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Background: Osteosarcoma (OSA) in dogs is an aggressive bone tumor with frequent chemotherapy failure and translational relevance for human health. Hypothesis/Objectives: We hypothesized that dogs with OSA could be treated safely by ex vivo activated T‐cells that were generated by autologous cancer vaccination and supported by interleukin‐2 (IL‐2) treatment with survival more than twice that reported for amputation alone. Animals Osteosarcoma‐bearing dogs (n = 14) were enrolled in a single‐arm prospective trial after complete staging before amputation. Four healthy dogs also were treated in a safety study. Methods: Autologous cancer cell vaccinations were administered intradermally and dogs underwent leukapheresis. Mononuclear cell products were stimulated ex vivo with a T‐cell‐activating agent. Activated product was transfused and 5 SC IL‐2 injections were administered q48h. Dogs were monitored for metastasis by thoracic radiography every 3 months. Results: Autologous cancer cell vaccine and activated cellular therapy (ACT) products were successfully generated. Toxicity was minimal after premedicants were instituted before ACT. With premedication, all toxicities were grade I/II. Median disease‐free interval for all dogs was 213 days. One dog developed cutaneous metastasis but then experienced spontaneous complete remission. Median survival time for all dogs was 415 days. Five dogs survived >730 days. Conclusions and Clinical Importance: This immunotherapy protocol without cytotoxic chemotherapy is safe and tolerable. Compared to historical amputation reports, survival was notably prolonged in this group of patients. Additional prospective studies are warranted to elucidate active immunologic mechanisms and further improve disease response and survival. [ABSTRACT FROM AUTHOR]