TAAs expressed by tumors can elicit a very specific immune response; therefore, a vaccine is appealing in that it represents a nontoxic therapeutic modality with great specificity. HER2 is one such TAA. Several peptides capable of inducing CD8+ T cells (also referred to as CTL) have been described from the HER2 protein including E75 (Nelipepimut-S), a 9 amino acid peptide derived from the HER2 protein’s extracellular domain (aa: 369-377:KIFGSLAFL), which is the most studied in laboratory and clinical investigation (reviewed by Mittendorf EA, et al.28). Initial studies investigating E75 in vitro and in an in vivo animal model showed that it was capable of inducing a peptide-specific, CTL-mediated immune response.29,30 This then led to a number of clinical trials including phase I/II studies conducted by our group evaluating E75+GM-CSF in the adjuvant setting. 9 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 The initial trial, which enrolled node-positive breast cancer patients with tumors expressing any degree of HER2 (immunohistochemistry [IHC] 1+, 2+ or 3+), was a phase I two-stage safety trial designed with escalating doses of E75 peptide in the initial stage followed by schedule alterations in the second stage.31 A trial enrolling high-risk node-negative patients was designed to delineate optimal biologic dosing. Both trials transitioned to phase II with disease recurrence as the primary efficacy endpoint. Patients in both studies had received standard therapy including surgery, chemotherapy, and radiation therapy as appropriate and were without evidence of disease at the time of enrollment. Because of the human leukocyte antigen (HLA)-restriction of the E75 peptide, patients were HLA typed after enrollment and then placed in either the treatment arm (HLA-A2+ or A3+ patients) or the observation group (HLA-A2- or A3- patients). The goals of these trials were to document safety, immunogenicity and clinical efficacy of the E75 vaccine. Because the node-positive and node-negative trials were run concurrently and the protocols were identical except for overlapping doses/schedules, the results were merged for analysis.32 At the time of an initial planned analysis of the combined results of the trials, a total of 171 patients were enrolled; 90 patients received the E75 vaccine while 81 were in the observation arms.32 Combined toxicity was minimal with local reactions being grade 1 (81%) and grade 2 (19%). Systemic toxicity was grade 0 (12%), 1 (71%), 2 (14%), and 3 (2%) (Figure1). The most common systemic toxicities were bone pain, flu-like symptoms and fatigue which are attributable to the GM-CSF. There were no grade 4 or 5 toxicities. All patients demonstrated in vitro immunologic responses and in vivo delayed type hypersensitivity (DTH) responses post-vaccination. At a median follow-up of 20 month, the recurrence rate seen in the vaccinated patients was 6% compared to 15% for patients in the observation group (p=0.04). In light of these encouraging data, the trial was continued and follow-up was extended to five years. With additional follow-up, late recurrences were seen in the vaccination arms of the trials which correlated with decreased levels of E75- specific CTL. A voluntary booster program was initiated and vaccinated patients ≥ 6 months from completion of their primary vaccination series (PVS) were eligible for participation. Patients enrolled onto the trial after initiation of the booster program were consented prospectively to 10 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 receive booster inoculations. An initial report of the booster inoculations showed them to be safe and effective in stimulating E75-specific immunity in patients who had waning levels of E75-specific CTL six months following completion of their PVS.33 When the length of follow-up for the trials was extended, additional analyses were incorporated to include evaluation of DFS at 24 and 60 months. With 60 month follow-up completed in all patients, we recently reported the final 5-year follow-up results. Among the 187 patients (108 HLA-A2/A3+ vaccinated; 79 HLA-A2/3- controls), the 60-month DFS were 90% for vaccinated patients versus 80% for controls (p=.08), a 50% reduction in the relative risk of recurrence.34 Because the trials began as dose- and schedule-finding trials, not all patients received the dose that was eventually determined to be optimal (1000µg E75 + 250µg GM-CSF). When outcomes were evaluated by dosing, the DFS rate was 95% for those who received the optimal dose (p=.05 versus unvaccinated controls) (Figure 2). When evaluated by whether patients received a booster inoculation, the DFS rate in the 53 boosted patients was 96% versus 83% in vaccinated patients that did not receive a booster inoculation (p=.04). E75 has been licensed to Galena Biopharma which has secured the nonproprietary drug name nelipepimut-S. NeuVax (nelipepimut-S + GM-CSF) is currently being evaluated in a multicenter, multinational, prospective, randomized, double- blind, controlled phase III study (NCT01479244; PI: Mittendorf). 35 The trial is enrolling NP breast cancer patients with HER2 1+/2+ tumors that are HLA-A2/3+ and are disease free after standard of care therapy. Patients are randomized to receive nelipepimut-S+GM-CSF or GM-CSF alone. The primary endpoint is 36-month DFS. Subsequently, E75 has also been found to bind to HLA-A24 and HLA-A26. Patients expressing these additional alleles have been included in another ongoing trial of NeuVax. Comparative clinical results between A2 and/or A3- positive patients and A24 and/or A26-positive patients has not been completed yet. 11 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 1.4 Combined HER2-directed therapy Nelipepimut-S and trastuzumab have been shown to have clinical benefit in patients with HER2-positive breast cancer. Although these agents share the same target, their mechanisms of action are different; therefore, the concept of combining these two immunotherapies is novel and worthy of investigation. Nelipepimut-S works by stimulating a HER2-specific CTL response. There have been multiple mechanisms of action described for trastuzumab including decreased signaling by prevention of HER2 dimerization, increased endocytic destruction of the HER2 receptor, and inhibition of extracellular domain shedding. 36 In addition, multiple immune-mediated mechanisms of action have been described. Trastuzumab is a human IgG antibody with a conserved Fc portion. Early in vitro and in vivo reports support an important role for antibody- dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer cells which have an Fc receptor (FcR) that binds the Fc portion of trastuzumab.37-39 More recently, an important role for adaptive immunity in mediating anti-HER2 therapy effects has been shown. Using a Rag-1-/- mouse that lacks T cells and B cells but does have FcR+ innate cells, Park et al. showed that HER2-targeted antibody treatment had minimal impact on tumor growth, suggesting that T cells are required for antibody activity.40 Consistent with this, when wild-type mice were administered a CD8-depleting antibody, the mice showed minimal initial responses to antibody therapy then relapsed quickly. In a small study of patients with HER2-positive breast cancer receiving trastuzumab, Taylor et al. demonstrated generation of a HER2-specific CD4+ T cell response in 6 of 10 evaluable patients. 41 This T cell response is in part due to increased antigen uptake through FcR on dendritic cells present in the tumor microenvironment resulting in increased antigen presentation for recognition by T cells. In that same study, investigators showed anti-HER2 antibodies in approximately 60% of 27 evaluable patients during treatment and found that these anti-HER2 humoral responses significantly increased during therapy and were associated with improvements in clinical response.41 Data presented by Knutson et al. at the 2013 annual meeting of the American Society of Clinical Oncology showed that patients treated on the trastuzumab arm of the NCCTG 9831 trial developed HER2-specific antibody responses.42 Cox modeling suggested that larger increases in antibody responses were associated with improved DFS (HR=0.23, p=.04). There is also data demonstrating an inverse relationship between HER2 and HLA class I expression suggesting that down-regulation of HER2 may increase antigen presentation by upregulating HLA molecules.43 Thus a complementary approach to immune-mediated therapy in breast cancer may provide synergism resulting in improved clinical outcomes. We have completed preclinical testing of the combination of trastuzumab with HER2-targeted vaccines.44 HER2-expressing tumor cells were incubated overnight with trastuzumab in concentrations of 10μg/mL and 50μg/mL. 12 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 Cytotoxicity of both pretreated and untreated tumor cells was measured using peripheral blood mononuclear cells (PBMC) from healthy HLA-A2+ donors as well as from patients vaccinated with nelipepimut-S. Cytotoxicity by peptide-specific CTL from non-vaccinated patients was increased by 5.6% (10μg /mL) and 15.3% (50μg /mL) (p=.002) in pretreated cells over untreated cells. Using PBMC from nelipepimut-S-vaccinated patients, peptide-specific cytotoxicity was 34% in untreated cells compared to 40% (10μg /mL) (p=.04) and 41% (50μg /mL) (p<.05) in pretreated cells. These findings suggest a synergism may exist with the combination of trastuzumab and vaccination. The safety of the combination of trastuzumab 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 patients with HER2-positive breast cancer (n=22) was found to be safe with over 99% of toxicities limited to grade 1 or 2.45 Median left ventricular ejection fraction (LVEF) was unchanged pre- to post-treatment, with three patients (15%) experiencing asymptomatic decreases in LVEF. Our group recently completed enrollment to a phase I trial evaluating the combination of trastuzumab and a CTL-eliciting HER2 peptide vaccine administered concurrently in the adjuvant setting to patients with HER2-positive breast cancer. All patients (n=18) have completed their vaccination series and we have not seen any cardiac toxicity with combination therapy during vaccine dose escalation. Finally, in preliminary results from our group’s ongoing phase II trial evaluating AE37 (HER2-derived CD4+ helper T cell eliciting vaccine) or GP2 (HER2-derived CTL-eliciting vaccine), no cardiac toxicity has been found in HER2-positive patients randomized to receive the vaccine after trastuzumab.46 These early clinical trials have shown the promise of efficacy in combination therapy as well. Disis and colleagues demonstrated prolonged increases in peptide-specific immunity after vaccination, as well as epitope-spreading within the HER2 protein and to other non-HER2 tumor antigens.47 In our studies, we investigated sequential therapy with trastuzumab followed by HER2 vaccination in the adjuvant setting. In our phase I/II studies of nelipepimut-S that enrolled patients with any degree of HER2 expression, 11 patients with HER2-positive tumors were vaccinated after standard of care trastuzumab. In our ongoing phase II study evaluating a second CTL-eliciting HER2-derived vaccine (GP2+GM-CSF) which has the same mechanism of action as nelipepimut-S, we have enrolled 78 HER2-positive patients who received standard of care trastuzumab; 44 were vaccinated after standard of care trastuzumab, 34 were randomized to no vaccine. Combining the patients from these two studies, after a median follow-up of 36 months, the DFS rate is 84% in the 34 patients randomized to no vaccine – comparable with reported rates of similarly staged and treated patients. In contrast, for the 55 patients who were vaccinated after completing trastuzumab therapy, the DFS rate is 100% (p=.012; unpublished 13 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 data) (Figure 3). These encouraging data suggest that synergism between trastuzumab and HER2 CTL- eliciting vaccines warrant further investigation. We believe that the patients with high-risk HER2- positive breast cancer defined as those who receive a neoadjuvant chemotherapy regimen that contains trastuzumab and who fail to achieve a pCR or those that undergo upfront surgery and are found to be node positive (≥ 4 positive lymph nodes regardless of hormone receptor status or 1-3 positive lymph nodes and are hormone receptor negative) represent the ideal population in which to further investigate this strategy. 2.0 OBJECTIVES In this study, we intend to assess the ability of the combination of trastuzumab and the HER2 vaccine nelipepimut-S (administered with the immunoadjuvant GM-CSF) given in the adjuvant setting to prevent recurrences in patients with high-risk HER2-positive breast cancer. High-risk is defined as those patients that do not achieve a pCR after neoadjuvant therapy with an approved regimen that includes trastuzumab and at least four cycles (12 weeks) of taxane-containing chemotherapy or those who undergo upfront surgery and are found to have ≥ 4 positive lymph nodes regardless of hormone receptor status or 1-3 positive lymph nodes and are hormone receptor negative. 2.1 Primary objective The primary objective is to compare invasive DFS between the two treatment groups from time of initiation of trastuzumab maintenance therapy (trastuzumab monotherapy) to time of invasive local, regional or distant recurrence, new primary, or death due to any cause. 2.2 Secondary objectives • Distant recurrence-free survival (DRFS). Time from initiation of trastuzumab maintenance therapy to distant recurrence, or death due to any cause (continuing follow-up on patients with local or regional 14 Protocol: 2014-0443 Version 2.1 Version Date: 11 April 2016 recurrence) • Assess local and systemic toxicities using standard NCI-CTCAE criteria V 4.03 • Evaluate in vivo and in vitro immune responses 3.0 STUDY DESIGN 3.1 Description of study This will be a multi-center, prospective, randomized, single-blinded, placebo- controlled phase II trial of trastuzumab + nelipepimut-S/GM-CSF versus trastuzumab + GM-CSF alone (Appendix A). The vaccine to be used in this study, nelipepimut-S, is investigational and will be used in combination with trastuzumab under the Investigational New Drug (IND) application BB-IND #14919.