Abstract Purpose: Hypersensitivity reactions (HSRs) were observed in three patients dosed in a phase I clinical trial treated with LOP628, a KIT targeted antibody drug conjugate. Mast cell degranulation was implicated as the root cause for the HSR. Underlying mechanism of this reported HSR was investigated with an aim to identifying potential mitigation strategies. Experimental Design: Biomarkers for mast cell degranulation were evaluated in patient samples and in human peripheral blood cell-derived mast cell (PBC-MC) cultures treated with LOP628. Mitigation strategies interrogated include pretreatment of mast cells with small molecule inhibitors that target KIT or signaling pathways downstream of FcϵR1, FcγR, and treatment with Fc silencing antibody formats. Results: Transient elevation of serum tryptase was observed in patients 1-hour posttreatment of LOP628. In agreement with the clinical observation, LOP628 and its parental antibody LMJ729 induced degranulation of human PBC-MCs. Unexpectedly, KIT small molecule inhibitors did not abrogate mast cell degranulation. By contrast, small molecule inhibitors that targeted pathways downstream of Fc receptors blunted degranulation. Furthermore, interference of the KIT antibody to engage Fc receptors by pre-incubation with IgG or using engineered Fc silencing mutations reduced or prevented degranulation. Characterization of Fcγ receptors revealed human PBC-MCs expressed both FcγRII and low levels of FcγRI. Interestingly, increasing the level of FcγRI upon addition of IFNγ, significantly enhanced LOP628-mediated mast cell degranulation. Conclusions: Our data suggest LOP628-mediated mast cell degranulation is the likely cause of HSR observed in the clinic due to co-engagement of the FcγR and KIT, resulting in mast cell activation. Clin Cancer Res; 24(14); 3465–74. ©2018 AACR.