Intratumoral coadministration of hyaluronidase enzyme and oncolytic adenoviruses enhances virus potency in metastatic tumor models

PURPOSE: Evaluate the codelivery of hyaluronidase enzyme with oncolytic adenoviruses to determine whether it improves the spread of the virus throughout tumors, thereby leading to a greater overall antitumor efficacy in tumor models. EXPERIMENTAL DESIGN: The optimal dose of hyaluronidase that provided best transduction efficiency and spread of a green fluorescent protein (GFP)-expressing adenovirus within tumors was combined with oncolytic viruses in tumor models to determine whether the combination treatment results in an improvement of antitumor efficacy. RESULTS: In mice injected with the adenovirus Ad5/35GFP and an optimal dose of hyaluronidase (50 U), a significant increase in the number of GFP-expressing cells was observed when compared with animals injected with virus only (P < 0.0001). When the oncolytic adenoviruses Ad5OV or Ad5/35 OV (OV-5 or OV5T35H) were codelivered with 50 U of hyaluronidase, a significant delay in tumor progression was observed, which translated into a significant increase in the mean survival time of tumor-bearing mice compared with either of the monotherapy-treated groups (P < 0.0001). Furthermore, the mice that received the combination of Ad5/35 OV and hyaluronidase showed the best antitumor efficacy. Importantly, the combination treatment did not increase the metastatic potential of the tumors. Lastly, the increase in virus potency observed in animals injected with both enzyme and virus correlated with enhanced virus spread throughout tumors. CONCLUSION: Antitumor activity and overall survival of mice bearing highly aggressive tumors are significantly improved by codelivery of oncolytic adenoviruses and hyaluronidase when compared with either of the monotherapy-treated groups, and it may prove to be a potent and novel approach to treating patients with cancer.     

Enhanced antitumor efficacy of a novel fiber chimeric oncolytic adenovirus expressing p53 on hepatocellular carcinoma

Oncolytic adenoviruses may offer a new treatment option and improve the prognosis for patients with hepatocellular carcinoma (HCC). However, the antitumor efficacy of oncolytic adenoviruses on HCC cells is compromised due to low expression of the adenovirus serotype 5 (Ad5) receptor on the target cells. In this study we showed that all HCC cell lines and clinical samples expressed high level of CD46, the receptor for Adenovirus 35 (Ad35) and constructed new fiber chimeric oncolytic adenoviruses with or without a p53 gene expression cassette, SG635-p53 and SG635, respectively. These variants were derived from the previously described Ad5 vectors SG600-p53 and SG600 by replacing the Ad5 fiber with a chimeric Ad5/35 fiber. It was found that the 5/35 fiber chimeric adenovirus vector (Ad5/35-EGFP) demonstrated significantly improved transduction in all tested HCC cell lines compared with the Ad5 vector (Ad5-EGFP). The new fiber chimeric oncolytic adenoviruses produced more progeny viruses in HCC cells than did the Ad5-based viruses but replicated weakly in normal fibroblast BJ cells. In addition, SG635-p53 mediated a higher level of transgenic expression than SG600-p53 in Hep3B and Huh7 cells and showed a markedly enhanced antitumor effect on HCC cells in vitro compared with SG635 or SG600-p53 without causing significant cytotoxicity to normal cells. Antitumor activity of SG635-p53 was shown in Hep3B subcutaneous xenograft tumor models following intratumoral injection, resulting in significant inhibition of tumor growth and prolonged survival of animals. Our data suggest that SG635-p53, as a fiber chimeric oncolytic adenovirus in combination with p53 expression, may serve as a novel, promising and safe anticancer agent for the treatment of HCC.     

Enhanced antitumor efficacy of fiber‐modified,midkine promoter‐regulated oncolytic adenovirus in human malignant mesothelioma

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.     

Enhanced gene transfer and oncolysis of head and neck cancer and melanoma cells by fiber chimeric oncolytic adenoviruses.

