Bright field (A), CFSE-immunofluorescence (B), and merged images showing CFSE-labeled DLD-1 inside macrophages as indicated by arrows (C).(TIF) pone.0201832.s002.tif (3.6M) GUID:?9B22D378-1FA4-415C-87EC-BC7EA35180D0 S3 Fig: ALX148 enhances antitumor therapy or on blood cell parameters in rodent and non-human primate studies. (3.6M) GUID:?9B22D378-1FA4-415C-87EC-BC7EA35180D0 S3 Fig: ALX148 enhances antitumor therapy or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograft models, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies. Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with ALX148 induces GSK2141795 (Uprosertib, GSK795) multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation, macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune cells, causing increased T cell effector function, pro-inflammatory cytokine production, and a reduction in the number of suppressive cells within the tumor microenvironment. Taken together, these results show that ALX148 binds and blocks CD47 with high affinity, induces a broad antitumor immune response, and has a favorable safety profile. Introduction A central question in the study of cancer is why the immune system sometimes fails to mount an effective antitumor response despite possessing the components needed to do so. One cause of this failure has become clear with the identification GSK2141795 (Uprosertib, GSK795) of checkpoint pathways, which are co-opted by tumors to inhibit their elimination by immune cells. This phenomenon has been best described for the adaptive component of the immune response, where cytotoxic T cell activity is usually suppressed by checkpoint signals originating from tumor and other cells in the tumor microenvironment . In the clinic, the CTLA-4 and PD-1 T cell checkpoint pathways have been validated as therapeutic targets, with their blockade leading to enhancement of the patients immune response and, in some cases, durable antitumor efficacy across several tumor types [2C4]. The CD47 pathway is an additional checkpoint that can suppress antitumor immunity [5, 6]. In contrast to previously identified checkpoint pathways that target the adaptive arm of the immune response, this pathway suppresses the activity of innate immune cells [7, 8]. CD47 is expressed on the GSK2141795 (Uprosertib, GSK795) surface of a broad range of cell types [9, 10], and this expression protects healthy cells from macrophage-mediated phagocytosis by interacting with its receptor, signal regulatory protein- (SIRP) [11, 12]. Engagement of SIRP triggers signaling through SIRP immunotyrosine inhibitory motifs (ITIMs), which inhibits phagocytosis and other components of macrophage function [13C21]. Analyses of human tumor tissue have implicated CD47 in cancer. High levels of CD47 expression GSK2141795 (Uprosertib, GSK795) have been observed in a variety of hematological and solid tumors [5, 22], and elevated CD47 expression is an adverse prognostic indicator for survival [22C25]. These findings indicate that tumor cells may utilize the CD47 pathway to evade macrophage surveillance. One component of this HsRad51 surveillance is GSK2141795 (Uprosertib, GSK795) usually Antibody-Dependent Cellular Phagocytosis (ADCP), in which antitumor antibodies initiate phagocytosis by binding tumor cells and engaging macrophage Fc gamma (Fc) receptors [26C28]. Blockade of the CD47-SIRP conversation enhances ADCP of tumor cells [24, 29C32], demonstrating that if unchecked, CD47 expression can safeguard tumor cells from macrophage phagocytosis. Similarly, CD47 blockade in mouse studies inhibits the growth of human tumor xenografts and promotes survival [22, 24, 25, 30, 33]. Notably, these xenograft studies utilized immunocompromised mice that lack most immune cell types other than macrophages. Thus, while these studies exhibited that CD47 blockade activates a.