Immunotherapy has emerged as a promising strategy against cancer, but many patients fail to achieve durable responses. Inefficiency of immunotherapy is often caused by the immunosuppressive tumor microenvironment. Previously, we demonstrated that treatment with an FcαRI-stimulating bi-specific antibody (BsAb), designed to recruit myeloid cells as cytotoxic effector cells, significantly decreased tumor growth in a murine cancer model. Nonetheless, complete tumor eradication was not achieved. In this study, we investigated if co-treatment with the pro-inflammatory cytokine TNF-α enhances the therapeutic efficacy of FcαRI BsAb. Although TNF-α did not affect antibody-dependent cellular phagocytosis (ADCP) of tumor cells, macrophage polarization, or antibody-dependent cellular cytotoxicity (ADCC) by natural killer cells, its combination with FcαRI BsAb increased tumor cell trogocytosis, neutrophil degranulation and tumor cell death. To exploit this synergy, we engineered a TNF-α x FcαRI bi-specific immunocytokine (FcαRI-TNF). Surface plasmon resonance and cellular binding assays demonstrated that FcαRI-TNF retained binding affinities for FcαRI, FcɣRIII, and the tumor-associated antigen EGFR comparable to FcαRI BsAb. Consistent with the combination of TNF-α and FcαRI BsAb, FcαRI-TNF neither influenced macrophage function nor polarization but enhanced neutrophil-mediated tumor killing in vitro. Intravital imaging in a murine MC38-cEGFR tumor model showed that FcαRI-TNF promoted in vivo neutrophil activation and swarming behavior. These findings suggest that FcαRI-TNF represents a promising candidate to improve neutrophil-driven immunotherapy of cancer.
