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

Human immune complexes, such as IgG1, bind to all Fc{gamma} receptors (Fc{gamma}R) found on myeloid cells, making it difficult to discern the specific role each Fc{gamma}R plays in Fc effector functions. Researchers have now engineered an Fc2KG, a Fc domain of aglycosylated human IgG1 that binds with near physiological affinity to Fc{gamma}RIIa/b without binding to any other Fc{gamma}Rs.

Crystallographic analysis revealed how mutations within the Fc domain compensate for the absence of N297 glycan and allow for selective binding. Single-cell phagocytosis assays demonstrated that particles opsonized with Fc-engineered antibodies formatted with Fc2KG or an Fc domain that only binds Fc{gamma}RI are ingested by THP-1 cells at nearly identical rates.

Furthermore, CD16+ primary human monocytes show that selective Fc{gamma}RII engagement is a key contributor to ADCP mediated by wild-type IgG1. Additionally, Fc2KG formatted antibodies induce high levels of GM-CSF. When tested on M1-like monocyte-derived human macrophages, trastuzumab formatted with wild-type IgG1 Fc, Fc2KG, or Fc5 demonstrated equal proficiency in trogocytotic killing of SK-BR-3 HER2+ cells.

However, only Fc{gamma}RI engagement led to the secretion of proinflammatory cytokines. These findings indicate that precisely engineered Fc antibodies can be utilized to elucidate the precise effector functions of human Fc{gamma}Rs, which is crucial for optimizing therapeutic antibodies.