 03 34; fax: +41 31 381 57 35. E-mail address: monique.vogel@iib.unibe.ch (M. Vogel). by 85–95%. The density of FcRI on basophils was simultane- ously declined [17]. Treatment led to lower exacerbations and reduced the need for systemic corticosteroid therapy in clinical trials [8,18–22]. However, as only 1000–1500 receptors need to be cross-linked to initiate an allergic reaction [17,23], high doses (ranging from 75 mg to 375 mg every two or four weeks, depend- ing on the patient’s body weight and total serum IgE levels) of omalizumab are required to achieve successful IgE neutralization, rendering this therapy useful and cost-effective only for a restricted group of patients with severe asthma [24]. Besides optimized cost- effectiveness, increased potency would not only allow the existing anti-IgE patients to be treated in a less-frequent and lower-dosing regimen, but also broaden the group of patients to those suffering from asthma but currently not matching the inclusion criteria for omalizumab treatment. Numerous attempts have been made to create molecules with higher potency, either by modifying the immunoglobulin structure directly [25] or by developing alternative binding scaffolds based on non-immunoglobulin molecules [26]. DARPins (designed ankyrin repeat proteins) represent a promising approach of such novel binding scaffolds [26,27]. DARPins are designed proteins derived from natural ankyrin repeat proteins. They are composed of several repeat subunits each representing a binding domain. The assem- bled protein consists of two capping repeats intervened by two or more binding modules that are randomly modified on their surface. Combinatorial libraries with DARPins of varying repeat numbers have been constructed and have been used for selections against a wide range of targets [28–34], reviewed recently [35,36]. In the field of allergy, we have recently characterized bispecific DARPins that 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.07.005