- Top of page
- Materials and Methods
- Disclosure Statement
Oncolytic virotherapy using adenoviruses has potential for therapeutic benefits in malignant mesothelioma. However, the downregulation of coxsackie virus/adenovirus receptor (CAR) expression is frequently a critical rate-limiting factor that impedes the effectiveness of adenovirus serotype 5 (Ad5)-based vectors in many cancer types. We evaluated CAR (Ad5 receptor) and CD46 (adenovirus serotype 35 [Ad35] receptor) expression in six human malignant mesothelioma cell lines. Very low CAR expression was observed in MSTO-211H and NCI-H2052 cells, whereas the other cell lines showed strong expression. In contrast, CD46 was highly expressed in all mesothelioma cell lines. On this basis, we replaced the CAR binding sequence of Ad5 with the CD46 binding sequence of Ad35 in the replication-defective adenoviruses and the tumor-specific midkine promoter-regulated oncolytic adenoviruses. By this fiber modification, the infectivity, virus progeny production, and in vitro cytocidal effects of the adenoviruses were significantly enhanced in low CAR-expressing MSTO-211H and NCI-H2052 cells, also resulting in similar or even higher levels in high CAR-expressing mesothelioma cell lines. In MSTO-211H xenograft models, the fiber-modified oncolytic adenovirus significantly enhanced antitumor effect compared to its equivalent Ad5-based vector. Our data demonstrate that Ad35 fiber modification of binding tropism in a midkine promoter-regulated oncolytic Ad5 vector confers transductional targeting to oncolytic adenoviruses, thereby facilitating more effective treatment of malignant mesothelioma.
Malignant mesothelioma, often caused by exposure to asbestos, is one of the most lethal cancers.[1, 2] Conventional therapies for malignant mesothelioma include surgical resection, chemotherapy, and radiotherapy, but are generally non-curative.[2, 3] Accordingly, new therapeutic options are urgently needed. Oncolytic virotherapy represents a highly promising approach to cancer treatment, and clinical trials of several replicating viruses and vector systems are ongoing.
A crucial factor in determining the therapeutic window for oncolytic virotherapy is the tumor selectivity of virus replication, which can be enhanced by substituting constitutively active native viral promoters with cellular promoters from genes highly upregulated in cancer cells. Midkine (MDK) is a basic heparin-binding growth factor and a developmentally important retinoic acid-responsive protein strongly induced during midgestation. MDK is implicated in cancer development because of its mitogenic effect,[6, 7] in the promotion of angiogenesis, and for its antiapoptotic,[9, 10] fibrinolytic, and transforming activities. MDK expression in normal human adult tissues is limited.[5, 13-15] In contrast, MDK is overexpressed in various human cancers,[13, 14, 16-20] compared to adjacent normal tissues. We previously showed that MDK expression is also highly upregulated in malignant mesothelioma compared to that in normal primary mesothelial and smooth muscle cells.
Accordingly, we found that the tumor-specific MDK promoter conferred tumor-selective transcriptional targeting on oncolytic adenoviruses, which proved highly effective in mesothelioma models. However, infection with conventional Ad5-based oncolytic vectors is limited by low physical binding efficiency because Ad5 uses CAR as its main cellular receptor[22-25] and, although CAR is ubiquitously expressed in normal epithelial cells, its expression is downregulated in many tumor types.[25, 26] Moreover, previous studies[26-28] suggest that tumor progression and aggressiveness correlates with suppressed CAR expression, so this limitation is exacerbated in advanced cancers. Downregulation of CAR expression in tumors therefore represents a significant obstacle to the use of Ad5 vectors for oncolytic virotherapy. Hence, modification of the adenovirus capsid, particularly of the viral fiber knob protein, which mediates primary binding of the virus to its cellular target, has been attempted to facilitate CAR-independent binding.
The adenovirus family comprises over 50 human serotypes. Among these, Ad35 uses the complement-regulating protein CD46 as its cellular receptor. CD46 is expressed on all nucleated cells at a low level, but its expression is greatly upregulated in cancer cells, including breast,[31-33] esophageal, colon,[31, 35] liver,[33, 36] pancreatic, prostate, and ovarian cancer cells. We evaluated CD46 and CAR expression in multiple mesothelioma cell lines. Subsequently, we investigated whether the antitumor efficacy of MDK-regulated Ad5-based oncolytic vectors improved in vitro and in vivo following modification of binding tropism through the replacement of the cellular binding domain of the Ad5 fiber knob with that of Ad35. Our results indicated that the dual-targeted adenovirus, combining cancer cell-selective binding tropism and transcriptional regulation, represents an effective strategy for malignant mesothelioma treatment by oncolytic virotherapy.
