Journal
FRONTIERS IN MOLECULAR BIOSCIENCES
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2022.823174
Keywords
structural comparison; anti-CD20 mab; molecular dynamics flexible fitting; small-angle X-ray scattering; full-length antibody
Categories
Funding
- European Union's Horizon 2020, Future and Emerging Technologies programme FET-OPEN, AMECRYS project [712965]
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The structure of rituximab, the first monoclonal antibody developed to target CD20 protein, has been investigated using small-angle X-ray scattering (SAXS), which revealed an asymmetric average conformation in solution. The flexibility of the antibody was found to depend on the relative orientations of its domains, with one of the Fab arms playing a major role. The structural characterization of rituximab can aid in the design of next-generation anti-CD20 antibodies and the optimization of its production.
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 angstrom and 197 angstrom, 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 degrees) in the more distant Fab unit than that in the less distant one (143 degrees). 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.
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