MERO 25 and II-45 respectively, were infected with In addition, the CMV promoter appeared to be more

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1 Gene Therapy (1997) 4, Stockton Press All rights reserved /97 $12.00 Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene MC Esandi 1, GD van Someren 1, AJPE Vincent 2, DW van Bekkum 3, D Valerio 1,31, A Bout 1,3 and JL Noteboom 1,4 1 Section Gene Therapy, Department of Medical Biochemistry, Leiden University, Rijswijk; 2 Department of Neurosurgery, University Hospital Rotterdam; and 3 IntroGene BV, Rijswijk, The Netherlands Replication-defective adenovirus vectors were generated inoculated on day 0 with 10 5 II-45 cells into the pleural in which the gene of interest (lacz, luciferase or HSV-tk) is cavity, received infectious particles of IG.Ad. driven by the adenovirus major late promoter (MLP) or CMV.TK on day 1, day 2, day 4 or day 8. One day after the human cytomegalovirus immediate early gene virus administration, 25 mg/kg GCV or PBS (controls) was promoter/enhancer (CMV). In vitro experiments with rat (II- injected i.p. (intraperitoneally) twice daily. On day 15, all 45) and human (MERO 25) mesothelioma cell lines animals were killed. Significant tumor regression, equivalrevealed that the CMV promoter was stronger than the ent to 5 log cell kill, occurred in the treated rats suggesting MLP promoter regarding levels of expression of the lucifer- an impressive bystander effect. In a survival study, animals ase reporter gene and ganciclovir (GCV) killing efficiency were treated 9 days after inoculation of 10 5 tumor cells with after tk gene transfer. Following administration of IG.Ad.CMV.TK and a 14 days course of GCV. This treat- IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) ment prolonged symptom-free survival time from 19 days into the pleural cavity of Fischer rats with established in the controls to 33 days in the treated group. These mesothelioma, a widespread distribution of infectious virus responses can be best explained by assuming continued particles through the thorax contents was demonstrated. tk expression in or around the tumor tissue during GCV However, a relatively small proportion of tumor cells were treatment. Our results confirm and extend earlier findings transduced. Nevertheless, a strong tumor growth inhibition with the same model and demonstrate the potential of the was observed following treatment with IG.Ad.CMV.TK herpes simplex virus thymidine kinase suicide gene recombinant adenovirus and GCV. Separate groups of rats therapy as a local treatment for malignant mesothelioma. Keywords: suicide gene therapy; malignant mesothelioma; recombinant adenovirus; HSV-thymidine kinase gene; promoter activity comparison Introduction inhibits DNA replication. Rapidly dividing cells were shown to be killed by this treatment while slowly rep- Malignant mesothelioma (MM) is a cancer of the meso- licating cells are less affected. 3 Thus, this strategy is conthelium most commonly occurring in the pleural cavity. sidered appropriate for the treatment of solid tumors that Its incidence is related to exposure to asbestos. 1 MM has are invading normal tissues constituted predominantly of a very poor prognosis. Despite intensive treatment with slowly or nondividing cells such as gliomas. In animal surgery, radiation therapy, or chemotherapy, the average tumors effective transfer of the HSVtk gene has been achisurvival time is only months from diagnosis. 2 eved with both recombinant adenovirus and recombinant Tumor growth is often limited to the thoracic cavity. retrovirus. Retroviral vectors mediate stable integration Most patients die of local extension of the disease rather into the genome exclusively in dividing cells but this systhan of metastases. This growth pattern suggests that tem has the limitation of low viral titers and low trans- MM may be a good candidate for local treatment. duction efficiency. 4 To overcome this limitation, murine One potentially useful strategy of local treatment is HSVtk retrovirus-producer cells have been injected transduction of tumor cells in vivo with suicide genes. directly into the cerebrospinal fluid of animals with The most widely explored of these suicide genes is the malignant leptomeningeal neoplasia 5 or into the tumor of herpes simplex virus thymidine kinase (HSVtk) gene. In rats carrying gliomas in the brain. 6,7 Since this last stratthe presence of the thymidine kinase, ganciclovir (GCV) egy has proven to be effective, trials in human patients is phosphorylated to a toxic nucleotide analogue which with gliomas have been initiated. In contrast to retrovirus, adenovirus vectors have been shown to be efficient tools for local gene transfer in vivo. Obvious advantages Correspondence: MC Esandi, Section Gene Therapy, Department of Medi- of recombinant adenovirus are that they have a broad cal Biochemistry, Leiden University, PO Box 3271, 2280 GG Rijswijk, range of target cells and no requirement of integration The Netherlands for expression. 8 4 Current address: Department of Clinical Oncology, Leiden University Efficient adenovirus-mediated gene Hospital, The Netherlands transfer of HSVtk in vivo has been described in trans- Received 2 October 1996; accepted 19 November 1996 plantable animal tumors such as melanoma, 9 glioma, 7

