HUMAN GENE THERAPY CLINICAL DEVELOPMENT 25:7–15 (March 2014) ª Mary Ann Liebert, Inc. DOI: 10.1089/humc.2013.181

Pre-Clinical Safety Studies

Preclinical Safety Assessment of Ad[I/PPT-E1A], a Novel Oncolytic Adenovirus for Prostate Cancer Ellen Schenk,1 Magnus Essand,2 Robert Kraaij,3 Rachel Adamson,4 Norman J. Maitland,4 and Chris H. Bangma1

Abstract

Prostate cancer is the most common malignancy in the Western world. Patients can be cured only when the tumor has not metastasized outside the prostate. However, treatment with curative intent fails in a significant number of men, often resulting in untreatable progressive disease with a fatal outcome. Oncolytic adenovirus therapy may be a promising adjuvant treatment to reduce local failure or the outgrowth of micrometastatic disease. Within the European gene therapy consortium GIANT, we have developed a novel prostate-specific oncolytic adenovirus: Ad[I/PPT-E1A]. This adenovirus specifically kills prostate cells via prostate-specific replication. This article describes the clinical development of Ad[I/PPT-E1A] with particular reference to the preclinical safety assessment of this novel virus. The preclinical safety assessment involved an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells, and a toxicity study in normal mice. These studies confirmed that Ad[I/PPT-E1A] efficiently kills prostate tumor cells in vivo, is not harmful to other organs, and is well tolerated in mice after systemic delivery. The safety, as well as the immunological effects of Ad[I/PPT-E1A] as a local adjuvant therapy, will now be studied in a phase I doseescalating trial in patients with localized prostate cancer who are scheduled for curative radical prostatectomy and can be used as an updated paradigm for similar therapeutic viruses.

Adenovirus-mediated gene therapy is being widely explored for prostate cancer and is attractive as an adjuvant treatment especially for localized disease because of the favorable safety profile and the easy accessibility of a prostate tumor (Schenk et al., 2010). It is well accepted that the efficacy of replication-deficient adenoviral vectors is too limited for the treatment of solid tumors and that targeted therapy with oncolytic adenoviruses now promises to be an effective antitumor treatment. In the past decade, a number of clinical trials for prostate cancer using the oncolytic adenoviruses Ad5-CD/TKrep (Freytag et al., 2002, 2003), Ad5-yCD/mutTKSR39rep-ADP (Freytag et al., 2007a), CV706 (DeWeese et al., 2001), and CG7870 (Small et al., 2006) have been reported from the United States. These viruses were all well tolerated as local salvage therapy for locally recurrent prostate cancer after radiotherapy (DeWeese et al., 2001; Freytag et al., 2002), for the systemic treatment of hormone-refractory metastatic prostate cancer (Small et al., 2006), and as a local adjuvant to radiotherapy in patients with a newly diagnosed disease

Introduction

P

rostate cancer is the most common malignancy and a major cancer-related cause of death in Western men (Ferlay et al., 2013). The disease can be cured only when the tumor is still localized to the prostate. However, therapies with curative intent for localized disease, including radical prostatectomy and radiotherapy, fail in a substantial number of patients because of insufficient treatment locally or because of the presence of micrometastases, resulting in biochemical recurrence and ultimately progressive disease with a fatal outcome (Djavan et al., 2003; Sukumar et al., 2013). Because of the widespread use of screening, more patients are currently being diagnosed with localized disease. As a result, there is a strong clinical need to improve the outcome of primary treatment of clinically staged localized prostate cancer. This may be achieved by adjuvant therapy aimed at the reduction of the local tumor size and the attack of presumed micrometastases before surgery or radiation, or at the treatment of recurrent disease.

Departments of 1Urology and 3Internal Medicine, Erasmus MC, 3000 CA Rotterdam, The Netherlands. 2 Rudbeck Laboratory, Department of Immunology, Genetics, and Pathology, Uppsala University, SE-75185 Uppsala, Sweden. 4 YCR Cancer Research Unit, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.

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(Freytag et al., 2003, 2007a). Data on the level of prostatespecific antigen (PSA), a biochemical measure for disease progression, and related parameters indicate direct antitumoral activity, even in the long-term. The 5-year follow-up outcome of patients treated with Ad5-CD/TKrep after local recurrence of prostate cancer after curative radiotherapy showed a significant increase in PSA doubling time from a mean of 17 months to 31 months and an average delay of 2 years for the need for androgen suppression therapy (Freytag et al., 2007b). The most likely explanation for this long-term clinical benefit as postulated by the investigators is the induction of antitumor immunity. Further research on the characteristics of the immune response triggered by oncolytic adenovirus therapy is needed to confirm this hypothesis. Oncolytic adenovirus therapy as an adjuvant for primary treatment of localized prostate cancer with curative intent has until now only been studied in combination with radiotherapy (Freytag et al., 2003, 2007a). To this date, no knowledge on the combined use of oncolytic adenovirus therapy and radical prostatectomy is available. Within the GIANT project, a European gene therapy consortium funded by the European Commission, a novel prostate-specific oncolytic adenovirus called Ad[I/PPT-E1A] has been developed that now will be clinically tested as an adjuvant treatment in patients with localized prostate cancer before surgery. Ad[I/PPT-E1A] is a prostate-specific replicating adenovirus whose growth is controlled by a synthetic regulatory sequence comprising the PSA enhancer, the prostate-specific membrane antigen (PSMA) enhancer, and the T-cell receptor gamma-chain alternate reading frame protein (TARP) promoter (Cheng et al., 2003, 2004, 2006). It has been demonstrated both in in vitro studies using prostate cancer cells and nonprostate cell lines, and in in vivo studies using mice with a subcutaneous xenograft derived from the LNCaP prostate cancer cell line that Ad[I/PPT-E1A] can specifically kill prostate tumor cells (Cheng et al., 2006). These promising preclinical results are now to be translated into the clinic. In a phase I dose-escalating trial, the safety and tolerability of Ad[I/PPT-E1A] as a local adjuvant before radical prostatectomy will be assessed, and the histopathological and immunological effects will be explored to get more insight in the mechanism of action of oncolytic adenovirus therapy. This article describes the clinical development of Ad[I/PPT-E1A] and focuses on the design and outcome of the preclinical safety studies that form the basis for the trial. Results and Discussion Clinical trial

The preclinical studies described in this article provide the basis for a phase I dose-escalating study. The primary objective of this trial is to assess the safety and tolerability of Ad[I/PPT-E1A], a novel prostate-specific oncolytic adenovirus (for further details on Ad[I/PPT-E1A] please refer to the Materials and Methods section, which is included in Supplementary Data [Supplementary Data are available online at www.liebertpub.com/humc]). A secondary objective is to explore the immunological effects induced by Ad[I/PPT-E1A] to get more insight in the mechanism of action of oncolytic adenovirus therapy. The study population will be composed of 12–18 men aged 35–70 years with localized prostate cancer scheduled

SCHENK ET AL.

for radical prostatectomy. Ad[I/PPT-E1A] will be administered 3 weeks before surgical removal of the prostate at 1 · 1011, 1 · 1012, or 5 · 1012 virus particles (VP) by an intraprostatic injection under guidance of transrectal ultrasonography in four equal deposits with a total volume of 1 ml. Patients will be intensively evaluated and monitored for toxicity and adverse events during the 3 weeks between virus injection and surgery. The main study parameter is dose-limiting toxicity (DLT) between 1 · 1011 and 5 · 1012 VP Ad[I/PPT-E1A], defined as any irreversible grade 3 or 4 toxicity. The first cohort of four patients will be treated with the lowest dosage of 1 · 1011 VP. If none of the patients experiences a DLT, the dose will be increased to 1 · 1012 VP for the second cohort of four patients, followed by 5 · 1012 VP for the third cohort of four patients. If a patient experiences a DLT, two additional patients will be enrolled at that dose level and the dose will be escalated if no more than one of six patients has a DLT. If DLT is observed in two patients at a dose level, that dose escalation will cease, and for the second and third cohort the previous dose level will be expanded to include a total of six patients. Secondary study parameters are immunological changes with respect to the systemic and local innate and adaptive immune system profile, and the presence of tumor-specific and adenovirus-specific T cells in blood and the prostate before, during, and after Ad[I/PPT-E1A] oncolytic adenovirus therapy. The trial is currently being conducted at the Erasmus MC Department of Urology, Rotterdam, The Netherlands (Principal Investigator C.H.B), and inclusion has started in autumn 2013. The clinical dossier was recently approved by the Dutch Central Committee on Research Involving Human Subjects (NL39923.000.12), and a compulsory permit for the deliberate release of a genetically modified organism into the environment has been obtained from the Dutch Ministry of Infrastructure and the Environment (IM 12-002). Objectives and study design

The strategy for the assessment of the preclinical safety of Ad[I/PPT-E1A] was designed based on discussions with the regulatory ethics agency. A major safety concern for the agency appeared to be the potential harmful effects of high systemic levels of Ad[I/PPT-E1A] because of leakage from the prostate or as a side effect of the virus administration procedure. The basis for this concern was the tragic event in the ornithine transcarbamylase trial in 1999, when a young patient died from acute toxicity after systemic administration of a high dose of an adenoviral vector. Since Ad[I/PPTE1A] is a novel virus not tested before in humans, favorable safety profiles in prostate cancer patients reported for comparable oncolytic adenoviruses could only be considered as circumstantial evidence. For the risk–benefit analysis in the planned first-use-in-man trial, the preclinical studies therefore had to address, on the one hand, the efficacy of the virus and, on the other hand, the effects of systemic Ad[I/ PPT-E1A]. As a result, the strategy designed for the preclinical safety assessment was based on the following three pillars: 1. Testing of the efficacy of Ad[I/PPT-E1A] in an animal model representing the prostate tumor characteristics of the patient population in the trial

