Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Large Nontransplanted Hepatocellular Carcinoma in Woodchucks: Treatment With Adenovirus-Mediated Delivery of Interleukin 12/B7.1 Genes

Large Nontransplanted Hepatocellular Carcinoma in Woodchucks: Treatment With Adenovirus-Mediated... Abstract Background: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. Methods: Large (2–5 cm), established intrahepatic tumors were given an injection once with 1 × 109 plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. Results: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1–2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P = .006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4+ and CD8+ T cells and interferon gamma (IFN γ). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4+ and CD8+ T cells and IFN γ) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (≥95%) 7 weeks after treatment. Conclusion: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans. Among the various strategies used in cancer gene therapy, activation of the immune system by vector-mediated transfer of immunomodulatory genes is a most promising approach because tumor development in immunocompetent hosts suggests that the host's immune system is failing to detect and eliminate malignant cells (1,2). This failure appears to result mainly from the evasive properties of the tumor (3), not from a lack of tumor antigens, because tumor cells transfected with immunomodulatory genes can induce specific antitumor immune responses resulting in tumor rejection (4,5). Adenoviral delivery of genes for cytokines and related immunomodulators, such as interleukin (IL) 2, IL-12, and IL-4, interferon gamma (IFN γ), and B7.1, can efficiently mediate tumor rejection and induce protective systemic immunity in several animal tumor models (6–12). Of these cytokines, IL-12 appears to possess the strongest antitumor activity. The mechanism for IL-12-mediated tumor killing includes the activation of natural killer cells, T cells, lymphokine-activated killer cells, and macrophages (13). IL-12 stimulates IFN γ production from natural killer and T cells, promotes the cellular immune response by facilitating the proliferation and activation of Th1 T-helper cells (14), and inhibits angiogenesis in vivo (15). IL-12 interacts synergistically with the costimulatory molecule B7.1/CD80, normally expressed on professional antigen-presenting cells, to enhance protective antitumor immunity (9,16–19). B7.1-mediated antitumor activity in these studies was largely attributed to the stimulation of natural killer and CD8+ T cells, whereas the requirement for CD4+ cells in tumor rejection is highly dependent on the tumor model (20). Previously, we constructed an adenovirus vector carrying genes for both IL-12 and B7.1, termed “AdIL-12/B7.1,” and demonstrated their synergistic effect on tumor growth by injecting the vector directly into breast adenocarcinomas transplanted from a polyoma middle-T-antigen (PyMT)-transgenic mouse model (9). In tumors derived from the transgenic model, one injection of the vector resulted in complete tumor regression in 70%–90% of the treated animals and induced long-term systemic immunity (9). However, the animal model in this study, as in the majority of cancer immunotherapy trials, is based on transplantation of established tumor cell lines into immunocompetent animals. In contrast, during carcinogenesis, a primary tumor develops from a single cell that becomes malignant in its natural environment (21), thus allowing the development of immune escape mechanisms that may negatively affect immunotherapeutic approaches in humans (21). To address this problem, we tested the ability of the AdIL-12/B7.1 vector to reduce the volume of primary, nontransplanted woodchuck hepatitis virus (WHV)induced hepatocellular carcinomas (HCCs) in woodchucks. WHV is closely related to the human hepatitis B virus in its structure, genomic organization, mechanism of replication, and the course of infection. Persistent WHV infection is associated frequently with the development of hepatic tumors (22). This experimental model has been applied extensively in the study of underlying mechanisms of human hepatitis B virus-induced hepatocarcinogenesis and in the development of prophylactic and therapeutic strategies of disease control (23,24). Materials and Methods Cell Culture The woodchuck liver cell line WH12/6 (25) was maintained in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS). Human MRC5 fibroblasts (cell line CCL 171; American Type Culture Collection, Manassas, VA) were grown in modified Eagle medium plus 10% FCS. All viruses were grown in 293 cells [adenovirus 5 E1-transformed human embryonic kidney cells (26)] maintained in modified Eagle medium/F-11 with 10% FCS. Media were supplemented with 2 mMl-glutamine, penicillin at 100 μg/mL, and streptomycin at 100 U/mL. Animals Adult American woodchucks (Marmota monax) were chronically infected with WHV, which is closely related to human hepatitis B virus. All chronic WHV carriers were provided by Marmotech, Inc., Ithaca, NY, and had one or more hepatic tumors that were 2 cm or larger in diameter, characterized by ultrasound and magnetic resonance imaging (MRI). In this study, a total of five animals have been used. Four animals were treated by intratumoral administration of Ad vectors after laparotomy, and one was given an injection under MRI guidance. Recombinant Adenovirus Vectors Construction of the recombinant adenovirus vector expressing both murine IL-12 subunits in early region 1 (E1) and B7.1 in early region 3 has been described previously (9). The control adenovirus AdEGFP encoding the enhanced green fluorescent protein was described (27). Adenovirus vectors were propagated, purified, and titrated as described previously (28,29). Enzyme-Linked Immunosorbent Assay and Flow Cytometry Analysis Hepatoma cells infected at a multiplicity of infection of 10 plaque-forming units (pfu) per cell were incubated in growth medium as indicated. Expression levels of secreted murine IL-12 were quantitated from infected cell supernatant by an enzyme-linked immunosorbent assay (ELISA) with the use of the mouse IL-12p70 DuoSet ELISA development system (R&D Systems Europe, Oxon, U.K.), according to the manufacturer's protocol. For B7.1 detection, cells were trypsinized, resuspended in ice-cold phosphate-buffered saline (PBS) containing 1% fetal bovine serum, incubated with the fluorescein isothiocyanate-conjugated anti-mouse B7.1 antibody (PharMingen, Hamburg, Germany) for 60 minutes on ice, washed twice in PBS, and analyzed for fluorescence with a FACSVantage flow cytometer (Becton Dickinson Biosciences, Heidelberg, Germany). Human MRC5 fibroblasts were used as a negative control for B7.1 expression because these cells do not react with the mouse B7.1 antibody. Ribonuclease Protection Assay Total RNA was extracted from frozen liver biopsy samples with the RNeasy Mini kit (Qiagen, Hilden, Germany) by following the manufacturer's directions. Ribonuclease protection assays (RPAs) for the analysis of cytokine and T-lymphocyte transcripts were performed with the RiboQuant™ RPA kit (PharMingen, San Diego, CA) according to the instruction manual. The protected RNA bands were quantitated on a Cyclone storage phospho screen imager (Packard, Dreiech, Germany). Intratumoral Administration of Adenovirus Vectors Hepatic tumors were selected preoperatively by ultrasound. Four woodchucks were anesthetized with 1 mL/kg of ketamine (Dr. Graeub, AG, Bern, Switzerland) and 0.3 mL/kg of xylazine (Bayer, Leverkusen, Germany). Median laparotomies were performed, and grossly identifiable tumors were located and measured with calipers before injection of adenovirus vectors and at various times after treatment. The tumor volume was calculated from the longest diameter and average width by assuming a prolate spheroid shape (6). Four woodchucks, each with two or more hepatic tumors that were 2.0 cm or larger in diameter, were treated with a single type of vector as follows: One woodchuck (animal 1) received multiple intratumoral injections (a total of 1 × 109 pfu of AdEGFP in 350 μL of PBS) in separate sites of one tumor nodule. One woodchuck (animal 2) received multiple intratumoral injections (a total of 1 × 109 pfu of AdIL-12/B7.1 in 350 μL of PBS) in separate sites of one tumor nodule. In two woodchucks (animals 3 and 4), two tumor nodules of equal size were treated with multiple intratumoral injections of either AdEGFP or AdIL-12/B7.1; each tumor nodule received a total of 1 × 109 pfu in 350 μL of PBS. Animals were killed on day 7 (animal 3), day 10 (animal 4), or day 14 (animals 1 and 2) after injection; the liver was exposed; and hepatic tumors were measured. Tumor and normal liver tissue samples were fixed in phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin–eosin for histologic examination. Samples of hepatic tumors and non-neoplastic liver were frozen in liquid nitrogen and stored at −70 °C for RPAs. Selective Adenovirus Vector Administration and Tumor Size Assessment by MRI Guidance For each MRI session, the woodchuck was fully anesthetized with ketamine (1 mL/kg) and xylazine (0.3 mL/kg). The tumor was selected, and the injection needle was placed directly into the tumor nodule under guidance by an MRI by use of a 1.5-tesla magnet (Magnetom Sonata; Siemens, Erlangen, Germany). The animal received a single intratumoral injection of 1 × 109 pfu of the AdIL-12/B7.1 vector in 350 μL of PBS. Other tumors identified served as controls. To maximize the signal-to-noise ratio, a standard head coil was used for data reception. The animal underwent axial MRI examinations of the liver with the following imaging parameters: 1) T1-weighted fast low-angle-shot (FLASH) sequence (162-ms/4.0-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 12-second acquisition time); 2) T2-weighted HASTE (half-Fourier-acquisition single-shot turbo-spin-echo) sequence (1000-ms/60-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 20-second acquisition time); and 3) T1-weighted FLASH sequence (162-ms/4.0-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 12-second acquisition time) with fat-saturation postintravenous application of 1 mL of Gd-diethylenetriaminepentaacetate; Magnevist; Schering, Berlin, Germany). Because tumors were most conspicuous on T1-weighted postcontrast images, this sequence was used to assess tumor volume and thus to monitor the response of tumor treatment. Therefore, region-of-interest measurements were performed on a computer workstation with the region-of-interest size adapted to encompass the tumor contours on the individual image sections. The calculated area was then multiplied by the craniocaudal extension of the tumor. All animal experiments were approved by and carried out according to guidelines set forth by the Animal Research Ethics Board of the University of Essen. Statistical Analysis The statistical significance of the relative increase in tumor size was calculated by paired Student's t test. All statistical tests were two-sided. Results Antitumor Activity of the AdIL-12/B7.1 Vector Against Nontransplanted WHV-Induced HCC In vivo IL-12 gene delivery by adenovirus vectors into established transplanted tumors can inhibit tumor growth and induce tumor regression in various mouse tumor models (8,30–34). We have demonstrated previously (9) that the antitumor activity of IL-12 in transplanted tumors can be increased substantially by also inserting B7.1. When we injected this vector into the transplanted tumors at 2.5 × 107 pfu, a low virus dose, we observed complete tumor regression and induction of systemic immunity in the majority of animals. To determine whether AdIL-12/B7.1 could be used to treat established nontransplanted (orthotopic) tumors, we used the woodchuck animal model. Before the vector was given to woodchucks, however, we infected WH12/6 cells in vitro and determined that murine IL-12 and B7.1 were expressed 3 and 5 days after infection (Fig. 1). Next, we selected large tumors in four woodchucks that were chronic WHV carriers and injected the tumors with AdIL-12/B7.1 or the control vector AdEGFP. The absolute tumor volumes of six tumor nodules, three injected with the AdIL-12/B7.1 virus and three treated with AdEGFP virus, are presented in Table 1 before and 7–14 days after treatment; the absolute tumor volumes of two untreated tumors are also presented after 14 days. On average, over a 2-week period, tumors given an injection intratumorally with AdIL-12/B7.1 showed an 80% reduction in volume from their original volume (Fig. 2, A), whereas untreated and AdEGFP-injected tumors showed an approximately twofold increase in volume (Fig. 2, A). Fig. 2, B, presents representative MRI images from one woodchuck at three representative time points (before treatment and 4 and 7 weeks after treatment), showing the long-term antitumor response after direct intratumoral injection of 1 × 109 pfu of AdIL-12/ B7.1 into a selected liver tumor nodule under MRI guidance. In this experiment, AdIL-12/B7.1 treatment had a pronounced effect on tumor growth over a 7-week period, leading to an approximately 95% reduction in tumor size from 8.6 cm3 at the time of injection to 0.5 cm3 by day 50 (Fig. 2, B-I to B-III). In contrast, no reduction in tumor size was noted in the surrounding untreated tumors. Thus, large established nontransplanted liver tumors can be treated efficiently by injection of an IL-12/B7.1-expressing adenovirus vector. Association of Regression of Primary HCC and an Increase in Immune Effector Cells and IFN γ Within the Tumor Gene transfer studies (5,35,36) in several mouse models have indicated that rejection of transplanted tumors expressing IL-12 and/or B7.1 is dependent on CD4+ and CD8+ T lymphocytes and natural killer cells. To analyze the immune response elicited by injection of AdIL-12/B7.1 into established nontransplanted liver tumors, we next examined liver and tumor biopsy samples from woodchucks 1–4 before and after treatment (Fig. 3, A). Histologic sections from HCCs showed the typical features of neoplastic hepatic cells forming pseudotubulary structures with striking changes in the nuclear morphology, characterized by condensed chromatin and macronucleoli (Fig. 3, A-III). In contrast to nodules injected with the control vector AdEGFP (Fig. 3, A-IV), necrosis and massive inflammatory infiltration were detected only in AdIL-12/B7.1-treated tumors (Fig. 3, A-V). In the nontumoral liver parenchyma, we found typical morphologic patterns of chronic hepatitis after persistent WHV infection, characterized by moderate fibrosis and mild mononuclear infiltrate of portal or periportal tracts with occasional necrosis [(37) and Fig. 3, A-I]. However, no evidence of adenovirus- induced hepatic toxicity was observed in normal liver tissue 10 days after intratumoral vector administration (Fig. 3, A-II). The histologic results were extended by RPA of liver and tumor biopsy samples obtained from woodchucks 1–4 (Fig. 3, B). Compared with the levels of T-lymphocyte-specific transcripts in hepatic tumors before treatment, RPA revealed an influx of CD4+ and CD8+ T cells into AdIL-12/B7.1-injected tumors (Fig. 3, B-II) that occurred at the peak of rejection. Compared with before-treatment samples, CD4+ lymphocytes showed an approximately sevenfold increase and CD8+ lymphocytes showed a moderate but important twofold increase. Moreover, AdIL-12/B7.1-injected HCC tissue showed an approximately twofold increase in the levels of IFN γ, the major determinant of the IL-12-mediated antitumor response. Thus, murine IL-12 expressed from an adenovirus vector is functional in woodchuck liver tumors. In contrast, no increase in CD4+ or CD8+ T cells or IFN γ occurred in tumors injected with control virus (Fig. 3, B-I) or in nontumoral liver samples (data not shown). Discussion Previously, we showed that intratumoral injection of an adenovirus coexpressing IL-12 and B7.1 is an extremely efficient method of inducing the rejection of transplanted PyMT-induced tumors (9). To determine whether it is also active against primary tumors, we selected the well-characterized model of WHV-induced HCC in woodchucks. This model is clinically important because WHV is one of the most efficient hepatocarcinogens in woodchucks (38). WHV-induced hepatocarcinogenesis shows strong similarity to hepadnavirus-induced carcinogenesis in humans (23,39). Because our previous data indicated that AdIL-12/B7.1-injected murine tumors were effectively rejected within approximately 2 weeks, the immune response against nontransplanted woodchuck liver tumors was first analyzed shortly after treatment. Consistent with the results obtained from the PyMT model (9), much of the tumor mass was rejected by day 14, suggesting that AdIL-12/B7.1 had a similar inhibitory effect on growth of transplanted and nontransplanted tumors at this early stage of treatment. The use of MRI guidance allowed us to locate and select large tumor nodules into which we could directly inject the vector, thereby avoiding the negative and stressful effects of surgery. MRI monitoring of a selected tumor revealed long-term tumor regression, resulting in nearly complete tumor elimination. However, there was a clear difference in the time course of tumor reduction between the tumor injected at one site under MRI guidance and the tumors injected at multiple sites during laparotomy. In murine models, intratumoral injection of adenovirus vectors carrying IL-12 and/or B7.1 can stimulate systemic immunity that can act effectively on distal tumors (7–9). No clear systemic effect, however, has been observed in the woodchuck model, which may reflect a variation in the immunogenicity of different tumor nodules (Roggendorf M: unpublished data). Because these tumors are not of clonal origin and might express different tumor antigens, an effect on surrounding tumors might be obtained when vascular anastomoses allow the virus to spread between the tumors. Therefore, a lack of anastomoses between the tumor and the normal liver in this model might contribute to the lack of systemic response. In addition, higher doses of cytokine vectors were more efficient in inhibiting tumor growth than lower doses (9,30,40). If the body weight of a woodchuck and the tumor size of 2–5 cm in diameter are considered, the virus dose of 1 × 109 pfu, used for woodchucks, is low compared with doses used in mice. Therefore, we cannot exclude the possibility that the IL-12/B7.1 virus might induce systemic immunity in primary cancer at higher vector doses. Our results suggest that regression of WHV-induced primary liver tumors is associated with massive intratumoral lymphocyte infiltration. We also show that intratumoral adenoviral IL-12/B7.1 expression in primary HCCs leads to increased intratumoral levels of IFN γ, consistent with results from a number of models indicating that the antitumor activity of IL-12 is IFN γ dependent (8,41,42). From recent observations, expression of major histocompatibility complex (MHC) class I molecules is altered in liver cells of animals chronically infected with WHV (43) and also commonly reduced on neoplastic cells of HCC nodules (Roggendorf M: unpublished data). Consequently, this reduction in MHC molecules may diminish the susceptibility of WHV-infected hepatocytes to virus-specific T cells, WHV-infected hepatocytes may evade the antiviral immunologic surveillance system, and thus liver damage may be perpetuated. The current model emphasizes the role of IFN γ for the modulation of peptide processing and stimulation of genes involved in antigen presentation by the MHC class I molecules (44). Thus, the activation of MHC class I-associated genes by local induction of IFN γ through tumor-infiltrating T lymphocytes may be a possible mechanism for efficient tumor rejection after IL-12/B7.1 immunotherapy in this model. However, our observation varies from the observations made in established