Epitope Characterization and Crystal Structure of GA101 Provide Insights into the Molecular Basis for Type I/II Distinction of CD20 Antibodies

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.