A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III-IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 10(9) virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe.