Eradication of Metastatic Renal Cell Carcinoma after Adenovirus-Encoded TNF-Related Apoptosis-Inducing Ligand (TRAIL)/CpG Immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC

Abstract 2271: Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists

Abstract Metastatic renal cell carcinoma (RCC) is incurable. IFNα or IL-2 immunotherapy has been used for RCC for more than 20 years, but response rates remain low and these cytokines can generate toxic. More recently, administration of multikinase inhibitors or anti-VEGF receptor mAb has become popular, but these agents are largely palliative and tumor regression is common. The shortcomings in current therapeutic options for patients with metastatic RCC provide the rationale for the development of novel treatment protocols. Triptolide, a diterpene triepoxide from the medicinal herb Tripterygium wilfordii, can suppress cell growth and induce apoptosis in a range of human tumor cells. Further, triptolide also sensitizes tumor cells to TNF-related apoptosis-inducing ligand (TRAIL). The objective of this study was to determine the effectiveness of combining triptolide with TRAIL receptor agonists against RCC. In vitro, the combination of triptolide and recombinant human TRAIL protein decreased cell viability in the human RCC cell line ACHN cells compared with either treatment alone. This decrease in viability correlated with increased induction of apoptosis (positive Annexin V staining) and caspase activation. The triptolide-induced sensitization was accompanied by decreased HSP70, HSP27, and HSF1 expression, as well as increased surface expression of TRAIL-R2. Similar in vitro treatment of the mouse renal cell adenocarcinoma cell line, Renca, demonstrated that triptolide enhanced the killing of these cells by membrane-bound TRAIL or agonistic anti-DR5 mAb. In vivo treatment of mice bearing orthotopic and metastatic Renca tumors showed that combination therapy consisting of minnelide (a water soluble version of triptolide) and agonistic anti-DR5 mAb resulted in significantly decreased tumor burden and increased animal survival compared to either therapy alone. Collectively, our data suggest that triptolide sensitizes RCC cells to TRAIL-induced apoptosis, which is accompanied by decreased heat shock protein expression and altered TRAIL receptor expression. These data also suggest that the combination of TRAIL receptor agonists and triptolide may prove to be an effective treatment for metastatic RCC, especially at doses that show suboptimal tumoricidal activity when used individually. Citation Format: Thomas S. Griffith, Erik L. Brincks, Tamara A. Kucaba, Britnie R. James, Veena Sangwan, Sulagna Banerjee, Ashok Saluja. Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2014-2271</jats:p

IR administration of Ad5mTRAIL+CpG stimulates CD8-dependent eradication of metastatic Renca tumors.

<p>(A) Parental Renca cells were given IR, followed by administration of PBS or Ad5mTR+CpG IR on d 7. Flow cytometric analyses examining T cell infiltration were performed on excised lungs on d 12. Numbers indicate the frequencies of live cells within the gated regions. (B) Mice were challenged as in (A), followed by PBS or Ad5mTR+CpG on d 7. Lungs were excised on d 21, and surface lung nodules were enumerated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031085#pone-0031085-g002" target="_blank">Figure 2</a> (n = 15 mice per group, combined from 3 independent experiments). (C, D) Renca-LUC cells were given IR, followed by administration of PBS or Ad5mTR+CpG IR on d 7. (C) Mean total light flux values from 5 mice per indicated treatment group are shown from d 6–21. The mean light flux value for 5 tumor-free mice is shown at d 23 to denote background level of detection. Light flux values for Ad5mTR+CpG-treated mice at d 21 are statistically insignificant to those from tumor-free mice (<i>p</i> = 0.182), suggesting tumor eradication was nearly complete. Ad5mTR+CpG vs PBS at d 21, <i>p</i> = 0.032. (D) Mean total light flux values measured on d 21 for 3–10 mice per group, combined from 2 individual experiments, are shown. Ad5mTR+CpG vs Ad5mTR+CpG with CD4 depletion <i>p</i> = 0.186; Ad5mTR+CpG vs Ad5mTR+CpG with CD8 depletion <i>p</i> = 0.015; Ad5mTRAIL+CpG – CD8 vs Ad5mTRAIL+CpG – CD4 <i>p</i> = .037; PBS vs Ad5mTR+CpG with CD8 depletion <i>p</i> = 0.573; PBS vs CpG <i>p</i> = 0.440. (E) Mice were challenged as in (A), followed by PBS or Ad5mTR+CpG on d 7. Survival data from 10 mice per group are shown through d 72. (F) Mice were challenged on d 0 with Renca-Luc, treated on d 7 with Ad5mTRAIL+CpG, then re-examined at d 102 by BLI for signs of tumor recurrence. Of 15 tumor-challenged mice, 11 survived to d 102, and 8 of these showed no evidence of tumor re-growth.</p

Ad5mTRAIL+CpG induces humoral immunity without autoimmunity.

<p>(A) Total serum IgG concentrations obtained on d 12 from the indicated treatment groups. Only Ad5mTR+CpC resulted in a significant increase in serum IgG versus tumor-free control mice. (B) Anti-dsDNA serum concentrations obtained on d 12, 20, and 48 from Ad5mTR+CpG-treated mice. (For both A and B, * indicates <i>p</i>, 0.05; ** indicates <i>p</i><0.01). (C, D) Representative photomicrographs of H&E stained sections of the contralateral kidney (C) and liver (D) taken from one Ad5mTR+CpG-treated mouse (of a total of 7 analyzed) at d 102 after IR tumor challenge. Scale bar indicates 200 µm.</p

Local combinatorial Ad5mTRAIL+CpG therapy leads to regression of primary renal tumors.

<p>(A) Parental Renca cells were injected IR, followed by PBS or Ad5mTRAIL (Ad5mTR) +CpG on d 7. Kidneys were excised on d 12 and analyzed by flow cytometry to assess T cell infiltration. Dot plots show the percentages of live CD8⁺ and CD4⁺ T cells per kidney, as well as expression of CD44 and CD62L on the gated CD8⁺ and CD4⁺ T cells. (B, C) Mice were challenged as in (A) and PBS, Ad5mTR and/or CpG was given on d 7. Tumor-challenged kidneys were excised and weighed on d 23. CD4 and CD8 depletions were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031085#s2" target="_blank">Methods</a>. Mean values from 4–10 mice per group, combined from 3 individual experiments, are shown. Asterisks indicate <i>p</i><0.05 for that treatment group vs normal, tumor-free kidney weights. Statistical <i>p</i> values are shown for PBS vs CpG, PBS vs Ad5mTR+CpG, Ad5mTR+CpG vs Ad5mTR+CpG with CD4 depletion, and Ad5mTR+CpG vs Ad5mTR+CpG with CD8 depletion. For PBS vs CpG alone or Ad5mTR alone <i>p</i> = 0.11, for Ad5mTRAIL+CpG –CD8 vs Ad5mTRAIL+CpG –CD4 <i>p</i> = .14. (C) In addition, the excised kidneys were processed for H&E staining. Areas of dense purple staining indicate renal tumors. Scale bars in upper panels = 2.0 mm.</p