Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab

Abstract

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.

Influence of Glycan Agents on Protein Crystallization with Ammonium Sulfate

“…The inclusion of glycan additives in protein solutions may occasionally cause a conformational change in protein structure. Belviso et al examined the conformation of a monoclonal antibody, Rituximab, using small-angle X-ray scattering (SAXS) measurement and MD simulation . The protein conformational changes are reflected in the R g analysis of MD simulation, and the changes can also be monitored by the experiments with SAXS.…”

“…Belviso et al examined the conformation of a monoclonal antibody, Rituximab, using small-angle X-ray scattering (SAXS) measurement and MD simulation. 60 The protein conformational changes are reflected in the R g analysis of MD simulation, and the changes can also be monitored by the experiments with SAXS. The conformational flexibility of the antibody molecule is high because the antibody comprised two Fab and one Fc domains.…”

B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients

“…Animal R.315 received a CD20 CAR with an antibody domain identical to the clinically-used CD20 antibody Rituximab. 48 The remaining animals (R.301-304) received a CD20 CAR-T cell product with a previously described CD20 CAR antibody domain. 23 All CAR-T cell vectors expressed a CD28 transmembrane domain, a 4-1BB costimulatory domain and the CAR construct utilized for animals R.301-304 additionally expressed EGFRt.…”

Biomolecular solution X-ray scattering at n2STAR Beamline

“…Understanding the structural characteristics of antibodies is a critical step for developing more competent therapeutic agents and SAXS could be utilized as a high throughput tool for skimming general structural characteristics of antibodies. In relation to singular characterization of antibodies, real time analysis of supplementation of additional compounds to determine the effects of these compounds or interacting particles paves the way for development of more efficient antibodies (Belviso et al, 2022). It is also shown that SAXS is capable of being a high throughput protein structure analysis tool with an automated system, without requiring extensive crystallization of multiple protein samples.…”