PROJECT SUMMARY/ABSTRACT Immune checkpoint therapies (ICTs) demonstrate the exciting efficacy of therapies that modulate the immune system to combat cancer. Compared to traditional chemotherapeutic strategies, which typically extend overall survival by a few months, ICTs can lead to durable anti-cancer responses that increase overall patient survival by years or more. However, 60-80% of patients are unresponsive to ICTs and new therapeutic strategies are necessary to treat these ICT-resistant tumors. ICTs modulate the adaptive immune system by acting on T cells. Agonists of toll like receptors (TLRs) act on the innate immune system and are an underexplored mechanism by which to drive an anti-cancer immune response. TLR5 agonists have been shown to be tolerable when administered subcutaneously and intravenously in clinical trials and flagellin, a TLR5 agonist, has demonstrated potent anti-tumor effects in animal models when administered by intra-tumoral injection. In addition, preliminary data has shown that intra-tumoral injection of flagellin can overcome ICT resistance and this effect is due to local modulation of the tumor immune microenvironment (TIME). However, as not all patient tumors are accessible by direct injection, new methodologies are needed to formulate and deliver flagellin as an anti-cancer therapeutic. As such, this proposal is focused on the development of a novel method to deliver flagellin directly to the tumor compartment and understanding the mechanisms by which tumor-targeted TLR5 activation overcomes ICT resistance. The central hypothesis of this proposal is that tumor-targeted activation of TLR5 of the TIME, via NF- κB signaling, will reprogram PMN-MDSCs in the TIME, relieving T cell suppression to overcome ICT resistance. This hypothesis will be investigated in the following aims. In Aim 1, the bio-distribution and pharmacokinetics of tumor-targeted delivery of flagellin will be assessed by PET. In Aim 2, the therapeutic efficacy of tumor-targeted TLR5 activation will be assessed as a standalone agent or in combination with ICT by treating ICT-resistant tumors. Finally, Aim 3 will investigate, the mechanisms by which tumor-targeted TLR5 activation reprograms PMN-MDSCs in the TIME to overcome ICT resistance. Understanding the mechanisms by which TLR5 activation can overcome ICT resistance and developing a mechanism to deliver flagellin directly to tumors will provide novel methodologies to combat ICT resistance and develop readily translatable therapeutics for patients with ICT-resistant tumors. In addition, completion of this proposal by the applicant at the MD Anderson Cancer Center in the Department of Cancer Systems Imaging will provide the applicant with exceptional training opportunities in cancer immunology and imaging, as well as enhance the applicant's training goals of becoming an independent investigator.