BACKGROUND The authors conducted exploratory phase 1–2 clinical trials vaccinating breast cancer patients with E75, a human leukocyte antigen (HLA) A2/A3–restricted HER-2/neu (HER2) peptide, and granulocyte-macrophage colony-stimulating factor. The vaccine is given as adjuvant therapy to prevent disease recurrence. They previously reported that the vaccine is safe and effective in stimulating expansion of E75-specific cytotoxic T cells. Here, they report 24-month landmark analyses of disease-free survival (DFS). METHODS These dose escalation/schedule optimization trials enrolled lymph node-positive and high-risk lymph node-negative patients with HER2 (immunohistochemistry [IHC] 1-3+) expressing tumors. HLA-A2/A3+ patients were vaccinated; others were followed prospectively as controls for recurrence. DFS was analyzed by Kaplan-Meier curves; groups were compared using log-rank tests. RESULTS Of 195 enrolled patients, 182 were evaluable: 106 (58.2%) in the vaccinated group and 76 (41.8%) in the control group. The 24-month landmark analysis DFS was 94.3% in the vaccinated group and 86.8% in the control group (P = .08). Importantly, because of trial design, 65% of patients received a lower than optimal vaccine dose. In subset analyses, patients who benefited most from vaccination (vaccinated group vs control group) had lymph node-positive (DFS, 90.2% vs 79.1%; P = .13), HER2 IHC 1+-2+ (DFS, 94.0% vs 79.4%; P = .04), or grade 1 or 2 (DFS, 98.4% vs 86.0%; P = .01) tumors and were optimally dosed (DFS, 97.3% vs 86.8%; P = .08). A booster program has been initiated; no patients receiving booster inoculations have recurred. CONCLUSIONS The E75 vaccine has clinical efficacy that is more prominent in certain patients. A phase 3 trial enrolling lymph node-positive patients with HER2 low-expressing tumors is warranted.

The hybrid AE37 vaccine seems safe and well tolerated with minimal toxicity if properly dosed. AE37 is capable of eliciting HER-2/neu-specific immune responses, even without the use of an adjuvant. This trial represents the first human experience with the Ii-Key modification, and to our knowledge, AE37 is the first peptide vaccine to show potency in the absence of an immunoadjuvant.

BACKGROUND: HER-2/neu, overexpressed in breast cancer, is a source of immunogenic peptides that include GP2 and E75. Phase 2 testing of E75 as an adjuvant vaccine has suggested a clinical benefit. GP2, derived from the transmembrane portion of HER-2/neu, has differing binding characteristics and may be more immunogenic than E75. Results of the first phase 1 trial of GP2 peptide vaccine are presented. METHODS: Disease-free, lymph node-negative, human leukocyte antigen (HLA)-A2 þ breast cancer patients were enrolled. This dose escalation trial included 4 groups to determine safety and optimal GP2 peptide/granulocyte-macrophage colony-stimulating factor (GM-CSF) dose. Toxicities were monitored. Immunologic response was assessed ex vivo via the HLA-A2:immunoglobulin dimer assay to detect GP2-specific CD8 þ T cells (and E75-specific CD8 þ T cells to assess epitope spreading) and in vivo via delayed type hypersensitivity (DTH) reaction (medians/ranges). RESULTS: Eighteen patients were enrolled. All toxicities were grade 2. Eight (88.9%) of 9 patients in the first 3 dose groups required GM-CSF dose reductions for local reactions !100 mm or grade !2 systemic toxicity. GM-CSF dose was reduced to 125 lg for the final dose group. All patients responded immunologically ex vivo (GP2-specific CD8 þ T cells from prevaccination to maximum, 0.4% [0.0%-2.0%] to 1.1% [0.4%-3.6%], P < .001) and in vivo (GP2 pre-to postvaccination DTH, 0 mm [0.0-19.5 mm] to 27.5 mm [0.0-114.5 mm, P < .001). E75-specific CD8 þ T cells also increased in response to GP2 from prevaccination to maximum (0.8% [0.0%-2.41%] to 1.6% [0.86%-3.72%], P < .001). CONCLUSIONS: The GP2 peptide vaccine appears safe and well tolerated with minimal local/systemic toxicity. GP2 elicited HER-2/neu-specific immune responses, including epitope spreading, in high-risk, lymph node-negative breast cancer patients. These findings support further investigation of the GP2 vaccine for the prevention of breast cancer recurrence. Cancer 2010;116:292-301. V C 2010 American Cancer Society.KEYWORDS: breast cancer, GP2, HER-2/neu, peptide, vaccine.Breast cancer is the most common malignancy in women (excluding cancers of the skin), and is the second leading cause of cancer mortality among women. 1 HER-2/neu is a tumor-associated antigen that is overexpressed in approximately 25% to 30% of breast cancer patients and correlates with more aggressive tumor behavior. 2,3 Immunogenic peptides derived from HER-2/neu can induce peptide-specific cytotoxic T lymphocytes (CTLs) that recognize tumor cells expressing these peptides complexed with major histocompatibility complex class I molecules. 4 Two such peptides include E75 and GP2. Our group has previously tested and reported on the E75 peptide used as a preventive clinical vaccine in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) and found it to be safe and effective in raising HER-2/neu immunity. Furthermore, this immunity may confer a clinical benefit in the adjuvant setting. 5

