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Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8(+) and CD4(+) T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8(+) T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.

In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8(+) T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8(+) effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.