All patients had previously undergone traditional therapy including surgery, chemotherapy, and/or radiation therapy as appropriate. At time of enrollment, all patients were without any evidence of disease. Because of the HLA-A2 restriction of the E75 peptide, the patients were HLA typed after enrollment and then placed in either the treatment arm (HLA-A2+) or the observation group (HLA-A2-). Interestingly, there seemed to be some differences in the two groups with HLA-A2+ patients having generally worse prognostic tumor characteristics (larger, higher histologic grade, less hormonal sensitivity). As a result of these differences, the treatment arm tended to have less hormonal therapy (a treatment known to reduce recurrence). These patients received the E75 peptide admixed with GM-CSF according to a two-stage design with escalating doses in the first stage and alterations in the vaccine schedule in the second stage. Again, this vaccination formula was shown to be safe and well tolerated. Clonal expansion of E75-specific CTL was noted along with CTL-mediated E75-directed lysis of HER2/neu-expressing tumors. Perhaps most striking, however, were the clinical results as related to disease recurrence. Keeping in mind the generally worse prognostic variables of the treatment arm, there were no deaths among those vaccinated versus two in the observation group. Furthermore, only two of 24 (8%) vaccinated patients developed recurrence versus six of 29 (21%) in the observation group (p=0.19). At a median follow-up of 22 months, the DFS among those vaccinated was 85.7% in contrast to 59.5% for those in the control group. These early results are compelling and indicate a tendency for this vaccination strategy to prevent recurrence among high-risk patients. The E75 vaccine was also evaluated in NN breast cancer patients in a separate but almost identical trial. In an analysis of the combined results of the two E75 trials to determine the safety and effectiveness of the E75 vaccine (Peoples et al. 2008), a total of 171 patients were enrolled with 90 in the NP trial and 81 in the NN trial. During this trial, the E75 peptide was determined to bind to the HLA-A3 molecule in addition to HLA-A2. As such, the vaccine was extended to HLA-A3+ patients as well. A total of 90 patients received the E75 vaccine (45 NP and 45 NN) while 81 were in the prospectively followed observation arm of the studies (45 NP and 36 NN). Combined toxicity was minimal with local reactions being grade 1 (86%) and grade 2 (14%). Systemic toxicity was grade 0 (16%), 1 (70%), 2 (13%), CONFIDENTIAL Page 12 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 and 3 (1%). All patients demonstrated in vitro immunologic responses and in vivo delayed type hypersensitivity (DTH) responses post-vaccination, though variable. For all 171 patients, the clinical recurrence rate for the vaccinated patients was 5.6% (5/90) compared to 14.8% (12/81) for the observation patients (p=0.04) at a median of 20 months. These combined results suggest that the E75 vaccine can be administered safely with minimal local or systemic toxicity. In vitro functional assays and in vivo DTH reactions indicate good immunologic responses to the vaccine, but most importantly, the E75 vaccine appears to reduce recurrences in disease-free, conventionally treated breast cancer patients. These results are similar to the Herceptin results with an approximate 50% reduction in recurrence rates in vaccinated NP and high-risk NN breast cancer patients. However, the vaccine was used in patients with all levels of HER2/neu expression, not only those with over-expression. Recently we have reported updated results from these two trials (Clifton et al.