- Top of page
- Materials and Methods
- Disclosure Statement
Tumor specificity and viral infectivity are critical to the success of oncolytic virotherapy. Even when replicating viruses are used, viral infectivity of target cells is a critical determinant of therapeutic efficacy. We recently showed that the MDK promoter confers tumor-selective transcriptional targeting on Ad5-based oncolytic adenoviruses, which proved highly effective in malignant mesothelioma models. In this previous study, we showed that MDK-regulated oncolytic adenoviruses exert a robust tumor-specific cytotoxicity in ACC-MESO-4 mesothelioma cells, and complete regression of s.c. tumor xenografts in athymic mice. This striking result may reflect the high permissivity of ACC-MESO-4 cells for MDK-regulated adenovirus infection, supported by the fact that ACC-MESO-4 cells showed the highest observed levels of MDK and CAR expression among a panel of six mesothelioma cell lines tested in the current study. However, in MSTO-211H and NCI-H2052 cells, which show high MDK but very low CAR expression, oncolytic effects of MDK-regulated oncolytic adenovirus with the wild-type Ad5 fiber were subtherapeutic relative to ACC-MESO-4 cells.
In the present study, we found that CAR expression levels varied significantly in six human mesothelioma cell lines in which the MDK promoter is highly activated. In contrast, CD46 was strongly expressed in all of these cell lines (Fig. 1). Accordingly, we investigated the use of the Ad35 fiber for modification of physical binding tropism (transductional targeting) combined with MDK promoter regulation (transcriptional targeting) of oncolytic Ad5 vectors. This dual-targeted adenovirus (Ad889) showed tumor cell-specific replication and cytotoxicity, but spared normal cells (Figs 4, 5). The dual-targeted virus also showed enhanced antitumor efficacy against CAR-low mesothelioma cells both in vitro and in vivo.
Unexpectedly, Ad35 fiber modification of the adenoviruses conferred more or comparable levels of infectivity, virus progeny production, and cytotoxicity in all mesothelioma cells (Figs 3-5). This may be caused by the higher level of CD46 expression in these cell lines, compared to CAR. Consequently, Ad35 fiber-modified, transcriptionally targeted oncolytic adenoviruses would be an ideal platform of virotherapy for malignant mesothelioma. In clinical situations, however, it will be feasible to evaluate CAR/CD46 and MDK expression in tumor specimens for selecting suitable oncolytic adenoviruses. Our MDK promoter-regulated oncolytic adenoviruses with wild-type Ad5 fiber and Ad5/F35 fiber may achieve tailor-made virotherapy for various but specific types of cancers with upregulated MDK expression.
Only a few studies have reported oncolytic adenoviruses with Ad35 fiber modifications. Sova et al. and Ni et al. constructed a capsid-modified Ad5-based oncolytic adenovirus containing short-shafted entire fibers derived from Ad35 and showed efficient infection of lung, liver, colon, prostate, ovarian, and cervical cancer cells. Similarly, Hoffmann et al. constructed an Ad5-based oncolytic adenovirus with the fiber shaft and knob domain of Ad35 and showed that the chimeric oncolytic vector has a significantly higher transduction efficiency of glioblastoma, sarcoma, and melanoma cells than the Ad5 vector. Furthermore, Yu et al.[32, 34] constructed a chimeric oncolytic adenovirus in which the Ad5 fiber knob was replaced with the Ad35 fiber knob, and showed enhanced transduction compared to that observed with the Ad5 fiber knob in esophageal, pancreatic, and breast cancer cells. Our study is the first to demonstrate an enhanced therapeutic effect of an Ad35 fiber-modified, transductionally targeted oncolytic adenovirus in malignant mesothelioma models.
To evaluate the in vivo therapeutic efficacy of fiber-modified Ad889 in CAR-low mesothelioma cells, it was necessary to use MSTO-211H cells, as NCI-H2052 cells are not tumorigenic in nude mice. However, in MSTO-211H cells, Ad888 with the wild-type Ad5 fiber also caused extensive cell death at an MOI of 10 (Figs 4, 5). Our previous work showed that high-dose MDK-regulated oncolytic adenoviruses with the wild-type Ad5 fiber did achieve significant tumor reduction in MSTO-211H xenograft mouse models. Taken together, the results from both studies suggest that CAR deficiency can be outweighed by other factors, such as coreceptors (e.g., integrins), the strength of the MDK-promoter activity, apoptotic death mechanisms, or innate immune responses, in MSTO-211H cells. Further enhancement of therapeutic efficacy may also be possible by incorporating suicide gene therapy, as we previously demonstrated that an MDK promoter-regulated oncolytic adenovirus armed with an HSV-TK suicide gene showed increased in vivo antitumor activity.
In conclusion, in the present study we have shown that Ad35 fiber modification of MDK-targeted oncolytic adenovirus vectors can increase viral infectivity and confer enhanced transduction efficiency and antitumor efficacy in malignant mesothelioma cells especially in which CAR is downregulated. As MDK and CD46 are both highly upregulated in a variety of cancers, including malignant mesothelioma, our dual-targeted oncolytic adenovirus may represent a highly promising general strategy for combined transcriptional and transductional targeting of oncolytic virotherapy.