Anti-CD20 Anticorps [MEM-97] (A85848)

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 antibody-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.

The results of previous biochemical studies indicated that a fraction of MHC class II proteins is associated with four proteins of the tetraspan family, CD37, CD53, CD81, and CD82, and possibly with other membrane components, at the surface of JY B lymphoma cells. In the present communication we used a biophysical technique, namely the flow cytometric energy transfer method, to demonstrate the proximity of these molecules at the surface of the cells. Significant energy transfer (and, therefore, proximity within the 2-10 nm range) was observed between fluorescently labeled mAbs to DR, DQ, and the tetraspan molecules CD53, CD81, and CD82. Moreover, two other B cell surface molecules, CD20 and MHC class I, were found to be close to each other and to MHC class II and the tetraspan proteins, based on the observed high energy transfer efficiencies between the relevant fluorescently labeled mAbs. The character of simultaneous energy transfer from CD20, CD53, CD81, and CD82 to DR suggests that all these molecules are in a single complex with the DR molecules (or a complex of several DR molecules) rather than that each of them is separately associated with different DR molecules. Based on these data and previous biochemical results, a model is proposed predicting that the B cell membrane contains multicomponent supramolecular complexes consisting of at least two MHC class I and at least one DR, DQ, CD20, CD53, CD81, and CD82 molecules. Closer analysis of the energy transfer efficiencies makes it possible to suggest mutual orientations of the components within the complex. Participation of other molecules, not examined in this study (CD19 and CD37), in these supramolecular structures cannot be ruled out. These large assemblies of multiple B cell surface molecules may play a role in signaling through MHC molecules and in Ag presentation to T cells.