Belviso Benny Danilo, Mangiatordi Giuseppe Felice, Alberga Domenico, Mangini Vincenzo, Carrozzini Benedetta, Caliandro Rocco
Institute of Crystallography, CNR, Bari, Italy.
Cineca, Casalecchio di Reno, Italy.
Front Mol Biosci. 2022 Apr 11;9:823174. doi: 10.3389/fmolb.2022.823174. eCollection 2022.
Rituximab, a murine-human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.
利妥昔单抗是一种鼠-人嵌合抗体，是首个开发用于靶向CD20蛋白的治疗性单克隆抗体（mAb）。其Fab结构域及其与CD20的相互作用已得到广泛研究，通过X射线衍射或冷冻电子显微镜获得的高分辨率原子模型也已存在。然而，由于蛋白质固有的灵活性阻碍了形成衍射良好的晶体以及三维显微镜图像的重建，全长抗体的结构仍然未知。本文通过小角X射线散射（SAXS）阐明了利妥昔单抗在稀溶液中的整体结构。通过密集的计算建模弥补了该技术可实现的有限数据分辨率，从而开发出一种新的有效程序来表征单克隆抗体的平均构象以及单个结构域的构象。SAXS数据表明，利妥昔单抗在溶液中采用不对称的平均构象，回转半径和最大线性尺寸分别为52 Å和197 Å。不对称主要是由于两个Fab单元相对于中央茎（Fc结构域）的排列不均匀，并且反映在各个单元的不同构象中。因此，发现对于抗原识别和结合至关重要的Fab肘部角度在较远的Fab单元中更大（169°），而在较近的Fab单元中较小（143°）。已发现抗体的整体灵活性强烈依赖于结构域间的相对取向，其中一个Fab臂起主要作用。在存在不同缓冲液和添加剂的情况下研究了平均结构和灵活性，并在升高温度直至抗体完全展开的过程中进行了监测。总体而言，利妥昔单抗的结构表征有助于设计下一代抗CD20抗体，并找到工业规模生产利妥昔单抗的更有效途径。
