SUMMARY Metastatic brain tumors are the most commonly observed intracranial tumors. Patients with advanced breast cancer have a high propensity to metastasize to the brain with human epidermal growth factor receptor (EGFR) positive and triple-negative breast cancer (TNBC; estrogen and progesterone receptor and Her2 negative) subtypes showing the highest incidence of brain metastases. To effectively treat multiple highly aggressive brain metastatic breast tumors (BMBT), there is an urgent need to develop therapeutics that target aberrant signaling pathways in tumor cells and the immune cells in the tumor microenvironment (TME) of BMBT. Recently, we have shown that intrathecal (IT) and intracarotid artery (ICA) injection of adult allogeneic “off the shelf” mesenchymal stem cells (MSC) expressing bifunctional protein simultaneously targeting EGFR and (DR)4/5), EvDRL have therapeutic efficacy in mouse models of BMBT that mimic clinical settings. These findings although promising, have raised fundamental questions on the potential of combining MSC released EvDRL induced tumor cell killing with therapeutic agents that simultaneously activate immune effector functions against BMBT. Our recently published studies indicate that DRL (TRAIL) component of EVDRL is the key driver of EVDRL mediated cell death in patient derived BMBT cells. Previous studies have shown that in addition to tumor cells, DRL induces apoptosis in myeloid derived suppressor cells (MDSC) and CD4+ CD25+ FoxP3+ Tregs and simultaneously increases recruitment of CD8+ T cells in the TME. Furthermore, clinical and pre-clinical studies using combined cytotoxic therapy and immune checkpoint (ICI) blockade have shown increased efficacy in breast metastatic tumors thus offering the potential to combine of MSC-EVDRL with immunomodulatory agents to treat BMBT. In this proposal, we will first evaluate the efficacy and influence of MSC-EVDRL induced tumor cell death in the TME in humanized (hu) NSG breast to brain metastatic tumor models generated from BMBT cells that have varying response to EVDRL mediated apoptosis. Next, we will create bimodal MSC releasing EVDRL and anti- programmed cell death protein (PD)-1 nanobodies (Nb-PD1) and evaluate the mechanism combined efficacy of engineered MSC in huNSG breast to brain metastatic tumor models. We hypothesize that human MSC-EvDRL will lead to specific killing of local and widely disseminated BMBT cells and Nb-PD1 will target T cells recruited to the TME. To ease clinical translation, we will incorporate activatable kill switch/PET imaging agent, herpes simplex virus-thymidine kinase (HSV-TK) into MSC and assess their fate by PET imaging and selective eradication mediated by HSV-TK activation. Given that engineered MSC are in phase I clinical trial in non-small cell lung cancer patients; a phase I/II trial using IT of anti-PD-1 antibody is currently ongoing in leptomeningeal metastatic patients; and our MSC-DRL therapy in primary brain tumor (GBM) pat...