Anti‐CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca2+ mobilization and cytotoxicity in human NK cells

NK cell‐mediated Ab‐dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc‐gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell‐mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low‐affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high‐affinity (VV) and VF SNP variants was two‐ to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low‐affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell‐mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy. Graphical Binding, Ca2+ signaling, and ADCC induced by anti‐CD20 rituximab in NK cells strongly depend on the IgG subclass.