CLINICAL DEVELOPMENT OF AD[I/PPT-E1A]

2. Testing of the specificity of replication and cytotoxicity of Ad[I/PPT-E1A] in a relevant series of primary cell cultures from human tissue 3. Testing of the safety of systemic Ad[I/PPT-E1A] in an animal model A description of the study designs as well as the rationale for the selected animal models and cell types is provided below for each preclinical study. Details on assays used in the preclinical studies are provided in Supplementary Data. Efficacy study in a human orthotopic xenograft mouse model. Ad[I/PPT-E1A] is a human adenovirus and will

replicate only in human prostate cells. Thus, preclinical efficacy of this virus can be established only in a human prostate model system. The human orthotopic PC346C xenograft mouse model was selected as the most appropriate in vivo model system. The PC346C xenograft closely resembles a clinically organ-confined prostate tumor, which is the targeted disease in the planned clinical trial. PC346C cells express the wild-type androgen receptor, are hormone dependent, and secrete large amounts of PSA (Marques et al., 2006; van Weerden et al., 2009; Maitland et al., 2010). The efficacy study was performed at Erasmus MC (by R.K.), Rotterdam, The Netherlands. Six male athymic NMRI nude mice bearing an orthotopic human PC346C tumor (size between 100 and 150 mm3) were treated with a single dose of 2 · 1010 VP of researchgrade Ad[I/PPT-E1A] by intratumoral injection of a total volume of 20 ll. This design mimicked the route of administration to be used in the clinical trial. As positive controls, six mice were injected with wild-type adenovirus (Ad[wt]) and another six mice with a conditionally replicating adenovirus controlled by the cytomegalovirus promoter (Ad[CMV-E1A]). As a negative control, six mice were treated with a replication-deficient adenovirus (Ad[mock]). Survival was assessed up to 63 days after treatment, and both tumor volumes and PSA levels in plasma were monitored. Serum levels of PSA were clinically used as a surrogate marker for biochemical recurrence after radical prostatectomy. In addition, histochemistry was conducted on tumor specimens to assess necrosis. The presence of Ad[I/PPTE1A] particles was tested by immunohistochemistry. Specificity study in human primary cells. The specificity of replication and cytotoxicity of clinical-grade Ad[I/PPTE1A] were tested in a panel of human primary cell cultures including vascular endothelial cells, bronchial epithelial cells, urothelial cells, and hepatocytes by the University of York (by N.J.M.), United Kingdom. These cell types were selected since they represent tissue potentially targeted in case of viral leakage from the prostate into the circulation (blood vessels) or into the urine (urinary tract), tissue for which Ad5 has a natural tropism (lungs) and tissue associated with the clearance of systemic adenovirus and as such with potential systemic toxicity (liver). Human primary prostate cells and the LNCaP prostate cell line were used as positive controls. An extensive description of the study recently was published (Adamson et al., 2012). Briefly, the cytotoxicity of Ad[I/PPT-E1A] was assessed at various time points after infection of Ad[I/PPT-E1A] at a multiplicity of infection

