Mitropoulou Alkistis N, Ceska Tom, Heads James T, Beavil Andrew J, Henry Alistair J, McDonnell James M, Sutton Brian J, Davies Anna M
Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, London SE1 1UL, UK.
UCB Celltech, 208 Bath Road, Slough SL1 3WE, UK.
Acta Crystallogr F Struct Biol Commun. 2020 Mar 1;76(Pt 3):116-129. doi: 10.1107/S2053230X20001466. Epub 2020 Mar 2.
Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI 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 FcεRI. 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.
免疫球蛋白E（IgE）在过敏反应中起核心作用，在该反应中，与FcεRI结合的IgE与变应原交联会触发肥大细胞和嗜碱性粒细胞脱颗粒以及炎性介质的释放。IgE与FcεRI之间的高亲和力相互作用长期以来一直是过敏性疾病治疗干预的靶点。奥马珠单抗是一种临床批准的抗IgE单克隆抗体，它也以高亲和力与游离IgE结合，阻止其与FcεRI相互作用。令人惊讶的是，所有试图使奥马珠单抗Fab与IgE的Fc区域（IgE-Fc）预先形成的复合物结晶以了解其作用机制的结构基础的尝试均告失败。相反，单独的Fab以不同的晶体形式选择性结晶，但其结构揭示了分子间Fab/Fab相互作用，这些相互作用显然足够强，足以破坏Fab/IgE-Fc复合物。其中一些相互作用在其他Fab晶体结构中也很常见。因此设计了突变以破坏在奥马珠单抗Fab晶体结构中观察到的两种反复出现的堆积相互作用，而不干扰奥马珠单抗Fab识别IgE-Fc的能力；这导致了Fab/IgE-Fc复合物的成功结晶和随后的结构测定。为实现这一结果而采用的诱变策略适用于其他难以处理的Fab/抗原复合物或以Fab作为结晶伴侣的系统。
