Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way.

Abstract 3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies. Disclosures: Niederfellner: Roche: Employment. Schwaiger: Roche: Employment. Georges: Roche: Employment. Wiechmann: Roche: Research Funding. Franke: Roche: Research Funding. Schaefer: Roche: Employment. Jenewein: Roche: Employment. Slootstra: Pepscan: Employment, Patents & Royalties. Moessner: Glycart: Employment, Equity Ownership, Patents & Royalties. Umana: Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner: Roche: Research Funding. Klein: Roche: Employment, Equity Ownership, Patents & Royalties.