Abstract
Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.
Highlights
In this study we investigated the combined roles of five factors affecting human NK cell–mediated Ab-dependent cellular cytotoxicity (ADCC), namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcgR polymorphism, 4) FcgR-blocking cytophilic Abs, and 5) Ab fucosylation
We demonstrate that the magnitude of NK cell–mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation
We demonstrate that the magnitude of NK cell–mediated ADCC response toward RBCs depends on a complex interplay between Ag, Ab glycosylation, target, and effector cell properties
Summary
RBCs with randomly coupled TNP triggered higher ADCC with fucosylated and hypofucosylated IgG1 compared with RhD-expressing R2R2 and R1r target cells under a similar level of opsonization (Fig. 1, Supplemental Fig. 2A, 2B). To define the influence of the FcgRIIIa–Val/Phe158 polymorphism on ADCC activity, NK cells from 18 FCGR3A-genotyped healthy volunteers were tested in the ADCC setup with R2R2 RBCs. When untreated RBCs with high RhD expression (R2R2) were used as target cells, the FcgRIIIa phenotype (from low to high affinity: Phe/Phe158, Val/Phe158, and Val/Val158) did significantly affect NK cell–mediated ADCC, both with fucosylated and hypofucosylated anti-RhD IgG1 (Fig. 3).
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