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(MOI) ranging between 5 and 5000, by determining the viability of the cells using the colorimetric MTS assay. The replication capacity of Ad[I/PPT-E1A] in the cell panel was assessed by determining the presence of active virus particles secreted into cell culture medium at various time points after infection of Ad[I/PPT-E1A] at an MOI of 500, and also in cell lysates at the final time point of the assay. The collected supernatants and cell lysates were titrated by infection of human embryonic kidney (HEK) 293 cells, and the infectious centers resulting from active Ad[I/PPT-E1A] virus were detected using immunostaining for the adenovirus hexon protein. Toxicity study in mice. In the clinical trial, Ad[I/PPTE1A] will be targeted to the prostate via a single intraprostatic injection at four deposits. There is a risk that Ad[I/ PPT-E1A] enters the circulation, either through leakage from the prostate or through the highly unlikely event of direct injection into the circulation during virus administration. To confirm that systemic Ad[I/PPT-E1A] is indeed not harmful, the toxicity of Ad[I/PPT-E1A] was assessed in mice. There is no preferred animal model available to test the safety of a prostate-specific oncolytic adenovirus. In general, all animal species are suboptimal because of the limited permissiveness to adenovirus when compared with humans. In addition, Ad[I/PPT-E1A] is selective for human prostatic cells only and as such the virus will not replicate at all in animal cells, neither in the prostate nor in other organs. The available orthotopic mouse models for prostate cancer are based on immune-deficient mice and are not representative of the patients eligible for the trial. Since an optimal animal model was not available, the mouse being a wellcharacterized and established immune-competent animal model for toxicity testing was selected as a species to study systemic effects by Ad[I/PPT-E1A]. To test the safety of circulating Ad[I/PPT-E1A] in a model in which Ad[I/PPTE1A] does not replicate, the virus was not injected in the prostate but was administered systemically. The Syrian hamster or the cotton rat, which are known to be semipermissive for human adenovirus replication and therefore can be considered to be more representative for the patient, were not selected as the animal model for the toxicity study for the following reasons. As stated above, Ad[I/PPT-E1A] will not replicate in any organ of a nonhuman species and as such semipermissiveness does not offer an advantage. Furthermore, at the time of the performance of the toxicity study, the Syrian hamster as well as the cotton rat were not an established animal model for toxicity testing under Good Laboratory Practice (GLP) conditions. A GLP single-dose toxicity study in mice was performed by the former contract research organization Visionar AB (Uppsala, Sweden). The study was designed to reflect a worst-case scenario, simulating that 10%, 100%, or 1000% of the maximum clinical dose (5 · 1012 VP) of Ad[I/PPTE1A] injected in the prostate would have leaked into the circulation. In summary, 146 male NMRI mice were injected once with 2 · 108, 2 · 109, or 2 · 1010 VP Ad[I/PPTE1A], corresponding to 0.1 · , 1 · , and 10 · the highest clinical dose based on weight/weight ratio, in a volume of 200 ll intravenously via tail veins. Controls were established by injecting mice with 200 ll of the buffer in which Ad[I/PPT-E1A] is stored (20 mM Tris, pH 8.0, 25 mM NaCl,

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2.5% glycerol). The animals were divided over 16 groups that were euthanized at 1, 4, 10, or 30 days. The weight and health status of the animals was recorded at day 0 (day of administration) and then at days 1, 4, 7, 10, 14, 21, and 30 after administration. Hematology, clinical chemistry, histopathology, and biodistribution parameters were assessed. The treatment and monitoring schedule is provided in Table 1. Summary of data Efficacy of Ad[I/PPT-E1A] in a human orthotopic xenograft mouse model. In the groups of mice treated with Ad[wt],

Ad[CMV-E1A], and Ad[mock], one mouse per group had to be excluded because of lethality not related to the tumor or the virus treatment. Mice injected with Ad[mock] all died within 44 days because of the tumor burden of maximum 1000 mm3. Five out of six mice treated with Ad[I/PPT-E1A] were still alive at the end of the study (day 63), and survival was comparable to survival of mice treated with Ad[wt] (Fig. 1). In contrast, only one out of five mice treated with Ad[CMV-E1A] survived during this study. We hypothesize that the difference in survival benefit induced by Ad[I/PPTE1A] and Ad[CMV-E1A] is related to the fact that the CMV promoter is too strong and therefore induces too high expression of E1A leading to apoptosis and less efficient replication of the virus. Previously, we have shown in cell lines that when the immediate early CMV promoter is used to control E1A gene expression the E1A protein level is much higher than when the recombinant I/PPT sequence is used (Cheng et al., 2006). In vitro, Ad[CMV-E1A] indeed kills prostate cancer cells much quicker than Ad[I/PPTE1A]. Since E1A is a potent proapoptotic protein, cell killing by Ad[CMV-E1A] is most likely a result of a combination of apoptosis and lysis. In vivo, the situation is very different since viruses must have the time to replicate inside infected host cells and release progeny viruses that can subsequently infect neighboring cells before the primary

infected host cells go into apoptosis. The positive effect on survival by Ad[I/PPT-E1A] observed in five mice coincided with a reduction of the tumor volume and of the plasma PSA levels in these mice (Fig. 2), indicating that the survival advantage was because of inhibition of tumor growth and tumor destruction by Ad[I/PPT-E1A]. To confirm that this effect could be ascribed to cytotoxicity, in another study with comparable conditions, histology of the prostate was performed at days 7, 14, and 21 after treatment with Ad[I/PPT-E1A], Ad[wt], Ad[CMV-E1A], or Ad[mock], and the level of necrosis was assessed (Fig. 3). The Ad[mock]-treated tumors showed a baseline level of necrosis of about 20%. The level of necrosis in Ad[CMVE1A]-treated tumors was slightly higher compared with necrosis in the Ad[mock]-treated tumors. In contrast, Ad[I/ PPT-E1A] induced massive cell death in a time-dependent manner, and the level of necrosis was comparable to that caused by Ad[wt]. Immunohistochemistry to assess the presence of Ad[I/PPT-E1A] particles in the tumor showed that the signal increased over time (Fig. 4), which is indicative of replication. This coincided with an expansion of the size of necrotic areas. All together, the results of this preclinical efficacy study confirmed that Ad[I/PPT-E1A] can kill human prostate tumor cells in vivo via cytotoxicity. Specificity of cytotoxicity and replication of Ad[I/PPT-E1A] in human primary cell cultures. The outcome of the speci-

