Abstract We have determined that IGFBP-2 is immunogenic in ovarian cancer. Our aim is to develop a multi-epitope vaccine that will elicit Th1 immunity to IGFBP-2. Antigen specific Th1 can modulate the tumor microenvironment to enhance cross priming, supporting the proliferation of cytotoxic T cells which are capable of eradicating ovarian cancer cells. Although some clinical benefit has been demonstrated with this strategy, recent ex vivo analyses of human PBMC have described the presence of antigen-specific CD4+ T regulatory cell (Tregs) in cancer patients that were not detected in healthy individuals. Thus, we questioned if we could optimize our vaccine to include only Th1-stimulating epitopes. Using a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes, we identified 14 IGFBP-2 peptides. Th1 immunogenicity (IFN-gamma) and potential immunosuppression (IL-10) was evaluated by ELISPOT for 40 different donors. Twenty-two percent of the donors only responded with IL-10 secretion to any peptide, 22% only responded with IFN-gamma and 53% of the patients had a mixed IFN-gamma and IL-10 response. To determine which peptides would induce a predominantly Th1 response in the greatest number of people, we used a ratio of IFN-gamma to IL-10 and analyzed both the magnitude and frequency of ELISPOT responses for each of peptides using the following algorithm: (corrected mean SPW) x (percent of responding donors). The peptides were then ranked from highest IL-10 response to highest IFN-gamma response. Interestingly, 6 of the 14 peptides demonstrated a preference to secrete IFN-gamma over IL-10 and are only located in the N-terminus (amino acids 1-163) of IGFBP-2; the remaining potentially immunosuppressive peptides are located in the C-terminus (amino acids 164-328). Vaccination with p1-163 in MMTV-neu mice demonstrated a robust Th1 response (p=0.03) and concomitant inhibition of tumor growth by 70% compared to adjuvant only control animals, p164-328-vaccinated or full length protein-vaccinated mice (p<0.001 for all). Vaccination with p164-328 or full length protein did not inhibit tumor growth. These data suggest that more effective vaccines can be designed when both Th1 epitopes and immunosuppressive epitopes are screened simultaneously and epitopes that are most likely to induce robust Th1 responses in the majority of individuals can be identified and included as vaccine components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1571. doi:1538-7445.AM2012-1571
Abstract The majority of patients with breast cancer have robust Type II immune responses directed against their tumors with little to no Type I immunity. The dominance of a Type II microenvironment is established early in breast tumorigenesis with a Type II immune signature prevalent even in pre-invasive lesions such as ductal carcinoma in situ. As a result, breast cancer is associated with abundant autoantibodies directed against tumor associated antigens and few infiltrating T-cells in the majority of patients. A recently reported inhibitor of protein and cellular fucosylation, 2-fluorofucose (2FF) has been shown to enhance immune cell function, in part through the generation of fucose-deficient antibodies which result in enhanced antibody dependent cell mediated cytotoxicity, as well as apparent modulation of T-cell dependent activity [Ca Res, Oct 1, 2014 74;2890]. For treatment studies, TgMMTV-neu (MMTVneu; luminal) and C31-Tag (C3T; triple negative) mice were treated orally with vehicle alone and 20mM 2FF when spontaneous tumor volume reached 75-100mm3 until sacrifice (n=20/grp). To assess the ability to prevent breast cancer development, mice were treated orally with vehicle alone, 20 or 50mM 2FF for up to 200 days starting at 6-8 weeks of age (n=20/grp). Tumor kinetics, disease free survival, and overall survival were calculated. Immune responses were evaluated by both DTH and IFN-gamma ELISPOT. To determine immune mechanism of action, NK, B, CD4, or CD8 cells were depleted concurrently with tumor implant. When tumor bearing mice were treated with 2FF, tumor growth was significantly inhibited in both groups over the course of therapy (MMTVneu p<0.0001 and C3T p<0.0001 compared to controls). In addition, survival was significantly prolonged in both models (MMTVneu p<0.0001 and C3T p=0.0014). In the prevention setting, 2FF significantly delayed the average age of tumor onset in both models at both doses with the higher does showing greater efficacy; MMTVneu 20mM, p=0.001, 50mM, p=0.0002. At the time of study termination (300d), 45% of 2FF-treated MMTVneu mice had no evidence of mammary tumors. 2FF also significantly improved the average age of tumor onset in C3T mice when comparing untreated to 20mM (p=0.007), and 50mM (p=0.0007) treated mice. At study termination, 33% of all 2FF-treated C3T animals were disease free. 2FF anti-tumor activity was associated with the induction of tumor antigen specific immunity. After treatment both the MMTVneu (p<0.0001) and C3T (p<0.0001) developed a DTH response to syngeneic tumor lysates. Of note, the DTH response was inversely associated with the rate of tumor growth (p<0.0001). 2FF anti-tumor activity was associated with the induction of tumor antigen specific immunity. Depletion of immune cell subsets using targeting antibodies demonstrated that the anti-tumor response was mediated in large part by CD4 T-cells which are involved in stimulating both humoral and CD8 T-cell responses. 2FF, a novel inhibitor of fucosylation has potent anti-tumor effects in 2 transgenic models of breast cancer; luminal and triple negative mammary tumors. The agent is active in these mouse models in both the treatment and prevention setting and thus may represent a rational therapeutic approach to evaluate in breast cancer patients. Citation Format: Disis ML, Rastetter L, Gad E, Koehnlein M, Senter PD, Gardai S, Okeley NM. Modulation of immunity with 2-fluorofucose (2FF) for breast cancer treatment and prevention. [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 PD3-08.
Abstract Tamoxifen is a standard treatment for estrogen receptor (ER)-positive breast cancer patients. However, acquired or de novo resistance to therapy is a major clinical problem. In hormone-resistant tumors, there is increased activation of insulin-like growth factor-1 receptor (IGF-IR) and subsequent downstream signaling molecules, such as AKT. We have determined that IGF-IR is immunogenic in breast cancer and is a potential target for active immunization. It has been demonstrated that natural immunogenic human epitopes can be predicted by high binding affinity across multiple class II alleles, thus, we used a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes of IGF-IR. Of the 20 potentially immunogenic peptides identified, five peptides (p1166-1181, p1212-1227, p1301-1316, 1307-1322 and p1311-1326) in the C-terminal domain were determined to elicit a predominantly inflammatory Th1 response compared to an immunosuppressive Th2 response in human PBMC. Overexpression of AKT induces tamoxifen resistance, while inhibition restores tamoxifen sensitivity. The tumor suppressor, PTEN, actively inhibits AKT. Data we have generated has demonstrated that vaccination restored PTEN activity in tumor cells. We questioned if modulation of PTEN could render tumors in the anti-estrogen-resistant MMTV-neu mouse model sensitive to tamoxifen therapy. Vaccination demonstrated a robust Th1 response (p<0.001) and concomitant inhibition of tumor growth by 85% compared to adjuvant only control animals (p<0.001). Treatment with tamoxifen in vaccinated tumor-bearing mice resulted in a further 38% reduction in growth (p=0.02). Thus, active immunization targeting IGF-IR may induce both immunologic and biologic effects resulting in the sensitization of the tumor to tamoxifen therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4392. doi:1538-7445.AM2012-4392
Meeting abstracts ELISPOT assays are routinely used to measure immune responses of T cells in fresh and frozen splenocytes preparations; however, standardized methods for murine ELISPOT are not widely available. Freezing cells can significantly impact the function of T cells. The aim of this study
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