Tumor irradiation followed by intratumoral cytokine gene ... - Nature

Cancer Gene Therapy (2004) 11, 61–72 All rights reserved 0929-1903/04 $25.00

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Tumor irradiation followed by intratumoral cytokine gene therapy for murine renal adenocarcinoma Gilda G Hillman,1 Philippe Slos,2 Yu Wang,1 Jennifer L Wright,1 Andrey Layer,1 Micael De Meyer,2 Mark Yudelev,1 Mingxin Che, 3 and Jeffrey D Forman1 1

Department of Radiation Oncology, Barbara Ann Karmanos Cancer Institute at Wayne State University School of Medicine and Harper Hospital, Detroit, Michigan 48201, USA; 2Transgene SA, 11 Rue de Molsheim, Strasbourg 67085, France; and 3Department of Pathology, Barbara Ann Karmanos Cancer Institute at Wayne State University School of Medicine and Harper Hospital, Detroit, Michigan 48201, USA. To circumvent the toxicity caused by systemic injection of cytokines, cytokine cDNA genes encoding the human interleukin IL-2 cDNA (Ad-IL-2) and murine interferon IFN-g gene (Ad- IFN-g) were inserted into adenoviral vectors. These constructs were used for intratumoral gene therapy of murine renal adenocarcinoma Renca tumors. Treatment with three doses of Ad-IL-2 or Ad- IFN-g, given a day apart, was more effective than single-dose gene therapy. We found that tumor irradiation enhanced the therapeutic efficacy of Ad-IL-2 and Ad-IFN-g intratumoral gene therapy. Tumor irradiation, administered 1 day prior to three doses of Ad-IL-2 treatment, was more effective than radiation or Ad-IL-2 alone, resulting in tumor growth arrest in all mice, increased survival and a consistent increase in complete tumor regression response rate. Complete responders rejected Renca tumor challenge and demonstrated specific cytotoxic T-cell activity, indicative of specific tumor immunity. The effect of radiation combined with three doses of Ad-IFN-g was less pronounced and did not lead to tumor immunity. Histological observations showed that irradiation of the tumor prior to gene therapy increased tumor destruction and inflammatory infiltrates in the tumor nodules. These findings demonstrate that tumor irradiation improves the efficacy of Ad-IL-2 gene therapy for induction of antitumor immune response. Cancer Gene Therapy (2004) 11, 61–72. doi:10.1038/sj.cgt.7700656 Keywords: adenovirus; IL-2; IFN-c; radiation; renal carcinoma

enal cell carcinoma (RCC) incidence has increased in recent years with approximately 31, 200 new cases R each year in the USA. This increased RCC incidence may 1

be linked to certain risk factors including smoking, obesity, high protein diets and hypertension.2,3 The disease is responsible for an estimated 12, 000 deaths each year.1 Nearly half of the patients present only with localized disease that can be treated by surgical removal.2,4,5 However, one third of the patients also present with metastatic disease and half of the patients treated for localized carcinomas subsequently develop metastatic disease.2,4 The median survival of patients with metastases is only 8 months, with a 5-year survival rate of less than 10%.2,4 Patients with metastatic RCC frequently present with pulmonary metastases that are poorly responsive to conventional treatment including most chemotherapeutic drugs, hormones and radiation therReceived April 29, 2003.

Adress correspondence and reprint requests to: Dr GG Hillman, Ph.D., Department of Radiation Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Hudson Weber Bldg, Room 515, 4100 John R., Detroit, MI 48201, USA. E-mail: [email protected]

This work was supported by Transgene SA

apy.2,4,5 The treatment of metastatic disease has been and remains a difficult clinical challenge. Immunotherapy is an alternate systemic approach for the treatment of metastatic cancers that is based on activation of a host immune response against the tumor cells using cytokines or activated immune cells.6 Cytokines are used in immunotherapy to enhance immune mechanisms directed against tumors.7 Cytokines are proteins produced following activation of lymphocytes, macrophages or other cells. Cytokines can induce a cascade of activation, proliferation and differentiation of immune cells and mount an antitumor immune response. The cytokine/lymphokine IL-2, produced by activated T lymphocytes, is critical for immune responsiveness and has been extensively used in clinical trials.2,4,8 Metastatic RCC can be treated with immunotherapy with interferons (IFN) and/or interleukin-2 (IL-2) with response rates of 15-27%.2,4,8 High dose of recombinant IL-2 is approved for the treatment of metastatic RCC but is associated with significant toxicity limiting its use to selected patients.4,8 Using various models of the Renca murine renal adenocarcinoma, we have investigated the interaction between local tumor irradiation and systemic IL-2 immunotherapy. In the kidney tumor model, a greater therapeutic effect was demonstrated on the primary

Radiation and gene therapy for murine renal carcinoma GG Hillman et al

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tumor and distant metastases by local irradiation of the tumor-bearing kidney followed by IL-2 therapy than with each modality by itself.9 In the pulmonary metastases model, irradiation of the left lung followed by systemic IL-2 therapy resulted in increased tumor reduction in both lungs, suggesting that radiation enhances the systemic effect of IL-2.10 We demonstrated the requirement for T cell and NK cell function in the mechanism of action of the combined therapy.10 In histological studies of lung tumor nodules, we found that radiation caused tumor cell apoptosis but also vascular damage allowing for an influx of macrophages and mononuclear cells in the tumor nodules and surrounding tissues.10,11 The combination of both therapies induced a greater vascular damage and massive infiltration of immune cells that could play a role in tumor destruction.10,11 Our data in the Renca models suggest that radiation therapy causes changes in the tumor cells and the tumor environment, which increase the tumor susceptibility to destruction by the immune system activated by IL-2. Evaluation of IL-4 and IFN-g in the Renca pulmonary metastases model showed that systemic treatment of either cytokine induced regression of pulmonary metastases in a dose-dependent manner and increased mouse survival.12,13 To circumvent the toxicity caused by systemic injection of cytokines, cytokine cDNA genes were inserted into adenoviral vectors or plasmid constructs for intratumoral gene therapy.14–20 We and others have shown that this approach can lead to tumor regression and induction of antitumor immune response.14–20 We have constructed E1/E3-deleted replication defective vectors encoding the human IL-2 cDNA under the control of RSV (Ad-pRSVIL-2) or CMV (Ad-pCMV-IL-2) promoters.20 We showed that intratumoral injection of Ad-pCMV-IL-2 in P815 mastocytoma tumors in B6D2 mice caused a greater increase in survival and tumor regression than that observed with Ad-pRSV-IL-2. Ad-pCMV-IL-2 intratumoral injections resulted in a higher production of IL-2 in the tumor than with Ad-pRSV-IL-2. Tumor-free mice following Ad-pCMV-IL-2 treatment exhibited systemic immunity and specific T-cell activity.20 Therefore, this construct was selected for subsequent studies in the Renca model. Based on our findings of increased efficacy of systemic IL-2 therapy by prior tumor irradiation in the Renca model and induction of systemic immunity by intratumoral injections of Ad-pCMV-IL-2 in P815 tumors, we have now investigated the combination of tumor irradiation with localized gene therapy delivered by intratumoral administration of Ad-pCMV-IL-2 in the Renca model. The effect of a new adenoviral IFN-g construct containing a CMV promoter for intratumoral gene therapy as a single modality or combined with tumor irradiation was also evaluated in Renca tumors. We found that radiation enhanced the therapeutic efficacy of Ad-IL-2 and Ad-IFN-g intratumoral gene therapy. Tumor irradiation, administered 1 day prior to initiation of Ad-IL-2 treatment, given over three consecutive days, was more effective than radiation alone or Ad-IL-2 gene therapy alone, resulting in tumor growth arrest in all mice

