Abstract
Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-A resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro(172)). The (170)ANPS(173) motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity.
Highlights
CD20 is a pan-B cell marker expressed from pre-B cells until B cells are differentiated into plasma cells [1]
Overall Structure of the Rituximab Fab-CD20 Epitope Peptide Complex—Recent studies have identified the epitope of CD20 recognized by Rituximab being a sequence motif located at the large extracellular loop of CD20 consisting of 170ANPS173 [4, 19, 23]
To mimic the conformation of the CD20 epitope, an intrachain disulfide bond was introduced between residues Cys167 and Cys183 of the synthesized peptide because such linkage has been found in human CD20 expressed in Escherichia coli and probably exists naturally [33]
Summary
Preparation of Antibody and Peptide—Rituximab was purchased from Roche. The Fab fragment was obtained by papain digestion of Rituximab and purified by cation exchange chromatography using SP-Sepharose FF (GE Healthcare) followed by hydrophobic interaction chromatography using phenylSepharose HP (GE Healthcare). Crystallization and Diffraction Data Collection—Initial crystallization trials of the Rituximab Fab fragment itself yielded large crystals that, did not diffract x-ray beyond 7-Å resolution and could not be used for structure determination. For co-crystallization experiments, the purified Rituximab Fab and the epitope peptide were mixed at a molar ratio of 1:5 at 4 °C for 12 h. Structure Determination, Refinement, and Analysis—The structure of the Rituximab Fab in complex with human CD20 epitope peptide was solved using the molecular replacement method as implemented in Phaser [25]. The structure of the Fab fragment of human mAb A5B7 [26] (Protein Data Bank code 1AD0) was used as the search model. Figures were prepared using programs Ribbons [31] and Pymol [32]
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