Background: GP2 is a 9 amino acid HLA-A2/A3 restricted HER2-derived peptide. GP2 + GM-CSF has been shown to be safe and effective in eliciting anti-HER2 immune response in breast cancer patients. Preclinical data has demonstrated that pretreatment of cells with trastuzumab (Tz) enhances susceptibility to lysis by GP2-specific cytotoxic T lymphocytes (CTLs). We conducted a phase Ib study to evaluate the combination of the GP2 vaccine and Tz. Methods: HLA-A2/A3 + patients with HER2 overexpressing breast cancer receiving Tz as standard therapy were enrolled. The study was designed as a 3+3 dose escalation trial with an expansion cohort evaluating 4 dose levels of the vaccine administered as 6 inoculations given every 3 weeks in combination with Tz (6mg/kg). Toxicity was graded 48-72 hr post vaccination using NCI Toxicity Criteria. Ejection fraction (EF) was monitored every 3 mo. Immunologic response was assessed in vivo by injection site local reaction (LR) and in vitro by quantifying the number of GP2-specific CTLs by HLA-A2: IgG dimer assays and their functional activity by ELISPOT. Results: 19 patients enrolled (median age 47 yr, mean tumor size 3.4 cm, 74% were grade 3, 53% ER/PR+, 63% node positive, 74% received anthracycline based therapy). Maximum local toxicities were grade 1 (77% of patients) and grade 2 (6%), and maximum systemic toxicities were grade 1 (24%) and grade 2 (5%). There were no grade 3 or 4 local or systemic toxicities. There was no significant change in EF at 3 mo (57± 1%, p=0.23) or 6 mo (59±1%, p=0.8) compared to baseline (58±0.9%). Mean post-vaccine series LR was significantly larger than initial vaccination LR (68.2 ± 8.6 mm vs 28.0 ± 10.3 mm, p=0.0004). In vitro assays demonstrated an increase in the maximal number of post- versus pre-vaccination GP2-specific CTLs by dimer assay (1.45 ± 0.19 vs 0.96 ± 0.19%, p=0.06) and increased ELISPOT activity [median 86 range (3-194) vs 34 (range 0-295) spots/10⁶ cells]. Conclusions: GP2 vaccine in combination with Tz is both safe and immunogenic in HER2-overexpressing breast cancer patients in the adjuvant setting. Toxicity was limited to mild local and systemic reactions. There were no dose limiting toxicities or cardiac events.