Abstract Adoptive cell transfer using autologous tumor infiltrating lymphocytes (TIL) represents a personalized cancer immunotherapy strategy targeting shared and unique tumor antigens expressed by a patient's cancer. Although originally developed for cutaneous melanoma therapy, we recently reported TIL therapy can be effective against metastatic uveal melanoma (UM), a cancer with low mutational burden and resistance to conventional immunotherapies. A significant limitation in improving upon these results is that the vast majority of antigen experienced TIL will undergo cell death shortly following infusion. Only a small subset persist as long-lived memory cells. This may stem from the limited metabolic reserve of highly differentiated effector T cells (TEFF). Chronic antigen exposure can result in deficiencies in nutrient sensing and flux through critical bioenergetic and biosynthetic pathways that support T cell activation, proliferation, and effector functions. Ex vivo metabolic reprograming could rescue “exhausted” TEFF and promote development of long-lived memory cells following adoptive transfer. Our long-term goal is to develop clinically relevant approaches that promote the metabolic fitness of human TIL following adoptive transfer. 4-1BB (CD137) co-stimulatory signaling can transiently improve the metabolic capacity of human tumor specific CD8+ TEFF. Our preliminary findings demonstrate that this metabolic improvement is dependent upon a novel mechanism employing the activity of the mitochondrial enzyme, arginase 2 (ARG2). Thus, the specific objective of this grant is to further characterize the role of ARG2 in reprogramming the fate and function of highly differentiated TEFF. Our central hypothesis is that ARG2 is the critical mediator of the improved metabolism found in TEFF following 4-1BB co-stimulation. Since 4-1BBL expression is often deficiently expressed by tumors, we, consequently, postulate that bioengineering TEFF to conditionally express ARG2 upon TCR stimulation will reprogram their cellular metabolism in the tumor microenvironment, improving in vivo persistence and function. We anticipate that these studies will enhance the metabolic fitness of TIL for future adoptive transfer clinical trials. To test our hypothesis, we propose the following Specific Aims: Aim 1. Identify key transcriptional regulators linking 4-1BB co-stimulation with ARG2 activity in human TEFF cells. Aim 2. Characterize the downstream metabolic and cellular effects of ARG2 expression in TEFF cells. Aim 3. Determine the safety and efficacy of adoptive transfer of human TIL bioengineered to express ARG2 in patient derived tumor xenograft models. Collectively, the completion of these studies will demonstrate the essential role of enhanced ARG2 expression in promoting and sustaining T cell metabolism. Bioengineered expression of ARG2 could markedly improve adoptive immunotherapy with TIL.