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Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.


INTRODUCTION
Over the past decade, tumor immunology has advanced in uncovering the molecular and cellular mechanisms responsible for cancer pathophysiology and for enhancing the immune response against tumors, yet there are still many unresolved problems concerning which tumors can escape, or suppress, the immune response. Therefore, immunotherapy for malignant diseases needs novel, improved clinical protocols that minimize tumor-induced immunological tolerance while simultaneously inducing tumor eradication. One potential strategy is a cytokine immunogene therapy that has a great potential for rejecting cancer cells by activating tumor-specific immune responses in cancer-bearing hosts.
Interleukin (IL)-12 is one of the most effective and promising antitumor cytokines. It is a heterodimeric cytokine composed of two different disulfide-linked subunits, designated p35 and p40, and is mainly produced by activated macrophages and dendritic cells (DCs). IL-12 stimulates interferon-g (IFN-g) production by natural killer (NK) cells, T cells, DCs and macrophages. Similarly, it facilitates T helper type 1 (Th1) differentiation and augments the cytolytic effect of NK cells and cytotoxic T lymphocytes (CTLs). Previous preclinical studies of IL-12 reported enhanced antitumor immunity. 1,2 More recently, we showed an enhanced antitumor effect of an oncolytic adenovirus (Ad) that expresses both IL-12 and B7-1 in a murine melanoma B16-F10 tumor model. 3 IL-18, IFN-g-inducing factor, is secreted mainly by activated macrophages and DCs. 4 IL-18 plays a significant role in cell-mediated immune responses and is capable of generating an antitumor immune response via induction of IFN-g production from, 5,6 enhancing the cytotoxicity of, 4,7 and stimulating the proliferation of T and NK cells. 6,8 Several studies have shown the antitumor efficacy of IL-18 in murine tumor models. For example, IL-18 exerted antitumor immunity by activating NK cells and establishing cytotoxic CD4 + T cells in Meth A sarcoma-bearing mice. 9 In addition, IL-18 was also shown to be a potent adjuvant, enhancing systemic antitumor immune responses and vaccine efficacy. 10 Therefore, IL-18 represents a promising therapeutic candidate for cancer immunogene therapy.
Previous studies reported that IL-18 is able to drive the Th2 immune response in an IL-4-dependent manner, but induces a Th1 immune response in the presence IL-12. 11,12 These results suggest that IL-18 alone could suppress the Th1 immune response via activation of Th2 immune response. Therefore IL-18, acting in concert with IL-12, may be important in effective immunotherapy. Consistent with this suggestion, an earlier study reported that IL-12 plus IL-18 polarized Th1 immunity, resulting in the generation of potent tumor-reactive T cells. 13 This combination also enhanced the cytolytic effect as well as IFN-g production from T and NK cells, as well as the proliferation in T cells. 11,14 Furthermore, previous studies reported that IL-12 in combination with IL-18 inhibited tumor angiogenesis, leading to tumor regression. 15 In this study, we show for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18. We also show the underlying mechanism of oncolytic Ad co-expressing IL-12 and IL-18, showing that antitumor immunity is associated with elevation of Th1/Th2 cytokine ratio and upregulation of IL-12, IL-18, IFN-g and granulocyte-macrophage colony-stimulating factor (GM-CSF) within the tumor tissues. Moreover, we show that the induction of an optimal tumor milieu, generating effective tumor-specific immunity through the upregulation of Th1 cytokines, enhances the generation of fully differentiated T cells expressing IL-12Rb2 or IL-18Ra.
