Two Structurally Different Rituximab-Specific CD20 Mimotope Peptides Reveal That Rituximab Recognizes Two Different CD20-Associated Epitopes

Peptide mimotopes of the CD20 epitope recognized by rituximab are useful tools for studying this therapeutic mAb's functional properties. We previously identified two structurally different peptides that are both effective mimotopes: a 7-mer cyclic peptide (Rp15-C) bearing the antigenic motif (a/sNPS) that matches 170(ANPS)173 of the extracellular loop of CD20, and a 12-mer linear peptide (Rp5-L) containing the antigenic motif (WPxWLE) lacking sequence homology to CD20. In this study, we investigated whether the different structures of Rp15-C and Rp5-L reflect the mimicry of the same or different CD20 epitopes recognized by rituximab. Using immunochemical methods, we found that, like Rp15-C, Rp5-L mimics the raft-associated form of CD20 (by inhibiting rituximab binding to CD20 in vitro). Rp5-L and Rp15-C elicit, in immunized mice, anti-CD20 Abs that stain CD20+ cells with a punctate pattern similar to that of rituximab. However, only anti-Rp5-L Abs recognize denatured CD20. When phage-display peptide libraries were panned with anti-Rp5-L, phage clones were enriched that expressed the consensus qWPxwL, similar to the antigenic motif (WPxWLE), but not matching (a/sNPS). Finally, (WPxWLE) and (ANPS) share some, but not all, contact sites within the rituximab Ag-combining site, indicating that (WPxWLE) is not an exact replica of Rp15-C (or CD20) (ANPS). Altogether, these results indicate that the two structurally different peptides are also conformationally different, and suggest that rituximab recognizes two different CD20-associated epitopes.