2 leptomeningeal neoplasia, 10 mesothelioma 11 and in human tumors growing in immunodeficient mice, eg head and neck squamous cell cancer 12 and hepatocellular carcinoma. 13 In the current study we investigated the therapeutic potential of two recombinant adenoviruses using the adenovirus type 2 major late promotor (MLP) and the cytomegalovirus immediate early promoter (CMV) respectively to drive the HSVtk gene. The CMV promoter has been found to be stronger than the MLP promoter in adenovirus harboring the p53 gene. 14 We were interested to know whether this difference would be reflected in more efficient cell kill after expression of the tk gene and treatment with GCV. The strength of the CMV promoter and the MLP promoter were compared by constructing vectors containing the luciferase gene and infecting human and rat mesothelioma cell lines with these vectors. The adeno-tk vectors were employed in combination with GCV to determine the cytotoxicity in vitro for both rat and human mesothelioma cell lines. Although the suicide effect was greater in vitro for the human cell line, we have used the rat mesothelioma for our in vivo studies. The human mesothelioma requires transplantation in immune deficient animals for in vivo studies. Since recent studies have suggested that cells transduced in vivo by adenovirus vectors are rapidly cleared by the immune system, 15 we preferred an immunocompetent animal model to study the in vivo potential of the suicide system because it mimics more closely the patient situation. The most effective of the two viruses, IG.Ad.CMV.TK, was used for treating mesotheliomas growing in the pleural cavity of rats. To study the distribution of transduced cells following intrapleural administration of recombinant adenovirus we applied an adenovirus vector encoding the lacz gene under the control of the CMV promoter. We compared the results of the in vivo treatment of rat mesothelioma with those reported by Elshami et Figure 1 Evaluation of luciferase activity after infection of human al 11 who treated the same tumor with an adenovirus vec- (MERO 25) and rat (II-45) mesothelioma cells with IG.Ad.CMV.luc and tor using a different promoter (RSV) to drive the HSVtk IG.Ad.MLP.luc. MERO 25 and II-45 cells were infected with gene. In our experience, in vivo treatment with the CMV IG.Ad.MLP.luc and IG.Ad.CMV.luc at MOI: 1, 10, 100. After 72 h, adenovirus was more tumoricidal than treatment with luciferase activity was measured in the lysates of the infected cells accord- ing to Materials and methods. (a) MERO 25 cells; (b) II-45 cells., the adeno-rsv virus as described by Elshami et al. 11 It is Ig.Ad.MLP.Luc;, IG.Ad.CMV.Luc. not clear whether that difference has to be ascribed to a more effective expression of the tk gene or to other factors, such as a faster growth rate of the mesothelioma in in both the human and the rat cell line after infection with our laboratory. Other differences between the effects of the CMV vector than with the MLP vector, indicating a the various viruses such as in vitro cytotoxicity and the reactions in normal tissues following injection into the pleural cavity are also discussed. Results In vitro studies stronger activity of the CMV promoter. In vitro comparison of IG.Ad.MLP.TK and IG.Ad.CMV.TK mediated tumor cell kill: GCV sensitivity of HSVtk-transduced mesothelioma cells was first tested in vitro. Human (MERO 25) or rat (II-45) cells were infected with either IG.Ad.MLP.TK or IG.Ad.CMV.TK at different multiplicities of infection (MOI). After 72 h of culture in the presence or absence of GCV, a clonogenic assay was performed. The results, presented in Figure 2, demonstrate Luciferase expression after infection of human and rat mesothelioma cells with either IG.Ad.MLP.luc or that there is an inverse relation between MOI and cell IG.Ad.CMV.luc: Human and rat mesothelioma cell lines, survival after GCV administration. MERO 25 and II-45 respectively, were infected with In addition, the CMV promoter appeared to be more IG.Ad.MLP.luc and IG.Ad.CMV.luc (Figure 1). With both effective than the MLP promoter in driving HSVtkrecombinant vectors the luciferase activity measured in mediated GCV toxicity both in human and rat cell lines. human cells was 100-fold higher than the activity measured The human MERO 25 cells were killed by GCV after in rat cells. These observations suggest higher susceptibility transduction at a MOI of 1 with the IG.Ad.CMV.TK vector of the human cells to infection by adenovirus vectors. whereas a MOI of 10 was required for a similar Furthermore, 10-fold higher expression was observed response with the IG.Ad.MLP.TK vector (Figure 2a and 281