To further investigate whether IL-12, IL-18, or IL-12 plus IL-18 expression would alter viral replication, RdB/IL-12, RdB/IL-18 or RdB/IL-12/IL-18 were evaluated for their capability to induce viral cytopathic effects in a variety of cell lines. Because murine cells tend to be less sensitive or even resistant to Ad infection compared with human cells, we employed three different types of human cancer cell lines (U343, C33A and A549) from varying histological types. Cells were infected with RdB (cognate oncolytic Ad), RdB/IL-12, RdB/IL-18 or RdB/IL-12/IL-18, along with Ad-DE1 as a negative control. The cells were then treated with crystal violet to show the relative extent of cell lysis. RdB/IL-12, RdB/IL-18 and RdB/IL-12/IL-18 generated cytopathic effects equivalent to RdB alone in all cell lines tested, showing that IL-12, IL-18, or IL-12 plus IL-18 expression does not decrease the viral replication ability of oncolytic Ad (Supplementary Figure 1). Oncolytic adenovirus co-expressing IL-12 and IL-18 I-K Choi et al Enhanced antitumor effect of IL-12-and IL-18-expressing oncolytic Ad To ascertain whether RdB/IL-12, RdB/IL-18 or RdB/IL-12/IL-18 induced the suppression of tumor growth in syngeneic mice, we injected 5Â10 5 cells of B16-F10 melanoma subcutaneously into C57BL/6 mice. When the tumor volume averaged 80 mm 3 , mice were injected intratumorally with phosphate-buffered saline (PBS), RdB, RdB/IL-12, RdB/IL-18 or RdB/IL-12/IL-18 once every other day for a total of three injections. As presented in Figure 2a Figure 2). Furthermore, RdB/IL-12/IL-18-treated mice showed higher survival rates compared with either RdB/IL-12 or RdB/IL-18 (Po0.01). All animals treated with RdB/IL-12/IL-18 remained viable 28 days after the beginning of the treatment, whereas only 16.7% of those treated with RdB/IL-18 and 50% of those treated with RdB/IL-12 survived for the same period of time (Figure 2b). Taken together, these results suggest that RdB/IL-12/IL-18 significantly prolonged survival and enhanced antitumor efficacy compared with either RdB/IL-12 or RdB/IL-18 in the B16-F10 murine melanoma model.
Increase of Th1/Th2 cytokine ratio An aberrant Th1/Th2 cytokine balance with elevated Th2 cytokine levels, which favors development and progression of cancers, was found in various tumor-bearing patients, and a correlation between cytokine levels and the efficacy of cancer treatments has also been reported. 16,17 Hence, we further investigated whether RdB/IL-12/IL-18, which produced markedly higher amounts of Th1 cytokines (IFN-g and GM-CSF) than any of the other groups, could converse from Th2 immunity toward Th1 immunity in tumor tissues. Tumor tissues from mice at 3 days following the final viral injection were harvested and analyzed with Th1/Th2/Th17 cytometric bead assay (CBA) kit (BD Biosciences PharMingen, San Diego, CA, USA). As IL-2, IL-4, IL-10 and IL-17 were not detected in the tumor tissues of all groups (data not shown), the ratio of Th1/Th2 cytokine was Generation of a tumor-specific immune response Results from enzyme-linked immunosorbent assay (ELISA) and CBA assay in tumor tissues indicate that RdB/IL-12/IL-18 might generate a tumor microenvironment that is more favorable to activate tumor-specific immune cells than an oncolytic Ad expressing either IL-12 or IL-18 alone. Therefore, we examined whether RdB/IL-12/IL-18 could enhance immune responses in vivo by assessing the number of immune cells expressing IFN-g, a cytokine secreted by activated T cells. Splenocytes from mice at 3 days following the final viral injection were harvested and co-cultured with irradiated B16-F10 tumor cells for 3 days in the presence of recombinant human IL-2.

Increased infiltration of CD4 + T, CD8 + T and NK cells in RdB/IL-12/IL-18-treated tumors
To analyze histological characteristics of tumor tissues following RdB/ IL-12/IL-18 treatment, we performed hematoxylin and eosin staining of tumor tissues. Histological evaluation of tumor sections revealed that large areas of tumors treated with RdB/IL-12/IL-18 were necrotic. In particular, tumors treated with RdB/IL-12 or RdB/IL-12/IL-18 were extensively infiltrated with immune cells, whereas tumors treated with RdB or RdB/IL-18 showed sparse infiltration. Moreover, denser immune cell infiltration was observed not only around, but also inside the remaining tumor tissues treated with RdB/IL-12/IL-18.
