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Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization

DOI: 10.1107/S2053230X20001466 DOI Help

Authors: Alkistis N. Mitropoulou (King's College London; Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma) , Tom Ceska (UCB Celltech) , James T. Heads (UCB Celltech) , Andrew J. Beavil (King's College London; Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma) , Alistair J. Henry (UCB Celltech) , James M. Mcdonnell (King's College London; Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma) , Brian J. Sutton (King's College London; Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma) , Anna M. Davies (King's College London; Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology Communications , VOL 76 , PAGES 116 - 129

State: Published (Approved)
Published: March 2020
Diamond Proposal Number(s): 1220 , 7656 , 9495

Open Access Open Access

Abstract: Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc∊RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc∊RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc∊RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

Journal Keywords: omalizumab; allergy; Fab; immunoglobulin E; antibody; protein engineering; X-ray crystallography.

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

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