nontransplanted 3-MC-induced mouse tumors after IL-7/B7.1 gene delivery (45). In that model, failure to induce tumor regression depends on the inability of T cells to infiltrate nontransplanted tumors. The observed difference might result from a difference in T-cell infiltration of tumors induced by viruses or by carcinogens rather than a general difference between transplanted and primary, nontransplanted malignant tumors. The animal model used in this study is expensive and difficult to use, and only a limited number of tumor-bearing WHV-infected woodchucks were available. Nevertheless, our results demonstrate that adenovirus vector-mediated immunotherapy is certainly not limited to artificial mouse tumor models and provide a powerful approach for the in vivo treatment of large, primary tumors in a model of hepadnavirus-induced carcinogenesis, reflecting the real-world scenario of HCC in humans. Direct injection into selected HCC nodules under MRI guidance appears to be a relatively nontoxic and feasible method for administrating viral vectors in human clinical trials. However, our data emphasize the need to evaluate the mechanisms involved in effective intratumoral penetration by immune effector cells as a prerequisite for successful cytokine treatment of natural tumors. Table 1. Tumor volume of established primary hepatocellular carcinomas after AdIL-12/B7.1 treatment Animal*  Treatment  Volume before treatment, cm3  Volume after treatment, cm3  Time, d†  *In woodchucks 3 and 4, one selected tumor nodule was treated with AdEGFP and one with AdIL-12/B7.1.  †Tumor size was determined at 7, 10, and 14 days after treatment.  3  AdEGFP  23.5  35.6  7  3  AdIL-12/B7.1  29.4  11.8  7  4  AdEGFP  2.8  7.45  10  4  AdIL-12/B7.1  37.8  6.8  10  1  AdEGFP  2.9  5.9  14  1  Untreated  5.9  10.6  14  2  AdIL-12/B7.1  3.6  0.25  14  2  Untreated  1.3  4.2  14  Animal*  Treatment  Volume before treatment, cm3  Volume after treatment, cm3  Time, d†  *In woodchucks 3 and 4, one selected tumor nodule was treated with AdEGFP and one with AdIL-12/B7.1.  †Tumor size was determined at 7, 10, and 14 days after treatment.  3  AdEGFP  23.5  35.6  7  3  AdIL-12/B7.1  29.4  11.8  7  4  AdEGFP  2.8  7.45  10  4  AdIL-12/B7.1  37.8  6.8  10  1  AdEGFP  2.9  5.9  14  1  Untreated  5.9  10.6  14  2  AdIL-12/B7.1  3.6  0.25  14  2  Untreated  1.3  4.2  14  View Large Fig. 1. View large Download slide View large Download slide Assessment of interleukin 12 (IL-12)/B7.1 production in vitro. Expression kinetics of murine IL-12 (A) and B7.1 (B) in WH12/6 cells before and 1, 3, and 5 days after infection with the adenovirus-based vector AdIL12-B7.1 at a multiplicity of infection of 10 is shown. The murine IL-12 (mIL-12) concentration in supernatant was determined by enzyme-linked immunosorbent assay by use of the anti-mouse IL-12p70 antibody. All samples were assayed in triplicate. The 95% confidence intervals are indicated. For B7.1 detection, cells were stained with fluorescein isothiocyanate-conjugated anti-mouse B7–1 antibody and analyzed by flow cytometry; uninfected WH12/6 cells were used as controls (center peak). Compared with the negative control (human MRC-5 fibroblasts, left peak), uninfected WH12/6 cells (center peak) scored almost negative for B7.1 expression (4% after 72 hours). B7.1-positive cells (right peak), labeled M1, were 65% of the total cells on day 1, 92% on day 3, and 95% on day 5. FL1 = fluorescence channel 1 (green). Fig. 1. View large Download slide View large Download slide Assessment of interleukin 12 (IL-12)/B7.1 production in vitro. Expression kinetics of murine IL-12 (A) and B7.1 (B) in WH12/6 cells before and 1, 3, and 5 days after infection with the adenovirus-based vector AdIL12-B7.1 at a multiplicity of infection of 10 is shown. The murine IL-12 (mIL-12) concentration in supernatant was determined by enzyme-linked immunosorbent assay by use of the anti-mouse IL-12p70 antibody. All samples were assayed in triplicate. The 95% confidence intervals are indicated. For B7.1 detection, cells were stained with fluorescein isothiocyanate-conjugated anti-mouse B7–1 antibody and analyzed by flow cytometry; uninfected WH12/6 cells were used as controls (center peak). Compared with the negative control (human MRC-5 fibroblasts, left peak), uninfected WH12/6 cells (center peak) scored almost negative for B7.1 expression (4% after 72 hours). B7.1-positive cells (right peak), labeled M1, were 65% of the total cells on day 1, 92% on day 3, and 95% on day 5. FL1 = fluorescence channel 1 (green). Fig. 2. View large Download slide View large Download slide Short- and long-term antitumor efficacy of AdIL-12/B7.1 on woodchuck hepatitis virus-induced primary liver tumors. Panel A: selected tumor nodules 2–5 cm in diameter were intratumorally injected with 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1 or the control virus AdEGFP (three tumors for each vector). The animals were killed within 14 days after treatment and were monitored for tumor regression relative to untreated tumors. The average increase in tumor volume is shown (shaded bars). Tumor volume before treatment was set as 1 (solid bars). The 95% confidence intervals (CIs) are indicated by error bars. The difference in the relative increase of the size of AdEGFP- versus AdIL12/B7.1-treated tumors was statistically significant (*) with P = .006: AdEGFP (2.1-fold [95% CI = 1.44-fold to 2.76-fold]; P = .084) versus AdIL-12/B7.1 (0.22-fold [95% CI = 0.03-fold to 0.41-fold]; P = .015). The absolute volumes from each injected tumor are shown in Table 1. Panel B: T1-weighted postcontrast magnetic resonance images of one representative woodchuck liver show neoplastic tumor nodules. One tumor indicated with white arrowheads was given a direct injection of 1 × 109 pfu of AdIL-12/B7.1 under magnetic resonance imaging guidance. The treated tumor is shown before (B-I) and 4 (B-II) and 7 (B-III) weeks after a single vector administration. Reductions in tumor size of 43% (4 weeks after injection) to 95% (7 weeks after injection) are shown in images B-II and B-III, respectively. Growth of the surrounding untreated nodules of equivalent size was unchanged. Fig. 2. View large Download slide View large Download slide Short- and long-term antitumor efficacy of AdIL-12/B7.1 on woodchuck hepatitis virus-induced primary liver tumors. Panel A: selected tumor nodules 2–5 cm in diameter were intratumorally injected with 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1 or the control virus AdEGFP (three tumors for each vector). The animals were killed within 14 days after treatment and were monitored for tumor regression relative to untreated tumors. The average increase in tumor volume is shown (shaded bars). Tumor volume before treatment was set as 1 (solid bars). The 95% confidence intervals (CIs) are indicated by error bars. The difference in the relative increase of the size of AdEGFP- versus AdIL12/B7.1-treated tumors was statistically significant (*) with P = .006: AdEGFP (2.1-fold [95% CI = 1.44-fold to 2.76-fold]; P = .084) versus AdIL-12/B7.1 (0.22-fold [95% CI = 0.03-fold to 0.41-fold]; P = .015). The absolute volumes from each injected tumor are shown in Table 1. Panel B: T1-weighted postcontrast magnetic resonance images of one representative woodchuck liver show neoplastic tumor nodules. One tumor indicated with white arrowheads was given a direct injection of 1 × 109 pfu of AdIL-12/B7.1 under magnetic resonance imaging guidance. The treated tumor is shown before (B-I) and 4 (B-II) and 7 (B-III) weeks after a single vector administration. Reductions in tumor size of 43% (4 weeks after injection) to 95% (7 weeks after injection) are shown in images B-II and B-III, respectively. Growth of the surrounding untreated nodules of equivalent size was unchanged. Fig. 3. View large Download slide View large Download slide Infiltration of immune effector cells in AdIL-12/B7.1-injected primary liver tumors. Panel A: representative liver and tumor sections from one woodchuck before (A-I and A-III) and after (A-II, A-IV, and A-V) treatment. One tumor nodule was given an injection of 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1, and one tumor nodule was given an injection of 1 × 109 pfu of the control vector AdEGFP. The animal was killed 10 days later and analyzed for treatment-associated morphologic changes. Liver parenchyma of chronic hepatitis after persistent woodchuck hepatitis virus infection before (A-I) and after (A-II) intratumoral injection are observed. No morphologic signs of adenovirus vector-related hepatic injury were detected. Pseudotubulary structures of neoplastic hepatocytes from untreated hepatocellular carcinoma (HCC) (A-III) and after injection with AdEGFP (A-IV) or AdIL-12/B7.1 (A-V) are shown. Several areas of necrosis and massive inflammatory infiltration are shown in A-V. (Hematoxylin–eosin staining. Scale bars = 100 μm.) Panel B: analysis of T-lymphocyte and interferon gamma (IFN gamma) messenger RNA (mRNA) levels by ribonuclease protection assay. The RNA was obtained from liver tumor biopsy samples before and 10 days after intratumoral injection of the control virus AdEGFP (I) or AdIL-12/B7.1 (II). The average increases in CD4, CD8, and IFN gammaγ mRNA levels after each treatment are presented. The mRNA levels before treatment were set as 1 for each parameter. Results are reported as the mean ± 95% confidence interval. Fig. 3. View large Download slide View large Download slide Infiltration of immune effector cells in AdIL-12/B7.1-injected primary liver tumors. Panel A: representative liver and tumor sections from one woodchuck before (A-I and A-III) and after (A-II, A-IV, and A-V) treatment. One tumor nodule was given an injection of 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1, and one tumor nodule was given an injection of 1 × 109 pfu of the control vector AdEGFP. The animal was killed 10 days later and analyzed for treatment-associated morphologic changes. Liver