To obtain more insight into the molecular mechanisms of cytokinemediated inhibition of tumor growth, we performed immunohisto-logical analysis. Immunohistochemical studies confirmed that the tumor-infiltrating immune cells were CD4 + T cells, CD8 + T cells and NK cells. Higher numbers of all three cell types were detected in wider areas of RdB/IL-12/IL-18-treated tumors than in tumors treated with either RdB/IL-12 or RdB/IL-18 ( Figure 6).
Increased IL-12Rb2 or IL-18Ra expression in RdB/IL-12/IL-18treated tumors To examine whether expression of IL-12Rb2 or IL-18Ra of T cells could be enhanced in RdB/IL-12/IL-18-treated tumor tissue, we performed immunofluorescence double staining using anti-CD4, anti-CD8, anti-IL-12Rb2 and anti-IL-18Ra antibodies. Sections were prepared from C57BL6 mice 3 days after the final virus injection and  (Figure 7). Effector T cells have to maintain both IL-12Rb2 and IL-18Ra expression for differentiating T cells. 18 Therefore, these data indicate that RdB/IL-12/ IL-18 treatment resulted in an improved generation of optimally differentiated T cells expressing IL-12Rb2 or IL-18Ra.

DISCUSSION
Therapeutic manipulation of immune responses by the use of cytokines is potent strategy for cancer immunotherapy and tumor growth suppression. 19 However, the systemic administration of cytokines at a therapeutic dose can result in serious toxicity in human cancer-bearing patients as well as in animal cancer models. 20,21 Some patients given IL-12 have even died following treatment. 22 Therefore, it is important to understand as to how to induce therapeutic levels of cytokines long enough to generate antitumor immunity effectively within the tumor microenvironment while simultaneously attenuating systemic toxicity. To address this issue, we have used an E1B-deleted and E1A-mutated oncolytic Ad, RdB, which exerts an antitumor effect in a cancer cell-specific manner as an intratumoral delivery system of therapeutic cytokines. 23 Moreover, this tumor-selective oncolytic adenoviral vector is competent at killing the tumor cells, as well as amplifying production of transgenes, by continuously replicating in tumor cells until the tumor tissues were eradicated. 24 Earlier data from our group showed that locally high expression of IL-12 in the tumor tissues by oncolytic Ad elicited potent antitumor effects without the induction of adverse effects by an increase in IL-12 Oncolytic adenovirus co-expressing IL-12 and IL-18 I-K Choi et al or IFN-g in the serum. 3 However, combination therapies of IL-12 and IL-18 have a more effective antitumor activity than either cytokine alone. 20,25,26 Several studies have shown that IL-18 and IL-12 synergistically mediate antitumor immunity in a number of animal tumor models. Oshikawa et al. 27 showed that IL-12-, pro-IL-18-and IL-1b-converting enzyme act synergistically to elicit an enhanced antitumor immune response in a murine mammary adenocarcinoma model via induction of cytotoxic CD8 + T cells and IFN-g. Similarly, recombinant IL-12 and IL-18 proteins were shown to significantly inhibit tumor growth and lymph-node metastasis in hepatoma tumor-bearing guinea-pigs. 28 Furthermore, in DC-based tumor vaccine therapy, DCs co-producing IL-12 and IL-18 induced complete tumor rejection through the generation of an effective Th1 immune response in a BALB/c sarcoma model. 29 In an effort to augment the efficiency of immune gene therapy in vivo, we explored the potential therapeutic benefit of an oncolytic Ad co-expressing IL-12 and IL-18. We first showed that RdB/IL-12/IL-18 resulted in not only enhanced and prolonged suppression of tumor growth and increased survival compared with either RdB/IL-12 or RdB/IL-18, but also a higher incidence of complete tumor regression (data not shown), suggesting that RdB/IL-12/IL-18 elicited an improved antitumor effect than RdB/IL-12 or RdB/IL-18 alone.
