Purpose: The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. Patients and Methods: Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. Results: Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. Conclusion: Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popula...

Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.

Background/Objectives: Regulatory T cells (Treg) suppress immunity in the tumor microenvironment, are linked to poor prognosis across breast cancer subtypes, and suppress the cytolytic function of cytotoxic CD8+ T cells. Denileukin diftitox, a diphtheria toxin (DT)/IL-2 fusion protein, targets and depletes Tregs. This Phase II study aimed to assess the safety of denileukin diftitox and its effect on Tregs and tumor growth in patients with advanced breast cancer. Methods: This single-arm Phase II study of denileukin diftitox enrolled patients with refractory stage IV breast cancer. Patients received denileukin diftitox 18 mcg/kg/day IV for Days 1–5 every 21 days for up to six cycles. Toxicity was assessed using CTCAE v3.0 and tumor response was evaluated per RECIST criteria. Blood samples were collected to analyze Tregs by flow cytometry and anti-DT antibodies by ELISA. Results: Fifteen patients with stage IV breast cancer were enrolled. Four patients completed all planned denileukin diftitox infusions and achieved stable disease (27%, 95% CI [0.08, 0.55]). Two patients (13%) discontinued due to toxicity, and nine patients (60%) discontinued due to progressive disease. Eleven patients experienced at least one grade 3 or 4 adverse event. Although there was a general reduction in peripheral blood Tregs, the difference in CD4+CD25+FOXP3+ Tregs levels post-treatment was not statistically significant (p = 0.10). Six patients (40%) achieved $\ge$25% reductions in Tregs. A significant increase in anti-DT IgG antibodies was observed post-treatment (p < 0.005). Conclusions: Denileukin diftitox demonstrated moderate toxicity in this advanced breast cancer cohort. Denileukin diftitox modulated regulatory T cells. However, the majority of patients experienced disease progression in the study.

Background: Breast cancer stem cells (CSCs), particularly those enriched in triple-negative breast cancer (TNBC), are key contributors to tumor recurrence, metastasis, and resistance to therapy. CSCs often undergo epithelial-to-mesenchymal transformation (EMT), enhancing their invasiveness. Immune-based strategies that selectively target CSC/EMT antigens offer a promising therapeutic approach. Methods: Twelve candidate CSC/EMT-associated proteins were identified through a systematic literature review. Human serum samples were assessed for antigen-specific IgG using ELISA. Th1/Th2 cytokine profiles, in response to predicted MHC II epitopes, were measured by ELISPOT in PBMCs. Epitope immunogenicity and tumor inhibition were evaluated in murine models, using either TNBC or luminal B syngeneic breast cancer cell lines. Results: Six of the candidate proteins (SOX2, YB1, FOXQ1, MDM2, CDH3, CD105) elicited antigen-specific IgG in human serum. Th1-selective epitopes, defined by high Th1/Th2 ratios, were identified for five of these proteins. Immunization of mice with peptide pools derived from CD105, CDH3, MDM2, SOX2, and YB1 induced significant antigen-specific IFN-γ responses. Tumor growth was significantly inhibited in the vaccinated mice across both the TNBC and luminal B breast cancer models, with mean tumor volume reductions ranging from 61% to 70%. Conclusions: CSC/EMT-associated antigens are immunogenic in humans and can be targeted using Th1-selective epitope-based vaccines. Immunization with these epitopes effectively inhibits tumor growth in multiple murine models of breast cancer. These findings support further clinical evaluation of CSC/EMT-targeted vaccines, especially for high-risk or advanced-stage breast cancer patients.

594 Background: Colon cancer (CRC) is an immunogenic tumor and adaptive immunity may play a role in inhibiting disease relapse. A vaccine to boost cellular immunity in CRC patients could prevent disease recurrence, but there have been few defined immunogenic proteins identified as vaccine candidates. TVG has shown that overexpressed tumor associated proteins, abundant in the cancer state but expressed at basal levels in normal cells, can be immunogenic. We hypothesized that ideal candidate antigens would be ones already validated as prognostic markers in multivariate analysis. Methods: From a directed literature review we selected antigens based on (1) incidence of expression, (2) independent predictor of poor prognosis or early disease recurrence, and (3) known biologic function in CRC pathogenesis. We identified peptides that were predicted to be high affinity binders across multiple HLA DR alleles. We evaluated immunogenicity of peptides with IFN-g ELISPOT assays in CRC and control patients. We then evaluated the efficacy of vaccination in reducing tumors in the AOM mouse model. We evaluated immunogenicity of peptides with IFN-g ELISPOT assays using vaccinated mice splenocytes. We quantified antigen protein expression in AOM tumors. Results: The eight proteins are: CDC25B, COX-2, EBAG9, EGFR, Fascin-1, IGF-1R, PRL-3 and VCP. For all candidate antigens there was a greater IFN-g response in CRC patients than in controls. Four antigens (COX-2 p<.0001, CDC25B p<.0002, and EGFR p=.01, Fascin-1 p=.05) demonstrated a statistically significant reduction in the development of colon tumors compared to vaccination with adjuvant alone. Conclusions: We conclude that: 1. biologically relevant CRC associated proteins can be identified that are associated with prognosis, 2. epitopes predicted to bind multiple DR alleles, derived from candidate antigens, elicit T cell responses greater in CRC patients than in normal controls, 3. vaccination with peptides derived from four antigens resulted in a statistically significant reduction in the development of colon tumors in a mouse model, and 4. these antigens may represent novel immunologic targets for CRC.