The Shaving Reaction. Rituximab/CD20 Complexes Are Removed from Mantle Cell Lymphoma Cells (Z138) and Chronic Lymphocytic Leukemia (CLL) Cells by Monocyte/Macrophages: In Vitro Studies and a SCID Mouse Model.

Abstract

Clinical investigations have revealed that infusion of immunotherapeutic monoclonal antibodies directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently we reported that Rituximab (RTX) treatment of CLL patients induced substantial loss of CD20 on B cells found in the circulation after RTX infusion, when RTX plasma concentrations were high. Antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been “shaved” of RTX/CD20 complexes by monocytes or macrophages of the mononuclear phagocytic system (MPS) in a reaction mediated by Fcγ receptors. Our in vitro model is based on reacting RTX-opsonized CD20+ cells with acceptor THP-1 monocytes differentiated to a macrophage-like phenotype. After 45 min at 37°C, ~50–80% of both RTX and CD20 are removed from opsonized cells, and both proteins are demonstrable on acceptor THP-1 cells, as ascertained by flow cytometry, fluorescence microscopy, and immunoblotting. Tests with inhibitors and use of F(ab')2 fragments of RTX indicate shaving is mediated by Fcγ receptors, and shaving occurs equally well in the presence and absence of complement. We developed a pre-clinical xenograft mouse model to replicate shaving: 5 X 106 Z138 cells were infused iv or sc, into SCID/CB17 mice, and ~25 days later animals were treated with varying doses of RTX. Z138 cells recovered from lungs (iv model) or tumors (sc model) of treated and control mice 30 min to 3 days after RTX infusion were analyzed by flow cytometry. Substantial loss of CD20 was demonstrable on RTX-targeted cells, and this loss was more pronounced at longer times after RTX infusion. Moreover, Z138 cells in untreated mice had moderate to high levels of CD20 and few cell-associated C3 fragments, but in RTX-treated mice the situation was reversed: the Z138 cells were CD20 poor (<20% of the CD20 levels compared to untreated mice) but had significant amounts of deposited C3dg. These findings replicate our earlier studies which demonstrated that treatment of CLL patients with RTX promotes loss of CD20 as well as complement activation and C3 fragment deposition on circulating B cells. It is likely that previously described antigenic modulation, usually reported for conditions of high circulating malignant cell burdens targeted by monoclonal antibodies, was also mediated by cells of the MPS via the shaving reaction. Our findings may have profound implications for the use of monoclonal antibodies in the immunotherapy of cancer.