To show the underlying mechanism of this enhanced RdB/IL-12/ IL-18-mediated antitumor effect, we next evaluated the characteristics of RdB/IL-12/IL-18-treated tumor tissues. Surprisingly, in contrast to our in vitro results, the expression levels of both IL-12 and IL-18 were markedly elevated in RdB/IL-12/IL-18-treated tumor tissues. More specifically, in vivo, RdB/IL-12/IL-18 elicited a 3.7-and 3.5-fold increase in IL-12 and IL-18 expression, whereas in vitro, RdB/IL-12/ IL-18 elicited a 3.2-and 2.6-fold decrease in IL-12 and IL-18 expression.
IL-18, in concert with IL-12, enhances IFN-g production from activated T and NK cells. 11,14 IFN-g in turn induces DC maturation and macrophages activation, resulting in the elevation of IL-12 and IL-18 expression in those cells. 18,30 This positive feedback loop could explain the upregulation of IL-12 and IL-18 in RdB/IL-12/IL-18treated tumor tissues. Furthermore, matured DCs and activated macrophages can themselves produce IFN-g as well as IL-12 and IL-18. 31,32 In accordance with this positive feedback loop, we found that expression levels of IFN-g, a Th1 cytokine, were considerably higher in RdB/IL-12/IL-18-treated mice than in RdB/IL-12-or RdB/ IL-18-treated mice (Figure 3c). IFN-g has a significant role in boosting cell-mediated antitumor immune responses by increasing cancer cell immunogenicity through enhancing antigen presentation as well as prompting antigen-presenting cells, NK cells, CTLs and Th1 T cells. 18 Yang et al. 33 have shown that co-administration of IFN-g or IL-12 with the recombinant Ad into the airway of C57BL/6 mice prevents the activation of Th2 cells and formation of neutralizing antibody, allowing for efficient re-administration of recombinant Ad. In agreement with such findings, our study shows that RdB/IL-12/IL-18 generates increased Th1/Th2 cytokine ratio with the upregulation of IL-12, IL-18, IFN-g and GM-CSF in tumor tissues. Taken together, these results suggest that RdB/IL-12/IL-18 administered intratumorally may be competent at killing the tumor cells, as well as amplifying production of cytokines, by continuously replicating in tumor cells regardless of Ad-specific neutralizing antibody levels.
GM-CSF expression was also dramatically increased in tumor tissues treated with RdB/IL-12/IL-18 compared with those treated with RdB/IL-12 or RdB/IL-18, supporting the importance of IFN-gand GM-CSF-mediated antitumor immune response. GM-CSF, a Th1 cytokine, stimulates an antitumor cell-mediated immune response by facilitating not only the recruitment, maturation and function of DCs, but also by enhancing the cross-presentation of DCs, which is important for recognizing tumor cells. [34][35][36] Recently, we showed that intratumoral delivery of GM-CSF can promote antigen presentation and elevate the tumor-specific CTL response. 37 Therefore, we suggest that upregulation of GM-CSF in the tumor microenvironment may enhance antigen presentation, leading to the generation of a tumorspecific immune response.
Tumor microenvironment has been shown to establish immunosuppressive cytokine networks that favor the suppression of an antitumor immune response and eventually generate tumor proliferation, angiogenesis and metastasis. Therefore, it is critical to understand the immunosuppressive environment at the tumor site to manipulate and generate antitumor immunity effectively within the tumor microenvironment. To this end, our data suggest that RdB/IL-12/IL-18 generated a tumor microenvironment more favorable to activate tumor-specific immune cells via upregulation of IL-12, IL-18, IFN-g and GM-CSF in tumor tissues. Moreover, the manipulation of a tumor milieu more favorable to activate tumor-specific immune cells by RdB/IL-12/IL-18 was further supported by the observation that Th1/Th2 cytokine ratio was markedly elevated in RdB/IL-12/IL-18treated tumor tissues (Figures 4a and b).
