In vitro evidence that combination treatment with anti-PD-1 and anti-CD47 immunotherapy may not be efficient in human soft tissue sarcoma.

Abstract

e23560 Background: Soft tissue sarcomas (STSs) are rare mesenchymal tumors. With more than 70 histological subtypes of STSs, studies defining robust biomarkers of therapeutic value are lacking. Even with the improvements in local control rates, metastases and death occur in 50% of patients that are diagnosed with high-risk STSs. Across nearly all sarcoma types, macrophages dominate the immune landscape of STSs. While immune checkpoint inhibitors (ICIs) represent the major immunotherapeutic approach in malignancies with a high load of T cells, the anti-CD47 anti-cancer therapy mostly promotes macrophage-mediated phagocytosis. The efficacy of both therapies is critically dependent on the composition of the tumor microenvironment. Methods: In our study, we evaluated the potential additive effects of combining the anti-PD-1 immunotherapy and anti-CD47 immunotherapy ex vivo. The proportions of tumor cells and tumor-infiltrating lymphocytes were characterized ex vivo from resected tumors of 73 patients who underwent surgery for STS. The expression of CD47 on tumor cells was analyzed by immunohistochemistry. Cell suspensions were obtained from freshly resected tumors and the proportions of CD45+, CD45-, CD3+, CD4+, and CD8+ cells were measured by flow cytometry. The effect of the anti-PD-1 blockade, anti-CD47 blockade, and the combinatorial application of anti-PD-1 and anti-CD47 therapies on the immune cell activation in vitro was investigated by culturing of cell suspensions at a concentration 0.3x106/ul in the presence/absence of therapeutic monoclonal antibody (Nivolumab 2 ug/ml; anti-CD47 clone MIAP410 20ug/ml) and evaluated by a cytokine Luminex assay (Milliplex® MAP Human Cytokine Bead Panel (Sigma Aldrich) for TNFα, IFNγ, and IL-2 cytokines analysis). Results: The addition of anti-PD1 to CD3/CD28 stimulation drastically increased the production of all cytokines analyzed. This increase in pro-inflammatory cytokine secretion was even more pronounced after adding an anti-CD47 monoclonal antibody to the cell culture. However, a combinatorial application of anti-PD-1 and anti-CD47 agents in vitro was found to limit rather than potentiate the immune activation in the TME of human STS. On an individual level, the most profound response to anti-PD-1 was observed in high-grade leiomyosarcoma and the most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma. Conclusions: Our data suggest that combinatorial strategies based on ICIs and anti-CD47 blockade may not provide the desired outcome in STSs. Both therapies, anti-PD-1, and anti-CD47 alone, could however bear a therapeutic potential in selected tumors after close evaluation of the CD47 and PD-1 expression status, as well as the extent of T cell/macrophage infiltration.