Background: Vaccination with the intracellular domain (ICD) of HER2 in pre-clinical models is both immunogenic and protective against the development of mammary tumors. This study was designed to examine the safety and immunogenicity of a DNA-based vaccine encoding the HER2 ICD in subjects with HER2⁺ breast cancer. Methods: Sixty-six patients with stage III or IV breast cancer in remission were enrolled sequentially into three vaccine arms: 1 (10µg), 2 (100µg) and 3 (500µg). Vaccines were admixed with 100µg GM-CSF and administered i.d. monthly for three immunizations. Endpoints included safety and immunogenicity (optimum dose). Toxicity and HER2 specific IFN-gamma immune responses were evaluated and DNA persistence at the vaccine site was assessed. Results: Sixty-four of the 66 (97%) patients enrolled completed all vaccinations. Vaccination was associated with minimal toxicity. All three doses of vaccine were able to significantly induce or boost HER2-specific T cell responses to ICD, however the incidence of subjects with augmented immunity was greatest in Arm 2 (14/22, 64%) when compared to Arm 1 (9/17, 53%) and Arm 3 (8/22, 36%). Patients in Arms 2 and 3 had immune responses to HER2 ICD of higher magnitude than patients in Arm 1, however, only patients in Arm 2 retained immunity at 1 year after vaccination. DNA persistence in the vaccine site was related to dose being found most frequently in Arm 3 patients. DNA persistence was associated with a decrease in magnitude of HER2 ICD response and long term immunity. At a median follow-up of 68 months after vaccination, patients in Arm 2 demonstrate superior overall survival (p=0.02) as compared with patients in arm 3. Conclusions: Immunization with a DNA-based HER2 ICD vaccine is safe and can generate robust T-cell responses in treated patients. The resulting immune response is dose-dependent, with 100µg as the optimal dose. Higher vaccine doses may have a detrimental effect on the long term retention of immunity. Clinical trial information: NCT00436254.