Purpose: HER2/neu, a source of immunogenic peptides, is expressed in >75% of breast cancer patients. We have conducted clinical trials with the HER2/neu E75 peptide vaccine in breast cancer patients with varying levels of HER2/neu expression. Vaccine response based on HER2/neu expression level was analyzed. Experimental Design: Patients were stratified by HER2/neu expression. Low expressors (n = 100) were defined as HER2/neu immunohistochemistry (IHC) 1 + to 2 + or fluorescence in situ hybridization < 2.0. Overexpressors (n = 51) were defined as IHC 3

BACKGROUND:The authors are conducting clinical trials of the HER-2/neu E75-peptide vaccine in clinically diseasefree breast cancer (BC) patients. Their phase 1-2 trials revealed that the E75 þ granulocyte-macrophage colony-stimulating factor (GM-CSF) vaccine is safe and effective in stimulating clonal expansion of E75-specific CD8 þ T cells.They assessed the need for and response to a booster after completion of primary vaccination series. METHODS: BC patients enrolled in the E75 vaccine trials who were !6 months from completion of their primary vaccination series were offered boosters with E75 þ GM-CSF. Patients were monitored for toxicity. E75-specific CD8 þ T cells were quantified using the human leukocyte antigen-A2:immunoglobulin G dimer before and after boosting. RESULTS: Fiftythree patients received the vaccine booster. Median time from primary vaccination series was 9 months (range, 6-35 months), and median residual E75-specific immunity was 0.70% (range, 0-3.49%) CD8 þ lymphocytes. Elevated residual immunity (ERI) (CD8 þ E75-specific T cells >0.5%) was seen in 94.4% of patients at 6 months from primary vaccination series versus 48% of patients at >6 months (P ¼ .002). The booster was well tolerated, with only grade 1 and 2 toxicity observed. Local reactions were more robust in patients receiving the booster at 6 months from primary vaccination series compared with those at >6 months (99.4 AE 6.1 mm vs 81.8 AE 4.1 mm, P ¼ .01). In patients lacking ERI, 85% had increased ERI after vaccination (P ¼ .0014). CONCLUSIONS: The HER-2/neu E75 peptide vaccine E75 stimulates specific immunity in disease-free BC patients. However, immunity wanes with time. A vaccine booster is safe and effective in stimulating E75-specific immunity in those patients without ERI. These results suggest that the booster may be most effective at 6 months after completion of the primary vaccination series. Peptide-based vaccines are being used in clinical trials either to treat 1,2 or to prevent recurrence of malignancy. 3,4Peptide-based vaccination techniques involve inoculating patients with immunogenic epitopes from tumor-associated antigens, typically given with an immunoadjuvant, to stimulate proliferation of peptide-specific lymphocytes. Peptidespecific lymphocytes then identify and eliminate tumor cells presenting the vaccine-targeted epitope. Peptide-based vaccines are attractive immunotherapeutic options because they have no malignant potential, have low toxicity profiles, are simple and inexpensive, are easily monitored and studied, and eventually will be easily exportable to the community. The individual peptides used in cancer vaccine preparations are often major histocompatibility complex (MHC) class-restricted and human leukocyte antigen (HLA) type-specific, thus stimulating either CD4 þ or CD8 þ lymphocytes and sometimes limiting the applicability of peptides to patients with the correct HLA type. Peptide vaccination techniques

BACKGROUND.E75, a HER‐2/neu‐derived peptide, was administered as a preventive vaccine with granulocyte‐macrophage–colony‐stimulating factor (GM‐CSF) in disease‐free lymph node‐positive (NP) and lymph node‐negative (NN) breast cancer (BCa) patients. The optimal biologic dose (OBD) was determined based on toxicity and immunologic response.METHODS.Patients were vaccinated over 6 months (3, 4, or 6 times) with different doses of E75 plus GM‐CSF. Toxicities were graded per National Cancer Institute Common Terminology Criteria. GM‐CSF was reduced for significant toxicity. Immunologic response was measured by delayed type hypersensitivity test (DTH), and E75‐specific CD8+ T‐cells were quantified with human leukocyte antigen‐A2:immunoglobulin G dimer and flow cytometry.RESULTS.Ninety‐nine patients (48 NP and 51 NN) were vaccinated in 7 dose groups. The OBD was 1000 μg E75 plus 250 μg GM‐CSF monthly × 6. The optimal dose group (ODG, n = 29) experienced similar toxicities to the suboptimal dose group (SDG, n = 70), which was comprised of the remaining 6 groups. The ODG demonstrated a trend toward an increase in the average postvaccine dimer (0.87 ± 0.10% vs 0.67 ± 0.05%; P = .07), a significantly larger DTH response (21.5 ± 2.5 mm vs 11.3 ± 1.3 mm; P = .0002), and a trend toward decreased recurrences (3.4% vs 12.9%; P = .27). Compared with the SDG, the ODG had larger tumors (percentage ≥T2: 55% vs 23%; P = .004), more positive lymph nodes (percentage NP: 76% vs 37%; P = .001), and higher grade tumors (percentage grade 3: 52% vs 30%; P = .07), but a shorter median follow‐up time (20 months vs 32 months; P < .001).CONCLUSIONS.Compared with suboptimally dosed patients, the optimally dosed E75 vaccine in disease‐free BCa patients had similar toxicity but enhanced HER‐2/neu‐specific immunity that may lead to decreased recurrences with additional follow‐up. Cancer 2008. Published 2008 by the American Cancer Society.

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Clinical trials have shown the AE37 vaccine to be safe and capable of generating peptide-specific, durable immune responses. This has been shown in patients with any level of HER-2/neu expression. Early clinical findings suggest there may be benefit to AE37 vaccination in preventing breast cancer recurrence.