Rituximab binds to a discontinuous epitope within the extracellular domain of CD20. (A) Phages displaying both parts of the presumed epitope bind stronger to rituximab than phage displaying ANPS or YCYSI alone. Phages (5 × 107 TU) displaying no peptide insert (fd-tet) or the indicated peptides, respectively, were incubated both on immobilized rituximab and control antibody basiliximab. Bound phages were recovered by K91 bacterial infection. Transduced bacteria were grown on LB plates containing tetracycline to determine the number of TUs by colony counting. TU counts are shown relative to the number of TUs recovered from wells incubated with fd-tet. Data are means from triplicate platings ± SEM. (B) GST fusion proteins block binding of CWWEWTIGC phages to rituximab depending on their mutational status. CWWEWTIGC phages or insertless fd-tet phages (5 × 107 TU), respectively, were incubated on immobilized rituximab in the presence or absence of excess molar concentrations (0.2 mg/mL) of GST fusion proteins containing the presumed wild-type or mutated sequences shown in Table 2. Bound phages were recovered by bacterial infection. Transduced bacteria were grown on LB plates containing tetracycline to determine the number of TUs by colony counting. TUs are indicated relative to binding of WWEWTIG phages in the absence of GST fusion protein. Data are means from triplicate platings ± SEM. Phage binding is significantly blocked by GST-CD20 (P = .018), GST-CD20 M1 (P = .023) and GST-CD20 M4 (P = .029) compared with the GST control. (C) Model of the transmembrane and the extracellular domain of CD20 (based on Ernst et al5 ) with the suspected discontinuous epitope marked in blue. The disulfide bridge between C167 and C183 (Ernst et al5 ) is marked in yellow.
