PROJECT SUMMARY/ABSTRACT Advances in immunotherapy, especially PD1/PD-L1 immune-checkpoint inhibitors (ICI), are revolutionizing the approach to personalized treatment for head and neck squamous cell carcinomas (HNSCC) with significant positive clinical results. However, a large percentage of HNSCC patients still receive no benefit from this treatment due to undefined mechanisms of resistance. Designing personalized targeted therapies with verified biomarkers has been challenging due to the complex molecular etiology of the disease. Thus, there is an urgent need to define resistance mechanisms operating within the tumor microenvironment (TME) to develop new therapeutic paradigms that can effectively and safely treat HNSCC. Our long-term goal is to understand the roots of pathogenic signaling during HNSCC progression. This proposal is based substantial published and preliminary data showing that cadherin desmoglein 2 (Dsg2) modulates cell growth, survival, and metastasis and is upregulated in many cancers including HNSCC where Dsg2 is often found overexpressed at the aggressive leading edge of the tumors. Most importantly, mAb targeting Dsg2 inhibits tumor development in xenograft model. Using these mAb we recently generated the anti-Dsg2 scFv CAR construct and the resultant Dsg2 CAR- T cells not only recognized SCC cells but had robust in vitro and in vivo antitumor activity. Mounting evidence suggests that exosomes or small extracellular vesicles (sEV) regulate local and systemic processes impacting tumor progression and therapeutic outcomes. This proposal is based on solid preliminary data obtained from an ICI clinical trial where sEV were purified from patients’ plasma and subjected to miRNAseq revealing an increase in miR-155-5p (miR-155) in HPVneg patients that responded to treatment. miR-155 targets Dsg2 and PD-L1 mRNAs and their translation, suggesting that miR-155-loaded sEV could have antitumor activity. Our working hypothesis is that engineered extracellular vesicles (eEV) carrying the Dsg2scFv-CD63 fusion proteins on the surface and loaded with miR-155 can be harnessed for therapeutic treatment of Dsg2-overexpressing cancers. The hypothesis and goal will be addressed in two Specific Aims designed to: 1) Engineer Dsg2scFv/miR-155 eEV and 2) Determine the antitumor activity of Dsg2scFv/miR-155 eEV. This proposal will deliver innovations in defining blood-borne molecules that modulate ICI resistance in HNSCC patients, along with an opportunity to rationally leverage this new knowledge through a unique proof-of-concept sEV therapy which represents a cutting edge of the field.