Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor In determining their efficacy because evidence from anti body-blocking studies Indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs Is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x Indicates the Identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions In the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 In the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 In an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was Indicated by marked differences In their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts. (C) 2002 by The American Society of Hematology.