Cooperation Between the CD20 Receptor and hIFN-Alpha Receptor in Overriding the Blocked CD20 Cell-Signaling in Rituximab-Resistant B-NHL By the Fusion Protein Anti-CD20-hIFN-Alpha

Introduction: The standard treatment of B-NHL consists of rituximab in combination with CHOP (RCHOP) and results in a significant clinical response. Rituximab inhibits cell-proliferation and inhibits cell survival/anti-apoptic signaling pathways. A subset of patients does not initially respond and a subset of responding patients develops resistance to RCHOP. The genetic engineering of a fusion protein, α-CD20-hIFN-α, was found to be active in the rituximab-resistant B-NHL cell lines.

Objective: To investigate the underlying mechanism by which α-CD20-hIFN-α signals in the resistant lines.

Hypothesis: We hypothesized that the treatment with the α-CD20-hIFN-α may result in the cooperation of both α-CD20 and hIFN-α and their interactions with corresponding receptors that will result in overriding α-CD20 blocked cell signaling.

Methods: Rituximab-resistant cell lines, R-2F7 and R-Ramos, were used as models. Cell signaling was determined by western. Sensitivity to drug-induced apoptosis was done by activation of caspase 3 by flow cytometry.

Results: Treatment of the R lines with α-CD20-hIFN-α resulted in the inhibition of cell growth and sensitization to doxorubicin-induced apoptosis. Treatment with single agents alone or combination was not effective. Treatment with the α-CD20-hIFN-α resulted in the inhibition of the NFκB and the p38 MAPK pathways. In addition, the hIFN-mediated signaling pathway, namely, PKC-d, was also inhibited by the α-CD20-hIFN-α.The role of PKC-d in drug sensitization was corroborated by the use of the specific inhibitor, Rotterin, which reversed the drug sensitization by α-CD20-hIFN-α and doxorubicin

Conclusion: The ability of the α-CD20-hIFN-α to inhibit cell survival and anti-apoptotic pathways, that was not achieved with single agents or combination, suggested that there may be a crosslinking of the CD20 and hIFN-α receptors by α-CD20-hIFN-α and results in triggering the cells via both receptors and inhibiting intracellular survival pathways and sensitization to drug apoptosis.

Clinical Implication: The findings also suggest the potential therapeutic application of the combination of α-CD20-hIFN-α and drugs for the treatment of patients resistant to RCHOP.