Effective Phagocytosis of Low Her2 Tumor Cell Lines with Engineered, Aglycosylated IgG Displaying High FcγRIIa Affinity and Selectivity

Abstract

Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.

Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis

“…ADCP results from the binding of Abs to antigens on the surface of target cells and the binding of their Fc moieties to FcgRs on the surface of effector cells (11,18). There are more than five different isotypes of FcgRs, and the individual isotypes are expressed at various amounts on different effector cells involved in different effector functions (4 6).…”

Antibody Engineering and Therapeutics

“…He noted that the absence of the N297 glycan normally results in high flexibility in the CH2 and CH3 portions of IgG. Prof. Georgiou’s group applied bacterial display to identify a palette of engineered aglycosylated Fc domains displaying high selectivity toward specific Fcγ receptors or C1q, potentially by stabilizing a conformer that binds only to a particular Fcγ receptors or C1q with high selectivity 98 . Finally, Prof. Georgiou noted that he is looking for a partner to further the development of the described antibody platforms.…”

Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding

“…5,6 Concurrently, IgG variants have also been generated to enhance ADCP and complement-dependent cytotoxicity (CDC) by increasing the Fc domain's affinity to FcgRIIa or C1q respectively. [7][8][9] These technologies are similar in that they attempt to enhance existing interactions between the IgG-Fc and immune receptors rather than introduce novel effector functions to IgG.…”