3 282 Figure 2 Comparison of in vitro efficacy of IG.Ad.MLP.TK and IG.Ad.CMV.TK transduction and GCV treatment. Human and rat mesothelioma cells were infected with the recombinant virus (MOI: 0, 1, 3, 10, 100) and cultured in the presence or absence of GCV. After 4 days a clonogenic assay was performed (see Materials and methods). (a) MERO 25 cells infected with IG.Ad.CMV.TK; (b) MERO 25 cells infected with IG.Ad.MLP.TK; (c) II-45 cells infected with IG.Ad.CMV.TK; (d) II-45 cells infected with IG.Ad.MLP.TK. (o: No colonies seen; bars, s.d.). b). At the highest MOI (100) IG.Ad.MLP.TK induced only mals 1, 4, 8, 13 and 15 days after inoculation of 10 5 tumor limited GCV sensitivity in II-45 rat cells, while with cells into the pleural cavity. The tumor growth as meas- IG.Ad.CMV.TK more than 90% cell kill was seen at an ured by the weight of the total thoracic organs is shown MOI of 30 and a weak response at an MOI of 10 (Figure in Figure 3. After 8 days, small disseminated tumor nod- 2c and d). These differences between the two promoters ules were visible in the pleural cavity but this was not are quantitatively similar to the difference of a factor of reflected by an increased weight of the thoracic organs. 10 observed following luciferase transductions (Figure 1). By the first time-point at which an increase was recorded, The data in Figure 2 also reveal that in terms of MOI tumor growth was already apparent macroscopically the rat cells are less permissive to transduction by both throughout the pleural cavity. At this point, day 13, the recombinant viruses than the human cells; by a factor of tumor mass has attained an average weight of 2.4 g 100 in the case of the CMV vector. Using the MLP vector which roughly represents cells. Thus, during the this difference could not be calculated accurately because first 13 days the inoculum of 10 5 tumor cells has of the weak response of the rat cells at an MOI of 100, expanded fold. This expansion requires at least but it is definitely more than 30-fold (Figure 2, compare between 14 and 15 cell doublings. Taking into account a a with c and b with d). certain amount of tumor cell loss, the cell cycle time of In the human mesothelioma cells both recombinant this tumor has to be less than 1 day. For this and the viruses were cytotoxic in the absence of GCV. This effect following calculations we have disregarded the contribution was less pronounced in the case of IG.Ad.MLP.TK. of the stroma to the tumor weight, as the microwas Neither of the two viruses were cytotoxic for cultured scopical inspection of these tumors shows an overwhelming rat cells at the MOIs tested, which may reflect the lower predominance of mesothelioma cells (Figure 4c). permissiveness of rat cells to adenovirus as compared with human cells. Efficiency of in vivo gene transfer: To evaluate in vivo gene transfer efficiency and the distribution of the recombinant In vivo studies adenovirus through the thorax contents, infectious units of IG.Ad.CMV.lacZ were injected intrapleurally into Tumor growth rate: The tumor growth characteristics of rats bearing 1- or 10-day-old malignant mesotheliomas. II-45 cells in F344 rats were determined by killing the ani- Control animals received IG.Ad.CMV. TK. Three days after

4 283 Figure 3 Tumor growth presented as the weight of the contents of the thoracic cavity. The growth of MM was evaluated by weighing the thoracic contents of rats 1, 4, 8, 13, 15 days after injection of 10 5 II-45 cells in the pleural cavity, four animals per group. (, mean; bars, s.d.). virus injection, the rats were killed and the thoracic cavity stained with X-gal solution to monitor lacz expression. At the time of death the tumors had been growing for 4 and 13 days, respectively. In the former group, macroscopical tumors could not be found, but at day 13 the thoracic cavity contained a large tumor mass. In both groups of rats the parietal and visceral pleura were stained uniformly blue. Microscopic inspection of paraffin sections showed a homogeneous distribution of blue cells in the mesothelium (Figure 4a and b). In the day 13 group a patchy distribution of blue cells mostly in the superficial cell layers of the tumor tissue was observed (Figure 4c). The pleural surfaces were normal, no inflammatory reaction of the mesothelium was observed. Figure 4 In vivo IG.Ad.CMV.lacZ-mediated gene transfer into intrapleural MM. Microscopic picture of the -galactosidase activity. Blue nuclear Treatment of malignant mesotheliomas in rats with staining indicates -galactosidase activity. (a) Heart and pericardium IG.Ad.CMV.TK/GCV (HPS stained, 400); (b) lung and pleura (HPS stained, 400); (c) lacz After confirmation of the susceptibility of HSVtk expressing expression in tumor tissue (HPS stained, 200). mesothelioma cells to GCV