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and an increase in mice responding with complete tumor regression. These mice rejected Renca tumor challenge and showed T-cell activity, indicative of specific tumor immunity. The effect of radiation combined with three doses of Ad-IFN-g was not as pronounced as that observed with radiation combined with Ad-IL-2 and did not lead to tumor immunity.

Materials and methods

Tumor model Renca, a murine RCC line, of spontaneous origin in a Balb/c mouse was maintained in vivo by serial intraperitoneal (i.p.) or subcutaneous (s.c.) passages.9,10 Renca cells were also cultured in vitro in culture medium (CM) consisting of RPMI 1640 medium (Gibco Laboratories, Grand Island, NY), supplemented with 10% heatinactivated newborn bovine serum (NBS) (Gibco), 2 mM glutamine, 100U/ml penicillin G, 100 mg/ml streptomycin, 0.5 mg/ml fungizone, 50 mg/ml gentamicin (Gibco), 1mM sodium pyruvate (Sigma, St Louis, MO), 0.1 mM nonessential amino acids (Gibco), and 10 mM Hepes buffer.10 For in vivo implantation, Renca cells were washed in Hank’s balanced salt solution (HBSS) and injected s.c. at 2 105 cells in 0.1 ml HBSS, in 4–6-weekold female Balb/c mice (Harlan Sprague Dawley Inc, Indianapolis, IN). To fit the radiation apparatus, cells were injected in the middle of the back, at 1.5 cm from the tail. Mice were shaved prior to injection for accurate location of the injection site and for monitoring tumor growth. Mice were housed and handled in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care. The animal protocol was approved by Wayne State University Animal Investigation Committee.

Adenoviral vectors All viral genomes were constructed as infectious plasmids by homologous recombination in Escherichia coli as previously described.21 The vectors AdTG13383 and AdTG14254 contain the cDNA for human IL-2 (Ad-IL2) and murine IFN-g (Ad-IFN-g), respectively, under the control of the human cytomegalovirus immediateearly enhancer/promoter region and the simian virus 40 late (SV40) or bovine growth hormone polyadenylation sequences in an adenovirus type 5 backbone (Ad5), which contains a deletion in E1 from nucleotide (nt) 455 to 3511 and in E3 from nt 28593 to 30470, according to the nucleotide numbering of Chroboczek.22 In this background (DE1DE3), the vector AdTG6401 without transgene served as internal negative control (Ad-null). The E1/E3-deleted vectors were grown on PER.C6.23 Virus propagation, purification and titration of infectious units (i.u./ml) by indirect immunofluorescence of the DNA binding protein (DBP) were performed as previously described.24 The number of total particles (TP) was measured using anion exchange HPLC.


Transduction of Renca cells with Ad-IFN-g in vitro: expression of MHC molecules by immunofluorescence Renca cells were plated at 2 105 cells/well in six-well plates. After 24 hours incubation at 371C in 5% CO2 incubator, cells were washed twice with PBS and Ad-IFNg vector was added in 0.5 ml PBS at different multiplicity of infection (m.o.i). Cells were infected for 1 hour at 371C and 5.5 ml CM was added. Cells were further incubated for 48–96 hours and tested for expression of MHC molecules by immunofluorescence. Cells were washed with HBSS and removed using 1:5000 versene. The cells were washed twice with HBSS. For cell surface expression of MHC Class I and Class II molecules, cells were labeled with fluorescein isothiocyanate (FITC)-conjugated antibodies. Anti-mouse H-2Kd monoclonal antibodies (mAb) directed against MHC Class I alloantigen or anti-mouse IA/I-E mAb specific to MHC Class II alloantigens (Pharmingen, San Diego, CA) were used in a direct immunofluorescence assay.13 Cells were labeled for 45 minutes on ice, then washed twice in HBSS. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Gates were set for nonspecific binding using cells labeled with FITC mouse IgG2a to control for MHC Class I mAbs or FITC rat IgG2a to control for MHC Class II mAb.

Radiation A radiation apparatus, developed for prostate tumors,25 was adapted for the radiation of s.c. tumors located in the middle of the back, at 1.5 cm of the tail. Acrylic jigs were designed to place anesthetized mice in the supine position with their fore and hind limbs restrained by posts for reproducible and accurate positioning of the s.c. tumor on the back as previously described.25 Three jigs were positioned on an aluminum frame mounted on the Xray machine to irradiate three mice at a time.25 Lead shields of 6.4 mm thickness were designed with three cutouts for the three mice to expose the area of the tumor to photon irradiation while shielding the rest of the mouse body.25 The radiation dose to the tumor and the scattered dose to areas of the mouse outside of the radiation field were carefully monitored. Photon irradiation was performed with a Siemens Stabilipan X-ray set (Siemens Medical Systems, Inc) operated at 250 kV, 15 mA with 1 mm copper filtration at a distance of 47.5 cm from the target.

