Rituximab (chimeric anti-CD20 mAb) is currently being used in the therapy of Non-Hodgkin’s B-cell Lymphoma (NHL). It has also been reported that the combination of rituximab and chemotherapy is effective in patients with NHL. Several proposed mechanisms have been suggested for rituximab-mediated effects in vivo and include ADCC, CDC and apoptosis. In addition, we have reported that rituximab can trigger the tumor cells and modifies several intracellular survival signaling pathways resulting in the sensitization of tumor cells to various chemotherapeutic drugs (

Alas et al., Clin. Cancer Res. 8:836, 2001
;
Jazirehi et al., Mol. Cancer Therapy 2:1183, 2003
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Vega et al., Oncogene 23:3530, 2004
). Rituximab-mediated signaling might have resulted from directly triggering CD20 alone or in conjunction with triggering FcRs on the tumor cells via the Fc fragment of rituximab. This study was designed to distinguish between these two possibilities. Since the CH2 domain in Fc is responsible for its interaction with the Fc receptor, we have genetically engineered a rituximab mAb in which the CH2 domain was deleted. The CHO cells secreted one monomeric form with properly formed disulfides rituximab (CH2−) and a dimeric form rituximab (CH2−) Dimer. The functions of the two CH2-deleted domain antibodies were examined in several systems and compared with those of the native rituximab. We used Ramos and 2F7 B-NHL cell lines as model systems. As expected, we demonstrate that the CH2-deleted antibodies are defective in their ability to mediate ADCC and CDC. However, their biological functions and their ability to signal the cells were not altered and were similar to those observed with rituximab. Hence, the CH2-deleted antibodies inhibited cell proliferation, induced cell aggregation in culture, and induced apoptosis when cross-linked with a secondary anti-human IgG. In addition, the CH2-deleted antibodies triggered the cells similar to rituximab and inhibited the Src kinase pLyn, the downstream p38 MAP kinase, the transcription factors NF-κB, YY1, SP1, and STAT3, and Bcl-2/Bcl-xL expression. These modifications resulted in the sensitization of B-NHL cell lines to both chemotherapeutic drugs and Fas-induced apoptosis. Altogether, these findings demonstrate that rituximab-mediated signaling and modification of intracellular signaling pathways can take place in the absence of any contribution of the Fc fragment cross-linked to the cell Fc receptors. These studies suggest that chemo and immuno-sensitization by rituximab can take place in vivo in the absence of Fc-FcR contributions and thus are of potential clinical relevance.

Topics:
cd20 antigens, cell lines, cross-linking, family-centered rounds, neoplasms, rituximab, signal transduction, antibodies, fc receptors, tumor cells

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2005, The American Society of Hematology
2004
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