Binder Mascha, Otto Florian, Mertelsmann Roland, Veelken Hendrik, Trepel Martin
Department of Hematology and Oncology, University of Freiburg Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
Blood. 2006 Sep 15;108(6):1975-8. doi: 10.1182/blood-2006-04-014639. Epub 2006 May 16.
Rituximab is a monoclonal antibody widely used in the treatment of malignant lymphoma and autoimmunity. Its epitope within the B-cell antigen CD20 is largely unknown. We used phage display libraries to select peptides binding to rituximab. Enriched peptides showed 2 sequence patterns: one motif (CALMIANSC) is related to (170)ANPS(173) within CD20, while another motif (WEWTI) may mimic the CD20 segment (182)YCYSI(185). Phages displaying either motif specifically bound rituximab. Binding to rituximab by the CD20 peptides ANPS and YCYSI was weak when used separately and enhanced when both peptides were linked. Recombinant CD20 extracellular loop proteins blocked binding of the selected CWWEWTIGC phage to rituximab, suggesting that CWWEWTIGC mimics the epitope. Blocking capacity was strongly reduced upon mutation of the CD20 strings ANPS or YCYSI. We conclude that rituximab binds a discontinuous epitope in CD20, comprised of (170)ANPS(173) and (182)YCYSI(185), with both strings brought in steric proximity by a disulfide bridge between C(167) and C(183).
利妥昔单抗是一种广泛用于治疗恶性淋巴瘤和自身免疫性疾病的单克隆抗体。其在B细胞抗原CD20上的表位在很大程度上尚不清楚。我们利用噬菌体展示文库筛选与利妥昔单抗结合的肽段。富集的肽段显示出两种序列模式：一种基序（CALMIANSC）与CD20内的（170）ANPS（173）相关，而另一种基序（WEWTI）可能模拟CD20片段（182）YCYSI（185）。展示这两种基序的噬菌体均能特异性结合利妥昔单抗。CD20肽段ANPS和YCYSI单独使用时与利妥昔单抗的结合较弱，而当两种肽段连接在一起时结合增强。重组CD20细胞外环蛋白可阻断所选的CWWEWTIGC噬菌体与利妥昔单抗的结合，这表明CWWEWTIGC模拟了表位。当CD20序列ANPS或YCYSI发生突变时，阻断能力大幅降低。我们得出结论，利妥昔单抗结合CD20中的一个不连续表位，该表位由（170）ANPS（173）和（182）YCYSI（185）组成，这两个序列通过C（167）和C（183）之间的二硫键在空间上接近。
