1999). In the trastuzumab q3 weekly monotherapy study (Baselga et al. 1998), 105 patients with HER2+ metastatic breast cancer were treated, with an objective response rate of 19% (23% in patients with measurable centrally confirmed HER2+ disease). The median baseline LVEF was 63%, which did not significantly change during the course of the study. One patient experienced symptomatic CHF, which resolved with medical treatment for CHF and discontinuation of trastuzumab. In the study of q3 weekly trastuzumab and paclitaxel (Leyland-Jones et al. 2000), 32 patients were treated with an investigator-assessed response rate of 59%. Ten patients had a decrease in LVEF of 15% or greater. One patient experienced symptomatic CHF, which improved symptomatically after medical therapy for CHF and discontinuation of trastuzumab. 1.6 E75 BACKGROUND E75 is a nine amino-acid peptide derived from the HER2/neu protein (369-377) (Fisk et al. 1995) and is the most studied CTL epitope from this tumor-associated antigen both in vitro and in vivo. Zaks and Rosenberg, in an early study, looked at vaccinating patients with the E75 peptide in an effort to determine its efficacy (Zaks et al. 1998). In this study, metastatic breast, ovarian, and colorectal cancer patients were given 1 mg of peptide mixed with incomplete Freud’s adjuvant subcutaneously every 3-4 weeks for a total of four doses. Three of four patients demonstrated peptide-specific CTL responses; however, these cells did not lyse HER2-positive tumor cells. No clinical response data was disclosed. Knutson and Disis also investigated the E75 peptide but mixed with GM-CSF which was chosen as an adjunct to stimulate an immune response based on previous animal studies (Disis et al. 1996). It is thought to potentiate the immunologic response by promoting the transformation of dermally present Langerhans cells into mature antigen-presenting dendritic cells (DC). The E75 + GM-CSF vaccine was given to six metastatic breast and ovarian cancer patients (Knutson et al. 2002). The vaccine resulted in E75-specific CTL responses in two of four evaluable patients. No clinical response information was given. Murray et al then used the same E75 epitope combined with GM-CSF as an adjuvant in a Phase I trial to examine the toxicity of this formulation as well as its ability to promote a detectable CTL-mediated E75-specific immune response (Murray et al. 2003). A total of 14 patients with stage IV breast or ovarian carcinoma underwent vaccination with escalating doses of the peptide combined with 250 mcg GM-CSF. The formulation was found to be safe with no grade 3 or 4 toxicity reported. Eight of those receiving the vaccine were evaluated for CTL activity directed against the E75 peptide, and four of the eight patients showed CTL-mediated lytic activity. Furthermore, these CTL were shown to remain for between one and 12 months following completion of the vaccine. Of interest, these advanced stage patients continued to undergo disease surveillance at short intervals CONFIDENTIAL Page 11 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 throughout the study. Despite the promising immunologic response, no clinical response was noted, but there was a trend toward delayed time to progression. All of the previously described studies were performed primarily in patients with metastatic disease and known residual tumor burden. While they demonstrate that the desired immune response could be elicited in the majority of patients, minimal clinical results were obtained or reported. This is likely related to the advanced stage of disease in the patients in whom these trials were conducted. Our approach has shifted focus from the treatment of late-stage disease to disease prevention among breast cancer patients considered to be at high risk for recurrence. E75 peptide admixed with GM-CSF has been previously shown to be a safe formulation which can induce an immune response. Because of its effectiveness and simplicity of design, this vaccine strategy was investigated as a possible adjunct after standard therapy to help reduce the risk of recurrence. A total of 53 patients with NP breast cancer were reported initially in our first adjuvant vaccine study (Peoples et al. 2005).