Synergistic Effect of IFN-γ on Breast Cancer Targeted Therapy Yasuhiro Nagai1, Hiromichi Tsuchiya2, Aaron E. Runkle1, Peter D. Young1,Mei Q. Ji1, Hongtao Zhang1 and Mark I. Greene1 1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 2Department of Pharmacology, Showa University of Medicine, 1-5-8 Hatanodai, Tokyo, 142-8555, Japan ErbB2 is amplified in ~30% of breast cancer patients, and its amplification is associated with poor prognosis of survival. We examined the synergistic effect of interferon-γ (IFN-γ) with anti-erbB2/neu mAb on an erbB2-positive cancer model. IFN-γ showed marginal effects on its own, indicating that immune therapies mediated by this cytokine alone are unlikely to be beneficial. However, we discovered that treatment of the tumors with anti-erbB2/neu mAb concomitant with IFN-γ led to dramatic inhibition of in vivo tumor growth in the syngeneic tumor model and the MMTV-neu transgenic mouse tumor prevention model. We noted an increase of M1 macrophage accumulation in the tumor tissues as well as diminished myeloid derived suppressor cells, and increased CD8+ T cell cytotoxicity against tumor cells in the IFN-γ plus anti-erbB2/neu mAb treated mice. The tumor cells treated with both mAb and IFN-γ underwent changes in the tumor stem cell marker ALDH-1, indicating a loss of stem cell-like properties; while mAb treatment alone did not lead to this phenotypic change. In addition, tumors resistant to the combination therapy had several mutations on antigen processing and presenting pathway genes, suggesting tumor immunity is important for this therapy. We found that IFN-γ treatment significantly increased tumor PD-L1 expression, so we included anti-PD-1 or PD-L1 antibody with this combination therapy. Interestingly, anti- PD-L1 antibody further reduced tumor growth, while anti-PD-1 had negligible effects. These results indicate that treatment with IFN-γ can improve targeted therapy and further induce PD-L1 expression on tumor cells, creating a new target on the tumor cells for anti-PD-L1 therapy.