parenchyma of chronic hepatitis after persistent woodchuck hepatitis virus infection before (A-I) and after (A-II) intratumoral injection are observed. No morphologic signs of adenovirus vector-related hepatic injury were detected. Pseudotubulary structures of neoplastic hepatocytes from untreated hepatocellular carcinoma (HCC) (A-III) and after injection with AdEGFP (A-IV) or AdIL-12/B7.1 (A-V) are shown. Several areas of necrosis and massive inflammatory infiltration are shown in A-V. (Hematoxylin–eosin staining. Scale bars = 100 μm.) Panel B: analysis of T-lymphocyte and interferon gamma (IFN gamma) messenger RNA (mRNA) levels by ribonuclease protection assay. The RNA was obtained from liver tumor biopsy samples before and 10 days after intratumoral injection of the control virus AdEGFP (I) or AdIL-12/B7.1 (II). The average increases in CD4, CD8, and IFN gammaγ mRNA levels after each treatment are presented. The mRNA levels before treatment were set as 1 for each parameter. Results are reported as the mean ± 95% confidence interval. Editor's note: B. Tennant owns an equity interest in Marmotech, Inc., Ithaca, NY, the company that donated the woodchucks used in this study. Supported in part by grant PU188/2–1 from the Deutsche Forschungsgemeinschaft and by the Internal Research Support Program from the University of Essen, Germany (to B. M. Pützer). We thank Silke Bosk for excellent technical assistance in performing the magnetic resonance tomography, Li Jun for surgical assistance, and Michael Lowak for help in animal handling. References 1 Chouaib S, Asselin-Paturel C, Mami-Chouaib F, Caignard A, Blay JY. The host–tumor immune conflict: from immunosuppression to resistance and destruction. Immunol Today  1997; 18: 493–7. Google Scholar 2 Pettit SJ, Seymour K, O'Flaherty E, Kirby JA. Immune selection in neoplasia: towards a microevolutionary model of cancer development. Br J Cancer  2000; 82: 1900–6. Google Scholar 3 Gilboa E. How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother  1999; 48: 382–5. Google Scholar 4 Davidoff AM, Kimbrough SA, Ng CY, Shochat SJ, Vanin EF. Neuroblastoma regression and immunity induced by transgenic expression of interleukin-12. J Pediatr Surg  1999; 34: 902–6; discussion 906–7. Google Scholar 5 Sun Y, Qian C, Peng D, Prieto J. Gene transfer to liver cancer cells of B7–1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone. Hum Gene Ther  2000; 11: 127–38. Google Scholar 6 Addison CL, Braciak T, Ralston R, Muller WJ, Gauldie J, Graham FL. Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in murine breast cancer model. Proc Natl Acad Sci U S A  1995; 92: 8522–6. Google Scholar 7 Addison CL, Bramson JL, Hitt MM, Muller WJ, Gauldie J, Graham FL. Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors. Gene Ther  1998; 5: 1400–9. Google Scholar 8 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther  1996; 7: 1995–2002. Google Scholar 9 Putzer BM, Hitt M, Muller WJ, Emtage P, Gauldie J, Graham FL. Interleukin 12 and B7–1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression. Proc Natl Acad Sci U S A  1997; 94: 10889–94. Google Scholar 10 Putzer BM, Bramson JL, Addison CL, Hitt M, Siegel PM, Muller WJ, et al. Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer. Hum Gene Ther  1998; 9: 707–18. Google Scholar 11 Emtage PC, Wan Y, Bramson JL, Graham FL, Gauldie J. A double recombinant adenovirus expressing the costimulatory molecule B7–1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model. J Immunol  1998; 160: 2531–8. Google Scholar 12 Addison CA, Gauldie J, Muller WJ, Graham FL. An adenovirus vector expressing interleukin-4 modulates tumorigenicity and induces regression in a murine breast cancer model. Int J Oncol  1995; 7: 1253–60. Google Scholar 13 Naume B, Gately M, Espevik T. A comparative study of IL-12 (cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-induced effects on immunomagnetically purified CD56+ NK cells. J Immunol  1992; 148: 2429–36. Google Scholar 14 Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today  1993; 14: 335–8. Google Scholar 15 Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst  1995; 87: 581–6. Google Scholar 16 Zitvogel L, Robbins PD, Storkus WJ, Clarke MR, Maeurer MJ, Campbell RL, et al. Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors. Eur J Immunol  1996; 26: 1335–41. Google Scholar 17 Chong H, Todryk S, Hutchinson G, Hart IR, Vile RG. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte–macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity. Gene Ther  1998; 5: 223–32. Google Scholar 18 Guckel B, Meyer GC, Rudy W, Batrla R, Meuer SC, Bastert G, et al. Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma. Cancer Gene Ther  1999; 6: 228–37. Google Scholar 19 Kuiper M, Sanches R, Bignon YJ, Farzaneh F. B7.1 and cytokines. Synergy in cancer gene therapy. Adv Exp Med Biol  2000; 465: 381– 90. Google Scholar 20 Wu TC, Huang AY, Jaffee EM, Levitsky HI, Pardoll DM. A reassessment of the role of B7–1 expression in tumor rejection. J Exp Med  1995; 182: 1415–21. Google Scholar 21 Kiessling R, Wasserman K, Horiguchi S, Kono K, Sjoberg J, Pisa P, et al. Tumor-induced immune dysfunction. Cancer Immunol Immunother  1999; 48: 353–62. Google Scholar 22 Gerin JL, Cote PJ, Korba BE, Miller RH, Purcell RH, Tennant BC. Hepatitis B virus and liver cancer: the woodchuck as an experimental model of hepadnavirus-induced liver cancer. In: Hollinger FB, Lemon SM, Margolis H, editors. Viral hepatitis and liver disease. Baltimore (MD): Williams & Wilkins; 1991. p. 556–9. Google Scholar 23 Roggendorf M, Tolle TK. The woodchuck: an animal model for hepatitis B virus infection in man. Intervirology  1995; 38: 100–12. Google Scholar 24 Gouillat C, Manganas D, Zoulim F, Vitrey D, Saguier G, Guillaud M, et al. Woodchuck hepatitis virus-induced carcinoma as a relevant natural model for therapy of human hepatoma. J Hepatol  1997; 26: 1324–30. Google Scholar 25 Toshkov I, Hacker HJ, Roggendorf M, Bannasch P. Phenotypic patterns of preneoplastic and neoplastic hepatic lesions in woodchucks infected with woodchuck hepatitis virus. J Cancer Res Clin Oncol  1990; 116: 581– 90. Google Scholar 26 Graham FL, Smiley J, Russell WC, Nairn R. Characterization of a human cell line transformed by DNA from human adenovirus 5. J Gen Virol  1977; 36: 59–74. Google Scholar 27 Putzer BM, Stiewe T, Crespo F, Esche H. Improved safety through tamoxifen-regulated induction of cytotoxic genes delivered by Ad vectors for cancer gene therapy. Gene Ther  2000; 7: 1317–25. Google Scholar 28 Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol  1995; 3: 207–20. Google Scholar 29 Hitt M, Bett AJ, Prevec L, Graham FL. Construction and propagation of human adenovirus vectors. In: Celis JE, editor. Cell biology: a laboratory handbook. San Diego (CA): Academic Press; 1994. p. 479–90. Google Scholar 30 Drozdzik M, Qian C, Xie X, Peng D, Bilbao R, Mazzolini G, et al. Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma. J Hepatol  2000; 32: 279–86. Google Scholar 31 Mazzolini G, Qian C, Xie X, Sun Y, Lasarte JJ, Drozdzik M, et al. Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12. Cancer Gene Ther  1999; 6: 514–22. Google Scholar 32 Gambotto A, Tuting T, McVey DL, Kovesdi I, Tahara H, Lotze MT, et al. Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12. Cancer Gene Ther  1999; 6: 45–53. Google Scholar 33 Bramson JL, Hitt M, Gauldie J, Graham FL. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Ther  1997; 4: 1069–76. Google Scholar 34 Caruso M, Pham-Nguyen K, Kwong YL, Xu B, Kosai KI, Finegold M, et al. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci U S A  1996; 93: 11302–6. Google Scholar 35 Qin Z, Richter G, Schuler T, Ibe S, Cao X, Blankenstein T. B cells inhibit induction of T cell-dependent tumor immunity. Nat Med  1998; 4: 627–30. Google Scholar 36 Horiguchi S, Petersson M, Nakazawa T, Kanda M, Zea AH, Ochoa AC, et al. Primary chemically induced tumors induce profound immunosuppression concomitant with apoptosis and alterations in signal transduction in T cells and NK cells. Cancer Res  1999; 59: 2950–6. Google Scholar 37 Frommel D, Crevat D, Vitvitsky L, Pichoud C, Hantz O, Chevalier M, et al. Immunopathologic aspects of woodchuck hepatitis. Am J Pathol  1984; 115: 125–34. Google Scholar 38 Buendia MA. Mammalian hepatitis B viruses and primary liver cancer. Semin Cancer Biol  1992; 3: 309–20. Google Scholar 39 Ponzetto A, Forzani B. Animal models of hepatocellular carcinoma: hepadnavirus-induced liver cancer in woodchucks. Ital J Gastroenterol  1991; 23: 491–3. Google Scholar 40 Noguchi Y, Richards EC, Chen YT, Old LJ. Influence of interleukin 12 on p53 peptide vaccination against established Meth A sarcoma. Proc Natl Acad Sci U S A  1995; 92: 2219–23. Google Scholar 41 Zitvogel L, Tahara H, Robbins PD, Storkus WJ, Clarke MR, Nalesnik MA, et al. Cancer immunotherapy of established tumors with IL-12. Effective delivery by genetically engineered fibroblasts. J Immunol  1995; 155: 1393– 403. Google Scholar 42 Tannenbaum CS, Wicker N, Armstrong D, Tubbs R, Finke J, Bukowski R, et al. Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12. J Immunol  1996; 156: 693–9. Google Scholar 43 Michalak TI, Hodgson PD, Churchill ND. Posttranscriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection. J Virol  2000; 74: 4483–94. Google Scholar 44 Fruh K, Yang Y. Antigen presentation by MHC class I and its regulation by interferon gamma. Curr Opin Immunol  1999; 11: 76–81. Google Scholar 45 Willimsky G, Blankenstein T. Interleukin-7/B7.1-encoding adenoviruses induce rejection of transplanted but not nontransplanted tumors. Cancer Res  2000; 60: 685–92. Google Scholar © Oxford University Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI: Journal of the National Cancer Institute Oxford University Press

Large Nontransplanted Hepatocellular Carcinoma in Woodchucks: Treatment With Adenovirus-Mediated Delivery of Interleukin 12/B7.1 Genes

Download PDF
8 pages

Loading next page...