ficity study has been reported by Adamson et al. (2012). Summarized, replication and cytotoxicity of Ad[I/PPT-E1A] was confirmed in the human primary prostate cells and the LNCaP prostate cell line. No replication of Ad[I/PPT-E1A] was observed in any of the nonprostate primary cell cultures, in contrast to wild-type Ad5 that efficiently replicated in all cell types and therefore killed the cells. Ad[I/PPTE1A] was not toxic to human primary bronchial epithelial cells, human primary umbilical vein endothelial cells, and

Table 1. Treatment and Monitoring Schedule in the Preclinical Toxicity Study Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Treatment Vehicle Vehicle Vehicle Vehicle 2 · 108 VP 2 · 108 VP 2 · 108 VP 2 · 108 VP 2 · 109 VP 2 · 109 VP 2 · 109 VP 2 · 109 VP 2 · 1010 VP 2 · 1010 VP 2 · 1010 VP 2 · 1010 VP

Day of termination Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day

1 4 10 30 1 4 10 30 1 4 10 30 1 4 10 30

Monitoring Biodistribution Hematology, clinical Hematology, clinical Hematology, clinical Biodistribution Hematology, clinical Hematology, clinical Hematology, clinical Biodistribution Hematology, clinical Hematology, clinical Hematology, clinical Biodistribution Hematology, clinical Hematology, clinical Hematology, clinical

chemistry chemistry, biodistribution, histopathology chemistry, biodistribution, histopathology chemistry chemistry, biodistribution, histopathology chemistry, biodistribution, histopathology chemistry chemistry, biodistribution, histopathology chemistry, biodistribution, histopathology chemistry chemistry, biodistribution, histopathology chemistry, biodistribution, histopathology

Number of mice 5 8 8 8 5 8 13 13 5 8 13 13 5 8 13 13

The vehicle was composed of 20 mM Tris, pH 8.0, 25 mM sodium chloride, and 2.5% glycerol. Doses of Ad[I/PPT-E1A] tested were 2 · 108 (0.1 · highest clinical dose), 2 · 109 (1 · highest clinical dose) and 2 · 1010 (10 · highest clinical dose). Details on the hematology and clinical chemistry parameters as well as the organs used for histopathology and biodistribution analysis are provided in Supplementary Data. VP, virus particles.

CLINICAL DEVELOPMENT OF AD[I/PPT-E1A]

FIG. 1. Survival of mice bearing a human orthotopic PC346C xenograft after a single intratumoral injection of 2 · 1010 VP Ad[I/PPT-E1A] (C, n = 6), Ad[wt] (-, n = 6), Ad[CMV-E1A] (:, n = 6), and Ad[mock] (>, n = 6), in a total volume of 20 ll phosphate buffered saline when tumors reached a size of 50–100 mm3 (15–20 days after inoculation with PC346C cells). Long-term survival was statistically analyzed by Logrank tests. Differences in survival compared with the Ad[mock]-treated animals were considered statistically significant when p-values were lower than 0.05. *p = 0.0025, **p = 0.0007. VP, virus particles.

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human primary urothelial cells. Up to an MOI of 50, Ad[I/ PPT-E1A] was not toxic to human primary hepatocytes. At higher levels, cytotoxicity was observed in these cells. No potential association of this cytotoxicity with replication of Ad[I/PPT-E1A] in human primary hepatocytes was confirmed, using a variety of tests. Besides the negative outcome of the replication assay, no I/PPT promoter activity was detected in these cells using serotype 5 adenoviruses expressing the green fluorescent protein. In addition, immunogold labeling in combination with transmission electron microscopy of Ad[I/PPT-E1A] confirmed the absence of replicating rafts of Ad[I/PPT-E1A] in the nuclei of the primary hepatocytes, which were, however, observed in hepatocytes infected with wild-type Ad5, and also showed a large number of Ad[I/PPT-E1A] particles sequestered only in the hepatocyte cytoplasm. It was concluded that the cytotoxic effects in human primary hepatocytes observed at high levels of Ad[I/PPT-E1A] were most likely because of sequestration of viral particles, potentially via the coxsackie adenovirus receptor that was found to be abundantly expressed on these cells, in combination with a fragile condition of cultured human primary hepatocytes. In summary, the specificity of replication of Ad[I/PPT-E1A] and the resulting cytotoxicity toward prostate cells was confirmed in the primary cell studies. Toxicity of Ad[I/PPT-E1A] in mice after systemic administration. Treatment of male NMRI mice with systemic

delivery of Ad[l/PPT-E1A] at a dose of 2 · 108, 2 · 109, or 2 · 1010 VP was well tolerated at all dosages. Analysis of hematology parameters showed a statistically significant difference only between groups with regard to the white

