Redirecting a patient’s T-cells against cancer shows tremendous clinical responses in certain tumor types, but potent therapies for ovarian cancer remain limited. Here we describe the preclinical development of three novel cancer immunotherapy platforms. We first isolated an ErbB2(369-377)-specific T-cell receptor (TCR) from a patient who was previously vaccinated against ErbB2, a protein ubiquitously overexpressed in ovarian cancer. We hypothesized that an ErbB2(369-377)-specific TCR can recognize endogenously processed ErbB2 protein in human cancer. This strategy re-directed human T-cells against ErbB2(369-377), conferring recognition of ErbB2(+) HLA-A2(+) tumor cell lines in vitro and in vivo. Together, our results provide a potential therapeutic for adoptive immunotherapy of ErbB2-expressing malignancies. We next targeted B7-H4, a protein highly expressed in cancer with low expression in healthy human tissues. We engineered T-cells with novel B7-H4-specific chimeric antigen receptors (CAR) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T-cell therapy can be applied safely in preclinical models. B7-H4 CAR T-cells displayed anti-tumor reactivity against B7-H4(+) human ovarian tumor xenografts followed by delayed, lethal toxicity. Comprehensive study of murine B7-H4 protein distribution uncovered expression in multiple tissues that correlated with widespread histologic lesions. We concluded that long-term engraftment of B7-H4 CAR T-cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. While preclinical murine models of CAR T-cell therapy are widely applied, they are limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. Therefore, we established a large, outbred canine model of CAR therapy to test the hypothesis that functional tumor-specific CAR T-cells can be generated and applied in patient dogs with spontaneous cancer. Anti-canine CD20 (cCD20) mRNA CAR electroporated T-cells exhibited antigen-specific recognition and lysis of cCD20(+) targets. In a first-in-canine study, autologous transfer of cCD20-Zeta CAR T-cells was well tolerated in a dog with relapsed B cell lymphoma. However, anti-tumor activity was transient, suggesting that product optimization is needed. Our study demonstrates feasibility for the use of CAR therapy in companion dogs to evaluate safety and efficacy prior to application in humans.