Abstract 1571: Developing antigen-specific Th1 epitope based vaccines by excluding immunosuppressive peptide sequences

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&amp;lt;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</jats:p

Tumor-associated autoantibodies from mouse breast cancer models are found in serum of breast cancer patients.

B cell responses to tumor antigens occur early in breast tumors and may identify immunogenic drivers of tumorigenesis. Sixty-two candidate antigens were identified prior to palpable tumor development in TgMMTV-neu and C3(1)Tag transgenic mouse mammary tumor models. Five antigens (VPS35, ARPC2, SERBP1, KRT8, and PDIA6) were selected because their decreased expression decreased survival in human HER2 positive and triple negative cell lines in a siRNA screen. Vaccination with antigen-specific epitopes, conserved between mouse and human, inhibited tumor growth in both transgenic mouse models. Increased IgG autoantibodies to the antigens were elevated in serum from women with ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). The autoantibodies differentiated women with DCIS from control with AUC 0.93 (95% CI 0.88-0.98, p \u3c 0.0001). The tumor antigens identified early in the development of breast cancer in mouse mammary tumor models were conserved in human disease, and potentially identify early diagnostic markers in human breast tumors

Elimination of IL-10–Inducing T-Helper Epitopes from an IGFBP-2 Vaccine Ensures Potent Antitumor Activity

Immunization against self-tumor antigens can induce T-regulatory cells which inhibit proliferation of Type I CD4(+) T-helper (Th1) and CD8(+) cytotoxic T-cells. Type I T-cells are required for potent anti-tumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting IGFBP-2 and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and IL-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly Th1 while the C-terminus stimulated Th2 and mixed Th1/Th2 responses. Epitope-specific Th2 demonstrated a higher functional avidity for antigen than epitopes which induced IFN-γ (p=0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T-cell lines expanded from the C-terminus vaccinated animals secreted significantly more Type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T-cells secreted Th1 cytokines and significantly inhibited tumor growth, as compared with naïve T-cells, when adoptively transferred (p=0.005). To determine whether removal of Th2 inducing epitopes had any effect on the vaccinated anti-tumor response, we immunized mice with the N-terminus, C-terminus and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (p<0.001) as compared to the C-terminus vaccine which had no anti-tumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the anti-tumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes

Dendritic Cell–Activating Vaccine Adjuvants Differ in the Ability to Elicit Antitumor Immunity Due to an Adjuvant-Specific Induction of Immunosuppressive Cells

PURPOSE: We questioned whether the vaccine adjuvant combination of TLR7 ligand agonist, imiquimod, with GM-CSF would result in enhanced dendritic cell recruitment and activation with increased antigen-specific immunity as compared with either adjuvant used alone. EXPERIMENTAL DESIGN: The adjuvant effects of GM-CSF and imiquimod were studied in OVA and MMTVneu transgenic mice using peptide-based vaccines. Type I immunity, serum cytokines, MDSC, and Treg cells levels were examined. RESULTS: Both GM-CSF and imiquimod equally induced local accumulation and activation of dendritic cells. Both adjuvants effectively enhanced OVA specific T-cell responses. We further evaluated the anti-tumor efficacy of adjuvant GM-CSF and imiquimod immunizing against murine IGFBP-2, a non-mutated oncoprotein overexpressed in the tumors of MMTVneu transgenic mice. Tumor growth was significantly inhibited in the mice receiving IGFBP-2 peptides with GM-CSF (p=0.000), but not in imiquimod-vaccine treated groups (p=0.141). Moreover, the addition of imiquimod to GM-CSF negated the anti-tumor activity of the vaccine when GM-CSF was used as the sole adjuvant. While GM-CSF stimulated significant levels of antigen specific Th1, imiquimod induced elevated serum IL-10. Both MDSC and Treg cells were increased in the imiquimod-treated but not GM-CSF-treated groups (p=0.000 and 0.006 respectively). Depleting MDSC and Treg in animals immunized with imiquimod and IGFBP-2 peptides restored anti-tumor activity to the levels observed with vaccination using GM-CSF as the sole adjuvant. CONCLUSION: Adjuvants may induce regulatory responses in the context of a self-antigen vaccine. Adjuvant triggered immune suppression may limit vaccine efficacy and should be evaluated in pre-clinical models especially when contemplating combination approaches