FIG. 2. Tumor volume and plasma PSA levels in individual mice bearing a human orthotopic PC346C xenograft after a single intratumoral injection of 2 · 1010 VP Ad[mock] (n = 5), Ad[CMV-E1A] (n = 5), Ad[I/PPT-E1A] (n = 6), and Ad[wt] (n = 5). Tumor volumes were measured once a week by transrectal ultrasonography and mice were euthanized when tumor volumes exceeded 1000 mm3 or after 63 days. PSA, prostate-specific antigen.

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FIG. 3. Comparative quantitation of tumor necrosis in individual mice bearing a human orthotopic PC346C xenograft after 7, 14, and 21 days of a single intratumoral injection of 2 · 1010 VP Ad[mock], Ad[CMV-E1A], Ad[I/PPT-E1A], and Ad[wt]. At each time point, two mice per group were euthanized. Necrosis was assessed in three sections per tumor, and is expressed as necrotic surface per total tumor surface of the respective section (mean – SEM). blood cell count. The magnitude of the difference was small and was considered to have limited biological relevance. Minor significant differences between groups were found with respect to the clinical chemistry parameters albumin, potassium, sodium, aspartate aminotransferase, and lactate dehydrogenase. Since the differences were within the range of normal variation, these findings were considered of limited toxicological relevance. At a dose of 2 · 109 and 2 · 1010 VP, comparable to 1 · and 10 · the highest clinical dose to be administered locally, lymphoid hyperplasia in the spleen was observed after 10 and 30 days. This may be indicative of an immune response to Ad[I/PPT-E1A]. Increased glycogen in the liver was observed at 30 days after administration of these same doses. Based on the outcome of the toxicity study, it was concluded that NMRI mice tolerated treatment with Ad[I/PPTE1A] administered systemically in doses of 0.1 · , 1 · , and 10 · , the maximum clinical dose intended to be injected intraprostatically in patients. Very high circulating concentrations, representing that >100% of the highest dose of Ad[I/PPT-E1A] that will be intraprostatically administered in patients participating in the clinical trial enters the blood stream, may cause an immune response, splenic hyperplasia, and increased glycogen depots in the liver.

Biodistribution of Ad[I/PPT-E1A] in mice after systemic administration. At days 1, 10, and 30 after systemic admin-

istration of 0.1 · , 1 · , and 10 · the highest clinical dose, virus DNA was found in liver and spleen (Table 2). With increasing dose, virus was detected in other organs as well such as the kidneys, lungs, and heart. At the highest dose, virus DNA was present in prostate and testis (days 1, 10, and 30), and in blood and plasma at day 1 after administration. It is likely that the amount of virus DNA is high in liver and spleen because of high blood perfusion in these organs. Higher doses of Ad[I/ PPT-E1A] may saturate the binding capacity in these organs, resulting in the spread of the virus to other organs. Conclusions

The results from the preclinical studies, showing specific cytotoxicity of Ad[I/PPT-E1A] in prostate cells and the absence of toxic effects in other organs, were favorable for the risk–benefit analysis of our trial. The capability of Ad[I/ PPT-E1A] to destroy human prostate (tumor) cells as previously demonstrated in cell lines (Cheng et al., 2006) was now confirmed in the human orthotopic PC346C xenograft mouse model as well as in human primary prostate cells. Furthermore, the preclinical data showed that in case of

FIG. 4. Illustration of Ad[I/PPT-E1A] immunohistochemistry of the prostate tumor in an orthotopic PC346C xenograft mouse 7, 14, and 21 days after intratumoral injection with 2 · 1010 VP Ad[I/PPT-E1A]. Tumor slices were stained with an antiadenoviral hexon protein antibody and with hematoxilin. Dark-brown spots indicate the presence of Ad[I/PPT-E1A] particles. Light-brown areas represent sticking of the antihexon antibody to necrotic tissue. Purple areas are viable tissue. At day 21, an intense dark-brown signal is visible that is indicative of large necrotic areas harboring Ad[I/PPT-E1A] particles. Color images available online at www.liebertpub.com/humc

CLINICAL DEVELOPMENT OF AD[I/PPT-E1A]

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Table 2. Biodistribution of Ad[I/PPT-E1A] in Mice After Systemic Delivery Vehicle

Brain Day 1 Day 10 Day 30 Liver Day 1 Day 10 Day 30 Heart Day 1 Day 10 Day 30 Spleen Day 1 Day 10 Day 30 Kidney Day 1 Day 10 Day 30 Lung Day 1 Day 10 Day 30 Prostate Day 1 Day 10 Day 30 Testis Day 1 Day 10 Day 30 Blood Day 1 Day 10 Day 30 Plasma Day 1 Day 10 Day 30

