Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

Abstract

Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

Cellular Cytotoxicity of Next-Generation CD20 Monoclonal Antibodies

“…The primary in vivo mechanism of cellular cytotoxicity of circulating CD20 mAb-opsonized cells in mice is ADCP by fixed macrophages in the liver (13)(14)(15)17). However, limited data on human in vivo CD20 mAb-mediated cellular cytotoxicity of circulating or tissue resident B cells are available.…”

“…Subsequent studies showed that natural killer (NK) cells are the primary peripheral blood effector cells performing ADCC, whereas monocytes and neutrophils have minimal cytotoxic activity (10,11). In contrast, in vivo murine studies of CD20 mAb-mediated clearance of circulating B cells show a major role for ADCP by fixed hepatic macrophages (Kupffer cells), with minimal NK cell-mediated cytotoxicity (12)(13)(14)(15)(16)(17). These data suggest that clearance of opsonized B cells could be similar to that of autoantibody opsonized blood cells, which are efficiently cleared by ADCP in the liver and spleen in human autoimmune hemolytic anemia and immune thrombocytopenia (18,19).…”

Maxed out macs: physiologic cell clearance as a function of macrophage phagocytic capacity

“…The cytotoxicity of many of these mAb is primarily mediated by the innate immune system . More recently, the use of intravital imaging and macrophage depletion strategies have revealed that ADCP by tissue‐resident macrophages, particularly Kupffer cells in the liver, are the major mediator of the therapeutic effect of these drugs, especially when used to treat B‐cell malignancies .…”

“…Exhaustion in the context of ADCP has been modeled in vitro using human monocyte‐derived macrophages cultured in the presence of excess numbers of IgG‐opsonized lymphocytes. Under these conditions, maximal clearance is achieved after 4 h, with very little additional engulfment beyond this time . Moreover, the presence of excess IgG‐opsonized lymphocytes on macrophages for 24 h leads to a sharp decrease in their phagocytic activity upon refeeding with fresh targets compared to previously unfed macrophages .…”

CIMT 2018: Pushing frontiers in cancer immunotherapy — Report on the 16th Annual Meeting of the Association for Cancer Immunotherapy

“…In order to understand the contribution of tissue-resident versus bone marrow-derived macrophages for cytotoxic antibody activity (e.g. IgG-mediated B cell depletion in the liver), 31 he and colleagues designed a model system involving chimeric mice expressing organ-specific FcγR. When organ-resident FcγR ± macrophages were depleted by clodronate liposomes, repopulation of the liver by Kupffer cells was observed to be bone marrow-dependent.…”