EHA Library - The official digital education library of European Hematology Association (EHA)

CD20 monoclonal antibodies (mAb) are a standard of care for B-lymphoid malignancies. Yet, their clinical efficacy is quite variable and many patients relapse, while their malignant cells express very low density of CD20 on the cell surface. In spite of being used for 20 years as a therapy target, little is known about the biology and regulation of CD20 inside the cell.

We show that treatment of B cells with different CD20 mAbs initiates a signaling cascade within the cells that is partially distinct from classical B-cell receptor signaling machinery and does not involve BCR proximal proteins. Importantly, it results in a prompt downregulation of CD20 expression. Through chronic exposure to gradually increasing doses of monoclonal antibodies, we have generated cell lines that are resistant to additional treatment with mAb. Notably, these cells are resistant also to any other of the available anti-CD20 antibodies even at very high concentrations as shown by dose-response experiments. This resistance is sustained for long period and maintained even upon many rounds of cell passages. We could confirm that these cells have downregulated CD20 protein from the cell surface and that this effect was not just due to its internalization. Instead, we detected a defect in CD20 transcription as measured by quantitative real-time PCR. Flow cytometry analysis of other surface markers showed a strong upregulation of CD55 and CD59, known inhibitors of complement activation. The combination of CD20 loss together with the increase of CD55 and CD59 is responsible for the complete resistance to the mAbs.

We have then analyzed changes in overall gene expressions by performing RNA sequencing and quantitative real-time PCR. We have identified several interesting genes whose expression was altered in our resistant cells when compared to their control counterparts. Among the most interesting hits was a strong downregulation of the transcription factor NFkB, which was expressed more than 10-fold lower in the rituximab or ofatumumab resistant cells. We could confirm this result in multiple independent experiments. We have postulated that anti-CD20-triggered signaling results in the inactivation of NFkB and this may lead to the block in CD20 transcription. To confirm our hypothesis, we have treated the cells with phorbol ester PMA, which nonspecifically activates NFkB. Indeed, cells treated with PMA managed to rapidly upregulate CD20 on their cell surface.

In summary, CD20 triggering by therapeutic mAbs initiates complex intracellular changes that result in downmodulation of CD20 expression. Further analysis of detailed intracellular mechanisms regulating CD20 is warranted in order to propose novel interrogation nodes that might modulate CD20 surface density and thereby enhance the therapeutic potential of CD20 monoclonal antibodies.