Tumor treatment with Ad-vectors and/or radiation Mice were injected s.c. with Renca cells at 2 105 cells in 0.1 ml HBSS. To apply intratumoral cytokine gene therapy or radiation or both combined, we have selected the time at which the tumor reached a size between 0.3 0.4 and 0.3 0.5 cm, that is a volume of 13–15 mm3; these tumors were found to contain about 1–5 105 cells. Variations in time of treatment initiation between experiments were due to slight differences in time of tumor nodule appearance. Established tumors were treated with selective tumor irradiation administered at

a single dose of 8 Gy photons as described above. A day later, intratumoral injections of Ad-IL-2, Ad-IFN-g or Ad-null vectors were initiated using a concentration of 1.5 1010 TP in 50 ml PBS. Various schedules of Advector administration were tested. Experimental groups consisted of 7–9 mice/group. Mice were monitored for tumor growth and survival. Tumors were measured in three dimensions, three times a week, with a caliper. Tumor volume was calculated using the formula: 0.5236 length width height. When tumors reached 1.5 cm in greatest diameter or 1 cm with ulceration, mice were moribund and killed in accordance with animal facilities regulations.

In vivo expression of cytokines Renca tumors were intratumorally injected with Ad-IL-2 or Ad-IFN-g or Ad-null vectors at a dose of 1.5 1010 TP. (equivalent to 5 108 i.u.) in 50 ml PBS. At least five mice were killed at each individual time point: the tumor was extracted, weighted and mechanically disrupted in 1 ml of PBS with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Boerhinger Mannheim). The suspension was centrifuged (12,000 g, 2 minutes, 41C) and the supernatant was frozen at 201C.20 Samples were assayed for human IL-2 or murine IFN-g using the Quantikine Human Immunoassay from R&D system.20

Cytolysis assay Splenocytes were isolated from the spleens of mice treated with intratumoral Ad-vectors and/or radiation or following challenge with Renca cells as well as from naı¨ ve mice. Splenocytes at 5 106/ml were stimulated with 105/ml mitomycin C-treated Renca cells (50:1 ratio) in 24-well plates for 5 days. These stimulated splenocytes were used as effector cells in a standard 4 hours 51Cr release assay, that is cell-mediated lympholysis (CML) assay, to measure the cytolysis of Renca cells.20 Briefly, effector cells were plated in 96-well plates and two-fold serially diluted in triplicate wells to result in effector to target ratio of 100–0.15:1. Renca cells target were labeled with 250 mCi 51Cr and used at 5000 cells/well. Following 4 hours incubation, release of 51Cr was measured in the cell supernatant using a gamma counter (Packard, Downers Grove, IL). The percent cytotoxicity was calculated using the mean of the triplicate wells taking into account 51Cr spontaneous release obtained from target cells incubated alone in medium and 51Cr maximal release obtained by detergent lysis of target cells.

Histology Tumors were resected and fixed in 10% buffered formalin. These tissues are embedded in paraffin, sectioned and processed for staining with H&E.11, 25

Statistical analysis Differences in expression of cytokines in tumors and in tumor growth curves were analyzed by two-tailed unpaired t-test at different time points. Survival curves

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Results

Upregulation of MHC molecules by Ad-IFN-g transduction of Renca cells in vitro As previously shown, Renca tumor cells are positive for expression of MHC Class I molecules but do not express Class II MHC molecules (Table 1).13.We had shown that Renca treatment with 100 U/ml recombinant murine IFN-g caused upregulation of MHC class I molecules and induction of MHC Class II molecules.13 To test for the biological activity of Ad-IFN-g, Renca cells were transduced with Ad-IFN-g and incubated for 48–96 hours. Transduction of Renca cells with Ad-null did not modify Renca expression of MHC molecules (Table 1). An increase in fluorescence intensity was observed for expression of MHC Class I molecules by 48 hours incubation with Ad-IFN-g, indicating upregulation of MHC Class I molecules by gene transduction (Table 1). Induction of MHC Class II molecules expression was detected at 72 hours post-transduction in a small percent of the cells, but increased to 60% of the cells by 96 hours. These data confirm the biological activity of the IFN-g gene inserted in the adenoviral vector and corroborate the findings observed by treatment of Renca cells with mIFNg protein.13

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Table 1 Upregulation of MHC Class I and Class II molecules in Renca cells by transduction with Ad-IFN-g vector Class I

tumor at 24 hours after Ad-IFN-g injection lasting only for 3 days (Fig 1a). IFN-g production was statistically significant in tumors treated with Ad-IFN-g compared to tumors treated with Ad-null on days 1 and 2 after vector injection (Po0.05). Injection of Ad-null led to a low baseline of cytokine production both for IFN-g and IL-2 (Fig 1a, b). Expression of IL-2 transgene was more significant than that of IFN-g transgene. The peak of IL-2 expression was detected at 24 hours after intratumoral injection and reached about 15 pg/mg of tumor (Fig 1b).

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were constructed using the Kaplan–Meier product limit method. Comparisons of survival curves were carried out using the log-rank test.25

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Renca cells were transduced in vitro for 48–96 hours with 100 m.o.i. Ad-IFN-g or Ad-null. Then the cells were labeled with mAbs specific to MHC class I (H-2Kd) and Class II (I-A/I-E) molecules and analyzed by flow cytometry. The percent of cells positive for MHC class I and Class II antigens is reported. MFI represents the mean fluorescence intensity.

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Figure 1 Cytokine levels after injection of Ad-IFN-g and Ad-IL-2 vectors. Established s.c. Renca tumors were injected with a single intratumoral administration of 1.5 1010 TP in 50 ml PBS of Ad-IFN-g (circles) or Ad-null vectors (squares) (a, b) and Ad-IL-2 vectors (triangles) (b). At different time points, tumors were resected and processed to measure either IFN-g or IL-2 using an ELISA assay as described in Materials and methods. The levels of cytokines were normalized to the tumor mass and expressed as picograms of cytokine per mg of tumor (mean of five mice7SEM).


Established s.c. Renca tumors were treated either with intratumoral injections of Ad-vectors or with radiation followed a day later by Ad-vectors administration.