exposure in vitro, we investigated the effect of administration of the IG.Ad.CMV.TK virus followed by treatment with GCV and subsequently received GCV, presented small tumor in pleural mesotheliomas in rats. After inoculation of 10 5 deposits in several sites within the thoracic cavity. How- II-45 cells into the right pleural cavity on day 0, groups ever, these tumor masses were much smaller than the of four rats received a single injection of infectious ones found in the control group receiving PBS, as is particles at the site of tumor inoculation either on day 1, reflected in the weight of the thoracic contents (groups 4 2, 4 or 8, respectively. One day after the injection of the and 8 respectively, Table 1). These small tumor deposits virus, i.p. treatment with GCV was started until the ani- could not be dissected out accurately, so that the total mals were killed on day 15. The macroscopical appearance weight could not be estimated. An approximation is pro- and the weight of the thoracic contents are listed in vided by subtracting the average thoracic content weight Table 1. In the animals of groups 1, 2 and 3 macroscopic of groups 1 to 3 (3.03 g) from that of group 4, which tumor growth was not observed inside the thorax, but in yields 0.17 g. The treated animals did not show any clinical five out of 12 rats tumor was present in the scar of the abnormalities at any time during the treatment. The thoracotomy. This may be due to leakage of tumor cells pleural surfaces of these animals were macroscopically into the wound bed. Presently, we have replaced the thoracotomy normal, but microscopic inspection revealed a mild by needle injection into the pleural cavity; this inflammatory reaction of the mesothelium with fibrosis procedure minimizes tumor growth in scar tissue. The and infiltration by macrophages and lymphocytes (Figure animals that were treated with IG.Ad.CMV.TK at day 8 5). This reaction was seen in both the parietal and the

5 284 Table 1 In vivo effect of the IG.Ad.CMV.TK/GCV treatment of II-45 mesotheliomas in Fisher rats Group (n = 4) IG.Ad.CMV.TK on day Treatment Weight (g) of thoracic Fraction of animals with contents on day 15 macroscopic tumor 1 1 GCV 2.9 (±0.2) a 1/4 b 2 2 GCV 3.2 (±0.4) 1/4 b 3 4 GCV 3.0 (±0.4) 3/4 b 4 8 GCV 3.2 (±0.3) 4/4 5 1 PBS 6.2 (±1.3) 4/4 6 2 PBS 6.7 (±1.1) 4/4 7 4 PBS 5.7 (±0.6) 4/4 8 8 PBS 7.6 (±0.5) 4/4 9 (No tumor) 2 GCV 2.8 (±0.4) 0/4 a Data are mean ± s.d., n = 4. b Tumor only present in the scar of the thoracotomy. trols. As a result of the treatment with GCV the symptom-free survival was significantly prolonged to 33 ± 1.8 days as compared to 19.2 ± 1.2 days (Figure 6, log rank test P 0.004). The tumor weight at the time of death as calculated from comparison of the weights of the thoracic contents with that of age-matched nontumor bearing animals was 2.9 ± 1.1 g in the GCV treated rats and 4.4 ± 0.8 g in the controls, the difference being not statistically significant. Discussion The in vitro data indicate that the CMV vectors (IG.Ad.CMV.luc, IG.Ad.CMV.TK) are about 10-fold more effective than the respective MLP vectors in the induction of luciferase expression and in inducing sensitivity to GCV-mediated toxicity. The latter suggests higher expression levels of tk following infection with the CMV virus. A similar difference was reported by Wills et al 14 for vectors carrying the p53 gene with the CMV and the MLP promoter, respectively. The CMV virus, as well as the MLP virus, exhibited cytotoxicity in the absence of GCV for the human cell line. Figure 5 Histologic section of the mesothelium after treatment with IG.Ad.CMV.TK/GCV. (a) Heart and pericardium of a rat treated with IG.Ad.CMV.TK on day 2, followed by treatment with GCV until death on day 14 (HPS stained, 400). (b) Heart and pericardium of a nontreated animal (HPS stained, 400). visceral pleura as well as in the pericardium. In the animals treated with PBS the pleurae were completely infiltrated with tumor tissue, so that it could not be established whether the inflammation noted in the GCV treated animals was caused by the recombinant virus or the combination of GCV and recombinant virus. Survival following treatment with IG.Ad.CMV.TK and GCV In a separate experiment two groups of eight rats each received the recombinant virus 8 days after inoculation of 10 5 tumor cells. Treatment with GCV was started the following day in one group and continued for 14 days. The other group was treated with PBS and served as con- Figure 6 Survival curves of rats with established mesothelioma treated with recombinant adenovirus and GCV. Rats (n = 16) were injected with IG.Ad.CMV.TK 9 days after tumor implantation. Twenty-four hours later 25 mg/kg of GCV or 1 ml of PBS was administered i.p. twice a day for 14 days. Rats were killed when moribund or when dyspnea developed. Log-rank statistical analysis revealed that the two survival curves were different (P 0.04).

