The clinical management of RCC is highly variable, and depends on tumor staging at the time of diagnosis. For localized renal tumors with no evidence of metastases or with a single metastasis, radical nephrectomy with surgical resection of the metastatic lesion is the standard therapeutic approach, and 5-year survival rates for these patients are high 
. However, nearly 30% of patients have multiple metastases at diagnosis, and an equal percentage will develop metastatic tumor recurrence following nephrectomy. Metastatic RCC is largely thought of as an incurable disease, with a median survival time of only 18 months 
. Administration of multikinase inhibitors such as sunitinib and sorafenib, or antibodies against vascular endothelial growth factor (VEGF) receptors are largely palliative options, since complete remissions in response to these agents are rare 
. In a small subset of patients the combination of radical nephrectomy plus high-dose IL-2 can be curative, but this approach is contraindicated in most individuals due to the severe toxicities associated with IL-2 administration 
. Shortcomings in current therapeutic options provide the rationale for continued attempts to identify novel treatment options for patients with metastatic RCC. At the same time, durable responses to IL-2 therapy illustrate that immunotherapy can be effective, and suggest that less-toxic immunotherapies, given either with or without radical nephrectomy, could be beneficial for a greater number of patients.
In our current study, we used a single IR administration of Ad5mTRAIL+CpG to induce protective T cell immunity against metastatic RCC. Of note, the adenoviral vector we used is replication-deficient and encodes a membrane-bound form of murine TRAIL. Consequently, we expected only limited direct killing of tumor cells within the kidney due to the lack of dissemination of the vector-derived TRAIL protein or the vector itself via replicative spread. Despite these limitations, IR Ad5mTRAIL+CpG injection gave rise to systemic T cell responses that were needed to fully suppress local and metastatic tumor outgrowth, as well as a humoral immune response characterized by elevated total serum IgG, anti-adenovirus IgG, and anti-dsDNA Ab. To our knowledge, this is the first time that local administration of adenoviral-encoded TRAIL has been shown to elicit systemic immune responses in an orthotopic, spontaneously metastasizing tumor model. Other reports investigating the efficacy of TRAIL-based therapies against localized or metastatic tumors have been published, but these have focused on examining the direct tumoricial activity of the therapy when administered locally or systemically (such as i.v. injection of agonistic TRAIL antibodies 
). It is important to keep in mind that there are strengths and weaknesses to all experimental tumor models used depending on the specific question(s) being asked; we were particularly interested in evaluating the contributions of the systemic antitumor immune response induced subsequent to Ad5mTRAIL+CpG administration to the overall control of the tumor burden.
As both Ad5humanTRAIL and CpG have shown minimal toxicity as single agents in Phase I clinical trials, they are excellent candidates for antitumor immunotherapies 
. We explored the potential of Ad5mTRAIL+CpG as a stand-alone therapy when the primary renal tumors were still quite small, but tumors cells had already spread to the lungs (). It was important to demonstrate that tumor metastasis to the lung had occurred at the time of therapy, to eliminate concern that any decrease in lung tumor burden was due to prevention of metastasis, rather than clearance of developing lung metastases. However, in a clinical context, it is tempting to speculate that AdTRAIL+CpG could also be used as an adjunct therapy prior to nephrectomy in cases of advanced RCC. Thus, future studies examining the combination of surgery plus immunotherapy for treatment of larger tumors are warranted. Use of Ad5mTRAIL+CpG as an adjunct therapy to nephrectomy would require careful examination of the timing between immunotherapy administration and surgery, as TRAIL-induced apoptosis of tumor cells, apoptotic body uptake by local DC, and activation of naive tumor antigen-specific T cells would need to occur prior to removal of the tumor-bearing kidney.
Although the Renca tumor line has been widely used to model RCC in mice, other murine kidney cancer cell lines do exist 
. These less-studied lines may have variable primary growth and/or metastatic properties that could influence the efficacy of experimental immunotherapies. Despite this fact, the majority of studies have used Renca administered either s.c., which only gives rise to localized tumors, or i.v., which produces experimental metastases 
. Much has been learned from studies using the s.c. and i.v. tumor challenge routes, but these models have important limitations. Consequently, use of the orthotopic Renca model is becoming more common, as this places primary tumors in the correct anatomical location that will spontaneously give rise to lung metastases 
. As the lung is one of the primary sites of metastasis in human RCC 
, the orthotopic Renca model also provides another degree of clinical relevance that is lacking in other models. In addition, our use of Luciferase-expressing Renca cells represents an advance in the pre-clinical modeling of metastatic RCC, as it allowed us to sensitively and accurately detect tumor cells in the kidneys and lungs of mice as early as d 7 post-tumor challenge () and to quantitate the kinetic differences in total tumor burdens that occur in the presence and absence of immunotherapy ().
