Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses

Abstract

Antibodies play a major role in clinical diagnostics and biopharmaceutical analysis, but are also a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design mAb mimics taking advantage of amino acids residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received a significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallography study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four β-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.Biomolecular recognition is central to many biological processes governing cell fate. In this context, the monoclonal antibodies (mAbs) are able to bind to a specific antigen triggering the death of the targeted cell. 1 To date, mAbs are increasingly used for treatment of a variety of diseases. 2 In particular, rituximab (RTX), first FDA (Food and Drug Administration) approved therapeutic mAb, is routinely used for the treatment of several types of lymphoma as well as some autoimmune disorders. 3 RTX was shown to target a transmembrane protein named CD20 (cluster of differentiation 20), which is highly expressed on most healthy and malignant B cells but not on precursor B cells. 4 The identification of CD20 recognized by Rituximab has raised great interest in recent years especially to better apprehend RTX mechanism and action. Additionally, understanding molecular recognition could pave the way for structure-based design of bioactive molecules. Very recently, it has been shown by using cryo-electron microscopy (cryo-EM) that the native CD20 structure is a compact dimeric form allowing RTX cross-links that can induce complement recruitment. 5,6 This result corroborates other studies indicating that CD20s are not expressed in monomeric form, but organized into supramolecular protein complexes. 7,8 In parallel, our group has studied the effect of CD20 density on the recognition by RTX, which is known to be a critical point for the therapeutic response. 9 An average critical inter-CD20 spacing of nearly 2 nm confers the best conditions for RTX binding. This value is in excellent agreement with cryo-EM structures. 5,6 It is important to note that this finding suggests RTX contacts with two different epitopes. 5 Regarding the identification of the antigen-binding site of the antibody, a pioneer crystal structure of the RTX antigen-binding fra...

Resemblance-Ranking Peptide Library to Screen for Binders to Antibodies on a Peptidomic Scale

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Mechanisms of Action and Limitations of Monoclonal Antibodies and Single Chain Fragment Variable (scFv) in the Treatment of Cancer

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