Glycoengineered CD20 Antibody Obinutuzumab Activates Neutrophils and Mediates Phagocytosis Through CD16B More Efficiently Than Rituximab
Authors: Golay Josée , Da Roit Fabio , Bologna Luca , Koller Claudia Ferrara , Leusen Jeanette H. W. , Rambaldi Alessandro , Klein Christian , Introna Martino 
Published: October 2019

Background
Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody).

Methods
Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry.

Results
Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation.

Conclusions
We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101.

Disclosures:
Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.