Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells

Abstract

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fc; receptors (Fc;R). Although an early goal has been enhanced Fc;RIIIa 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 Fc;RIIa. Here, we describe a set of engineered Fc variants with diverse Fc;R affinities, including a novel substitution G236A that provides selectively enhanced binding to Fc;RIIa relative to Fc;RIIb. Variants containing this substitution have up to 70-fold greater Fc;RIIa affinity and 15-fold improvement in Fc;RIIa/Fc;RIIb 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 Fc;R to effector functions mediated by NK cells and macrophages. These experiments show that Fc;RIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor Fc;RIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

“…Blocking FcgRII (CD32) or FcgRIII (CD16) reduced ADCP to some extent, whereas blocking FcgRI (CD64) had no effect. This indicates that both CD16 and CD32 are involved in ADCP, whereas previously only CD32 was thought to be mainly involved (Richards et al, 2008). The CD16 antibody (3G8 clone) used for blocking does not, however, distinguish between FcgRIIIa and FcgRIIIb.…”

Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40

“…For SGN-40, weaker binding to mouse relative to human CD64 (FcgR1) and lack of binding to mouse CD16 (FcgRIII) were also noticed. Although Fcg-receptor interactions are essential for effector cell activation, no direct correlation between binding affinities of therapeutic antibodies and activation of effector cells were reported (Richards et al, 2008), because these responses are ultimately regulated by the balance between activating and inhibitory signals delivered through Fcg receptors. Therefore, the differences in binding affinity of SGN-40 between Fcg-RI þ III are not predictive for antitumour activities in vivo.…”

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…[30][31][32][33][34][35] Furthermore, they may elicit antibody-dependent cell-mediated cytotoxicity or phagocytosis (ADCC or ADCP, respectively) via Fcg receptors (FcgRs), which is important for cancer biotherapeutics. 3,[36][37][38][39] Fully functional Fc domains may also confer the potential to trigger the classical pathway of complementdependent humoral response, and thereby elicit complementdependent cytotoxicity (CDC). 40,41 To overcome major limitations of existing bispecific biotherapeutics and to create a universally applicable format that can be fine-tuned in multiparametric drug optimization, we designed the CODV format to serve as a versatile platform for the development of bispecific agents.…”