Tom van Meerten | PubMed Health

PubMed2006

Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity.

PURPOSE: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. EXPERIMENTAL DESIGN: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. RESULTS: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. CONCLUSIONS: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

Antibodies, Monoclonal↗

PubMed2006

The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20.

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

Amino Acid Sequence↗