” Abstract: The clinical benefit of anti-EGFR antibody, cetuximab, remains suboptimal among patients with oral cancer, and one underlying cause is the failure of this antibody to induce potent anticancer immunity due to immunosuppressive mechanisms within the tumor microenvironments. Elevated levels of adenosine is considered a major immunosuppressive mechanism in healthy tissues and tumors, and it may cause resistance to cetuximab by suppressing innate and adaptive antitumor immunity induced by the antibody. Adenosine deaminase (ADA) catalyzes the degradation of adenosine to inosine and is capable of reversing immunosuppressive effects of adenosine. However, systemic enzyme administration causes autoimmune disorders in normal tissues, where adenosine protects the host from excessive immune activation. We hypothesize that targeted delivery of ADA into oral tumors overcomes adenosine-mediated immunosuppression precisely in the tumors, enhancing the efficacy and safety of cetuximab therapy. Our preliminary studies have shown that targeted delivery of ADA led to localization of ADA on the cell surface EGFR+ oral cancer cells, and the surface-anchored ADA could deplete extracellular adenosine. In an orthotopic mouse model of oral cancer, the targeted delivery resulted in substantial increase in tumoral ADA level compared to non-targeted delivery. In tumor-bearing syngeneic mice, targeted ADA delivery led to reprograming of tumor microenvironments into an immunogenic landscape and resulted in immune-mediated tumor regression when combined with radiation therapy. In this proposal, we will explore immunostimulatory mechanisms of this targeted delivery approach (Aim 1) and will further optimize it by using a bi-targeting method (Aim 2). This project can be directly translated into a targeted cancer immunotherapy to treat patients with oral cancer. By addressing lack of cancer specificity, a main obstacle to the development of next-generation cancer treatments, we will contribute to the use of precision medicine for cancer immunotherapy.