Refer to Parent Grant SUMMARY AND SPECIFIC AIMS OF PARENT GRANT (R21CA282629, PI: KATZ). Although T cell mediated immune responses are critical for successful immunotherapy, T cells associated with malignant lesions are typically dysfunctional and fail to control tumor growth. Treatment with tumor infiltrating lymphocytes (TIL) that are isolated, activated, and expanded ex vivo has proven effective in some melanoma patient populations. However, many patients do not respond, presumably due to one of several host immune factors. Current understanding of TIL mechanism of action suggests both an early robust expansion of tumor-specific effector T cells and transfer of less differentiated cells with long-term survival capacity are key to successful therapy. Evidence for the former includes the need for high dose exogenous IL-2 at the time of TIL infusion, correlation of response with a high frequency of effector T cells, and majority of tumor killing occurring early after the initiation of therapy. Evidence for the latter is found in many pre-clinical experiments as well as clinical observations where the presence of TILs from the central memory subset in the infusion product correlates with tumor regression. Our overall goal is to improve TIL therapeutic efficacy through generation of TIL products with both the transient ability to effectively immediately kill tumor cells and the long-term ability to persist and maintain durable antitumor responses. We have developed robust methods to reprogram TILs with mRNA-mediated gene therapy. The use of our mRNA approach has the advantages of increased safety, high efficiency, rapid production, tightly controlled expression levels, and simultaneous multi-factor reprogramming. In preliminary work, we developed a system that increases mRNA lifespan by an order of magnitude. Our single cell analysis of patient TILs preand post-expansion has identified two specific pathways deficient in the expanded TIL product that likely contribute to their poor immediate efficacy and absence of memory fate. Both will be augmented by TIL mRNA reprogramming. By leveraging our multidisciplinary team and existing infrastructure, the knowledge gained from this project will be quickly translatable into clinical trials to examine the efficacy of mRNA reprogramming of TIL to improve treatment of patients with advanced melanoma. We will complete the following Specific Aims: Aim 1. Determine the ability to enhance post-expansion TIL survival. 1.1. Increase TIL durability by expression of an anti-apoptotic protein with a novel long-lived (loli) mRNA expression system. 1.2. Augment TIL efficacy by reprogramming with supportive cytokine mRNAs. 1.3. Analyze efficacy of TILs reprogrammed with survival and cytokine mRNAs in patient derived xenografts. Hypothesis: TILs encounter a large bottleneck of elimination upon infusion as they adapt from ex vivo culture conditions to the harsh tumor microenvironment. By reprog...