PURPOSE: The purpose of this study was to evaluate a fiber knob replacement strategy to improve infectivity and efficacy of Ad5 fiber chimeric oncolytic viruses for treatment of melanoma and head and neck cancers (HNC). EXPERIMENTAL DESIGN: Adenoviral receptors and transduction levels were used to determine the level of infectivity of fiber-modified, green fluorescent protein-expressing, replication-deficient viruses in a panel of melanoma and HNC cell lines in vitro. Virus yield and cytotoxicity assays were used to determine the tumor specificity and virus replication-mediated cytotoxicity of the fiber-modified oncolytic viruses in the same panel of melanoma and HNC in vitro. Xenograft tumor models were used to assess the antitumor activity of those fiber-modified chimeric viruses compared with the parental virus. RESULTS: Marker gene expression following gene transfer of the fiber chimeric vectors in melanoma and HNC cell lines was approximately 10-fold higher than that obtained with parental Ad5 vector. The fiber chimeric oncolytic variants mediated killing of melanoma and HNC cells that was 2- to 576-fold better than with the parental virus. In addition, fiber chimeric variants produced 2- to 7-fold more progeny virus in tumor cells than the parental virus. Moreover, a high multiplicity of infection was needed for the fiber chimeric viruses to produce cytotoxicity in normal cells. A significantly stronger antitumor response and survival advantage were shown in the tested melanoma and HNC xenograft models following i.t. injections. CONCLUSIONS: In vitro and in vivo studies showed the improved transduction, replication, cytotoxicity, antitumor efficacy, and survival advantage in melanoma and HNC tumor models, suggesting a potential use of these oncolytic agents for the treatment of melanoma and HNCs.     

Macrophage metalloelastase (MME) as adjuvant for intra-tumoral injection of oncolytic adenovirus and its influence on metastases development

Oncolytic adenoviruses are a promising treatment alternative for many advanced cancers, including colorectal cancer. However, clinical trials have demonstrated that single-agent therapy in advanced tumor masses is rarely curative. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy. As oncolytic viruses kill preferentially cancer cells, high extracellular matrix (ECM) content constitutes potential barriers for viral penetration within tumors. In this study, the ECM-degrading proteases relaxin, hyaluronidase, elastase and macrophage metalloelastase (MME) were tested for their antitumor efficacy alone and in combination with oncolytic adenovirus. MME improved the overall antitumor efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intra-tumoral treatment of primary tumors from HT29 cells with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison with either monotherapy alone. Contradictory results of effects of MME on tumorigenesis and metastasis formation have been reported in the literature. This study demonstrates for the first time in a metastatic animal model that MME, as a monotherapy or in combination with oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization aiming at clinical applications for metastatic colorectal cancer.     

A fiber-modified mesothelin promoter-based conditionally replicating adenovirus for treatment of ovarian cancer

PURPOSE: Recently, virotherapy has been proposed as a new therapeutic approach for ovarian cancer. Conditionally replicative adenoviruses (CRAd) may contain tumor-specific promoters that restrict virus replication to cancer cells. Mesothelin, a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal ovarian tissues. The purpose of this study was to explore the therapeutic utility of a mesothelin promoter-based CRAd in a murine model of ovarian cancer, using noninvasive in vivo imaging. EXPERIMENTAL DESIGN: We constructed a mesothelin promoter-based CRAd with a chimeric Ad5/3 fiber (AdMSLNCRAd5/3) that contains an Ad5 tail, Ad5 shaft, and an Ad3 knob. Previously, a chimeric Ad5/3 fiber has shown improved infectivity in many ovarian cancer cells. Viral replication and oncolysis were assessed in a panel of ovarian cancer cell lines. To test the oncolytic efficacy of AdMSLNCRAd5/3 in a murine model, bioluminescence imaging of tumor luciferase activity and survival analysis were done. RESULTS: AdMSLNCRAd5/3 achieved up to a 10,000-fold higher cell killing effect and up to 120-fold higher levels of viral replication in all human ovarian cancer cells, compared with wild-type Ad5. AdMSLNCRAd5/3 significantly inhibited tumor growth as confirmed by in vivo imaging (P < 0.05). Survival with AdMSLNCRAd5/3 was significantly enhanced when compared with no virus or with a wild-type Ad5-treated group (P < 0.05). CONCLUSIONS: The robust replication, oncolysis, and in vivo therapeutic efficacy of AdMSLNCRAd5/3 showed that this CRAd is a promising candidate for treating ovarian cancer. Importantly, we have applied in vivo imaging that has allowed repeated and longitudinal measurements of tumor growth after CRAd treatment.     

Oncolytic adenovirus based on serotype 3

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.     

