Oncolytic adenovirus Ad5/3-Δ24 and chemotherapy for treatment of orthotopic ovarian cancer

Abstract

Objective.

Oncolytic adenoviruses capable of replication selectively in tumor cells are an appealing approach for the treatment of neoplastic diseases refractory to conventional therapies. The aim of this study was to evaluate the effect of dose and scheduling of a tropism-modified, adenovirus serotype 3 receptor-targeted, Rb/p16 pathway-selective replication-competent adenovirus, Ad5/3-Δ24, against human ovarian adenocarcinoma. As oncolytic viruses and chemotherapy can have synergistic interactions, the antitumor efficacy of Ad5/3-Δ24 was also studied in combination with epirubicin and gemcitabine, common second-line treatment options for platinum-resistant ovarian cancer.

Methods.

Orthotopic murine models of peritoneally disseminated ovarian cancer were utilized to compare survival of mice treated with either a single viral dose or weekly delivery. The lowest effective dose of intraperitoneal Ad5/3-Δ24 was determined. Combinations of Ad5/3-Δ24 and gemcitabine or epirubicin were studied in vitro as well as in vivo.

Results.

Treatment outcome after administration of a single dose of Ad5/3-Δ24 was as effective as delivery of several weekly doses. Our results also demonstrate that a single intraperitoneal injection of 100 viral particles significantly increased the survival of mice compared to untreated animals. Further, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone.

Conclusion.

These preclinical data suggest that Ad5/3-Δ24 represents a promising treatment strategy for advanced ovarian cancer as a single agent or in combination with chemotherapy.

Introduction

Despite improvements in the treatment of ovarian carcinoma in the last three decades, it remains the leading cause of death from gynecological malignancies in developed countries [1]. Median survival has improved due to multi-modality treatments often including a combination of primary cytoreductive surgery with platinum and taxane compounds. Nonetheless, early diagnosis continues to be challenging, and about 70% of patients present with disseminated disease for which the 5-year survival rate is approximately 30%. Therefore, novel treatment approaches such as targeted therapies are needed [2], [3]. Ovarian cancer spreads preferentially throughout the peritoneal cavity (stage III) and, since the peritoneal cavity is easily accessible and offers a degree of compartmentalization, these patients represent an attractive target for regional therapy. Of note, there is growing evidence that patients with microscopic residual disease might benefit from intraperitoneal (i.p.) treatment [4].

Oncolytic adenoviruses represent an attractive modality for cancers refractory to conventional therapies [5], [6], [7]. Such viruses are modified to take advantage of tumor-specific alterations that allow viral replication preferentially in cancer cells. Replication causes oncolytic death of the cell, resulting in release of viral progeny to surrounding cells. Amplification of the virus leads to more efficient intratumoral penetration and spread within solid tumor masses when compared to replication-deficient adenovirus vectors, which are traditionally used in gene therapy approaches. One strategy to limit replication of oncolytic adenoviruses to tumor tissues is to engineer specific deletions into the viral genome. These deletions abrogate replication in normal cells but can be transcomplemented by tumor-specific factors. Ad5-Δ24 (a.k.a. dl922–947) carries a 24-bp deletion in the constant region 2 domain of the adenoviral E1A gene [8], [9]. Thus, the E1A protein is unable to bind the retinoblastoma (Rb) tumor suppressor protein for release of E2F and subsequent effective viral replication in non-cycling normal cells. Therefore, the virus replicates selectively in cells deficient in the Rb/p16 pathway, including most if not all cancer cells including ovarian cancer cells [10], [11]. Tumor specificity of these adenoviral mutants has been previously demonstrated [8], [9].

The efficacy of oncolytic adenoviruses can be compromised if they cannot enter target cells effectively [12]. Most adenoviral gene therapy strategies are based on serotype 5 (Ad5), which binds to the coxsackie-adenovirus receptor (CAR). Unfortunately, expression of CAR is frequently down-regulated in many types of advanced cancers including ovarian cancer [6], and may be a general phenomenon related to carcinogenesis. However, lack of CAR can be circumvented by substituting the knob domain of Ad5 with the corresponding domain of serotype 3 (Ad3). Utilization of such 5/3 fiber chimeras allows virus binding and entry through the Ad3 receptor, which is expressed to high degree on ovarian cancer cells [13]. The preclinical safety and normal tissue biodistribution of this approach seems to be comparable to wild type Ad5 virus [14].

Ad5/3-Δ24 is an Rb binding deleted oncolytic adenovirus retargeted to the Ad3 receptor. It has been previously shown that this agent delivers a powerful antitumor effect to ovarian cancer cells in vitro, to clinical ovarian cancer specimens, and in orthotopic models of ovarian cancer [15], [16]. However, when combined with gemcitabine for intraperitoneal treatment of SKOV3.ip1 carcinomatosis, certain schedules resulted in liver toxicity, while giving virus 24 h after gemcitabine resulted in dramatically enhanced survival [17]. Therefore, we sought to investigate if these were model or chemotherapy agent-specific findings. Also, for optimizing protocols for upcoming clinical trials, it is of interest if weekly administration is superior over a single dose. Each ovarian cancer cell infected in vitro with Ad5/3-Δ24 rapidly produces thousands of new virions. Therefore, we performed in vivo dose de-escalation to test if virus amplification would translate into efficacy with very small doses, which might be promising with regard to clinical trials with patients with advanced ovarian cancer.