144 SIRP? deficient CAR-Macrophages exhibit enhanced anti-tumor function and bypass the CD47 immune checkpoint

Background Adoptive macrophage cell therapy represents a novel approach for cancer immunotherapy. Macrophages engineered to express chimeric antigen receptors (CAR-M) have shown promising pre-clinical results against solid tumors by improving tumor clearance, overall survival and facilitating the remodeling of microenvironment induce potent adaptive immune response. CD47 is well-established checkpoint molecule that over-expressed on cells. binds signal regulatory protein ? (SIRP?) limit phagocytosis effector functions. In this study we evaluated impact CAR-M activity showed CD47-resistant targeted mediates enhanced anti-tumor activity. Methods CRISPR/Cas9 was used deplete cognate receptor SIRP? from primary human macrophages (>90% efficiency >90% viability) increase function. To assess CAR-M, < i >in vitro</i > co-culture assays were established with an anti- epidermal growth factor 2 (HER2) CAR HER2+ lines. Macrophage killing target cells quantified in real-time using genetically encoded fluorophore (to monitor growth) or pH-sensitive dye phagocytic acidification). parallel, phenotypic characterization surface molecules, cytokine secretion levels, biochemical analysis downstream signaling molecules response purified HER2 stimulation evaluated. Results knockout (KO) alone failed cytotoxicity but This demonstrated reduced time required kill 50% 2-fold activity, indicating synergy between KO stimulation. Furthermore, absence SIRP?, cytokine/chemokine secretion, polarization, observed. Conclusions We show first feasibility generating gene edited therapeutic purposes, demonstrate deletion enhances CAR-M.