Effect of Ad-null control vector. To assess the effect of Ad-null control adenoviral vector on tumor growth, Renca tumors were treated with three intratumoral injections of Ad-null vector (1.5 1010 TP/injection) given a day apart on day 14, 15 and 16. The growth kinetics of Ad-null vector-treated tumors was comparable to that of control tumors treated with PBS with most of the tumors growing significantly by day 25 (Fig 2 a, b). Two of six mice show tumor growth delay until day 40 when treated with Ad-null (Fig 2b) compared to one of six in PBS-treated mice (Fig 2a), but all these mice ultimately developed large tumors. These data show that the empty adenoviral vector has limited therapeutic efficacy. Treatment of Renca tumors with a single dose of Adcytokine vectors alone and combined with radiation. To assess the efficacy of adenoviral-cytokine vectors, Ad-IL2 and Ad-IFN-g, different schedules and doses of these vectors were tested for intratumoral administration in established Renca tumors as single gene therapy or combined with prior tumor irradiation. Representative experiments are presented in Figures 3 and 4. Administration of a single dose of 1.5 1010 TP of AdIL-2 or Ad-IFN-g to established Renca tumors delayed tumor growth in three of six mice compared to control treated mice but by day 40 all tumors grew (Fig 3 a–c). Only one mouse treated with Ad-IL-2 showed complete tumor regression by day 30 but tumor regrowth was observed on day 41. Radiation of Renca tumors with 8 Gy photons caused a slight delay in tumor growth (Fig 3d). Treatment of Renca tumors with 8 Gy photons followed a day later by a single intratumoral injection of Ad-IL-2 (1.5 1010 TP) induced complete tumor regression in four of six mice between day 30 and 40 post cell injection (about 2–3 weeks following treatment) (Fig 3e). Longterm follow-up of these mice showed that tumor regrowth was observed in three of these mice by day 54, 68, and 82, whereas one mouse remained tumor free. Radiation followed by a single intratumoral injection of Ad-IFN-g (1.5 1010 TP) caused tumor growth delay in all mice until about day 40 but this effect was transient and tumor regrowth occurred (Fig 3f). Only one mouse showed complete tumor regression by day 23 but tumor reappeared by day 37 (Fig 3f). These data suggest that

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The production of IL-2 in Renca tumors treated with AdIL-2 was statistically significant from that of Ad-nulltreated tumors on days 1, 2 and 3 (Po0.05). This IL-2 production progressively declined but was detectable for up to 7 days. Cytokine production was undetectable in untreated tumors.

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Figure 2 Growth curves of Renca tumors treated with Ad-null vector. Established s.c. Renca tumors were injected intratumorally with 50 ml PBS (a) or Ad-null control vector (b) at 1.5 1010 TP in 50 ml PBS for three consecutive days. Each treatment group consisted of six mice. Each symbol represents the tumor volume of one individual mouse at different time points post cell injection.

tumor irradiation increased the efficacy of a single dose of Ad-IL-2 gene therapy and delayed tumor growth when combined with Ad-IFN-g.

Treatment of Renca tumors with multiple doses of Adcytokine vectors alone and combined with radiation. The combination of radiation with multiple doses of Advectors was more effective than with single-dose gene therapy for the treatment of s.c. Renca tumors (Fig 4 e, f). Three intratumoral treatments of Ad-IL-2 or Ad-IFN-g in established Renca tumors were given on day 11, 12 and 13 post tumor cell injection, at a dose of 1.5 1010 TP per injection. Following three doses of Ad-IL-2, two of eight mice showed complete tumor regression by day 19 (1 week post-treatment) and remained free of tumor until

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Figure 3 Treatment of Renca tumors with irradiation followed by one intratumoral injection of Ad-IL-2 or Ad-IFN-g vectors. Established s.c. Renca tumors were treated either with PBS (a), Ad-IL-2 (b) or Ad-IFN-g (c) or with radiation alone (d), radiation þ Ad-IL-2 (e) or radiation þ AdIFN-g (f). Radiation was administered at 8 Gy photons on day 15 followed by one intratumoral injection of Ad-IL-2 or Ad-IFN-g at 1.5 1010 TP in 50 ml PBS, on day 16. Each treatment group consisted of six mice. Each symbol represents the tumor volume of one individual mouse at different time points post cell injection.

day 69 when they were killed to check for T-cell activity (Fig 4b). In this treatment group, three of eight mice showed tumor growth delay up to about 35 days followed by tumor regrowth and two of eight mice showed rapid tumor growth comparable to that of control mice (Fig 4a, b). Treatment with three doses of Ad-IFN-g caused tumor delay in most of the mice (eight of nine) between 30 and 40 days but tumor regrowth occurred in all mice and no tumor regression was observed (Fig 4c). When tumor irradiation was given at 8 Gy on day 10 to established Renca tumors in the same experiment, tumor delay was observed in five of nine mice for about 20 days (by day 30 post cell injection), but this effect was transient and all tumors grew to large sizes (Fig 4d). Tumor irradiation given on day 10 followed by Ad-IL-2 gene therapy on days 11, 12 and 13 induced the best antitumor response with complete and lasting tumor regression in four of nine mice (Fig 4e). In these mice, tumors stopped growing as soon as the treatment was given and disappeared completely by day 30. In the other five mice from this group, tumor growth was delayed until 30–35 days (Fig 4e). The effect of radiation combined with three injections of Ad-IFN-g was not as pronounced (Fig 4f). Only one mouse of nine showed a complete tumor regression, whereas most mice had tumor growth delay comparable to that of Ad-IFN-g alone (Fig 4c). Comparisons between the various treatment groups showed that initially, tumor growth was significantly inhibited in all

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treatment groups versus control group due to transient tumor growth inhibition for up to 32–35 days following radiation, adeno-cytokine vectors or both combined (Po0.05). No differences were found between these treatment groups except between radiation þ Ad-IL-2 compared to radiation alone (Po0.05). At later time points, a mixed pattern of responses was observed with tumor regrowth in some mice, whereas other mice showed complete tumor regression. The survival follow-up of this experiment showed that most of the mice in the control group were dead by day 30, except one mouse in which tumor growth was slower leading to death by day 45 (Fig 5). Ad-IL-2 gene therapy alone or radiation alone caused an increase in median survival to 38–41 days compared to 25 days in control mice (Fig 5a). Combination of radiation with Ad-IL-2 caused a further increase in median survival to 62 days and in overall survival compared to radiation or Ad-IL-2 alone and control mice (Po0.01) (Fig 5a). In the combined treatment group, 44% of the mice remained tumor free for up to 92 days until killed for T-cell cytotoxic assays. Ad-IFN-g gene therapy alone or combined with radiation showed an increase in survival compared to control mice (Po0.01) with a median survival of about 42 days, but no significant differences were observed between the groups receiving Ad-IFN-g and radiation þ Ad-IFN-g (P40.25) (Fig 5b). The mouse survival corroborates the findings of tumor growth depicted in Fig 4. The proportion of


