PROJECT SUMMARY Efficacy of CAR T cell therapies against solid tumors has been limited by a lack of expansion in vivo, low levels of tumor trafficking, poor functionality/persistence and by suppression in the tumor micro-environment (TME). Conventionally prepared CAR T cells are unable to retain sufficient differentiational plasticity to undergo natural pathways of T cell proliferation/differentiation/persistence in vivo for sustained, effective cytotoxic anti-tumor activity. Oncolytic viruses (OV) selectively replicate in tumor cells to generate a highly inflammatory TME which may enhance CAR T cell recruitment to, and function within, solid tumors. Our goal is to develop a novel regimen by which oncolytic virotherapy can be used as a potent immunological adjuvant to improve the efficacy of CAR T cell therapy against solid tumors. By loading the OV on the CAR T cells ex vivo, we achieved highly significant improvements in tumor therapy compared to virus or CAR T cells alone. Mice treated with completely systemically delivered CAR T and VSV developed a population of CD8+ CAR T cells with T Cell Receptor (TCR) specificity for the immunodominant H2Kb VSV N52-59 epitope of VSV which selectively expanded in vivo to very high frequency. This population of dual-specific (DS) (virus-specific and CAR T antigen-specific) memory-like (TM) CAR T cells 1). persisted in mice for much longer than conventional CAR T, 2). was significantly more functional against tumor, and 3). could be rapidly re-activated in vivo against tumor by a secondary systemic boost with homologous virus, resulting in long-term tumor cures. These therapeutic effects were observed with two different OV (VSV and reovirus) and across tumor sites (subcutaneous and intra-cranial). Therefore, here we propose a completely novel approach to expand the scope of CAR T cell therapy against solid tumors in which dual specific CAR T cells with improved activity against tumors are generated by using co-administered OV to induce a recapitulation of the physiological pathways that lead to optimal (CAR) T cell activation, proliferation and differentiation in vivo in response to virus infection. We have formulated three Specific Aims: 1) To define the molecular mechanisms by which TCR engagement of DS CAR T cells determines their phenotype, improved persistence and function; 2) To generate in vivo expanded populations of DS CAR T cells with TCR specificity against either tumor associated, or viral recall, antigens and to determine their therapeutic activity against established tumors; 3) To optimize in vivo expansion, persistence/longevity and re-activation of DS CAR T cells through novel boost and rest strategies targeting either the TCR, CAR or both. This will lead to implementation of fully systemic protocols for the combination of CAR T cells with OV which do not require any access to tumors.