Therefore, we intend to also collect clinical data on the use of the vaccine in these two new patient populations. 1.7 HER2-DIRECTED THERAPIES IN HER2 LOW-EXPRESSING TUMORS While Herceptin has shown significant clinical benefit in the adjuvant setting for breast cancer patients with HER2 3+ expressing tumors, there has been some suggestion that patients with HER2 2+ expressing tumors may benefit as well from adjuvant Herceptin. In an exploratory analysis of the NSABP trial B-31 (Romond et al. 2005), 174 patients who CONFIDENTIAL Page 13 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 were subsequently found to be HER2-negative (negative on FISH and less than IHC 3+) may have derived a benefit from adjuvant Herceptin therapy with a hazard ratio for disease recurrence of 0.34 (95% CI, 0.14 to 0.80; p=0.014) (Paik et al, 2008). This finding has resulted in the initiation of the NSABP trial B-47 to evaluate the efficacy of Herceptin in HER2 low-expressing tumors. HER2-derived, CD8-eliciting, peptide-based vaccines also appear to have benefit in patients with HER2 low-expressing tumors. Patients from our E75 vaccine trials were stratified by HER2 expression levels (Benavides et al. 2009). Patients with HER2 low-expressing tumors (IHC 1+, IHC 2+ or FISH<2.0) (n=100; 56 vaccine, 44 control) were compared with HER2 overexpressors (n=51; 29 vaccine, 22 control). Low-expressors had larger maximum immunological responses to E75 vaccination than over-expressors (p=0.04). This analysis also found that vaccinated IHC 1+ patients had increased long-term immunity (p=0.08). In addition to improved immunity compared to over-expressors, patients with low-expressing tumors had a mortality benefit compared to low-expressing control patients (p=0.08), a benefit present even in IHC 1+ vaccinated patients (p=0.05). 1.8 COMBINED HER2-DIRECTED THERAPY While both Herceptin and E75 each have been shown to have clinical benefit in HER2-expressing breast cancer, the novel concept of combining these two immunotherapies is appealing and worthy of investigation. While Herceptin and E75 share the same immunologic target, the specific immunologic mechanisms of action for these therapies are different. This complementary approach to immune-mediated therapy in breast cancer may provide a clinically beneficial synergism. The combination of Herceptin with HER2-targeted vaccines has been evaluated in preclinical testing (Mittendorf et al. 2006). HER2-expressing tumor cells were incubated overnight with Herceptin in concentrations of 10 mcg/mL and 50 mcg/mL. Cytotoxicity of both pretreated and untreated tumor cells was measured using peripheral blood mononuclear cells from healthy, HLA-A2+ donors as well as from patients vaccinated with E75. Cytotoxicity by peptide-specific cytotoxic T lymphocytes from non-vaccinated patients was increased by 5.6% (10 mcg/mL) and 15.3% (50 mcg/mL) (p=0.002) in pretreated cells over untreated cells. Using peripheral blood mononuclear cells from E75-vaccinated patients, peptide-specific cytotoxicity was 34.2% in untreated cells compared to 40.6% (10 mcg/mL) (p=0.035) and 40.7% (50 mcg/mL) (p=0.0005) in pretreated cells. These findings suggest a synergism may exist with the combination of Herceptin and HER2 vaccination. The safety of the combination of Herceptin and HER2-derived peptide vaccines has been evaluated in early clinical trials. The administration of trastuzumab concurrently with a HER2 CD4+ helper T cell-eliciting vaccine composed of multiple peptides (one of which contains the E75 epitope) to metastatic breast cancer patients over-expressing HER2 (n=22) was found to be safe (Disis et al. 2009).