Human Fcγ receptors compete for TGN1412 binding which determines the antibody's effector function.

The first-in-human clinical trial of the CD28-specific monoclonal antibody (mAb) TGN1412 resulted in a life-threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fc gamma receptor (Fc gamma R) interactions, these interactions contributed to TGN1412-induced T-cell activation. Using cell lines (TFs) expressing human Fc gamma RI, -IIa, -IIb, or -III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by Fc gamma Rs as it can be deduced from literature. However, upon coculture of TGN1412-decorated T cells with TFs or human primary blood cells, we observed that binding capacities by Fc gamma Rs do not correlate with the strength of the mediated effector function. Fc gamma RIIa and Fc gamma RIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high-affinity Fc gamma RI, exhibiting strong TGN1412 binding, mediated hardly any T-cell proliferation. These findings are of biological relevance because we show that Fc gamma RI binds TGN1412, thus prevents binding to Fc gamma RIIa or Fc gamma RIIb, and consequently disables T-cell proliferation. In line with this, Fc gamma RI(-)Fc gamma RII+ but not Fc gamma RI(+)Fc gamma RII+ monocytes mediate TGN1412-induced T-cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the Fc gamma R-mediated effector function, which has major implications for the design of therapeutic mAbs.