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Figure 4 Treatment of Renca tumors with irradiation followed by three intratumoral injections of Ad-IL-2 or Ad-IFN-g vectors. Established s.c. Renca tumors were treated either with PBS (a), Ad-IL-2 (b) or Ad-IFN-g (c) or with radiation alone (d), radiation þ Ad-IL-2 (e) or radiation þ AdIFN-g (f). Radiation was administered with 8 Gy photons on day 10 followed by three intratumoral injections of Ad-IL-2 or Ad-IFN-g at 1.5 1010 TP in 50 ml PBS on day 11, 12 and 13. Each treatment group consisted of eight to nine mice. Each symbol represents the tumor volume of one individual mouse at different time points post cell injection.

tumor-free mice at the end of the observation period, on day 69, was 0/8 in the PBS control group, 2/8 in Ad-IL-2 group, 0/9 in Ad-IFN-g group, 0/9 in radiation group, 4/9 in radiation þ Ad-IL-2 group and 1/9 in radiation þ AdIFN-g group. In separate experiments, the dose and schedule of gene therapy were further investigated. Spacing the three doses of Ad-IL-2 or Ad-IFN-g 2 days apart instead of a day apart did not result in greater therapeutic effect when this schedule was given alone or combined with radiation. In addition, a lower antitumor response and shorter survival were caused by two injections of Ad-cytokine vectors alone or combined with tumor irradiation compared to three injections of Ad-vectors þ / radiation. No complete responses were observed with radiation combined with two injections of Ad-IL-2. Finally, simultaneous injections of Ad-IL-2 mixed with Ad-IFN-g showed a therapeutic effect comparable to that of Ad-IL-2 alone.

T-cell activity and response to challenge tumor in mice treated with radiation and gene therapy Some of the surviving mice from the experiment shown in Figures 4, 5 were tested for T-cell activity prior to tumor challenge and some post tumor challenge. Mice tested

prior to tumor challenge were killed on day 69 for CTL assay and included a tumor-bearing mouse treated with radiation and three tumor-free mice treated with Ad-IL-2, radiation þ Ad-IL-2 and radiation þ Ad-IFN-g, respectively (Fig 6a). The splenocytes from these mice and from naı¨ ve mice were isolated and stimulated, in vitro, with mitomycin C-treated Renca cells for 5 days. Cytotoxicity of stimulated splenocytes was tested in a CML assay against 51Cr-labeled Renca cells. From all the mice shown in Figure 6a, only the mouse treated with radiation alone showed a remaining tumor, the other mice were clear of tumor. Radiation alone did not induce cytotoxic activity against Renca. Splenocytes from mice treated with Ad-IL2 or radiation þ Ad-IL-2 showed cytotoxic activity against Renca target (Fig 6a). Radiation combined with Ad-IFN-g induced only a low level of cytotoxic activity against Renca of 10% compared to 30% with radiation þ Ad-IL-2 at 100:1 E/T ratio (Fig 6a). Splenocytes from naı¨ ve mice stimulated with Renca cells did not kill Renca target cells (Fig 6 a, b). Three mice rendered tumor free by radiation þ Ad-IL-2 in the experiment shown in Figures 4, 5 were rechallenged with s.c. injection of Renca cells at 1 105/0.1 ml HBSS in the flank on day 78 post initial Renca injection. These mice did not develop s.c. tumors at the challenge site by

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−10 1

b

100

10

100

10

100

Untreated Ad-IF Nγ

b

40

Radiation 80

Rad+Ad-IF Nγ

60

% cytotoxicity

Present Survival

30

40

20

20

10

0 10

20

30

40

50

60

70

0


Figure 5 Survival of mice treated with tumor irradiation followed by intratumoral injections of Ad-IL-2 or Ad-IFN-g vectors. Survival of mice from experiment shown in Fig. 4 is reported, following PBS, AdIL-2, radiation or radiation þ Ad-IL-2 treatment (a) and following PBS, Ad-IFN-g, radiation or radiation þ Ad-IFN-g treatment (b). Although these treatments groups were tested in the same experiment, the survival curves were drawn separately for Ad-IL-2 þ /radiation and Ad-IFN-g þ /radiation for presentation clarity.

day 14, whereas naı¨ ve control mice developed tumors by days 8–10. On day 14 post challenge, splenocytes were isolated and stimulated with mitomycin C-treated Renca cells for 5 days, then tested in a CML assay against 51Cr labeled Renca cells. Splenocytes from mice treated with radiation and Ad-IL-2, which rejected challenge tumor, showed CTL activity against Renca target (Fig 6b) comparable to that of mice responding to Ad-IL-2 or radiation þ Ad-IL-2 and not rechallenged with Renca (Fig 6a). In separate experiments, mice rendered tumor –free by treatment with Ad-IL-2 or radiation þ Ad-IL-2 and

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−10 1

E/T Ratio

Figure 6 Cytotoxic activity of splenocytes from mice treated with Adcytokine vectors and/or radiation. Surviving mice from experiment shown in Figures 4, 5 were tested for T-cell activity against Renca target. (a) On day 69, splenocytes were isolated from mice treated with Ad-IL-2 (open diamonds), radiation (open squares), radiation þ Ad-IL-2 (closed squares) or radiation þ Ad-IFN-g (closed triangles) and stimulated with mitomycin C-treated Renca cells for 5 days in vitro. These cells were assayed against 51Cr-labeled Renca target cells in a CML assay. (b) The cytotoxic activity of three mice treated with radiation þ Ad-IL-2 and rechallenged with Renca cells on day 78 (closed squares, closed triangles). Splenocytes were isolated 14 days after tumor challenge, stimulated with Renca cells and tested in CML assay against Renca target. Splenocytes from naı¨ve Balb/c mice (open circles, a and b) were also stimulated with Renca cells and tested in the CML assays. In (a) and (b), each curve represent data from an individual mouse and each point on the curves is the mean percent cytotoxicity of triplicate wells at each E/T ratio. The standard deviation (SD) of the mean of triplicate wells was lower than 10%.