 
/lp/oxford-university-press/large-nontransplanted-hepatocellular-carcinoma-in-woodchucks-treatment-IQpnlt08OM

References (48)

Publisher
Oxford University Press
Copyright
© Oxford University Press
ISSN
0027-8874
eISSN
1460-2105
DOI
10.1093/jnci/93.6.472
Publisher site
See Article on Publisher Site

Abstract

Abstract Background: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. Methods: Large (2–5 cm), established intrahepatic tumors were given an injection once with 1 × 109 plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. Results: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1–2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P = .006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4+ and CD8+ T cells and interferon gamma (IFN γ). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4+ and CD8+ T cells and IFN γ) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (≥95%) 7 weeks after treatment. Conclusion: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans. Among the various strategies used in cancer gene therapy, activation of the immune system by vector-mediated transfer of immunomodulatory genes is a most promising approach because tumor development in immunocompetent hosts suggests that the host's immune system is failing to detect and eliminate malignant cells (1,2). This failure appears to result mainly from the evasive properties of the tumor (3), not from a lack of tumor antigens, because tumor cells transfected with immunomodulatory genes can induce specific antitumor immune responses resulting in tumor rejection (4,5). Adenoviral delivery of genes for cytokines and related immunomodulators, such as interleukin (IL) 2, IL-12, and IL-4, interferon gamma (IFN γ), and B7.1, can efficiently mediate tumor rejection and induce protective systemic immunity in several animal tumor models (6–12). Of these cytokines, IL-12 appears to possess the strongest antitumor activity. The mechanism for IL-12-mediated tumor killing includes the activation of natural killer cells, T cells, lymphokine-activated killer cells, and macrophages (13). IL-12 stimulates IFN γ production from natural killer and T cells, promotes the cellular immune response by facilitating the proliferation and activation of Th1 T-helper cells (14), and inhibits angiogenesis in vivo (15). IL-12 interacts synergistically with the costimulatory molecule B7.1/CD80, normally expressed on professional antigen-presenting cells, to enhance protective antitumor immunity (9,16–19). B7.1-mediated antitumor activity in these studies was largely attributed to the stimulation of natural killer and CD8+ T cells, whereas the requirement for CD4+ cells in tumor rejection is highly dependent on the tumor model (20). Previously, we constructed an adenovirus vector carrying genes for both IL-12 and B7.1, termed “AdIL-12/B7.1,” and demonstrated their synergistic effect on tumor growth by injecting the vector directly into breast adenocarcinomas transplanted from a polyoma middle-T-antigen (PyMT)-transgenic mouse model (9). In tumors derived from the transgenic model, one injection of the vector resulted in complete tumor regression in 70%–90% of the treated animals and induced long-term systemic immunity (9). However, the animal model in this study, as in the majority of cancer immunotherapy trials, is based on transplantation of established tumor cell lines into immunocompetent animals. In contrast, during carcinogenesis, a primary tumor develops from a single cell that becomes malignant in its natural environment (21), thus allowing the development of immune escape mechanisms that may negatively affect immunotherapeutic approaches in humans (21). To address this problem, we tested the ability of the AdIL-12/B7.1 vector to reduce the volume of primary, nontransplanted woodchuck hepatitis virus (WHV)induced hepatocellular carcinomas (HCCs) in woodchucks. WHV is closely related to the human hepatitis B virus in its structure, genomic organization, mechanism of replication, and the course of infection. Persistent WHV infection is associated frequently with the development of hepatic tumors (22). This experimental model has been applied extensively in the study of underlying mechanisms of human hepatitis B virus-induced hepatocarcinogenesis and in the development of prophylactic and therapeutic strategies of disease control (23,24). Materials and Methods Cell Culture The woodchuck liver cell line WH12/6 (25) was maintained in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS). Human MRC5 fibroblasts (cell line CCL 171; American Type Culture Collection, Manassas, VA) were grown in modified Eagle medium plus 10% FCS. All viruses were grown in 293 cells [adenovirus 5 E1-transformed human embryonic kidney cells (26)] maintained in modified Eagle medium/F-11 with 10% FCS. Media were supplemented with 2 mMl-glutamine, penicillin at 100 μg/mL, and streptomycin at 100 U/mL. Animals Adult American woodchucks (Marmota monax) were chronically infected with WHV, which is closely related to human hepatitis B virus. All chronic WHV carriers were provided by Marmotech, Inc., Ithaca, NY, and had one or more hepatic tumors that were 2 cm or larger in diameter, characterized by ultrasound and magnetic resonance imaging (MRI). In this study, a total of five animals have been used. Four animals were treated by intratumoral administration of Ad vectors after laparotomy, and one was given an injection under MRI guidance. Recombinant Adenovirus Vectors Construction of the recombinant adenovirus vector expressing both murine IL-12 subunits in early region 1 (E1) and B7.1 in early region 3 has been described previously (9). The control adenovirus AdEGFP encoding the enhanced green fluorescent protein was described (27). Adenovirus vectors were propagated, purified, and titrated as described previously (28,29). Enzyme-Linked Immunosorbent Assay and Flow Cytometry Analysis Hepatoma cells infected at a multiplicity of infection of 10 plaque-forming units (pfu) per cell were incubated in growth medium as indicated. Expression levels of secreted murine IL-12 were quantitated from infected cell supernatant by an enzyme-linked immunosorbent assay (ELISA) with the use of the mouse IL-12p70 DuoSet ELISA development system (R&D Systems Europe, Oxon, U.K.), according to the manufacturer's protocol. For B7.1 detection, cells were trypsinized, resuspended in ice-cold phosphate-buffered saline (PBS) containing 1% fetal bovine serum, incubated with the fluorescein isothiocyanate-conjugated anti-mouse B7.1 antibody (PharMingen, Hamburg, Germany) for 60 minutes on ice, washed twice in PBS, and analyzed for fluorescence with a FACSVantage flow cytometer (Becton Dickinson Biosciences, Heidelberg, Germany). Human MRC5 fibroblasts were used as a negative control for B7.1 expression because these cells do not react with the mouse B7.1 antibody. Ribonuclease Protection Assay Total RNA was extracted from frozen liver biopsy samples with the RNeasy Mini kit (Qiagen, Hilden, Germany) by following the manufacturer's directions. Ribonuclease protection assays (RPAs) for the analysis of cytokine and T-lymphocyte transcripts were performed with the RiboQuant™ RPA kit (PharMingen, San Diego, CA) according to the instruction manual. The protected RNA bands were quantitated on a Cyclone storage phospho screen imager (Packard, Dreiech, Germany). Intratumoral Administration of Adenovirus Vectors Hepatic tumors were selected preoperatively by ultrasound. Four woodchucks were anesthetized with 1 mL/kg of ketamine (Dr. Graeub, AG, Bern, Switzerland) and 0.3 mL/kg of xylazine (Bayer, Leverkusen, Germany). Median laparotomies were performed, and grossly identifiable tumors were located and measured with calipers before injection of adenovirus vectors and at various times after treatment. The tumor volume was calculated from the longest diameter and average width by assuming a prolate spheroid shape (6). Four woodchucks, each with two or more hepatic tumors that were 2.0 cm or larger in diameter, were treated with a single type of vector as follows: One woodchuck (animal 1) received multiple intratumoral injections (a total of 1 × 109 pfu of AdEGFP in 350 μL of PBS) in separate sites of one tumor nodule. One woodchuck (animal 2) received multiple intratumoral injections (a total of 1 × 109 pfu of AdIL-12/B7.1 in 350 μL of PBS) in separate sites of one tumor nodule. In two woodchucks (animals 3 and 4), two tumor nodules of equal size were treated with multiple intratumoral injections of either AdEGFP or AdIL-12/B7.1; each tumor nodule received a total of 1 × 109 pfu in 350 μL of PBS. Animals were killed on day 7 (animal 3), day 10 (animal 4), or day 14 (animals 1 and 2) after injection; the liver was exposed; and hepatic tumors were measured. Tumor and normal liver tissue samples were fixed in phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin–eosin for histologic examination. Samples of hepatic tumors and non-neoplastic liver were frozen in liquid nitrogen and stored at −70 °C for RPAs. Selective Adenovirus Vector Administration and Tumor Size Assessment by MRI Guidance For each MRI session, the woodchuck was fully anesthetized with ketamine (1 mL/kg) and xylazine (0.3 mL/kg). The tumor was selected, and the injection needle was placed directly into the tumor nodule under guidance by an MRI by use of a 1.5-tesla magnet (Magnetom Sonata; Siemens, Erlangen, Germany). The animal received a single intratumoral injection of 1 × 109 pfu of the AdIL-12/B7.1 vector in 350 μL of PBS. Other tumors identified served as controls. To maximize the signal-to-noise ratio, a standard head coil was used for data reception. The animal underwent axial MRI examinations of the liver with the following imaging parameters: 1) T1-weighted fast low-angle-shot (FLASH) sequence (162-ms/4.0-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 12-second acquisition time); 2) T2-weighted HASTE (half-Fourier-acquisition single-shot