0.1 · max clin dose

1 · max clin dose

10 · max clin dose

Pos

Neg

Pos

Neg

Pos

Neg

Pos

Neg

0

5

0 0 0

5 5 5

0 0 0

5 4 5

0 0 0

5 5 5

0

5

5 5 5

0 0 0

5 4 5

0 0 0

5 5 5

0 0 0

0

5

0 0 0

5 5 5

3 3 0

2 1 5

4 5 5

1 0 0

0

5

5 5 4

0 0 1

5 4 5

0 0 0

5 5 5

0 0 0

0

5

0 0 0

5 5 5

4 3 3

1 1 2

5 5 5

0 0 0

0

5

0 0 0

5 5 5

4 3 5

1 1 0

5 5 5

0 0 0

0

5

0 0 0

5 5 5

0 0 0

5 5 5

5 5 5

0 0 0

0

5

0 0 0

5 5 5

0 0 0

5 5 5

2 4 1

3 1 4

0

5

0 0 0

5 5 5

0 0 0

5 5 5

5 0 0

0 5 5

0

5

0 0 0

5 5 5

0 0 0

5 5 5

5 0 0

0 5 5

The presence of Ad[I/PPT-E1A] was assessed by real-time polymerase chain reaction using Ad[I/PPT-E1A]-specific primers (see Supplementary Data for further details). Results are presented as positive (light gray for organs where Ad[I/PPT-E1A] was detected at all doses, medium gray for organs where Ad[I/PPT-E1A] was detected at the two highest doses, and dark gray for organs where Ad[I/PPTE1A] was detected at the highest dose) or negative (uncolored). The strength of the signal, which is a measure for the amount of viral DNA, in the individual organs has not been taken into consideration. Neg, negative; Pos, positive.

infection of other organs, the risks of harmful effects should be negligible. First of all, extreme systemic levels of Ad[I/ PPT-E1A] up to 10 · the maximum clinical dose of 5 · 1012 VP were well tolerated by mice. In addition, the study in human primary cells confirmed that the mechanism of action of Ad[I/PTT-E1A], that is, cytotoxicity caused by replication, is selective for prostate cells only and that the virus is not harmful to other tissues and organs. The observed cytotoxicity in primary hepatocytes at an MOI above 50 was carefully considered in the risk assessment. The data

were supportive for the uptake of large numbers of viral particles in combination with a fragile condition of cultured human hepatocytes as the most likely explanation for this observation, but a definite conclusion could not be made. The in vitro results were therefore extrapolated to the in vivo situation, considering a worst-case scenario in which all prostate epithelial cells would be infected by Ad[I/PPTE1A], one infected cell would generate 1000 infectious progeny, and 2% of the maximum amount of virus generated in the prostate would leak into the circulation. It should

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be noted that this scenario ignored the fact that four deposits of 0.25 ml will not be sufficient to infect the entire prostate (Barton et al., 2008) and that leakage of a maximum of 2% of intraprostatically administered oncolytic adenovirus reported in literature was based on data from polymerase chain reaction analysis that were not confirmed to correspond to intact and infectious virus particles (DeWeese et al., 2001). Importantly, in two trials on another oncolytic adenovirus, the appearance of virus DNA in the circulation after intraprostatic injection was not found to be associated with the presence of circulating infectious virus particles (Freytag et al., 2002, 2003). Even in this worst-case scenario, an MOI above 50 could never be achieved in the liver because of the excess of hepatic cells. Indeed, the toxicity study in mice showed that uptake of circulating Ad[I/PPT-E1A] by the liver was not associated with liver pathology, even after systemic administration of 10 · the maximum clinical dose. It was therefore concluded that the observed in vitro cytotoxicity in hepatocytes is not relevant in the setting of the trial. A theoretical argument used to support the conclusion that circulating Ad[I/PPT-E1A] will not be harmful to a patient is the known neutralization of adenovirus by natural human defense mechanisms involving the immune system via its neutralizing antiadenovirus antibodies and antiadenovirus T cells, and inactivation through binding to erythrocytes via CAR and the complement receptor CR1 (Carlisle et al., 2009). Indeed, systemic adenovirus has only been reported to cause serious organ damage in severely immunocompromised individuals at levels of 5 · 1012 to 5 · 1013 VP (Lion et al., 2003; Seidemann et al., 2004). Finally, the fact that systemic oncolytic adenovirus therapy at comparable doses has been well tolerated by cancer patients (Nemunaitis et al., 2001; Small et al., 2006; Au et al., 2007) is supportive for the safety of Ad[I/PPT-E1A] within the design of the planned trial. The clinical product of Ad[I/PPT-E1A] has been produced in HEK293 cells. It is known that production of adenovirus in HEK293 cells can result in recombination events between the adenoviral DNA and the HEK293 genome, resulting in active impurities. The clinical-grade Ad[I/PPTE1A] product was therefore characterized for the presence of recombinants containing the E1B gene, and a level of 1 VP per 1 · 106 VP Ad[I/PPT-E1A] was found (see Supplementary Data). This finding was carefully considered in the risk–benefit analysis. One of the main functions of E1B is to prevent apoptosis of the infected cell via inactivation of the p53 pathway, and as such introduction of the E1B gene will not result in a modification of the mechanism of action. Some of the E1B-containing recombinants might have the wild-type E1A promoter because of recombination of the entire E1 gene from the 293 genome, which could affect the specificity of the virus. Overall, the effect of an E1B-containing recombinant, either with the I/PPT promoter or with the wild-type E1A promoter, in the prostate upon local administration will be comparable to that of parental Ad[I/PPT-E1A]. In case of systemic leakage, no adverse effects are expected for both variants because of rapid neutralization of circulating virus. Furthermore, as described above, even in a worst-case scenario, extrapolated levels of systemic Ad[I/PPT-E1A] because of leakage from the prostate or of direct injection

