Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies

• Published in: Blood 2011-07, Vol.118 (2), p.358-367
• Main Authors: Niederfellner, Gerhard, Lammens, Alfred, Mundigl, Olaf, Georges, Guy J., Schaefer, Wolfgang, Schwaiger, Manfred, Franke, Andreas, Wiechmann, Kornelius, Jenewein, Stefan, Slootstra, Jerry W., Timmerman, Peter, Brännström, Annika, Lindstrom, Frida, Mössner, Ekkehard, Umana, Pablo, Hopfner, Karl-Peter, Klein, Christian
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - classification; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived - chemistry; Antibody Specificity; Antigens, CD20 - chemistry; Antigens, CD20 - genetics; Antigens, CD20 - immunology; Biological and medical sciences; Cell Line; Crystallography, X-Ray; Epitope Mapping - methods; Epitopes - analysis; Epitopes - chemistry; Hematologic and hematopoietic diseases; Humans; Medical sciences; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Protein Structure, Secondary; Rituximab
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2010-09-305847

CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.