Because existing therapeutic cancer vaccines provide only a limited clinical benefit, a different vaccination strategy is necessary to improve vaccine efficacy. We developed a nanoparticulate cancer vaccine by encapsulating a synthetic long peptide antigen within an immunologically inert nanoparticulate hydrogel (nanogel) of cholesteryl pullulan (CHP). After subcutaneous injection to mice, the nanogel-based vaccine was efficiently transported to the draining lymph node, and was preferentially engulfed by medullary macrophages but was not sensed by other macrophages and dendritic cells (so-called "immunologically stealth mode"). Although the function of medullary macrophages in T cell immunity has been unexplored so far, these macrophages effectively cross-primed the vaccine-specific CD8(+) T cells in the presence of a Toll-like receptor (TLR) agonist as an adjuvant. The nanogel-based vaccine significantly inhibited in vivo tumor growth in the prophylactic and therapeutic settings, compared to another vaccine formulation using a conventional delivery system, incomplete Freund's adjuvant. We also revealed that lymph node macrophages were highly responsive to TLR stimulation, which may underlie the potency of the macrophage-oriented, nanogel-based vaccine. These results indicate that targeting medullary macrophages using the immunologically stealth nanoparticulate delivery system is an effective vaccine strategy.

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Purpose: Blockade of CTL-associated antigen-4 (CTLA-4), an inhibitory immunomodulatory molecule on T cells, has been shown to enhance T-cell responses and induce tumor rejection, and a number of clinical trials with anti-CTLA-4 blocking monoclonal antibody (mAb) are under way. However, accumulating evidence indicates that anti-CTLA-4 mAb increases the number of CD4+CD25+Foxp3+ regulatory T cells (Treg) and that anti-CTLA4 mAb alone is often insufficient to reject established tumors in mice and humans. Thus, finding maneuvers to control Tregs and other immunosuppressive mechanisms remains a critical challenge. Experimental Design: The potential to enhance antitumor immune responses by combining anti-CTLA-4 mAb with anti–glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) mAb, a costimulatory molecule that abrogates directly/indirectly Treg-mediated immune suppression or anti-CD25 mAb that depletes Tregs was analyzed with two tumor models, CT26 (a murine colon carcinoma cell line) and CMS5a (a murine fibrosarcoma cell line). Results: Anti-CTLA-4/anti-GITR mAb combination treatment exhibited far stronger antitumor effects compared with either antibody alone. This strong antitumor effect was attributed to (a) increased numbers of CD8+ T cells infiltrating tumor sites in anti-CTLA-4 mAb–treated mice and (b) increased cytokine secretion and Treg resistance of tumor-specific CD8+ T cells with strongly upregulated CD25 expression in anti-GITR mAb–treated mice, indicating distinct quantitative/qualitative changes induced by modulating CTLA-4 and GITR signaling. Conclusions: This study shows that combined treatment with different immune modulators can augment antitumor immune responses and provides justification for exploring anti-CTLA-4/anti-GITR mAb combination treatment in the clinic. Clin Cancer Res; 16(10); 2781–91. ©2010 AACR.