6 Again, based on MOI the CMV virus was more toxic than group 4 were exposed to GCV at day 9. As was calculated the MLP virus. The viruses were not toxic for rat cells but in the Results section, at day 15 tumor weight was 0.17 this may well be due to the lower susceptibility of rat cells g that is equivalent to tumor cells. Based on this to infection by adenovirus, which we found to be about last point, a regrowth curve was constructed for the 100-fold for all the various end points studied (Figures 1 treated rats of group 4, Table 1 (Figure 7, curve B). The and 2). We have observed a similar dose-dependent toxic fraction of tumor cells killed by the treatment can be cal- effect of adeno-tk virus in other human cell lines including: culated assuming an immediate cytotoxic effect on day 9 glioma cells (U251), small cell lung carcinoma cells (GLC- followed by a regrowth at the same rate as represented 01), non-small cell lung carcinoma cells (A549) and mela- by the growth curve for nontreated tumors (Figure 7, noma cells (518 A2), but not in the rat glioma cell line 9L. 16 curve A) and comparing the number of cells for both This toxic effect seems to be related to the levels of curves on day 9. By these means of comparison the result expression of the transgene in the infected cells; we also of the treatment can be expressed as about 5 log cell kill. observed cytotoxicity related to higher expression of the The tumor growth curve of the treated animals in this lacz gene after recombinant adenovirus infection of human experiment has shifted 9.7 days (Figure 7, curve B). cells. 16 It is important to know whether these cytotoxic Accordingly these animals were expected to die on day effects in vitro haverelevancefortheclinicaluseofadenoviral 29. If we compare this with the survival time of the rats vectors for gene therapy. In the present study, we treated for 14 days with GCV (Figure 6), the latter ani- observed a mild inflammation of the normal mesothelium mals survived for 33 days, that is 4 days longer than after treatment with IG.Ad.CMV.TK and GCV. This process expected. Since the only difference was the longer is not related to tumor growth because it was also observed exposure to GCV it has to be assumed that the tumor in animals without tumor that received the same treatment. response is determined by the duration of the GCV treatment. This reaction could be caused by overexpression of the tk It is therefore worth investigating longer exposure gene. More extensive toxicity studies are clearly needed to times than presently employed. Apart from this issue, the elucidate whether this reaction is caused by the recombinant tumor response in our survival experiment seems to sig- adenovirus, the GCV or the combination of both nificantly exceed expectations based on the distribution agents. More relevant is whether this treatment-related of the transduced cells as demonstrated with the lacz inflammation will occur in patients. The only way to inves- recombinant virus. In that experiment only a few per cent tigate this issue is dose-finding studies in patients. of the tumor cells were transduced. Apparently, the so- The results of the two in vivo experiments using suicide called bystander effect in this tumor is much greater than gene therapy provide some data to evaluate the resulting so far assumed. It seems unlikely that the bystander effect tumor inhibition in a quantitative way. Firstly, a theoreti- could persist beyond the few days of GCV treatment. By cal growth curve can be constructed based on three measured that time the vast majority of transduced tumor cells tumor weights obtained at day 13: 2.4 g, day 15: should have entered cell division with suicide as a conse- 4.6 g (both from Figure 3: weight of thoracic organs of quence. Then, the only other source of thymidine kinase tumor-bearing rats minus weight of thoracic organs of to produce phosphorylated GCV is nondividing transduced rats 1 day after injection of tumor cells) and day 19: 4.4 g cells. Such cells could be tumor cells that are (from survival experiment data, control group). These are resting. Alternatively as we observed that a large proportion converted to number of tumor cells considering 1 g equal of normal mesothelium cells were also transconverted to 10 9 cells. The other informative point is the inoculum duced in the lacz experiment (Figure 4a and b), it is of 10 5 tumor cells at day 0. A Gompertz-fitted growth tempting to speculate that these cells are an additional curve 17 was