ABSTRACT Cytomegalovirus (CMV) infection is virtually unavoidable, with ~35% of the US population 6-11 years old seropositive for CMV antigens, increasing to >90% of those over 80 years old. While the infection rarely causes morbidity in healthy individuals, active CMV in the very young, very old or immunocompromised may result in significant morbidity and mortality. To avoid the long-term use of antivirals, which have significant toxicities, there is interest in adoptive cell therapy (ACT) targeting CMV infected cells for at risk populations. In particular, ACT is attractive for transplant recipients who are prone to post-transplant CMV resulting from latently infected donor tissue provided to a CMV-negative and immune suppressed recipient. This strategy can suppress CMV in stem cell transplant patients when CMV specific T cells are isolated from HLA-matched donors (most often the original stem cell donors), but relies on the very limited availability of donor-sourced T cells and is less available to solid organ recipients. Newer approaches, as yet untested in CMV but effective in blood cancers, address this shortcoming by introducing recombinant T-cell receptors (TCRs) or chimeric antigen receptors (CARs) into patient effector T cells to target diseased cells. In the long-term, we aim to test our hypothesis that CAR-based ACT, which is generalizable and does not require HLA-matching, can be as effective for transplant patients as TCR-based approaches. In order to meet this goal, we currently propose engineering a panel of improved human anti-pp65 TCRs and anti-gB CARs for in vitro evaluation of their abilities to target cells harboring CMV. In Aim 1, we will build on our early work in developing anti-pp65 TCRs over a wide affinity range (<10 nM – 30 M) using mammalian cell display and flow cytometric sorting of CDR libraries. The relationship between TCR affinity and activity is complex and this panel will allow us to compare these TCRs for optimal function in vitro now and in vivo models in subsequent projects. In Aim 2, we will generate anti-gB CAR libraries to screen for truly antigen-dependent, robust T cell activation. We will screen directly for Jurkat T cell activation in a new system based on our previous mammalian display work. Previous CAR development efforts indicate that CAR T cells experience exhaustion as a result of tonic signaling in the absence of target antigen. Our screening in both the presence and absence of gB-expressing cells will allow us to select for minimized antigen- independent activation. Finally, Aim 3 directly compares the ability of TCR and CAR expressing PBMC-derived T cells to kill and suppress viral spread in CMV infected fibroblast cells. Major products from this work will include (1) affinity and functional screening tools and methods for TCR and CAR engineering, (2) insights into the impact of affinity on pp65 CMV TCR activation and function, (3) tools to reduce tonic signaling in CARs, and (4) quantitati...