69

rechallenged 5–9 months later with Renca cells rejected the challenge tumor. Owing to a higher complete tumor regression response rate in the groups treated with radiation þ Ad-IL-2, additional experiments were conducted in which established Renca s.c. tumors were treated with 8 Gy photon radiation followed a day later by intratumoral injections of Ad-IL-2 for 3 consecutive days as described in Materials and methods. Complete tumor regression was observed in nine of 10 mice treated with radiation þ AdIL-2 compared to four of nine mice treated with Ad-IL-2 alone. These data confirm that tumor irradiation prior to Ad-IL-2 gene therapy significantly enhances the response rate compared to Ad-IL-2 therapy alone (Po0.05). Tumor-free mice were immune to rechallenge with parental Renca tumor cells. In a separate experiment, six mice rendered tumor free by radiation þ Ad-IL-2 were rechallenged with unrelated D2F2 mammary tumor cells syngeneic to Balb/c mice given s.c. at 1 105 cells in the flank. Four of six mice developed D2F2 tumors by 15–18 days similarly to five naı¨ ve mice injected with 1 105 D2F2 cells.

Histology of Renca tumors treated with photon radiation and Ad-IL-2 or Ad-IFN-g Established Renca s.c. tumors were selectively irradiated with 8 Gy photons on day 12 and treated on days 13, 14 and 15 with intratumoral injections of Ad-vectors. A week after the end of gene therapy, on day 21, tumors were resected and processed for H&E staining to assess in situ alterations induced by each modality and combined modalities. Untreated Renca tumors presented as large and well-vascularized nodules consisting of sheets of pleomorphic epithelial cells with prominent nucleoli (Fig 7a). Frequent mitosis, a few giant cells and only few apoptotic cells were observed (Fig 7a). Tumors treated with Ad-null vectors also presented as large nodules showing similar histological findings as those seen in untreated tumors (Fig 7, inset). Following Ad-IL-2 treatment, much smaller tumor nodules were observed with extensive geographic necrosis in most of the nodule and only focal viable tumor areas (Fig 7b). Hemorrhages and numerous inflammatory cells surrounding necrotic tumors in junction areas on edge of tumor nodule and inside remaining tumor areas consisted of active, large lymphocytes, histiocytes, neutrophils and granulocytes (Fig 7b). Areas with more intense apoptosis and nuclear debris were found. Ad-IFN-g-treated tumors presented as smaller nodules than control, with reduced vascularization, looking ischemic. Histologically, patchy areas of apoptotic cells and necrotic areas were seen among large areas of tumor cells (Fig 7c). A few inflammatory cells were seen surrounding the tumor with almost none inside tumor. Following radiation, tumor nodules were small and showed focal hemorrhages. A larger number of giant cells than in untreated tumors were seen including cells with abnormal mitosis, or eccentric nuclei and large vacuoles that are characteristic of radiation-induced cell alterations (Fig 7d). Areas of tumor destruction, apopto-

Figure 7 Histology of Renca tumors treated with radiation and Adcytokine vectors. Established s.c. Renca tumors were selectively irradiated with 800 cGy photons on day 12 and treated over 3 days with Ad-vectors. Tumors sections, obtained on day 21, were stained with H&E. The main findings were labeled on the prints with T for tumor, N for necrosis, A for apoptosis, H for hemorrhages, G for giant cells, M for mitosis and I for inflammatory cells. (a) Untreated tumor, sheets of pleomorphic epithelial cells with prominent nucleoli and frequent mitosis. (a) inset, Ad-null control vector-treated tumor presenting like untreated tumor. (b) Ad-IL-2-treated tumor, showing extensive necrosis with apoptotic cells, multifocal hemorrhages and inflammatory cells with small viable tumor areas. (c) Ad-IFN-gtreated tumor, patchy areas of apoptotic cells and necrotic areas seen among large areas of tumor cells. (d) Radiation-treated tumor with areas of apoptosis, numerous giant cells with abnormal mitosis, eccentric nuclei or large vacuoles and hemorrhages. (e) Radiation þ Ad-IL-2-treated tumor showing large areas of necrosis with extensive apoptosis, multifocal hemorrhages and inflammatory cells. Note areas of remaining tumor with high frequency of giant cells and abnormal mitosis (M). (f) Radiation þ Ad-IFN-g-treated tumor showing necrotic areas and remaining tumor areas with giant cells and abnormal mitosis (M), apoptotic cells and surrounding inflammatory cells. All magnifications, 50.

sis and necrosis with a few inflammatory cells in areas of tumor destruction were also seen (Fig 7d). Treatment with radiation followed by Ad-IL-2 resulted in small tumor nodules with large areas of necrosis in the center of the nodule covering 80% of the nodule, extensive apoptosis and focal hemorrhages (Fig 7e). Inflammatory cells were prominent surrounding and infiltrating tumors with neutrophils and active lymphocytes. Areas of the remaining tumor showed radiation-induced changes including a high frequency of giant cells and abnormal

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Table 2 Summary and quantitation of histological observations

Control Ad-null Ad-IL-2 Ad-IFN-g Radiation Radiation+Ad-IL-2 Radiation+Ad-IFN-g

% Surviving tumor cells

Apoptosis

Inflammatory infiltrates

80 80 20 70 60 20 35

+ + +++ ++ ++ +++ +++

7 7 +++ + + +++ ++

The extent of viable tumor cells, apoptosis and inflammatory infiltration, observed in the histological analysis depicted in Figure 7, were graded. The degree of apoptosis and inflammatory infiltrates was scaled from mild (7) to heavy (+++).

mitosis (Fig 7e). Treatment with radiation followed by Ad-IFN-g caused small tumor nodules with focal hemorrhages showing necrosis and apoptosis (Fig 7f). Inflammatory cells were seen surrounding the nodule with some invading remaining tumor areas (Fig 7f). Radiation induced giant cells and abnormal mitosis were observed. Quantitation of the histological findings showed that 80% of the tumor nodules consisted of viable tumor cells in control and Ad-null tumor sections (Table 2). Treatment with Ad-IL-2 was more effective at tumor destruction than Ad-IFN-g with only about 20% remaining tumor compared to 70%, respectively, when tumors were assessed at an early time point of 21 days (Table 2). Radiation caused tumor destruction but at a lower extent than Ad-IL-2 with 60% remaining tumor. Combination of radiation with Ad-IL-2 resulted in comparable tumor destruction, apoptosis and inflammatory infiltrates than Ad-IL-2 alone; however, surviving tumor cells were giant and abnormal, presenting features of radiation-induced alterations (Table 2). Radiation and Ad-IFN-g did decrease by half the extent of surviving tumor cells compared to each modality alone and caused an increase in apoptosis and inflammatory infiltrates (Table 2). For apoptosis, a semiquantitative method was used based on morphological recognition of apoptotic cells with typical fragmented and condensed nuclei and shrinkage of cytoplasm (Table 2). To grade the extent of apoptotic cells, the entire tumor area on the slide was screened. The lowest grade þ represented individual scattered apoptotic cells and the highest grade þ þ þ was given to tumors showing large areas with numerous apoptotic cells as shown in Figure 7b, e. The intermediate grade þ þ represented small aggregates of apoptotic cells scattered in different regions of the tumor typical of that seen with Ad-IFN-g (Fig 7c).