estimated using these points (Figure 7, curve and long-lasting source of toxic GCV metabolites that can A). In the experiment described in Table 1 the rats of be transfered to nontransduced tumor cells. Another hypothesis could be an immunological reaction against the tumor. These issues are presently under investigation. Our in vivo results largely confirm the data published recently by Elshami et al 11 on treatment of MM. However, there are some differences. First, we found a longer survival time after treatment with IG.Ad.CMV.TK/GCV, 14 days against 8 days reported by Elshami et al. 11 Moreover, we did not observe differences in tumor weight at the time of death in our survival study while Elshami et al reported lower tumor weights in the treated animals. Both groups of investigators used the same tumor model, similar techniques for implantation of the tumor cells and treated similar tumor sizes. However Elshami et al 11 started the treatment 4 days after injection of 10 6 II-45 cells and in our case we started 8 days after injection of 10 5 cells. Apparently, the growth characteristic of the tumor is different between the two groups. In our hands the tumor cell doubling time is about 24 h while for Elshami et al it is longer, approximately 2 days. This discrepancy could explain why we did not find a marked difference in tumor weight at the time Figure 7 Gompertz fitted growth curve for rat mesothelioma. Curve A ( ): estimated curve of tumor growth in nontreated animals. Curve of death in our survival experiment. Both groups applied B (-- --): estimated curve of tumor growth in animals treated according similar recombinant adenovirus doses, but it is difficult to to Table 1, group 4. compare them since the procedures for determining virus 285

7 286 Gene therapy for malignant mesothelioma titers were different. The recombinant adenovirus vectors are also different: in our case the promoter that drives the HSVtk gene is the CMV promoter and Elshami et al used a construct with the RSV (Rous Sarcoma virus) promoter. Moreover, we retained in our vector the E3 region whose function in the wild-type virus is to evade an immune response against infected cells. This may have caused longer HSV-tk expression and may have resulted in more effective therapy. We can not provide a conclusive explanation for the observed difference in outcome between the two laboratories. Both the different vectors as well as the faster tumor growth may have contributed to the higher sensitivity of the tumor cells to the HSV-tk/GCV treatment in our hands. Clearly, the responses observed in the present experiments were obtained with tumor masses that are many orders of magnitude smaller than those to be encountered in patients with MM. On the other hand, human tumor cells seem to be more permissive to adeno-tk. In analogy with the strategy successfully developed for con- ventional treatment modalities, it should be determined if successive cycles of gene transfer/gcv improve the response rate of larger tumors. In addition, new ways of distributing the virus throughout the deeper layers of a tumor, have to be developed. Figure 8 Physical map of pcmv.tk, the plasmid used for the generation of IG.Ad.CMV.TK. HSV-TK: herpes simplex virus thymidine kinase; CMV: cytomegalovirus immediate early gene promoter and enhancer; SD-SA: 180 bp region of the SV40 genome containing late viral protein gene 16s/19s splice donor and acceptor signals; SV40: Simian virus 40 polyadenylation sequence (nt of the SV40 genome); BglII ScaI fragment of aden- ovirus type 5: nt of the adenovirus type 5 genome. Materials and methods IG.Ad.CMV.luc. The MERO 25 human mesothelioma and II-45 rat mesothelioma cells were plated in 24-well culture Cell lines and culture conditions dishes (Costar Europe, Badhoevedorp, The MERO 25, a human mesothelioma cell line, was kindly Netherlands) at a density of 10 4 cells per well. The cells provided by Dr M Versnel (University of Rotterdam, The were infected in triplicate with IG.Ad.MLP.luc or Netherlands). II-45, a cell line isolated from asbestosthe infected cells were prepared 72 h after infection to IG.Ad.CMV.luc at an MOI of 0, 1, 10 and 100. Lysates of induced rat mesothelioma 18 was a gift from Dr Ch Walker (Anderson Cancer Center, Houston, TX, USA). measure luciferase activity according to Fortunati et al. 19 Tumor cells were cultured in Dulbecco s modified Eagle s To study the suicide gene efficacy a clonogenic assay was medium supplemented with 10% fetal calf serum, nonculture