Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis whereasAntigenic Modulation Limits Type I Monoclonal Antibody Efficacy

摘要

Rituximab is an anti-CD20 monoclonal antibody (mAb) used in the treatment of B-cell malignancies. Loss of surface CD20 antigen from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalised in a B-cell intrinsic process; and shaving, where mAb:CD20 complexes undergo trogocytic removal by effector cells such as macrophages. However, there are conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. Here we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex-vivo macrophages with varying concentrations of non-Fc gamma receptor (FcγR)-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localisation of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation suggesting that the two compete for surface rituximab:CD20. Under all conditions tested modulation predominated in rituximab loss whilst shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the non-modulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared to type II BHH2 human IgG1 wild-type mAb. Therefore shaving may represent an important mechanism of resistance when modulation is curtailed and glycoengineering mAb to increase affinity for FcγR may enhance resistance due to shaving.