Abstract
The structure of CD20 with the antibody drug rituximab showed the binding mode and 2:2 stoichiometry.
Major Finding: The structure of CD20 with the antibody drug rituximab showed the binding mode and 2:2 stoichiometry.
Concept: Cryo-electron microscopy revealed a previously unrecognized second epitope and Fab–Fab interactions.
Impact: This structure provides a basis for further exploration of how CD20 antibodies deplete B cells.
Multiple monoclonal antibodies targeting CD20, an integral membrane protein specific to B cells, are approved for the treatment of B-cell malignancies and autoimmune disorders. These therapies are effective because they result in B-cell depletion, but there appear to be several mechanisms of action, and a lack of high-resolution structural data on CD20 has hindered understanding of how anti-CD20 therapies work at the molecular level. Rougé and colleagues used cryo-electron microscopy to determine the structure of CD20 bound to the therapeutic antibody rituximab at a resolution of 3.3 Å. Inspection of the structure revealed that rituximab-bound CD20 existed as a dimer, with each dimer being bound by two antigen-binding fragments (Fab) from two separate rituximab molecules. Further analysis of the structure showed that CD20 had a unique fold characterized by four tightly packed antiparallel transmembrane helices and two extracellular loops (ECL), one of which (ECL2) contained a highly solvent-accessible region that bears the epitope recognized by most CD20 antibodies. In addition to this known epitope on ECL2, the structure also contained a secondary epitope on ECL1, which was mainly recognized by light-chain residues and, based on the contact surface area, likely contributed substantially to the affinity of rituximab for CD20. Fab–Fab interactions between the two rituximab molecules in each complex were facilitated by proximity between the primary epitopes of each CD20 molecule, further strengthening the tetrameric structure. Combined with the results of some biochemical experiments, these structural details explain why rituximab has such high affinity (in the nanomolar range) for CD20 despite its low affinity for the primary epitope found on ECL2. Interestingly, molecular modeling of an assembly of CD20 and rituximab molecules based on the structure revealed by this study suggested how rituximab facilitates cell-surface CD20 clustering and complement recruitment. In summary, this study provides an unprecedented view into the structure of the therapeutic target CD20 and imparts hints about the mechanism of action of rituximab.
Note: Research Watch is written by Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details. For more Research Watch, visit Cancer Discovery online at http://cancerdiscovery.aacrjournals.org/CDNews.
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