The role of TRAIL in Renca tumor clearance has been investigated in several previous studies 
, and our current work complements and builds on these findings. For example, one study using an agonistic mAb against DR5 induced only minimal apoptosis of Renca cells in vitro
and in vivo
after i.v. tumor challenge, but robust in vivo
protection was observed when combined with the proteasome inhibitor bortezomib 
. Another study showed that while TRAIL impaired the growth of Renca hepatic metastases in vivo
, it was not involved in the protective response against i.v.-induced Renca lung metastases 
. Our data shows that administration of Ad5mTRAIL+CpG at the primary tumor site not only reduces the primary tumor burden, but also protects against the outgrowth of metastatic lung tumors (). The difference between our finding and the previous reports may stem from the fact that the tumor challenge and treatment routes were different, leading to qualitatively different responses.
We showed previously that Ad5mTRAIL can bring about apoptotic cell death in Renca cells in vitro
, and demonstrated the ability of Ad5mTRAIL+CpG to clear localized s.c. Renca tumors 
. In the latter study, we found that depletion of CD4 T cells had a protective effect in mice that received Ad5mTRAIL+CpG, leading to prolonged survival relative to intact mice that received the same therapy. This finding suggested that regulatory CD4 T cells were suppressing CD8 T cell-mediated tumor clearance. In contrast, we now find that in mice with orthotopic Renca tumors, depletion of CD4 T cells in Ad5mTRAIL+CpG-treated mice results in larger tumor burdens ( and ), indicating that CD4 T cells are directly contributing to tumor clearance and/or providing the necessary help to generate an optimal CD8 T cell response. These disparate findings imply that orthotopic tumor challenge and IR therapy administration give rise to fundamentally different tumor microenvironments and immune responses than what occur with standard s.c. tumor challenges.
In addition to being given as a therapeutic agent, TRAIL can be used by endogenous T cells as an effector mechanism to bring about tumor cell death. It was shown previously by Seki et al. that cytotoxic T lymphocytes did not use TRAIL or perforin to kill Renca tumor cells, but instead relied primarily upon FasL in vivo
, particularly when the cognate antigen levels were low (determined with HA-expressing Renca-HA cells 
). A preliminary assessment of cytolytic mechanisms in our system supported these findings, in that Ad5mTRAIL+CpG-mediated renal tumor clearance proceeded normally in mice that were lacking either TRAIL or perforin (data not shown).
Our current study demonstrates the feasibility of using T cell stimulation as a means to protect against primary renal and metastatic tumors in mice. Despite the fact that T cell-mediated eradication of advanced RCC has been documented in cancer patients, this is still a rare event 
. RCC is an immunogenic tumor, yet most patients with advanced disease fail to respond objectively to immunotherapy. One explanation might be the inability of activated T cells to overcome tumor-derived immune suppression. Several reports have shown that the accumulation of tumor-induced suppressor cells, including myeloid-derived suppressor cells and regulatory T cells, is an impediment to the induction of protective antitumor immunity in RCC patients 
and in murine Renca models 
. We applied Ad5mTRAIL+CpG therapy to mice in which the primary renal tumors were left intact. This immunotherapy led to a striking reduction in body-wide tumor burdens through d 21–23 (), but we did observe tumor regrowth in approximately 50% of mice (). It is possible that combining Ad5mTRAIL+CpG with low-dose cyclophosphamide or sunitinib to remove suppressive cell populations would lead to complete tumor eradication in a larger percentage of mice.
Our data demonstrate that combinatorial immunotherapy consisting of adenovirus-encoded TRAIL+CpG1826 in an orthotopic RCC model in mice was effective in inducing a systemic T cell response that contributed to reducing local and metastatic tumors. While a humoral immune response was also evident in treated mice, our studies were not designed to examine the contribution of the humoral response to the eventual reduction in tumor burden. The abrogation of tumor clearance after depletion of CD8+
cells would suggest that a protective anti-tumor humoral response was not present, but formal investigation would need to be done to confirm this conclusion. Perhaps more importantly, the transient increases in serum IgG and anti-dsDNA did not lead to development of autoimmunity, further implying that this immunotherapeutic approach has the potential for clinical translation. Our conclusion is supported by the two prior reports on tumor-induced anti-dsDNA Ab, in which it was suggested that these antibodies had tumoricidal functions in vitro and in vivo, and did not trigger autoimmunity 
. In particular, Ad5TRAIL+CpG may be a suitable treatment alternative for patients with inoperable, advanced RCC, in that IR administration of Ad5TRAIL+CpG may stimulate systemic cellular anti-tumor immunity that can target any residual primary tumor not directly killed by TRAIL as well as distal metastases.