turbo-spin-echo) sequence (1000-ms/60-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 20-second acquisition time); and 3) T1-weighted FLASH sequence (162-ms/4.0-ms repetition time/echo time, 256 × 256 matrix, 5-mm slice thickness, 24-cm2 × 14-cm2 field of view, one signal acquired, 12-second acquisition time) with fat-saturation postintravenous application of 1 mL of Gd-diethylenetriaminepentaacetate; Magnevist; Schering, Berlin, Germany). Because tumors were most conspicuous on T1-weighted postcontrast images, this sequence was used to assess tumor volume and thus to monitor the response of tumor treatment. Therefore, region-of-interest measurements were performed on a computer workstation with the region-of-interest size adapted to encompass the tumor contours on the individual image sections. The calculated area was then multiplied by the craniocaudal extension of the tumor. All animal experiments were approved by and carried out according to guidelines set forth by the Animal Research Ethics Board of the University of Essen. Statistical Analysis The statistical significance of the relative increase in tumor size was calculated by paired Student's t test. All statistical tests were two-sided. Results Antitumor Activity of the AdIL-12/B7.1 Vector Against Nontransplanted WHV-Induced HCC In vivo IL-12 gene delivery by adenovirus vectors into established transplanted tumors can inhibit tumor growth and induce tumor regression in various mouse tumor models (8,30–34). We have demonstrated previously (9) that the antitumor activity of IL-12 in transplanted tumors can be increased substantially by also inserting B7.1. When we injected this vector into the transplanted tumors at 2.5 × 107 pfu, a low virus dose, we observed complete tumor regression and induction of systemic immunity in the majority of animals. To determine whether AdIL-12/B7.1 could be used to treat established nontransplanted (orthotopic) tumors, we used the woodchuck animal model. Before the vector was given to woodchucks, however, we infected WH12/6 cells in vitro and determined that murine IL-12 and B7.1 were expressed 3 and 5 days after infection (Fig. 1). Next, we selected large tumors in four woodchucks that were chronic WHV carriers and injected the tumors with AdIL-12/B7.1 or the control vector AdEGFP. The absolute tumor volumes of six tumor nodules, three injected with the AdIL-12/B7.1 virus and three treated with AdEGFP virus, are presented in Table 1 before and 7–14 days after treatment; the absolute tumor volumes of two untreated tumors are also presented after 14 days. On average, over a 2-week period, tumors given an injection intratumorally with AdIL-12/B7.1 showed an 80% reduction in volume from their original volume (Fig. 2, A), whereas untreated and AdEGFP-injected tumors showed an approximately twofold increase in volume (Fig. 2, A). Fig. 2, B, presents representative MRI images from one woodchuck at three representative time points (before treatment and 4 and 7 weeks after treatment), showing the long-term antitumor response after direct intratumoral injection of 1 × 109 pfu of AdIL-12/ B7.1 into a selected liver tumor nodule under MRI guidance. In this experiment, AdIL-12/B7.1 treatment had a pronounced effect on tumor growth over a 7-week period, leading to an approximately 95% reduction in tumor size from 8.6 cm3 at the time of injection to 0.5 cm3 by day 50 (Fig. 2, B-I to B-III). In contrast, no reduction in tumor size was noted in the surrounding untreated tumors. Thus, large established nontransplanted liver tumors can be treated efficiently by injection of an IL-12/B7.1-expressing adenovirus vector. Association of Regression of Primary HCC and an Increase in Immune Effector Cells and IFN γ Within the Tumor Gene transfer studies (5,35,36) in several mouse models have indicated that rejection of transplanted tumors expressing IL-12 and/or B7.1 is dependent on CD4+ and CD8+ T lymphocytes and natural killer cells. To analyze the immune response elicited by injection of AdIL-12/B7.1 into established nontransplanted liver tumors, we next examined liver and tumor biopsy samples from woodchucks 1–4 before and after treatment (Fig. 3, A). Histologic sections from HCCs showed the typical features of neoplastic hepatic cells forming pseudotubulary structures with striking changes in the nuclear morphology, characterized by condensed chromatin and macronucleoli (Fig. 3, A-III). In contrast to nodules injected with the control vector AdEGFP (Fig. 3, A-IV), necrosis and massive inflammatory infiltration were detected only in AdIL-12/B7.1-treated tumors (Fig. 3, A-V). In the nontumoral liver parenchyma, we found typical morphologic patterns of chronic hepatitis after persistent WHV infection, characterized by moderate fibrosis and mild mononuclear infiltrate of portal or periportal tracts with occasional necrosis [(37) and Fig. 3, A-I]. However, no evidence of adenovirus- induced hepatic toxicity was observed in normal liver tissue 10 days after intratumoral vector administration (Fig. 3, A-II). The histologic results were extended by RPA of liver and tumor biopsy samples obtained from woodchucks 1–4 (Fig. 3, B). Compared with the levels of T-lymphocyte-specific transcripts in hepatic tumors before treatment, RPA revealed an influx of CD4+ and CD8+ T cells into AdIL-12/B7.1-injected tumors (Fig. 3, B-II) that occurred at the peak of rejection. Compared with before-treatment samples, CD4+ lymphocytes showed an approximately sevenfold increase and CD8+ lymphocytes showed a moderate but important twofold increase. Moreover, AdIL-12/B7.1-injected HCC tissue showed an approximately twofold increase in the levels of IFN γ, the major determinant of the IL-12-mediated antitumor response. Thus, murine IL-12 expressed from an adenovirus vector is functional in woodchuck liver tumors. In contrast, no increase in CD4+ or CD8+ T cells or IFN γ occurred in tumors injected with control virus (Fig. 3, B-I) or in nontumoral liver samples (data not shown). Discussion Previously, we showed that intratumoral injection of an adenovirus coexpressing IL-12 and B7.1 is an extremely efficient method of inducing the rejection of transplanted PyMT-induced tumors (9). To determine whether it is also active against primary tumors, we selected the well-characterized model of WHV-induced HCC in woodchucks. This model is clinically important because WHV is one of the most efficient hepatocarcinogens in woodchucks (38). WHV-induced hepatocarcinogenesis shows strong similarity to hepadnavirus-induced carcinogenesis in humans (23,39). Because our previous data indicated that AdIL-12/B7.1-injected murine tumors were effectively rejected within approximately 2 weeks, the immune response against nontransplanted woodchuck liver tumors was first analyzed shortly after treatment. Consistent with the results obtained from the PyMT model (9), much of the tumor mass was rejected by day 14, suggesting that AdIL-12/B7.1 had a similar inhibitory effect on growth of transplanted and nontransplanted tumors at this early stage of treatment. The use of MRI guidance allowed us to locate and select large tumor nodules into which we could directly inject the vector, thereby avoiding the negative and stressful effects of surgery. MRI monitoring of a selected tumor revealed long-term tumor regression, resulting in nearly complete tumor elimination. However, there was a clear difference in the time course of tumor reduction between the tumor injected at one site under MRI guidance and the tumors injected at multiple sites during laparotomy. In murine models, intratumoral injection of adenovirus vectors carrying IL-12 and/or B7.1 can stimulate systemic immunity that can act effectively on distal tumors (7–9). No clear systemic effect, however, has been observed in the woodchuck model, which may reflect a variation in the immunogenicity of different tumor nodules (Roggendorf M: unpublished data). Because these tumors are not of clonal origin and might express different tumor antigens, an effect on surrounding tumors might be obtained when vascular anastomoses allow the virus to spread between the tumors. Therefore, a lack of anastomoses between the tumor and the normal liver in this model might contribute to the lack of systemic response. In addition, higher doses of cytokine vectors were more efficient in inhibiting tumor growth than lower doses (9,30,40). If the body weight of a woodchuck and the tumor size of 2–5 cm in diameter are considered, the virus dose of 1 × 109 pfu, used for woodchucks, is low compared with doses used in mice. Therefore, we cannot exclude the possibility that the IL-12/B7.1 virus might induce systemic immunity in primary cancer at higher vector doses. Our results suggest that regression of WHV-induced primary liver tumors is associated with massive intratumoral lymphocyte infiltration. We also show that intratumoral adenoviral IL-12/B7.1 expression in primary HCCs leads to increased intratumoral levels of IFN γ, consistent with results from a number of models indicating that the antitumor activity of IL-12 is IFN γ dependent (8,41,42). From recent observations, expression of major histocompatibility complex (MHC) class I molecules is altered in liver cells of animals chronically infected with WHV (43) and also commonly reduced on neoplastic cells of HCC nodules (Roggendorf M: unpublished data). Consequently, this reduction in MHC molecules may diminish the susceptibility of WHV-infected hepatocytes to virus-specific T cells, WHV-infected hepatocytes may evade the antiviral immunologic surveillance system, and thus liver damage may be perpetuated. The current model emphasizes the role of IFN γ for the modulation of peptide processing and stimulation of genes involved in antigen presentation by the MHC class I molecules (44). Thus, the activation of MHC class I-associated genes by local induction of IFN γ through tumor-infiltrating T lymphocytes may be a possible mechanism for efficient tumor rejection