Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

Abstract

Anti-CD20 monoclonal antibody (mAb) represents an effective strategy for the treatment of B cell malignancies that may involve complement activity, antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP). While ADP mediated by Kupffer cells is essential to deplete circulating tumors, the relative contribution of each mechanism to the elimination of non-circulating targets has yet to be clarified. Using intravital imaging in a model of MYC-driven B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To quantify the contribution of different cytotoxic mechanisms in situ, we designed a dual fluorescent reporter to track phagocytosis and apoptosis in real-time. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting only in partial depletion. Moreover, macrophage density was strongly reduced in tumor-rich regions. Given their sessile phenotype, macrophages primarily targeted neighboring tumors, resulting in a substantial spatial constraint. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy represents a critical path towards the design of optimized therapies. Key pointsO_LIFunctional intravital imaging establishes antibody-dependent phagocytosis as the major mechanism acting at the tumor site during anti-CD20 therapy. C_LIO_LIA transient wave of phagocytosis and a limited macrophage density restrict the efficiency of anti-CD20 anti-tumor activity. C_LI