title: Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies.
abstract: 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.
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, Penzberg, Germany.
authors: Gerhard
authors: G
authors: Niederfellner
authors: Alfred
authors: A
authors: Lammens
authors: Olaf
authors: O
authors: Mundigl
authors: Guy J
authors: GJ
authors: Georges
authors: Wolfgang
authors: W
authors: Schaefer
authors: Manfred
authors: M
authors: Schwaiger
authors: Andreas
authors: A
authors: Franke
authors: Kornelius
authors: K
authors: Wiechmann
authors: Stefan
authors: S
authors: Jenewein
authors: Jerry W
authors: JW
authors: Slootstra
authors: Peter
authors: P
authors: Timmerman
authors: Annika
authors: A
authors: Brännström
authors: Frida
authors: F
authors: Lindstrom
authors: Ekkehard
authors: E
authors: Mössner
authors: Pablo
authors: P
authors: Umana
authors: Karl-Peter
authors: KP
authors: Hopfner
authors: Christian
authors: C
authors: Klein
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000145:classification*; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D000918:Antibody Specificity; D018951:Antigens, CD20 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D018360:Crystallography, X-Ray; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000939:Epitopes; D000069283:Rituximab; C543332:obinutuzumab