after IL-12/B7.1 immunotherapy in this model. However, our observation varies from the observations made in established nontransplanted 3-MC-induced mouse tumors after IL-7/B7.1 gene delivery (45). In that model, failure to induce tumor regression depends on the inability of T cells to infiltrate nontransplanted tumors. The observed difference might result from a difference in T-cell infiltration of tumors induced by viruses or by carcinogens rather than a general difference between transplanted and primary, nontransplanted malignant tumors. The animal model used in this study is expensive and difficult to use, and only a limited number of tumor-bearing WHV-infected woodchucks were available. Nevertheless, our results demonstrate that adenovirus vector-mediated immunotherapy is certainly not limited to artificial mouse tumor models and provide a powerful approach for the in vivo treatment of large, primary tumors in a model of hepadnavirus-induced carcinogenesis, reflecting the real-world scenario of HCC in humans. Direct injection into selected HCC nodules under MRI guidance appears to be a relatively nontoxic and feasible method for administrating viral vectors in human clinical trials. However, our data emphasize the need to evaluate the mechanisms involved in effective intratumoral penetration by immune effector cells as a prerequisite for successful cytokine treatment of natural tumors. Table 1. Tumor volume of established primary hepatocellular carcinomas after AdIL-12/B7.1 treatment Animal*  Treatment  Volume before treatment, cm3  Volume after treatment, cm3  Time, d†  *In woodchucks 3 and 4, one selected tumor nodule was treated with AdEGFP and one with AdIL-12/B7.1.  †Tumor size was determined at 7, 10, and 14 days after treatment.  3  AdEGFP  23.5  35.6  7  3  AdIL-12/B7.1  29.4  11.8  7  4  AdEGFP  2.8  7.45  10  4  AdIL-12/B7.1  37.8  6.8  10  1  AdEGFP  2.9  5.9  14  1  Untreated  5.9  10.6  14  2  AdIL-12/B7.1  3.6  0.25  14  2  Untreated  1.3  4.2  14  Animal*  Treatment  Volume before treatment, cm3  Volume after treatment, cm3  Time, d†  *In woodchucks 3 and 4, one selected tumor nodule was treated with AdEGFP and one with AdIL-12/B7.1.  †Tumor size was determined at 7, 10, and 14 days after treatment.  3  AdEGFP  23.5  35.6  7  3  AdIL-12/B7.1  29.4  11.8  7  4  AdEGFP  2.8  7.45  10  4  AdIL-12/B7.1  37.8  6.8  10  1  AdEGFP  2.9  5.9  14  1  Untreated  5.9  10.6  14  2  AdIL-12/B7.1  3.6  0.25  14  2  Untreated  1.3  4.2  14  View Large Fig. 1. View large Download slide View large Download slide Assessment of interleukin 12 (IL-12)/B7.1 production in vitro. Expression kinetics of murine IL-12 (A) and B7.1 (B) in WH12/6 cells before and 1, 3, and 5 days after infection with the adenovirus-based vector AdIL12-B7.1 at a multiplicity of infection of 10 is shown. The murine IL-12 (mIL-12) concentration in supernatant was determined by enzyme-linked immunosorbent assay by use of the anti-mouse IL-12p70 antibody. All samples were assayed in triplicate. The 95% confidence intervals are indicated. For B7.1 detection, cells were stained with fluorescein isothiocyanate-conjugated anti-mouse B7–1 antibody and analyzed by flow cytometry; uninfected WH12/6 cells were used as controls (center peak). Compared with the negative control (human MRC-5 fibroblasts, left peak), uninfected WH12/6 cells (center peak) scored almost negative for B7.1 expression (4% after 72 hours). B7.1-positive cells (right peak), labeled M1, were 65% of the total cells on day 1, 92% on day 3, and 95% on day 5. FL1 = fluorescence channel 1 (green). Fig. 1. View large Download slide View large Download slide Assessment of interleukin 12 (IL-12)/B7.1 production in vitro. Expression kinetics of murine IL-12 (A) and B7.1 (B) in WH12/6 cells before and 1, 3, and 5 days after infection with the adenovirus-based vector AdIL12-B7.1 at a multiplicity of infection of 10 is shown. The murine IL-12 (mIL-12) concentration in supernatant was determined by enzyme-linked immunosorbent assay by use of the anti-mouse IL-12p70 antibody. All samples were assayed in triplicate. The 95% confidence intervals are indicated. For B7.1 detection, cells were stained with fluorescein isothiocyanate-conjugated anti-mouse B7–1 antibody and analyzed by flow cytometry; uninfected WH12/6 cells were used as controls (center peak). Compared with the negative control (human MRC-5 fibroblasts, left peak), uninfected WH12/6 cells (center peak) scored almost negative for B7.1 expression (4% after 72 hours). B7.1-positive cells (right peak), labeled M1, were 65% of the total cells on day 1, 92% on day 3, and 95% on day 5. FL1 = fluorescence channel 1 (green). Fig. 2. View large Download slide View large Download slide Short- and long-term antitumor efficacy of AdIL-12/B7.1 on woodchuck hepatitis virus-induced primary liver tumors. Panel A: selected tumor nodules 2–5 cm in diameter were intratumorally injected with 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1 or the control virus AdEGFP (three tumors for each vector). The animals were killed within 14 days after treatment and were monitored for tumor regression relative to untreated tumors. The average increase in tumor volume is shown (shaded bars). Tumor volume before treatment was set as 1 (solid bars). The 95% confidence intervals (CIs) are indicated by error bars. The difference in the relative increase of the size of AdEGFP- versus AdIL12/B7.1-treated tumors was statistically significant (*) with P = .006: AdEGFP (2.1-fold [95% CI = 1.44-fold to 2.76-fold]; P = .084) versus AdIL-12/B7.1 (0.22-fold [95% CI = 0.03-fold to 0.41-fold]; P = .015). The absolute volumes from each injected tumor are shown in Table 1. Panel B: T1-weighted postcontrast magnetic resonance images of one representative woodchuck liver show neoplastic tumor nodules. One tumor indicated with white arrowheads was given a direct injection of 1 × 109 pfu of AdIL-12/B7.1 under magnetic resonance imaging guidance. The treated tumor is shown before (B-I) and 4 (B-II) and 7 (B-III) weeks after a single vector administration. Reductions in tumor size of 43% (4 weeks after injection) to 95% (7 weeks after injection) are shown in images B-II and B-III, respectively. Growth of the surrounding untreated nodules of equivalent size was unchanged. Fig. 2. View large Download slide View large Download slide Short- and long-term antitumor efficacy of AdIL-12/B7.1 on woodchuck hepatitis virus-induced primary liver tumors. Panel A: selected tumor nodules 2–5 cm in diameter were intratumorally injected with 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1 or the control virus AdEGFP (three tumors for each vector). The animals were killed within 14 days after treatment and were monitored for tumor regression relative to untreated tumors. The average increase in tumor volume is shown (shaded bars). Tumor volume before treatment was set as 1 (solid bars). The 95% confidence intervals (CIs) are indicated by error bars. The difference in the relative increase of the size of AdEGFP- versus AdIL12/B7.1-treated tumors was statistically significant (*) with P = .006: AdEGFP (2.1-fold [95% CI = 1.44-fold to 2.76-fold]; P = .084) versus AdIL-12/B7.1 (0.22-fold [95% CI = 0.03-fold to 0.41-fold]; P = .015). The absolute volumes from each injected tumor are shown in Table 1. Panel B: T1-weighted postcontrast magnetic resonance images of one representative woodchuck liver show neoplastic tumor nodules. One tumor indicated with white arrowheads was given a direct injection of 1 × 109 pfu of AdIL-12/B7.1 under magnetic resonance imaging guidance. The treated tumor is shown before (B-I) and 4 (B-II) and 7 (B-III) weeks after a single vector administration. Reductions in tumor size of 43% (4 weeks after injection) to 95% (7 weeks after injection) are shown in images B-II and B-III, respectively. Growth of the surrounding untreated nodules of equivalent size was unchanged. Fig. 3. View large Download slide View large Download slide Infiltration of immune effector cells in AdIL-12/B7.1-injected primary liver tumors. Panel A: representative liver and tumor sections from one woodchuck before (A-I and A-III) and after (A-II, A-IV, and A-V) treatment. One tumor nodule was given an injection of 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1, and one tumor nodule was given an injection of 1 × 109 pfu of the control vector AdEGFP. The animal was killed 10 days later and analyzed for treatment-associated morphologic changes. Liver parenchyma of chronic hepatitis after persistent woodchuck hepatitis virus infection before (A-I) and after (A-II) intratumoral injection are observed. No morphologic signs of adenovirus vector-related hepatic injury were detected. Pseudotubulary structures of neoplastic hepatocytes from untreated hepatocellular carcinoma (HCC) (A-III) and after injection with AdEGFP (A-IV) or AdIL-12/B7.1 (A-V) are shown. Several areas of necrosis and massive inflammatory infiltration are shown in A-V. (Hematoxylin–eosin staining. Scale bars = 100 μm.) Panel B: analysis of T-lymphocyte and interferon gamma (IFN gamma) messenger RNA (mRNA) levels by ribonuclease protection assay. The RNA was obtained from liver tumor biopsy samples before and 10 days after intratumoral injection of the control virus AdEGFP (I) or AdIL-12/B7.1 (II). The average increases in CD4, CD8, and IFN gammaγ mRNA levels after each treatment are presented. The mRNA levels before treatment were set as 1 for each parameter. Results are reported as the mean ± 95% confidence interval. Fig. 3. View large Download slide View large Download slide Infiltration of immune effector cells in AdIL-12/B7.1-injected primary liver tumors. Panel A: representative liver and tumor sections from one woodchuck before (A-I and A-III) and after (A-II, A-IV, and A-V) treatment. One tumor nodule was given an injection of 1 × 109 plaque-forming units (pfu) of AdIL-12/B7.1, and one tumor nodule was given an injection of 1 × 109 pfu of