862 Targeting PSGL-1, a novel macrophage checkpoint, repolarizes suppressive macrophages, induces an inflammatory tumor microenvironment, and suppresses tumor growth

Background Macrophages play an important role in cancer by modulating both the innate and adaptive parts of the immune system. In non-pathological conditions, multiple subsets of macrophages balance the immune response. In cancer, M2-like immune-suppressive tumor-associated macrophages (TAMs) dominate the tumor microenvironment (TME). TAMs promote tumor growth, support neo-angiogenesis and enable metastasis formation. Macrophage modulators driving macrophage repolarization from the M2-like to a pro-inflammatory M1-like phenotype are an attractive novel class of cancer immunotherapy. Here we present identification, validation, and pre-clinical data of a novel macrophage checkpoint, PSGL-1, which supports targeting this molecule for immune-oncology. Methods To assess the therapeutic potential of using anti-PSGL-1 antibodies to convert macrophage phenotype and the tumor microenvironment toward a more inflammatory state, we employed in vitro primary macrophage and multi-cellular assays, ex vivo patient-derived tumor cultures, and a humanized mouse PDX model. Results Within the multiple subsets of macrophages, PSGL-1 is expressed at high levels on immune-suppressive TAMs and in vitro differentiated M2 macrophages. We show that targeting PSGL-1 via an antagonistic antibody repolarized M2 macrophages to a more M1-like state, both phenotypically and functionally as assessed in primary in vitro macrophage assays. Further, these repolarized M1-like macrophages enhanced the inflammatory response in complex multi-cellular assays, including SEB stimulated PBMC assays and mixed-lymphocyte reactions (MLRs). To establish a pre-clinical proof-of-concept for targeting PSGL-1, we turned to ex vivo cultures of fresh patient-derived primary tumors, where the complexity of the TME can be most preserved. RNA-seq data show that ex vivo cultures treated with anti-PD-1 antibody recapitulate TME changes in anti-PD-1 treated patients, including a strong T-cell IFN-gamma signature and a reduction in oncogenic pathway activation. Blocking PSGL-1 resulted in a robust pro-inflammatory signature driven by TNF-alpha/NF-kappa-B and chemokine-mediated signaling. The increase in TNF-alpha signaling was accompanied by reduction in oxidative phosphorylation and fatty acid metabolism. The increase in pro-inflammatory cytokine and chemokine production was confirmed by measuring secreted protein levels, further confirming the re-polarization of macrophages within a tumor setting. Lastly, we employed a humanized mouse PDX model of melanoma and show that anti-PSGL-1 treatment resulted in suppression of tumor growth favorably compared to anti-PD-1. At the cellular and molecular levels, anti-PSGL-1 treatment lead to a more enhanced inflammatory microenvironment, including a reduced M2:M1 macrophage ratio, increased antigen presentation, pro-inflammatory mediators, and effector T cell infiltration and activation. Conclusions Our data support anti-PSGL-1 as a macrophage repolarizing agent and an effective macrophage-targeted therapy for Immuno-Oncology.