SCHENK ET AL.

into the circulation are not associated with adverse effects in individuals with a healthy immune system. This theoretical argument was confirmed by the preclinical safety studies showing that the clinical Ad[I/PPT-E1A] product is not harmful to other organs even at extremely high levels. Therefore, the risk for harmful events because of the presence of E1B-containing recombinants was considered to be negligible. Based on the risk assessment, the risks of unwanted effects for patients treated in the trial are considered minimal and are reduced even further by a number of measures. Patients eligible for this trial should be immunocompetent and should have a detectable anti-Ad titer at the time of inclusion. In the event of introduction of Ad[I/PPT-E1A] into the circulation, the immune system of these patients will be capable of neutralizing the virus. The first group of patients will be treated with a conservative dose of 1 · 1011 VP, and the dose will be escalated to 1 · 1012 VP only if the 1 · 1011 VP dose is well tolerated. The same procedure will be applied for escalation to 5 · 1012 VP. Local administration by ultrasound-guided injection targets Ad[I/PPT-E1A] directly to the prostate and thereby reduces the risk on systemic effects. Finally, the prostate will be surgically removed around 3 weeks after injection of Ad[I/PPT-E1A], resulting in an activity time frame of only 3 weeks in total. The clinical development process described in this article has faced two major challenges. The first challenge concerned the design of the preclinical safety studies and the selection of the appropriate models. For human oncolytic adenoviruses, no animal model exists that is fully representative for a human being. Species selectivity of human adenovirus serotype 5 and differences between species with respect to the immune system severely hamper the preclinical prediction of efficacy and safety in humans (Seymour and Fisher, 2009; Maitland et al., 2010). By designing a preclinical strategy that consists of an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells, and a toxicity study in normal mice, and by supporting the outcomes with data from the literature, we have managed to obtain regulatory approval for the preclinical safety assessment of a novel oncolytic adenovirus. A second challenge concerned the production of clinical-grade virus by a non-European manufacturer. The clinical Ad[I/PPT-E1A] product was produced according to Good Manufacturing Practices (GMP) by the FDAapproved Vector Production Facility at the Center for Cell and Gene Therapy (CAGT) in Houston, TX. CAGT has a substantial track record of manufacturing clinical-grade adenoviral vectors and was a former National Gene Vector Laboratory. However, since there is no mutual recognition of GMP accreditation between Europe and the United States, compliance of the production and quality control testing with Annex 13 of the EU-GMP (Directive 91/356/ EEC) had to be demonstrated. This was achieved after a laborious desk audit. After overcoming the challenges described above, to the best of our knowledge we will now be the first in Europe to bring a novel oncolytic adenovirus for prostate cancer to the clinic in an investigator-initiated trial. The clinical development strategy as described in this article may be helpful for others to bring their gene therapy innovations to the patient.

CLINICAL DEVELOPMENT OF AD[I/PPT-E1A] Acknowledgments

The authors would like to acknowledge all members of the GIANT consortium. This work was supported by the European Union through the Sixth Framework Programme Integrated Project GIANT (Contract No. LSHB-CT-2004512087) and by the Dutch ZonMw Programme Translational Gene Therapy Research. N.J.M. acknowledges program support for prostate biology from Yorkshire Cancer Research. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Dr. Ellen Schenk Department of Urology Erasmus MC Room Na1707, P.O. Box 2040 3000 CA Rotterdam The Netherlands E-mail: [email protected] Received for publication September 21, 2013; accepted after revision February 14, 2014. Published online: February 17, 2014.