Discussion

We have previously shown that Renca pulmonary metastases are responsive to systemic administration of recombinant human IL-2 or recombinant murine

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IFN-g. 10,11,13 The effect of IL-2 therapy was enhanced by prior tumor irradiation.10,11 IFN-g caused a transient delay in tumor growth.13 To improve the efficacy of cytokine therapy and overcome the toxicity of systemic delivery of cytokine proteins, we have investigated intratumoral gene therapy with adenoviral (Ad)-cytokine vectors. The therapeutic effect of gene therapy with AdIL-2 and Ad-IFN-g for the treatment of RCC was tested in Renca s.c. tumors. Based on our previous studies on the interaction of tumor irradiation and immunotherapy and the studies of Zeng et al,26 showing increased transduction efficiency of adenoviral vectors by radiation, we have tested the combination of tumor irradiation with intratumoral administration of Ad-cytokine vectors. Intratumoral treatment of established s.c. Renca tumors with a single dose of 1.5 1010 TP of Ad-IL-2 or Ad-IFN-g induced transient tumor growth delay in 50% of the mice. Repeated administration of Ad-cytokine vectors, given a day apart, was more effective than singledose gene therapy. We found that treatment with three doses of Ad-IL-2, given over 3 consecutive days, caused tumor growth delay in the majority of the mice, by 1 week post treatment, and complete tumor regression in 25% of the mice. This increased efficacy may result from transduction of additional tumor cells after each Adcytokine vector injection and higher and constant production of cytokine in situ in the tumor. Indeed, we have previously shown that the efficacy of Ad-IL-2 (pCMV) for intratumoral treatment of P815 murine mastocytoma tumors correlated with the amount of IL-2 produced inside the tumor and this production lasted approximately 2 weeks.20 We also showed that constant production of IL-2 in the tumor nodule was needed for antitumor activity as intratumoral injections of recombinant IL-2 protein were not effective probably due to rapid clearance of IL-2 from the tumor site.20 In vivo, Renca tumors treated with a single injection of 1.5 1010 TP of Ad-IL-2 showed a similar profile of intratumoral IL-2 expression as that observed in P815 mastocytoma tumors, that is the cytokine production was detectable for 10 days. The peak of IL-2 expression was detected 24 hours after intratumoral injection and reached around 15 pg IL-2/mg of tumor, which is lower than 100pg IL-2/ mg of tumor observed as a maximum 24 hours after injection of P815 tumor.20 However, IFN-g expression was found to be extremely low in Renca tumors and reached only 1 pg IFN-g/mg of tumor 24 hours after injection and lasted only for 3 days. This observation may explain, in part, why the antitumoral activity of Ad-IFN-g is weak in the Renca model with a small increase in survival compared to control groups and no tumor eradication. Tumor irradiation given one day prior to a single intratumoral dose of Ad-IL-2 increased the efficacy of gene therapy by causing complete tumor regression in 66% of the mice at about 2–3 weeks post-Ad-IL-2 treatment. In a long-term follow up of 82 days, only one of six mice remained tumor free. Radiation also enhanced the effect of a single intratumoral dose of Ad-IFN-g by inducing tumor growth delay, but tumor


regrowth occurred in all mice. The radiation enhancing effect on antitumor activity was more pronounced when three injections of Ad-vectors were administered over 3 days after the tumor irradiation. Tumor irradiation followed a day later by initiation of Ad-IL-2 gene therapy, for 3 days, led to tumor growth arrest in all mice and complete tumor regression in four of nine mice, whereas radiation alone had only a transient effect on tumor growth. The effect of radiation combined with three doses of Ad-IFN-g was not as pronounced as that observed with radiation combined with Ad-IL-2, and resulted only in 1 tumor-free mouse out of nine. No additional increase in survival was observed in mice treated with radiation and Ad-IFN-g compared to radiation or Ad-IFN-g alone or radiation þ Ad-IL-2. The combination of radiation with Ad-IL-2 therapy caused a significant increase in mouse survival compared to Ad-IL-2 or radiation alone resulting in 44% of tumor-free mice. Repeated experiments demonstrated a two-fold increase in mice responding with long lasting complete tumor regression following radiation þ Ad-IL-2 compared to Ad-IL-2 alone, confirming that tumor irradiation enhanced the response rate to AdIL-2 therapy. Surviving mice, which had complete tumor regression following Ad-IL-2 gene therapy alone or combined with tumor irradiation, rejected Renca tumor challenge demonstrating induction of specific immunity. These data were reproduced in additional experiments with radiation þ Ad-IL-2 responding mice rejecting Renca tumor rechallenge but accepting unrelated D2F2 tumor cells. These recent data confirm that these mice developed a specific antitumor immune response. Splenocytes isolated from mice treated with Ad-IL-2 or radiation þ Ad-IL-2, before or after Renca challenge, demonstrated T-cell cytotoxic activity against Renca tumor cells following Renca stimulation in vitro. These data corroborate our findings on immunity to rechallenge and suggest that Ad-IL-2 gene therapy alone or combined with radiation induces a specific antitumor immune response. Rejection of tumor challenge and cytotoxic Tcell activity were also demonstrated in P815 tumors treated with Ad-IL-2, confirming induction of specific tumor immunity by Ad-IL-2 gene therapy.20 However, low cytotoxic activity against Renca target was observed in the splenocytes from a tumor-free mouse treated with radiation þ Ad-IFN-g, whereas no activity was obtained with splenocytes from naı¨ ve mice or one tumor-bearing mouse treated by radiation. In our previous studies on Renca lung tumors, systemic administration of murine recombinant IFN-g also induced partial tumor inhibition and increase in mouse survival and this effect was not abrogated by T-cell depletion.13 In the current study, we have demonstrated that even constant production for 3 days by Ad-IFN-g-transduced cells caused only tumor growth delay with no complete tumor regression. As mentioned above, local expression of IFN-g in Renca tumors reached 1 pg/mg of tumor, which is low compared to IL-2 levels in the range of 10 or 100 pg/ml for Renca and P815 tumors, respectively. Presumably, such a local

