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.

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.