Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Abstract

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radio-labeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca2+ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca2+, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bc1-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bc1-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials.

Apoptosis assays with lymphoma cell lines: problems and pitfalls

“…Must include negative control Ab. Annexin V staining (Shan et al, 2000;Darzynkiewicz et al, 2001;Cragg and Glennie, 2004) Subcellular fragments may obscure results. Must monitor size of objects counted in the FACS.…”

Novel antibodies as anticancer agents

“…A number of antibodies that are currently in use or in clinical testing for cancer treatment, including anti-CD20 antibody Rituximab, have been shown to directly induce apoptosis in tumor cells and this activity may contribute significantly to the clinical performance (Shan et al, 2000).…”

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells

“…Recently it was demonstrated that cross-linking of anti-CD20 antibodies on the surface of B-cells using F(ab′)2-fragments or Fc-receptor positive cells induces apoptosis in B-cells (Shan et al, 1998). The same group confirmed that protein tyrosine kinases, increased intracellular Ca 2+ -concentrations, and caspases are involved (Shan et al, 2000). In the latter paper it was also shown that CD95 (Fas) becomes up-regulated after cross-linking of anti-CD20-antibodies on B-cells, but that the Fas-FasL route is not important in the in vitro setting.…”

“…This characteristic is probably caused by the fact that unlike most other cancer cells, malignant B-cells may be capable to function as antigen-presenting cells (Chaperot et al, 2000). Also the recent finding that after cross-linking of anti-CD20-antibodies on B-cells Fas is up-regulated in these cells (Shan et al, 2000) could have major implications for T-cell retargeting-based lymphoma therapies, because this will make the B-cells even more sensitive for FasL on activated T-cells.…”

“…by Fc-receptor positive cells) on B-cells can lead to apoptosis of the target cell (Shan et al, 1998(Shan et al, , 2000. The latter effects are thought to contribute significantly to the efficiency of treatment with this 'signalling antibody' (Cragg et al, 1999).…”