expression of IFN-g in Renca tumors might be required for better antitumoral activity of IFN-g cytokine produced within the tumor. Since these mice died from large tumors, before the end of the 70-day observation period, no tumor rechallenge was given to them. Even though, IFN-g can upregulate expression of MHC Class I and Class II molecules at least in vitro, which should increase Renca immunogenicity, its effect on activation of the immune system did not lead to a complete antitumor immune response, and prior tumor irradiation had a limited effect on enhancing this response in contrast to IL-2. Histological observations of tumors, treated by radiation followed by three administrations of Ad-cytokine vectors over 3 days, correlated with our findings on tumor growth and mouse survival. By 1 week after treatment, most of the tumors resected from mice treated with gene therapy or radiation were much smaller in size than those of control mice, probably due to treatment-induced growth arrest, but marked histological differences were observed depending on the treatment. Irradiated tumors or Ad-IFN-g-treated tumors showed limited inflammatory infiltration and tumor destruction with necrotic areas and apoptotic cells but 60–70% of the tumor cells looked viable. In contrast, Ad-IL-2-treated tumors showed only focal viable tumor areas and extensive necrosis in most of the nodule associated with numerous inflammatory cells, apoptotic cells and hemorrhages. Irradiation of the tumor prior to gene therapy caused greater tumor destruction, vascular damage and inflammatory cell infiltration in the tumor nodule. Surviving tumor cells showed radiationinduced alterations including a high frequency of giant cells with eccentric nuclei, large cytoplasmic vacuoles or abnormal mitosis. In other studies, these cells were shown to be in a process of degeneration by 3 weeks after radiation.27 Such cells may be representative of a different mechanism of radiation-induced late cell death in addition to radiation-induced apoptosis and necrosis.27 The extent of tumor destruction caused by radiation þ Ad-IL2 was more pronounced than with radiation þ Ad-IFN-g. These observations correlate with the tumor growth inhibition and/or delay observed following these treatments. The histological findings, including enhanced tumor destruction, inflammatory response and vascular damage by combination of radiation with IL-2 cytokine gene therapy, are similar to those observed in previous studies in the Renca model and prostate tumor models treated with radiation followed by systemic IL-2 therapy. These findings suggest that radiation therapy causes changes in the tumor cells and the tumor environment, which increase the tumor susceptibility to destruction by the immune system activated by IL-2. The sequence of tumor irradiation first followed by gene therapy to trigger an immune response was selected based on our previous findings that radiation significantly decreased tumor burden and also induced an inflammatory response.10,11,25 This sequence takes advantage of (a) the presence of inflammatory cells in the vicinity of the tumor mobilized by radiation-induced tissue damage participating in the immune response triggered by the

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gene therapy,10,11,25 (b) the pool of tumor proteins and peptides generated from radiation-induced tumor apoptosis28 and (c) radiation also increases gene transduction efficiency and duration of expression following adenoviral vector delivery.26 Renca tumor irradiation alone caused a transient growth inhibition and increased host survival as shown previously in the Renca lung tumor model10 and other models.25 Radiation could have contributed by debulking the tumor and increasing IL-2 gene transduction of surviving cells, thus improving efficiency of in situ genetic modification leading to an immune response that eradicated remaining tumor cells. Recent preliminary data showed that IL-2 secretion in tumors and in the serum was higher and of longer duration if the tumors were first irradiated 1 day prior to intratumoral Ad-IL-2 treatment than in Ad-IL-2-treated mice with no radiation. In addition, radiation might have destroyed or limited suppressive immunoregulatory T cells.

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13. Hillman GG, Visscher D, Hamzavi F, et al. Inhibition of murine renal carcinoma pulmonary metastases by systemic administration of interferon gamma: mechanism of action and potential for combination with IL-4. Clin Cancer Res. 1997;3:1799–1806. 14. Addison CL, Braciak T, Ralston R, et al. Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in a murine breast cancer model. Proc Natl Acad Sci USA. 1995;92:8522–8526. 15. Addison CL, Bramson JL, Hitt MM, et al. 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;5:1400–1409. 16. Cordier L, Duffour MT, Sabourin JC, et al. Complete recovery of mice from a pre-established tumor by direct intratumoral delivery of an adenovirus vector harboring the murine IL-2 gene. Gene Ther. 1995;2:16–21. 17. Huang H, Chen SH, Kosai K, et al. Gene therapy for hepatocellular carcinoma: long-term remission of primary and metastatic tumors in mice by interleukin-2 gene therapy in vitro. Gene Ther. 1996;3:980–987. 18. Toloza EM, Hunt K, Swisher S, et al. In vitro cancer gene therapy with a recombinant interleukin-2 adenovirus vector. Cancer Gene Ther. 1996;3:11–17. 19. Saffran DC, Horton HM, Yankauckas MA, et al. Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+T-cell immunity. Cancer Gene Ther. 1998;5:321–330. 20. Slos P, De Meyer M, Leroy P, et al. Immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human IL-2 cDNA: induction of CD8(+) T-cell immunity and NK activity. Cancer Gene Ther. 2001;8:321–332. 21. Chartier C, Degryse E, Gantzer M, et al. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol. 1996;70:4805–4810. 22. Chroboczek J, Bieber F, Jacrot B. The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2. Virology. 1992;186: 280–285. 23. Fallaux FJ, Bout A, van der Velde I, et al. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum Gene Ther. 1998;9:1909–1917. 24. Lusky M, Christ M, Rittner K, et al. In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2a, or E1/E4 deleted. J Virol. 1998;72:2022–2032. 25. Hillman GG, Maughan RL, Grignon DJ, et al. Neutron or photon irradiation for prostate tumors: enhancement of cytokine therapy in a metastatic tumor model. Clin Cancer Res. 2001;7:136–144. 26. Zeng M, Cerniglia GJ, Eck SL, et al. High-efficiency stable gene transfer of adenovirus into mammalian cells using ionizing radiation. Hum Gene Ther. 1997;8:1025–1032. 27. Hillman GG, Maughan RL, Grignon DJ, et al. Responsiveness of experimental prostate carcinoma bone tumors to neutron or photon radiation combined with cytokine therapy. Int J Rad Onc Biol Phys. 2003;56:1426–1437. 28. Bellone M, Iezzi G, Rovere P, et al. Processing of engulfed apoptotic bodies yields T cell epitopes. J Immunol. 1997;159:5391–5399.