performed. MERO 25 and II-45 cells were seeded in 24-well dishes (Costar), 10 4 cells per well. After 3 h, the cells essential amino acids, penicillin (100 IU/ml; Gibco, Breda, The Netherlands) and streptomycin (50 g/ml; were infected in triplicate with IG.Ad.MLP.TK or Gibco, Breda, The Netherlands). All cell lines were mainively. The culture medium in each well was replaced with IG.Ad.CMV.TK at MOI of 0, 1, 3, 10, 30 and 100, respecttained at 37 C in a humidified atmosphere at 5% CO 2. medium 24 h later with or without 10 m of GCV (Syntex Adenovirus vectors BV, Rijswijk, The Netherlands). After 72 h the cells in the The construction of IG.Ad.MLP.TK and IG.Ad.MLP.luc control wells (MOI 0, without GCV) were counted and have been described in detail elsewhere. 7,10 IG.Ad.CMV.TK diluted in order to seed 200 cells in a 60 mm culture dish was made from the pcmv.tk plasmid (Figure 8), in which (Costar). The same dilutions were made for the cells of the the HSV-tk expression is under the control of the CMV prodishes other wells, thus identical volumes were placed in 60 mm moter and SV40 RNA splicing signals (180 bp) containing and the cells were cultured using the same con- splice donor and acceptor signals of the late viral genes 16s ditions as described previously. After 8 10 days, dishes and 19s. These sequences were isolated from pcmv were fixed with methanol (70% solution) and stained with NLS/lacZ, 19 obtained from Dr Fortunati (Erasmus Univer- a methylene blue solution; macroscopic colonies (more than sity,rotterdam,thenetherlands).theescherichia coli lacz 50 cells) were counted. marker gene preceded by a nuclear location signal and the luciferase gene were cloned in plasmids similar to In vivo protocols pcmv.tk called pcmv.lacz and pcmv.luc respectively. MM were established in syngeneic Fisher 344 rats (11- The adenovirus vectors IG.Ad.CMV.TK, IG.Ad.CMV.LacZ week-old, males, weighing g) by injecting 10 5 IIand IG.Ad.CMV.luc were generated by cotransfecting cells suspended in 500 l PBS into the pleural cavity cells with SalI linearized plasmids and the large ClaI fraglateral via thoracotomy between the eighth and ninth ribs at the ment of wild-type Ad5 DNA. Recombinant adenovirus side of the right thorax. To evaluate the tumor were plaque-purified twice, propagated and titrated accordafter growth rate, animals were killed 1, 4, 8, 13 or 15 days ing to standard procedures. The virus titers were determinated injection of the tumor cells, and the thoracic contents by end point cytopathogenic effect (CPE) assay. 20 (lungs, heart, mediastinum, trachea, and diaphragm) including tumor tissue were weighed. The average In vitro studies To compare the efficiency of gene transfer in vitro, mesothelioma cell lines were infected with IG.Ad.MLP.luc or weight of the thoracic contents of age-matched control rats were subtracted to arrive at an approximation of the tumor mass. Four rats were used per group.

8 In vivo gene transfer using lacz as gene marker was (Division of Health Research, TNO, The Netherlands) for evaluated as follows: on day 0, MM were established in evaluation of the histological preparations. Fisher rats (n = 16) by injecting 10 5 II-45 cells according to the procedure described above. On day 1 (n = 4) and References on day 10 (n = 4), the same procedure was used to admin- 1 Stanton MF, Wrench C. Mechanism of mesothelioma induction ister infectious particles of IG.Ad.CMV.LacZ in with asbestos and fibrous glass. J Natl Cancer Inst 1972; 48: 700 l of PBS. Control animals received an identical dose of IG.Ad.CMV.TK. Rats were killed 3 days after virus 2 Sugarbaker DJ et al. Extrapleural pneumonectomy, chemo- administration. The vascular system of the animals was therapy and radiotherapy in the treatment of diffuse malignant mesothelioma. J Thorac Cardiovasc Surg 1991; 102: first perfused with 10 ml of PBS and subsequently with 3 Moolten FL. Drug sensitivity ( suicide ) genes for selective can- 10 ml of 0.2% gluteraldehyde (Sigma, Axel, Belgium). The cer chemotherapy. Cancer Gene Ther 1994; 1: thoracic contents were removed and fixed in the 0.2% 4 Mulligan RC. The basic science of gene therapy. Science 1993; gluteraldehyde solution for 1 h, washed three times in 260: PBS and stained with X-gal (5-bromo-4-chloro-3-indolyl- 5 Ram Z et al. Intrathecal gene therapy for malignant leptomeningeal -galactopyranoside) solution (Molecular Probes Europe, neoplasia. Cancer Res 1994; 54: Leiden, The Netherlands) for 3 h at 37 C, as described by 6 Culver KW et