To activate tumor-specific immune cells, antitumor adaptive immunity in the tumor milieu should be optimally generated. Here, we showed that mice injected with RdB/IL-12/IL-18 had enhanced CTL activity, as well as an increase in tumor-specific IFN-g-secreting immune cells, than did those given RdB/IL-12 or RdB/IL-18. In accordance with these results, both histological and immunohistochemical results showed immense necrotic regions as well as infiltration of NK, CD4 + T and CD8 + T cells into the tumor tissues of RdB/ IL-12/IL-18-treated mice compared with those of RdB/IL-12-or RdB/ IL-18-treated mice. NK cells are known to play an important role in the promotion of Th1 responses by producing IFN-g and modulating DC activity. 38,39 This observation suggests that IL-12 plus IL-18 activate NK cells that mediate oncolysis and DC maturation that, in turn, enhance antigen presentation to induce tumor-specific T cells to differentiate from naïve T cells, leading to the generation of antitumor immunity.
Biological functionality of RdB/IL-12/IL-18-mediated IL-12 and IL-18 expression in tumor tissues depends on the expression level of IL-12Rb2 and IL-18Ra. Therefore, upregulation of IL-12Rb2 and IL-18Ra correlates with the capacity of IL-12 and IL-18 to bind and initiate the development of Th1 immunity. Reports from in vitro studies have shown that IL-18 enhances the production of IL-12Rb2 on T cells, whereas IL-12 mediates upregulation of IL-18Ra on T cells. 40,41 Here, we showed that the numbers of T cells expressing IL-12Rb2 or IL-18Ra were increased in tumor tissues of RdB/IL-12/ IL-18-treated tumors (Figure 7). Moreover, animals treated with RdB/ IL-12/IL-18 showed an elevated number of CD4 + and CD8 + T cells expressing IL-12Rb2 or IL-18Ra at the tumor tissues. The expression of IL-12Rb2 or IL-18Ra receptors are necessary for the differentiation of effector T cells. 18 Therefore, our finding implies that intratumoral delivery of IL-12 and IL-18 by RdB/IL-12/IL-18 resulted in improved generation of optimally differentiated CD4 + and CD8 + T cells expressing IL-12Rb2 or IL-18Ra. Our results further support the view put forth by Li et al. 13 that IL-12, in combination with IL-18, induces differentiation of the antitumor effector T cells CD4 + and CD8 + .
In conclusion, the finding in our report provides a novel underlying mechanism of combination therapies via IL-12 and IL-18 that promotes differentiation of T cells expressing IL-12Rb 2 or IL-18Ra. We also provide the first study where intratumoral expression of both IL-12 and IL-18 via oncolytic Ad vector results in the creation of more potent tumor-specific immunity. These results provide a new insight into therapeutic mechanisms through IL-12 plus IL-18, as well as a potential clinical cancer immunotherapeutic agent for the generation of improved antitumor immunity.

Animal studies
Six-to eight-week-old male C57BL/6 mice were obtained from Charles River Laboratories International Inc.
Ads or PBS were injected intratumorally (1Â10 8 plaque-forming unit per tumor in 30 ml of PBS) on days 0, 2 and 4. Control mice were injected with PBS only. Tumor volume was evaluated once every 2 days by electronic caliper measure: volume¼0.523LW 2 (L¼the length of tumors; W¼the width of tumors). Tumor responses to each treatment were compared using a log-rank test analysis (StatView software; Abacus Concepts Inc., Berkeley, CA, USA). The percentage of surviving mice was determined by monitoring the tumor growthrelated events (tumor size 42500 mm 3 ). The survival curve was then plotted against time after treatment. Differences in survival were considered statistically significant when P-values were o0.01.
