Recent trials of monoclonal antibody-based therapy have demonstrated clear anti-tumor activity with minimal toxicity in patients with low grade and follicular B-cell malignancies. In a recently completed trial, approximately 50 percent of patients that had refractory disease or relapsed following standard modalities of treatment responded to therapy with unlabeled chimeric anti-CD20. Although the assumption is that most of this anti-tumor activity is related to antibody dependent cellular cytotoxicity, there is little direct evidence for this mechanism. A growing body of data indicates that transmembrane signaling via CD20 plays a key role in the control of B-cell proliferation and differentiation. The applicants hypothesize that transmembrane signaling via CD20 may be important in the clinical response to anti-CD20 monoclonal antibody therapy. The current study is designed to assess whether anti-CD20 mediates transmembrane signaling in malignant lymphocytes obtained from patients scheduled to undergo anti-CD20 antibody therapy, and to determine whether signaling correlates with clinical response. Malignant lymphocytes will be harvested from patients enrolled in a clinical trial of anti-CD20 moAb therapy prior to the administration of mAb. Harvested lymphocytes will be treated in vitro with anti-CD20 or isotype-matched control antibody. Changes in a variety of parameters at different points along the signaling cascade will be assessed to determine whether transmembrane signaling has taken place, and whether it results in downstream changes. Parameters to be measured include tyrosine phosphorylation, intracellular calcium flux, Bcl-2, Bax and Bcl-XL message and protein expression, cell cycle analysis, proliferation, apoptosis and immunophenotype. Any observed changes will be correlated with clinical response to antibody therapy. The detection of a correlation between antibody-induced signaling and clinical response would supply strong evidence that transmembrane signaling is important clinically, and would impact on the design of the next generation of regimens that utilize this new therapeutic approach.