the control vector AdEGFP. The animal was killed 10 days later and analyzed for treatment-associated morphologic changes. Liver parenchyma of chronic hepatitis after persistent woodchuck hepatitis virus infection before (A-I) and after (A-II) intratumoral injection are observed. No morphologic signs of adenovirus vector-related hepatic injury were detected. Pseudotubulary structures of neoplastic hepatocytes from untreated hepatocellular carcinoma (HCC) (A-III) and after injection with AdEGFP (A-IV) or AdIL-12/B7.1 (A-V) are shown. Several areas of necrosis and massive inflammatory infiltration are shown in A-V. (Hematoxylin–eosin staining. Scale bars = 100 μm.) Panel B: analysis of T-lymphocyte and interferon gamma (IFN gamma) messenger RNA (mRNA) levels by ribonuclease protection assay. The RNA was obtained from liver tumor biopsy samples before and 10 days after intratumoral injection of the control virus AdEGFP (I) or AdIL-12/B7.1 (II). The average increases in CD4, CD8, and IFN gammaγ mRNA levels after each treatment are presented. The mRNA levels before treatment were set as 1 for each parameter. Results are reported as the mean ± 95% confidence interval. Editor's note: B. Tennant owns an equity interest in Marmotech, Inc., Ithaca, NY, the company that donated the woodchucks used in this study. Supported in part by grant PU188/2–1 from the Deutsche Forschungsgemeinschaft and by the Internal Research Support Program from the University of Essen, Germany (to B. M. Pützer). We thank Silke Bosk for excellent technical assistance in performing the magnetic resonance tomography, Li Jun for surgical assistance, and Michael Lowak for help in animal handling. References 1 Chouaib S, Asselin-Paturel C, Mami-Chouaib F, Caignard A, Blay JY. The host–tumor immune conflict: from immunosuppression to resistance and destruction. Immunol Today  1997; 18: 493–7. Google Scholar 2 Pettit SJ, Seymour K, O'Flaherty E, Kirby JA. Immune selection in neoplasia: towards a microevolutionary model of cancer development. Br J Cancer  2000; 82: 1900–6. Google Scholar 3 Gilboa E. How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother  1999; 48: 382–5. Google Scholar 4 Davidoff AM, Kimbrough SA, Ng CY, Shochat SJ, Vanin EF. Neuroblastoma regression and immunity induced by transgenic expression of interleukin-12. J Pediatr Surg  1999; 34: 902–6; discussion 906–7. Google Scholar 5 Sun Y, Qian C, Peng D, Prieto J. Gene transfer to liver cancer cells of B7–1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone. Hum Gene Ther  2000; 11: 127–38. Google Scholar 6 Addison CL, Braciak T, Ralston R, Muller WJ, Gauldie J, Graham FL. Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in murine breast cancer model. Proc Natl Acad Sci U S A  1995; 92: 8522–6. Google Scholar 7 Addison CL, Bramson JL, Hitt MM, Muller WJ, Gauldie J, Graham FL. Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors. Gene Ther  1998; 5: 1400–9. Google Scholar 8 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther  1996; 7: 1995–2002. Google Scholar 9 Putzer BM, Hitt M, Muller WJ, Emtage P, Gauldie J, Graham FL. Interleukin 12 and B7–1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression. Proc Natl Acad Sci U S A  1997; 94: 10889–94. Google Scholar 10 Putzer BM, Bramson JL, Addison CL, Hitt M, Siegel PM, Muller WJ, et al. Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer. Hum Gene Ther  1998; 9: 707–18. Google Scholar 11 Emtage PC, Wan Y, Bramson JL, Graham FL, Gauldie J. A double recombinant adenovirus expressing the costimulatory molecule B7–1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model. J Immunol  1998; 160: 2531–8. Google Scholar 12 Addison CA, Gauldie J, Muller WJ, Graham FL. An adenovirus vector expressing interleukin-4 modulates tumorigenicity and induces regression in a murine breast cancer model. Int J Oncol  1995; 7: 1253–60. Google Scholar 13 Naume B, Gately M, Espevik T. A comparative study of IL-12 (cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-induced effects on immunomagnetically purified CD56+ NK cells. J Immunol  1992; 148: 2429–36. Google Scholar 14 Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today  1993; 14: 335–8. Google Scholar 15 Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst  1995; 87: 581–6. Google Scholar 16 Zitvogel L, Robbins PD, Storkus WJ, Clarke MR, Maeurer MJ, Campbell RL, et al. Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors. Eur J Immunol  1996; 26: 1335–41. Google Scholar 17 Chong H, Todryk S, Hutchinson G, Hart IR, Vile RG. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte–macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity. Gene Ther  1998; 5: 223–32. Google Scholar 18 Guckel B, Meyer GC, Rudy W, Batrla R, Meuer SC, Bastert G, et al. Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma. Cancer Gene Ther  1999; 6: 228–37. Google Scholar 19 Kuiper M, Sanches R, Bignon YJ, Farzaneh F. B7.1 and cytokines. Synergy in cancer gene therapy. Adv Exp Med Biol  2000; 465: 381– 90. Google Scholar 20 Wu TC, Huang AY, Jaffee EM, Levitsky HI, Pardoll DM. A reassessment of the role of B7–1 expression in tumor rejection. J Exp Med  1995; 182: 1415–21. Google Scholar 21 Kiessling R, Wasserman K, Horiguchi S, Kono K, Sjoberg J, Pisa P, et al. Tumor-induced immune dysfunction. Cancer Immunol Immunother  1999; 48: 353–62. Google Scholar 22 Gerin JL, Cote PJ, Korba BE, Miller RH, Purcell RH, Tennant BC. Hepatitis B virus and liver cancer: the woodchuck as an experimental model of hepadnavirus-induced liver cancer. In: Hollinger FB, Lemon SM, Margolis H, editors. Viral hepatitis and liver disease. Baltimore (MD): Williams & Wilkins; 1991. p. 556–9. Google Scholar 23 Roggendorf M, Tolle TK. The woodchuck: an animal model for hepatitis B virus infection in man. Intervirology  1995; 38: 100–12. Google Scholar 24 Gouillat C, Manganas D, Zoulim F, Vitrey D, Saguier G, Guillaud M, et al. Woodchuck hepatitis virus-induced carcinoma as a relevant natural model for therapy of human hepatoma. J Hepatol  1997; 26: 1324–30. Google Scholar 25 Toshkov I, Hacker HJ, Roggendorf M, Bannasch P. Phenotypic patterns of preneoplastic and neoplastic hepatic lesions in woodchucks infected with woodchuck hepatitis virus. J Cancer Res Clin Oncol  1990; 116: 581– 90. Google Scholar 26 Graham FL, Smiley J, Russell WC, Nairn R. Characterization of a human cell line transformed by DNA from human adenovirus 5. J Gen Virol  1977; 36: 59–74. Google Scholar 27 Putzer BM, Stiewe T, Crespo F, Esche H. Improved safety through tamoxifen-regulated induction of cytotoxic genes delivered by Ad vectors for cancer gene therapy. Gene Ther  2000; 7: 1317–25. Google Scholar 28 Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol  1995; 3: 207–20. Google Scholar 29 Hitt M, Bett AJ, Prevec L, Graham FL. Construction and propagation of human adenovirus vectors. In: Celis JE, editor. Cell biology: a laboratory handbook. San Diego (CA): Academic Press; 1994. p. 479–90. Google Scholar 30 Drozdzik M, Qian C, Xie X, Peng D, Bilbao R, Mazzolini G, et al. Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma. J Hepatol  2000; 32: 279–86. Google Scholar 31 Mazzolini G, Qian C, Xie X, Sun Y, Lasarte JJ, Drozdzik M, et al. Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12. Cancer Gene Ther  1999; 6: 514–22. Google Scholar 32 Gambotto A, Tuting T, McVey DL, Kovesdi I, Tahara H, Lotze MT, et al. Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12. Cancer Gene Ther  1999; 6: 45–53. Google Scholar 33 Bramson JL, Hitt M, Gauldie J, Graham FL. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Ther  1997; 4: 1069–76. Google Scholar 34 Caruso M, Pham-Nguyen K, Kwong YL, Xu B, Kosai KI, Finegold M, et al. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci U S A  1996; 93: 11302–6. Google Scholar 35 Qin Z, Richter G, Schuler T, Ibe S, Cao X, Blankenstein T. B cells inhibit induction of T cell-dependent tumor immunity. Nat Med  1998; 4: 627–30. Google Scholar 36 Horiguchi S, Petersson M, Nakazawa T, Kanda M, Zea AH, Ochoa AC, et al. Primary chemically induced tumors induce profound immunosuppression concomitant with apoptosis and alterations in signal transduction in T cells and NK cells. Cancer Res  1999; 59: 2950–6. Google Scholar 37 Frommel D, Crevat D, Vitvitsky L, Pichoud C, Hantz O, Chevalier M, et al. Immunopathologic aspects of woodchuck hepatitis. Am J Pathol  1984; 115: 125–34. Google Scholar 38 Buendia MA. Mammalian hepatitis B viruses and primary liver cancer. Semin Cancer Biol  1992; 3: 309–20. Google Scholar 39 Ponzetto A, Forzani B. Animal models of hepatocellular carcinoma: hepadnavirus-induced liver cancer in woodchucks. Ital J Gastroenterol  1991; 23: 491–3. Google Scholar 40 Noguchi Y, Richards EC, Chen YT, Old LJ. Influence of interleukin 12 on p53 peptide vaccination against established Meth A sarcoma. Proc Natl Acad Sci U S A  1995; 92: 2219–23. Google Scholar 41 Zitvogel L, Tahara H, Robbins PD, Storkus WJ, Clarke MR, Nalesnik MA, et al. Cancer immunotherapy of established tumors with IL-12. Effective delivery by genetically engineered fibroblasts. J Immunol  1995; 155: 1393– 403. Google Scholar 42 Tannenbaum CS, Wicker N, Armstrong D, Tubbs R, Finke J, Bukowski R, et al. Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12. J Immunol  1996; 156: 693–9. Google Scholar 43 Michalak TI, Hodgson PD, Churchill ND. Posttranscriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection. J Virol  2000; 74: 4483–94. Google Scholar 44 Fruh K, Yang Y. Antigen presentation by MHC class I and its regulation by interferon gamma. Curr Opin Immunol  1999; 11: 76–81. Google Scholar 45 Willimsky G, Blankenstein T. Interleukin-7/B7.1-encoding adenoviruses induce rejection of transplanted but not nontransplanted tumors. Cancer Res  2000; 60: 685–92. Google Scholar © Oxford University Press

Journal

JNCI: Journal of the National Cancer Institute – Oxford University Press

Published: Mar 21, 2001

There are no references for this article.