Project Summary Most of the patients with malignant brain cancer or glioblastoma (GBM) do not live more than 20 months despite highly aggressive treatments, and those who do have a very high probability of tumor recurrence. State-of-the- art therapeutic strategies that instruct and/or help our body's immune system to fight against malignant cancers have been recently introduced and hope has been provided by success in clinical studies targeting non-brain cancers. However, this so-called immunotherapy, particularly checkpoint inhibition, has failed to show measurable benefits among patients with brain cancers in a number of recent clinical trials. This disappointing reality is largely attributed to the unique ability of brain cancers to resist immunotherapy or to suppress our defensive immune system. Specifically, transforming growth factor β (TGFβ) is highly upregulated and plays pivotal roles in promoting the immunosuppressive tumor microenvironment in a multi-pronged manner in GBM. Thus, we hypothesize that TGFβ blockade would mask the tumor immuno-suppression, thereby rescuing checkpoint inhibition as a viable and potent treatment for GBM. Indeed, the validity and potential impacts of this combined approach have been preclinically demonstrated in multiple non-brain malignant solid tumors. However, its realization in GBM is yet to be accomplished due to inability to achieve uniform and robust delivery of TGFβ inhibitors throughout the brain tumor tissue, including the particularly immunosuppressive hypoxic tumor areas. The tightly sealed blood-brain barrier (BBB) precludes extravasation of systemically-administered therapy into the brain tissue. Once beyond the BBB, therapy must percolate the highly dense and adhesive tumor extracellular matrix to spread throughout the tumor tissue and reach hypoxic tumor regions distanced from blood vessels. To this end, we propose to develop and evaluate innovative human ferritin protein nanocage-based delivery platform capable of overcoming these challenging biological barriers for widespread TGFβ blockade throughout the brain tumor tissue following systemic administration. We recently demonstrated that our prototype nanocage provides stable systemic circulation, efficient extravasation and tumor uptake, tumor tissue penetration as well as accumulation in hypoxic tumor regions. In addition, our pilot study shows that the nanocage specifically designed to carry TGFβ trap provides markedly enhanced ability to block the immunosuppressive TGFβ signaling in vitro compared to the clinically-relevant anti-TGFβ antibody. We also provide a proof-of-concept evidence suggesting that this nanocage enhances therapeutic efficacy of a clinically used checkpoint inhibitor in a mouse model of GBM. We thus expect that TGFβ-antagonizing nanocages to be further developed in this proposal will dismantle the notoriously immunosuppressive nature of GBM and thus make the otherwise refractory immune checkpoint inhibition highly effi...