Abstract
The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcgamma receptors (FcgammaR). Although an early goal has been enhanced FcgammaRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcgammaRIIa. Here, we describe a set of engineered Fc variants with diverse FcgammaR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb. Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcgammaR to effector functions mediated by NK cells and macrophages. These experiments show that FcgammaRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcgammaRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.
Highlights
It is well documented that the activating Fcg receptor (FcgR) FcgRIIIa plays an important role in the therapeutic activity of some monoclonal antibodies
R131 FcgRIIa is associated with greater susceptibility to infectious disease, a relationship that is hypothesized to be due to the critical role of IgG2 in fighting pathogens and the capacity of this isotype to mediate monocyte and neutrophil effector function only with the H131 form (14 – 16)
The therapeutic relevance of FcgRIIb is supported by the improved antibody antitumor activity [5] and greater B-cell depletion [7] observed in FcgRIIb-/mice, and the correlation observed between the anticancer activity of mouse IgG subclasses and their activating to inhibitory (A/I) ratios [17]
Summary
It is well documented that the activating Fcg receptor (FcgR) FcgRIIIa plays an important role in the therapeutic activity of some monoclonal antibodies. The therapeutic relevance of FcgRIIb is supported by the improved antibody antitumor activity [5] and greater B-cell depletion [7] observed in FcgRIIb-/mice, and the correlation observed between the anticancer activity of mouse IgG subclasses and their activating to inhibitory (A/I) ratios [17] These results have led to the hypothesis that A/I ratios are an important variable in determining antibody-mediated effector function [17]. 2518 Optimized FcgRIIa Binding Enhances Phagocytosis affinities and specificities, including selective engagement of FcgRIIIa and FcgRIIa relative to FcgRIIb. The enhancements in macrophage phagocytosis mediated by these novel variants show that Fc engineering could be used to increase the activity of effector cell types expressing both activating and inhibitory receptors. These variant provide the potential to improve the performance of monoclonal antibodies targeting cancers and infectious diseases and have enabled us to probe the roles of the different FcgR in the effector functions of different immune cell types
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