al. In vivo gene transfer with retroviral vector- Bout et al 21 Samples of lung, heart, mediastinum, dia- produced cells for treatment of experimental brain tumors. phragm, intercostal muscle and tumors were embedded Science 1992; 256: Vincent AJPE et al. Herpes simplex virus thymidine kinase gene in paraffin, 2 m sections were made and stained with therapy for rat malignant brain tumors. Hum Gene Ther 1996; 7: hematoxylin, pholin and saphrane (HPS) For the in vivo treatment of MM, infectious par- 8 Horwitz MS. Adenoviridae and their replication. In: Fields BN, ticles of IG.Ad.CMV.TK were introduced into the pleural Knipe DM (eds). Virology. Raven Press: New York, 1990, pp cavity of Fisher rats (n = 32) on day 1, 2, 4 or 8 after tumor cell implantation on day 0. Administration of GCV 9 Bonnekoh B et al. Inhibition of melanoma growth by adenoviralmediated (50 mg/kg/day) or PBS i.p. twice per day was started 24 HSV thymidine kinase gene transfer in vivo. J Invest h after the virus infection and continued until the animals Dermatol 1995; 104: were killed. Another group (n = 4) without tumor was 10 Vincent AJPE et al. Treatment of leptomeningeal metastases in a rat model using a recombinant adenovirus containing the treated with IG.Ad.CMV.TK on day 1 and received the HSV-tk gene. J Neurosurg 1996; 85: GCV treatment for 13 days. At day 15, all rats were killed 11 Elshami AA et al. Treatment of pleural mesothelioma in an and inspected for macroscopic tumor growth in the tho- immunocompetent rat model utilizing adenoviral transfer of the racic cavity. The total thoracic contents and the dia- herpes simplex virus thymidine kinase gene. Hum Gene Ther phragm including tumors were weighed. Histologic sec- 1996; 7: tions were made of the thoracic organs at the midway 12 O Malley BW, Chen S, Schwartz M, Woo SLC. Adenovirusmediated transverse level to include parts of the lungs, heart and gene therapy for human head and neck squamous cell mediastinum. The diaphragm was cut separately. The cancer in a nude mouse model. Cancer Res 1995; 55: tissues were embedded in paraffin, 2 m sections were 13 Wills KN et al. Gene therapy for hepatocellular carcinoma: made and stained with HPS. chemosensitivity conferred by adenovirus-mediated transfer of the HSV thymidine kinase gene. Cancer Gene Ther 1995; 2: For the survival study, 8 days after intrapleural implan tation of 10 5 II-45 tumor cells on day 0, Fisher rats (n = 14 Wills KN et al. Development and characterization of recombi- 16) were injected with infectious particles of IG.Ad. nant adenovirus encoding human p53 for gene therapy of can- CMV.TK. On day 9, eight rats received 50 mg/kg per day cer. Hum Gene Ther 1994; 5: GCV and another eight rats were injected with PBS for Yang Y et al. Inactivation of E2a in recombinant adenoviruses days. The rats were observed daily and killed when show- improves the prospects of gene therapy in cystic fibrosis. Nat ing dyspnea, which was accompanied by lethargy and Genet 1994; 7: weight loss. Thoracic contents were dissected and weighed. 16 Vincent AJPE et al. Preclinical testing of recombinant adenoviral The symptom-free period is presented in a survival curve. HSV-tk gene therapy for CNS malignancies. Neurosurgery (in The log rank test was applied for statistical analysis of press). 17 Simpson-Herren L, LLoyd HH. Kinetic parameters and growth symptom-free latency data. Gompertz-fitted curves were curves for experimental tumor systems. Cancer Chemother Rep calculated according to Simpson-Herren et al ; 54: Craighead JE, Akley NJ, Gould LB, Libbus BL. Characteristics of tumors and tumor cells cultured from experimental asbestosinduced mesotheliomas in rats. Am J Pathol 1987; 129: Acknowledgements 19 Fortunati E et al. In vitro and in vivo gene transfer to pulmonary This work was supported by the Foundation Banco del cells mediated by cationic liposomes. Biochim Biophys Acta 1996; 1306: Sud (MCE), The Netherlands Organization for Scientific 20 Precious B, Russell WC. Growth, purification and titration of Research and Het Preventie Fonds. The authors wish to adenoviruses. In: Mohy B (ed). Virology: A Practical Approach. thank HMJPM Brok, J van der Brugge and J de Vast for IRL Press: Oxford, 1985, pp expert technical assistance, J Jansen (TNO-CSD, The 21 Bout A et al. Lung gene therapy: in vivo adenovirus-mediated Netherlands) who kindly performed the Gompertz-fitted gene transfer to Rhesus monkey airway epithelium. Hum Gene curves constructions and calculations and Dr C Zurcher Ther 1994; 5: