Introduction: We are conducting a phase II clinical trial of the GP2+GM-CSF vaccine for the prevention of breast cancer recurrence in disease-free, node-positive or high-risk node negative patients (pts). GP2 is an HLA-A2+-restricted immunogenic peptide from the HER2 protein (aa: 654-662) capable of stimulating CD8+ cytotoxic T-lymphocytes (CTLs) to recognize and destroy HER2-expressing tumor cells. Here, we present the final pre-specified analysis of our study, disease free survival (DFS) at one year from last enrolled pt. Methods: The trial is a prospective, randomized, multi-center, placebo-controlled, single-blinded, phase I/II trial designed to evaluate the safety and clinical efficacy of GP2 in breast cancer patients. Pts with any level of HER2 (immunohistochemistry [IHC] 1-3+) expression were enrolled after standard of care therapy. Blinded HLA-A2+ pts were randomized 1:1 to receive GP2 + GM-CSF or GM-CSF alone. Pts in both treatment arms received 6 monthly intradermal inoculations during the primary vaccine series (PVS) followed by 4 booster inoculations administered every 6 months. Data was collected for demographic, safety, immunologic, and clinical recurrence. Per the statistical plan, DFS was analyzed for both intention-to-treat population (ITT; all randomized pts) and per-treatment population (PT; excluding recurrences during the PVS and second malignancies) using Kaplan-Meir methods. Continuous variables are compared using analysis of variance techniques and proportions compared with Fisher's exact test. Results: This trial enrolled 180 pts (vaccine group n=89 and control group n=91). There were no differences in age, grade, hormone receptor status, tumor size or nodal status between groups (all p>0.2). With median follow up of 36.2 months, the five-year DFS estimates did not demonstrate a difference between vaccinated (VG) and control groups (CG) with ITT (82.6% v 80.4%, p=0.96, respectively) or PT (88.9% v 84.3%, p=0.52, respectively). On further subset analysis, in HER2 3+ by IHC (or positive via FISH) pts, ITT analysis showed a DFS of 93.8% v 87.2% (VG, n=50 v CG, n=51; p=0.67, respectively) while the PT analysis revealed a DFS of 100% v 87.2% (VG, n=48 v CG, n=50; p=0.052, respectively). In ER/PR+ pts, ITT analysis showed a DFS of 88.1% v 80.6% (VG, n=54 v CG, n=60; p=0.41, respectively) while PT analysis demonstrated a DFS of 91.5% v 85.2% (VG, n=52 v CG, n=57; p=0.42, respectively). Conclusions: The final pre-specified analysis of this randomized phase II trial of the GP2+GM-CSF adjuvant breast cancer vaccine shows no statistical differences between treatment arms. However, it does identify a particular patient population where the vaccine may have efficacy; HER2 3+ patients appear to derive the greatest clinical benefit from GP2 vaccination after trastuzumab treatment with the PT analysis nearing statistical significance. Further studies are required to confirm these findings and investigate the potential link between hormone receptor status and the induction of clinically beneficial anti-tumor immunity with this vaccine. Citation Format: Greene JM, Schneble EJ, Perez S, Murray JL, Berry JS, Trappey AF, Hale DF, Vreeland TJ, Clifton GT, Ardavanis A, Litton JK, Shumway NM, Papamichail M, Peoples GE, Mittendorf EA. Final pre-specified analysis of the phase II trial of the GP2+GM-CSF peptide vaccine in high risk breast cancer patients to prevent recurrence. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-01.

Background: HER2 over-expression is associated with more aggressive malignant disease. The introduction of trastuzumab and other HER2-directed therapies, however, has led to improved prognosis for patients (pts) with HER2 over-expressing (OE) tumors. Currently, no HER2-targeted therapies are available for patients with HER2 low-expressing (LE) (1+, 2+ by IHC) tumors. We are conducting a randomized, controlled Phase II trial of multiple peptide vaccines enrolling patients with any level of HER2 expression (1+, 2+ and 3+). Here, we report survival data based on levels of HER2 expression in our unvaccinated, control pts. Methods: After standard of care therapy, disease-free, high-risk BCa pts were randomized to receive either peptide+GM-CSF (Vaccine Group, VG) or GM-CSF alone (Control Group, CG) in six, monthly doses followed by four boosters every six months. Pts were prospectively followed for recurrence. Demographic information was available for all pts and was compared between groups using chi square or fisher exact tests. Disease-Free Survival (DFS) was compared using log rank. Results: To date, we have enrolled 196 pts in the CG. 96 pts had HER2 OE tumors, 100 had LE tumors. The only significant demographic difference between the CG OE and LE groups was more ER/PR positive patients in LE (LE 72% vs OE 51%, p = 0.008). 83% of CG OE pts received trastuzumab, 3% of CG LE pts received trastuzumab. At a median f/u of 30 mo, DFS was significantly higher for CG OE vs CG LE (92.5% v 65.5%, p = 0.001). Conclusions: In the cohort of control pts from our ongoing vaccine trial, conducted in an era when Tz has been standard of care therapy for patients with HER2 OE tumors, we have shown that HER2 LE pts are at higher risk of recurrence than OE pts, despite having more ER/PR positive. This calls for increased efforts to develop novel therapies for patients with HER2 LE disease. We have previously shown a trend towards increased DFS with the HER2 vaccines, AE37 (p = 0.13, median f/u 22 mo) and E75 (p = 0.16, median f/u 60mo) in HER2 LE pts, suggesting that these vaccines may represent one such novel therapeutic approach. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-14-01.

BACKGROUND: HER2 is a commonly expressed tumor-associated antigen in breast (BrCa) and ovarian cancer (OvCa) and, therefore, an attractive target for immunotherapy. We have investigated HER2-derived peptides as vaccines mixed with GM-CSF to include GP2 (a HLA-A2 and HLA-A3 restricted, CD8+ eliciting epitope) and AE37 (a HLA unrestricted, MHC class II, CD4+ eliciting epitope). Both peptide vaccines (PV) have shown clinical promise individually. There is clear rationale for combining GP2 and AE37 to elicit a more robust immune response (IR) of both CD4+ and CD8+ T cells. Here, we summarize initial toxicity (tox) and in vivo IR data from a phase 1 trial of the combined PV. METHODS: The trial is being performed as a five cohort, 3+3 dose-escalation, safety trial. Clinically disease-free, HLA-A2+ and A3+, BrCa and OvCa patients with tumors expressing any level of HER2 (IHC 1-3+) and who have completed standard-of-care therapy are accrued. In the first cohort, three patients received six, monthly intradermal inoculations (R1-R6) of 100mcg of AE37, 100mcg of GP2, and 125mcg of GM-CSF or 100:100:125. The second cohort received 250mcg of AE37, 100mcg of GP2, and 125mcg of GM-CSF or 250:100:125. Three additional cohorts were vaccinated: 250:250:125, 500:250:125, and 500:500:125. Toxicity was graded 48-72 hours post vaccination using NCI Toxicity Criteria v4.0. After each inoculation, local reactions (LR) are measured via the sensitive ballpoint pen method and reported as the orthogonal mean (OM). IR is assessed in vivo by delayed type hypersensitivity (DTH) reactions with separate intradermal inoculations of AE37, AE36, and GP2 antigens, measured both pre-vaccination (R0) and after the vaccine series (R6) via the sensitive ballpoint pen method, and reported as the OM. Means were compared using paired t-tests. RESULTS: 28 patients enrolled; 8 withdrew consent, 1 recurred prior to completing R6, 3 had an intercurrent illness, 14 patients completed R1-R6, and the vaccine series is ongoing in 2 patients. Six patients did not receive any inoculations and, therefore, are not included in this safety analysis. In 22 patients, the vaccine was well tolerated (max local tox: 23% Grade (Gr) 1, 73% Gr 2, 4% Gr 3; max systemic tox: 14% Gr 0, 50% Gr 1, 36% Gr 2). No dose-limiting toxicity was observed. For the 14 patients who completed the VS, the median age was 51(35-83). Breast tumor size was 3.3±1.1cm and ovarian tumor size was 10.0±2.3cm. Compared to GP2 LR at R1 (15.5±4.1mm), LR increased at R2 (31.7±5.9mm), R3 (42.9±7.4mm), R4 (35.3±7.3mm), R5 (45.0±9.9mm), and R6 (25.9±6.7mm, p = 0.17). Compared to the AE37 LR at R1 (18.5±3.8mm), LR increased at R2 (37.3±6.7mm), R3 (36.4±4.6mm), R4 (42.2±5.9mm), R5 (46.0±8.9mm), and R6 (36.2±6.6mm). Unless stated, all LR p-values < 0.05. After the VS, AE37 DTH increased from 0.0±0.0mm to 19.6±6.7mm (p<0.01), AE36 DTH increased from 0.0±0.0mm to 10.3±3.9mm (p<0.01), and GP2 DTH reactions increased from 0.3±0.2mm to 4.1±2.0mm (p = 0.056). CONCLUSIONS: Initial results from a phase I trial of a vaccine combining GP2 and AE37 peptides show that dual administration of the peptides is well tolerated at all tested dosing levels. Additionally, the combination is capable of stimulating strong peptide-specific in vivo immune responses. Continued testing of this vaccination strategy is underway. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-02.