Fc Gamma Rll (CD32b) Expression Affects Anti-CD20 Monoclonal Antibody Cellular Responses and Is Down Regulated in Rituximab Resistant Pre-Clinical Models

Abstract

Rituximab's primary mechanisms-of-action include: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CMC), induction of apoptosis and anti-proliferation. The acquirement of phenotypic changes in cancer cells or host immune cells over time may affect rituximab responsiveness and underscores the complexity of the potential mechanisms-of-resistance to anti-CD20 monoclonal antibodies (mAbs). Laboratory studies had demonstrated the importance of FcγRIIa and FcγRIII activating receptors in the biological activity of rituximab and other monoclonal antibodies (mAbs) in cancer medicine. Recently, investigators had focused their efforts in the study of FcγRIIb function expressed primarily on normal and malignant B-cells and its role in inflammatory conditions or hematological malignancies. FcγRIIb is an inhibitory low affinity Fcg receptor capable to induce potent inhibitory (apoptotic) signals via immune receptor tyrosine-based inhibition motif (ITIM). Previously, we generated several rituximab-resistance lymphoma cell lines (RRCL) and demonstrated a global down-regulation of CD20. On the other hand, forced expression of CD20 in RRCL did not restore rituximab sensitivity suggesting the existence of additional mechanisms of resistance to mAbs targeting CD20 (Tsai et al Clin Cancer Res. 2012;18:1039-50). To this end, we evaluated the expression and significance of FcγRIIb (CD32b) in RRCL. FcγRIIb gene and protein expression levels were determined in a panel of rituximab sensitive (RSCL) or RRCL by quantitative polymerase chain reaction (qPCR) and western blotting respectively. Subsequently, we silenced FcγRIIb in RSCL using siRNA interference and evaluated changes in the capacity of several mAbs targeting CD20 (rituximab, ofatumumab, and obinotuzumab) of inducing CMC or ADCC in vitro. RRCL were found to have almost a complete loss in FcgRIIb mRNA and protein levels when compared to RSCL. In addition, silencing of FcγRIIb in RSCL decreased the capacity of rituximab (P=0.04 [ADCC] and P=0.15 [CMC]), ofatumumab (P=0.02 [ADCC] and P =0.10 [CMC]), and obinituzumab (P=0.02 [ADCC] and P<0.01 [CMC]) in inducing effective ADCC and CMC when compared to scramble transfected cells. Together our data suggests that the chronic exposure of lymphoma cells to rituximab results in the decreased expression of FcγRIIb. In addition, FcγRIIb low affinity inhibitory receptors appear to contribute to rituximab anti-tumor activity in vitro. Additional in vivo experiments are ongoing to further define the role of FcγRII in the anti-tumor activity of mAbs targeting CD20 and how it is down regulated in the context of acquired resistance to rituximab-based therapies. (Research, in part, supported by The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.