Evaluation of adenovirus vectors containing serotype 35 fibers for tumor targeting

There is growing evidence from in vitro studies that subgroup B adenoviruses (Ad) can overcome the limitations in safety and tumor transduction efficiency seen with commonly used subgroup C serotype 5-based vectors. In this study, we confirm that the expression level of the B-group Ad receptor, CD46, correlates with the grade of malignancy of cervical cancer in situ. We also demonstrate the in vivo properties of Ad5-based vectors that contain the B-group Ad serotype 35 fiber (Ad5/35) in transgenic mice that express CD46 in a pattern and at a level similar to humans. Upon intravenous and intraperitoneal injection, an Ad5/35 vector did not efficiently transduce normal tissue, but was able to target metastatic or intraperitoneal tumors that express CD46 at levels comparable to human tumors. When an oncolytic Ad5/35-based vector was employed, in both tumor models antitumor effects were observed. Furthermore, injection of Ad5/35 vectors into CD46 transgenic mice caused less innate toxicity than Ad5 vectors. Our data demonstrate that Ad vectors that target CD46 offer advantages over Ad5-based vectors for treatment of cancer.     

Targeting of adenovirus vectors to tumor cells does not enable efficient transduction of breast cancer metastases

Targeting of oncolytic adenoviruses to tumors can potentially increase their efficacy and safety profile after systemic application. We have developed recently a capsid-modified vector containing the adenovirus serotype 35 fiber shaft and knob inserted into an Ad5 capsid. This Ad5/35 vector infects cells via a coxsackievirus adenovirus receptor-independent pathway. Here we attempted to exploit this new tropism of Ad5/35 vectors for tumor-specific infection. In vitro, the Ad5/35 vector efficiently transduced human breast cancer cells that were refractory to infection with conventional Ad5-based vectors. Additionally, primary mouse hepatocytes were relatively refractory to Ad5/35 infection in vitro or after systemic vector application to mice. In an animal model of breast cancer metastasis, intraportal infusion of MDA-MB435 cells produced multiple hepatic metastases that were surrounded by extracellular matrix and developed blood vessels confined to the tumor stroma. Tail vein injection of a standard Ad5-based vector into tumor-bearing animals resulted in transduction of mouse hepatocytes but not metastases. However, the capsid-modified Ad5/35 vector transduced only approximately 8% of metastases. The metastases that were susceptible to Ad5/35 infection demonstrated blood vessels in close proximity to tumor nests without extracellular matrix separating endothelial and tumor cells. These findings indicate that transduction of liver metastases not only requires tumor-specific tropism but also new strategies to increase accessibility of tumor cells to systemically applied oncolytic adenoviruses.     

Comparison of herpes simplex virus- and conditionally replicative adenovirus-based vectors for glioblastoma treatment

In this study we compared side-by-side the anti-neoplastic activity of the oncolytic herpes simplex virus-1 (HSV-1) vector G47Delta with that of a conditionally replicative adenoviral vector for the treatment of glioblastoma. We analyzed the transduction efficiency of permanent glioblastoma cell lines and short-term cultures of glioblastoma cells with HSV.Luc and four adenovirus type 5 (Ad5)-based vectors that differed only in their fiber gene (Ad5.Luc, AdlucRGD, and the fiber chimeric vectors Ad5/3.Luc and Ad5/35.Luc). In the tested short-term cultures of glioblastoma cells the vectors Ad5/35.Luc and HSV.Luc had an equal transduction efficiency which was approximately 70% higher than that of Ad5.Luc. In a subcutaneous xenograft glioblastoma model in nude mice we observed a significantly higher local tumor control with the G47Delta vector compared to the conditionally replicative Ad5/35 adenovirus. We confirmed in glioblastoma that the intratumoral expression of measles virus fusogenic membrane glycoproteins (FMG) encoded by replication-defective Ad5/35 or HSV-1 amplicon vectors synergistically enhances chemotherapy with temozolomide. The anti-neoplastic effect was superior when the replication-defective FMG encoding vectors were trans-complemented for replication with the respective oncolytic vector. This approach was necessary due to packaging constraints of adenovirus. At day 100, of 6 treated animals 1 was alive that received the Ad5/35- and 3 that received the HSV-1-based triple therapy. In an intracranial glioblastoma xenograft model we demonstrated the applicability of this strategy. Due to the higher oncolytic efficacy and packaging capacity of the HSV-1 vectors compared to adenovirus, these vectors are promising for the treatment of glioblastoma.     

Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.     

Engineered adenoviruses combine enhanced oncolysis with improved virus production by mesenchymal stromal carrier cells

Oncolytic viruses have demonstrated in pre‐clinical and clinical studies safety and a unique pleiotropic activity profile of tumor destruction. Yet, their delivery suffers from virus inactivation by blood components and sequestration to healthy tissues. Therefore, mesenchymal stromal cells (MSCs) have been applied as carrier cells for shielded virus delivery to tumors after ex vivo infection with oncolytic viruses. However, infection and particle production by MSCs have remained unsatisfying. Here, we report engineered oncolytic adenoviruses (OAds) for improved virus production and delivery by MSCs. OAds are uniquely amenable to molecular engineering, which has facilitated improved tumor cell destruction. But for MSC‐mediated regimens, OAd engineering needs to achieve efficient infection and replication in both MSCs and tumor cells. We show that an Ad5/3 chimeric OAd capsid, containing the adenovirus serotype 3 cell‐binding domain, strongly increases the entry into human bone marrow‐derived MSCs and into established and primary pancreatic cancer cells. Further, we reveal that OAd with engineered post‐entry functions—by deletion of the anti‐apoptotic viral gene E1B19K or expression of the death ligand TRAIL—markedly increased virus titers released from MSCs, while MSC migration was not hampered. Finally, these virus modifications, or viral expression of FCU1 for local 5‐FC prodrug activation, improved tumor cell killing implementing complementary cytotoxicity profiles in a panel of pancreatic cancer cell cultures. Together, our study establishes post‐entry modification of OAd replication for improving virus delivery by carrier cells and suggests a panel of optimized OAds for future clinical development in personalized treatment of pancreatic cancer.     

Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM‐CSF: Results in vitro,in rodents and in humans

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune‐checkpoint‐inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno‐virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3‐D24‐GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma‐specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK‐MEL‐28 melanoma xenografts in nude mice when combined with low‐dose cyclophosphamide. Furthermore, virally‐expressed granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well‐tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3‐D24‐GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.     

Tissue-specific promoters active in CD44+CD24-/low breast cancer cells

It has been proposed that human tumors contain stem cells that have a central role in tumor initiation and posttreatment relapse. Putative breast cancer stem cells may reside in the CD44(+)CD24(-/low) population. Oncolytic adenoviruses are attractive for killing of these cells because they enter through infection and are therefore not susceptible to active and passive mechanisms that render stem cells resistant to many drugs. Although adenoviruses have been quite safe in cancer trials, preclinical work suggests that toxicity may eventually be possible with more active agents. Therefore, restriction of virus replication to target tissues with tissues-specific promoters is appealing for improving safety and can be achieved without loss of efficacy. We extracted CD44(+)CD24(-/low) cells from pleural effusions of breast cancer patients and found that modification of adenovirus type 5 tropism with the serotype 3 knob increased gene delivery to CD44(+)CD24(-/low) cells. alpha-Lactalbumin, cyclo-oxygenase 2, telomerase, and multidrug resistance protein promoters were studied for activity in CD44(+)CD24(-/low) cells, and a panel of oncolytic viruses was subsequently constructed. Each virus featured 5/3 chimerism of the fiber and a promoter controlling expression of E1A, which was also deleted in the Rb binding domain for additional tumor selectivity. Cell killing assays identified Ad5/3-cox2L-d24 and Ad5/3-mdr-d24 as the most active agents, and these viruses were able to completely eradicate CD44(+)CD24(-/low) cells in vitro. In vivo, these viruses had significant antitumor activity in CD44(+)CD24(-/low)-derived tumors. These findings may have relevance for elimination of cancer stem cells in humans.     

