Abstract ACT of T cell receptor (TCR) gene- modified T cells specific to tumor antigens (Ags) is currently in clinical trials for patients with advanced malignancies. Transduction (Tdx) of human and murine T cells with our retroviral vector encoding the TIL 1383I TCR genes allows for recognition and killing of HLA-A2+, Tyrosinase (Tyr)+ melanoma cells in vitro and in vivo. Limitations to the efficacy of autologous ACT include: less potent activity of transferred cells in vivo, possibly due to the immunosuppressive tumor microenvironment or to the altered biology of cells from a cancer patient. Another limitation is the monospecificity of the transferred cells, which could lead to immune escape variants; therefore, it is vital to build upon this therapy to achieve better and more durable clinical responses. Therapeutic efficacy of cancer vaccines has been shown to correlate with mounting a broad systemic anti-tumor response through induction of cross priming. A phase I clinical trial in collaboration with Dr. Keld Kaltoft, administered intratumoral injections of irradiated, allogeneic, MART-1-specific T cells into stage IV melanoma patients. Two of fifteen patients had regression of non-injected lesions and two patients developed vitiligo, suggesting induction of systemic anti-tumor immunity and generation of T cells specific to additional melanoma Ags. We propose to use intratumoral injection of Tyr TCR transduced (Td) allogeneic T cells as an alternate approach to systemic infusion of Tyr TCR Td autologous T cells to enhance host immune activation and overcome the immunosuppression that prevents destruction of tumors. We hypothesize that intratumoral injection of Tyr TCR Td allogeneic T cells will promote pro-inflammatory cytokine responses and lytic activity, leading to a favorable anti-tumor environment that facilitates epitope spreading and cross priming against additional melanoma Ags and results in regression of distant, untreated tumor lesions. In Aim II, to further enhance anti-tumor responses and elicit cross priming, we will genetically modify Tyr TCR Td T cells to overcome the immunosuppressive tumor microenvironment and to boost adaptive immune responses. By understanding mechanisms that maximize T cell function within the tumor microenvironment and generate systemic anti-tumor immunity, we can improve upon the therapeutic efficacy and safety of T cell-based immunotherapies.