Work

Antibodies with Engineered Fc Domains Having Absolute Binding Selectivity to either FcγRIIa or FcγRI Delineate the Respective Effector Phenotypes by Human Monocytes and Macrophages

Year: 2026

Type: preprint

Abstract: IgG1 immune complexes bind to all the Fcγ receptors (FcγR) expressed on myeloid cells, making it challenging to determine the precise role of each FcγR on Fc effector phenotypes. Here we report the engineering of Fc2 KG , an aglycosylated human IgG1 Fc domain that binds with near physiological affinity to FcγRIIa/b with no detectable binding to any other FcγRs. Crystallographic analysis elucidated the structural basis of how mutations in the Fc domain compensate for the absence of the N297 glycan and enable selective binding. Using single-cell phagocytosis assays, we show that particles opsonized with Fc-engineered antibodies formatted with Fc2 KG or with an Fc domain that binds only FcγRI (Fc5), are ingested by THP-1 cells with near identical kinetics. We find that with CD16 + primary human monocytes, selective FcγRII engagement is a major contributor to the ADCP mediated by wild-type IgG1. Additionally, we showed that Fc2 KG formatted antibodies induce high levels of GM-CSF. With M1-like monocyte-derived human macrophages, trastuzumab formatted with wild-type IgG1 Fc, with Fc2 KG or Fc5 were all equally proficient in the trogocytotic killing of opsonized SK-BR-3 HER2 + cells but only FcγRI engagement led to secretion of proinflammatory cytokines. Collectively, our results highlight how precisely tuned, Fc-engineered antibodies can be deployed to answer long-standing questions regarding the precise effector functions mediated by human FcγRs, information which is key for the optimization of therapeutic antibodies.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.08.31.748147