Stem Cells Engineered to Release IFNβ and Scfv-Pd1 Target Glioblastoma and Alter the Tumor Microenvironment

Highly malignant brain tumors, glioblastomas (GBM), are immunosuppressive, thereby limiting current promising immunotherapeutic approaches. In this study, we first created interferon receptor 1 (IFNAR1) knockout allogeneic mesenchymal stem cells (MSC) to secrete dual-function pro-apoptotic and immunomodulatory Interferon (IFN) β (MSCKO-IFNβ) using a single lentiviral vector CRISPR/Cas9 system, and tested their efficacy in mouse models of immunosuppressive GBM. We show that MSCKO-IFNβ induce apoptosis in GBM cells and upregulate the cell surface expression of programmed death ligand (PDL)-1 in tumor cells. Next, we engineered MSCKO to release a secretable single-chain variable fragment (scFv) to block programmed death (PD)-1 and show the ability of MSCKO-scFv-PD1 to enhance T cell activation and T cell-mediated tumor cell killing. We further tested the effect of MSCKO-scFv-PD1 in vivo to reduce the tumor growth and improve the survival of mice bearing GBMs after tumor sensitization with MSCKO-IFNβ. To simultaneously express both immune modulators, we engineer MSCKO-IFNβ to co-express scFv-PD1 (MSCKO-IFNβ-scFv-PD1) and show the expression of both IFNβ and scFv-PD1 in vitro leads to T cell activation and lowers the viability of tumor cells. Furthermore, to mimic the clinical scenario of GBM tumor resection and subsequent treatment, we show that hyaluronic acid (HA) gel encapsulated MSCKO-IFNβ-scFv-PD1 treatment of resected tumors results in the increase of CD4+ and CD8+ T cells, mature conventional Dendritic Cells type II (cDC2) and activation of microglia as compared to the control treatment group. Overall, these results show that MSCKO-IFNβ-scFv-PD1 have the ability to shape the tumor microenvironment and enhance therapeutic outcome in GBM.