Phosphatidylserine (PS)-Targeting Chimeric Interferon (IFN) Fusion Proteins for Anti-Tumour Applications

In viable cells, membrane phospholipids are asymmetrically distributed, whereby the anionic phospholipid phosphatidylserine (PS) is localized to the inner leaflet of the plasma membrane. During apoptosis, phospholipid asymmetry collapses, and PS is externalized to the external leaflet where it serves as an “eat-me” signal for clearance through efferocytosis, which drives immune suppression. PS is also externalized on tumor endothelial cells, stromal cells, and cancer cells in the tumor microenvironment (TME), reflecting a pathophysiological state of solid cancers that function to suppress host anti-tumor immunity. Here, in an attempt to modulate the TME and induce an immunogenic immune response, we generated novel recombinant fusion proteins containing type I and type III IFNs (IFN-β-IFN-λ) fused into a single polypeptide chain separated by a short linker. The IFN-β-IFN-λ fusion proteins retain the functions of both type I and type III IFNs but show combined effects to improve biological function as well as enhance anti-tumor activities. To localize IFNs to sites of externalized PS, we next fused the IFN-β-IFN-λ chimeric protein to the PS-targeting gamma-carboxyglutamic acid-rich (Gla) and EGF domains of Gas-6, rendering the IFN biologics as PS-targeting modalities. Gas6-IFN-β-IFN-λ proteins selectively bind to PS, including apoptotic cells and live PS-positive CDC50 mutant cells. _In vivo_, Gas6-IFN-β-IFN-λ retain strong anti-tumor activities in a syngeneic model when expressed ectopically in E0771 breast cancer and B16-F10 melanoma models. These studies support the utility of first-in-class IFN fusion proteins that target the immune-stimulatory features of IFNs to the exposed PS in the TME.