Quantification of cytokines and Th1/Th2/Th17 profile in tumor tissue Tumors were harvested 3 days after the last viral inoculation of the B16-F10 tumor-bearing mice and snap-frozen in liquid nitrogen. Tumor tissues were homogenized (ART-MICCRA D-8; ART moderne Labortechnik, Munchen, Germany) in ice-cold radio immunoprecipitation assay buffer (Elipis Biotech, Taejeon, South Korea) with a proteinase inhibitor cocktail (Sigma, St Louis, MO, USA). Homogenates were then centrifuged in a high-speed microcentrifuge for 10 min and determined for total protein content using a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Levels of IL-12, IL-18, IFN-g and GM-CSF in the tumor tissue extract were determined by ELISA according to the supplier's recommendations (IL-12 ELISA kit; Endogen, IL-18 ELISA kit; MBL, IFN-g ELISA kit; Endogen, GM-CSF ELISA kit; Endogen). In addition, Th1/Th2/Th17 type cytokine expression profiles in the tumor tissue extract were estimated using Th1/Th2/Th17 CBA kit (BD Biosciences PharMingen). ELISA and CBA results were normalized relative to the total protein concentration in each tumor and were calculated as picograms per milligram of total protein.

IFN-c ELISPOT assay in splenocytes
At 3 days following the last viral treatment, spleens were obtained aseptically from tumor-bearing mice, and unicellular splenocytes were prepared as described previously. 3 Prepared spleen cells were co-cultured with irradiated B16-F10 (5000 rad) tumor cells for 3 days in the presence of recombinant human IL-2 (100 U ml À1 ; R&D Systems, Minneapolis, MN, USA). An IFN-g ELISPOT assay was then carried out as described previously. 3 The spots were measured with a computer-based immunospot system (AID Elispot Reader System, version 3.4; Autoimmun Diagnostika GmbH, Strassberg, Germany).

Cytotoxicity assay in splenocytes
The cytolytic activity of tumor-specific CTL was evaluated by 4 h 51 Cr-release assay. Co-cultured spleen cells were prepared as described for the IFN-g ELISPOT assay. After co-culture, splenocytes as effector cells were recovered and incubated with 51 Cr-labeled B16-F10 or NIH3T3 cells as target cells at 100:1 E:T ratios for 4 h at 37 1C and 5% CO 2 . Thereafter, the supernatant was obtained and 51 Cr release was assessed. The percentage of specific lysis was calculated by the following formula: ((c.p.m. experimental releaseÀc.p.m. spontaneous release)/(c.p.m. maximum releaseÀc.p.m. spontaneous release))Â100. Spontaneous release was determined by incubation of the labeled target cells without effector cells. For maximum release, labeled target cells were treated with detergent.
Histology and immunohistochemistry for NK-1.1, CD4 and CD8 For histological study, tumor tissues were snap-frozen in OCT compound (Sakura Finetec, Torrance, CA, USA), and 10-mm sections were cut on a cryostat. Representative sections were stained with hematoxylin and eosin, and then investigated using light microscopy. To detect lymphocytes, tumor tissues were snap-frozen in OCT compound (Sakura Finetec), and 10-mm sections were cut, subjected to immunohistochemistry, and then counterstained with Meyer's hematoxylin. The sections were incubated with purified rat anti-mouse CD4 monoclonal antibody (BD Biosciences PharMingen), purified rat antimouse CD8 monoclonal antibody (BD Biosciences PharMingen) or purified mouse anti-mouse NK-1.1 monoclonal antibody (Biolegend, San Diego, CA, USA) as a primary antibodies, and then with goat anti-rat IgG (BD Biosciences PharMingen) or goat-anti mouse IgG (Southern Biotech, Birmingham,