376 Macrophages engineered to express synthetic cytokine switch receptors act as living microenvironment converters

Abstract

Background Cytokines in tissue microenvironments regulate the balance between pro-and anti-inflammatory signaling. Dysregulated cytokine expression causes deleterious immunosuppression or inflammation, underpinning the pathophysiology of numerous diseases. As examples, anti-inflammatory cytokines in solid tumors suppress immune activation and safeguard the tumor, whereas pro-inflammatory cytokines in rheumatoid arthritis drive chronic inflammation. Rebalancing inflammation/immunosuppression by targeting aberrant cytokine signaling offers a generalizable approach to treating many diseases, but systemic cytokine blockade carries risks such as increased risk of infection. Cellular immunotherapies may offer a localized platform that could activate in response to cytokines then proportionately remodel the microenvironment's inflammatory state as needed. Macrophages are tissueinfiltrating homeostatic regulators responsible for initiating and resolving inflammation. Engineered macrophages have demonstrated a promising ability to target tumor cells utilizing chimeric antigen receptors. Here, we leveraged the ability of macrophages to regulate inflammation by generating macrophages that express synthetic cytokine switch receptors. We termed this platform "Engineered Microenvironment Converters" (EM-C) and evaluated its modular ability to convert immunosuppressive M2 signals into pro-inflammatory M1 responses for solid tumor microenvironment conversion, or vice versa for inflammatory disease. Methods EM-C were generated by expressing switch receptors (SR) in primary human macrophages. To convert IL10, a prevalent immunosuppressive cytokine in the TME, into a proinflammatory signal, an IL10 SR was designed and delivered to macrophages using VPX-Lentiviral particles. The in vitro response of IL10 EM-Cs to IL10 was monitored using phenotypic characterization of surface molecules, measurement of cytokine production, and biochemical analysis of downstream signaling. To assess the ability of EM-Cs to alter the inflammatory status of their environment, in vitro co-culture assays were established with M2-polarized bystander cells, and the phenotype of bystanders and EM-Cs was assessed individually. Similarly, EM-Cs targeting TGFb or IFNg were characterized. Results IL10 EM-Cs converted IL10 into a pro-inflammatory signal. Unlike wildtype macrophages, IL10 EM-Cs treated with IL10 upregulated M1 markers and cytokines in a dose-dependent manner. Furthermore, IL10 EM-Cs repolarized bystander M2 macrophages towards a pro-inflammatory phenotype following co-culture. Additionally, TGFb EM-Cs were generated and converted TGFb to a pro-inflammatory signal. IFNg EM-Cs were generated and converted IFNg, a canonical M1 cytokine, into an M2 signal. Conclusions We present for the first time a novel immunotherapy platform that harnesses macrophages as "living converters" to locally regulate inflammation for oncology and inflammatory applications. By demonstrating EM-Cs in the M2-to-M1 and M1-to-M2 direction, this platform offers modularity in co...