Potent systemic antitumor activity from an oncolytic herpes simplex virus of syncytial phenotype

Conditionally replicating (oncolytic) viruses, which selectively replicate in tumor cells but not in normal cells, show great promise as antitumor agents for cancer therapy. The principal antitumor activity of these viruses derives from their replication within tumor cells, which results in cell destruction and the production of progeny virions that can spread to adjacent tumor cells. However, one potential limitation of this approach is that viral gene deletions conferring tumor selectivity also result frequently in reduced potency of the virus in tumors. Therefore, strategies designed to enhance the potency of current oncolytic viruses will likely increase their chance of clinical success. Here we report the construction of an oncolytic herpes simplex virus (HSV) of which the infection also causes strong cell membrane fusion (syncytial formation). In vitro characterization on a variety of human tumor cells of different tissue origins showed that the plaques from this virus (Fu-10) are phenotypically unique and are significantly larger than those from the parental G207 virus, a well-characterized oncolytic HSV lacking fusogenic function. Furthermore, the syncytial formation caused by this virus depended on HSV replication, indicating that cell membrane fusion will only occur in dividing cells (such as tumor cells) where the virus can undergo a full infection cycle but not in normal cells where the viral replication is restricted. Systemic administration of Fu-10 into mice with established lung metastatic breast cancer resulted in a dramatic therapeutic effect. These studies demonstrate that incorporation of fusogenic function into an oncolytic virus can significantly increase the potency of viral oncolysis; this may lead to an enhanced clinical performance, especially in late-stage cancer patients.     

Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad "detargeting" from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor-dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites.     

Transthyretin-driven oncolytic adenovirus suppresses tumor growth in orthotopic and ascites models of hepatocellular carcinoma

Strategies to increase antitumor efficacy of oncolytic adenoviruses are actively investigated. We have previously shown that E1B-55 kDa-deleted adenovirus, designated Ad5WS1, has therapeutic potential for treating hepatocellular carcinoma (HCC). To achieve HCC-restricted replication of oncolytic adenovirus, we generated Ad5WS2, an E1B-55 kDa-deleted adenovirus with its E1A gene driven by the liver-specific transthyretin promoter. Our results showed that Ad5WS2 could replicate within tumor cells where the transthyretin gene was expressed. Mouse transthyretin promoter was active in murine and human HCC cells, but relatively quiescent in cells of non-liver origin. Ad5WS2 caused severe cytolytic effect on HCC cells, but was much attenuated in non-HCC cells. Peritoneal administration of Ad5WS2 into mice bearing liver tumors grown in ascites resulted in enhanced survival. In an orthotopic HCC model, Ad5WS2, when systemically administered, exerted higher antitumor effects than Ad5WS1. Lack of viral replication in normal organs and minimal hepatic toxicity was noted after Ad5WS2 treatment. Furthermore, the antitumor effect of Ad5WS2 could be enhanced when combined with chemotherapeutic agent cisplatin in the ascites tumor model. These results suggest that E1B-55 kDa-deleted adenovirus driven by the transthyretin promoter may be a safer and more efficacious oncolytic agent for the treatment of primary and metastatic HCC. ( Cancer Sci 2009; 100: 537–545)     

Complementary adenoviral vectors for oncolysis

Replication-competent adenoviruses (Ads) were used for oncolytic virotherapy soon after they were discovered. Recently mutated and genetically engineered Ads have been shown to selectively lyse tumor cells. We have split the human Ad type 5 genome into two defective viruses that complement each other only in certain tumor cells. The genome of one of these vectors, GT5610, contains only the minimal viral elements required in cis for replication and packaging and the E1 viral genes with E1A under the control of the human alpha-fetoprotein promoter. This "controlled" vector has a capacity for 30 kilobases of foreign DNA. The supplemental vector, AdHbeta, contains all adenoviral genes except for E1. Both vectors were designed to carry heterologous reporter genes whose expression could be detected throughout the tumor. Coinfection of hepatocarcinoma cells that have the capacity to transcribe genes under the control of the alpha-fetoprotein promoter leads to cell lysis and copropagation. The oncolytic spread of these complementary vectors in vivo was demonstrated by the intratumoral injection of human hepatocarcinomas xenografted in severe combined immunodeficient (SCID) mice. This system presents safety and gene capacity features that could yield a therapeutic advantage over oncolysis by a single virus.     

A novel chromogranin-A promoter-driven oncolytic adenovirus for midgut carcinoid therapy.

PURPOSE: The use of replication-selective oncolytic adenoviruses is an emerging therapeutic approach for cancer, which thus far has not been employed for carcinoids. We therefore constructed Ad[CgA-E1A], a novel replication-selective oncolytic adenovirus, where the chromogranin A (CgA) promoter controls expression of the adenoviral E1A gene. EXPERIMENTAL DESIGN: The Ad[CgA-E1A] virus was evaluated for E1A protein expression, replication ability, and cytolytic activity in various cell lines. It was also evaluated for treatment of xenografted human carcinoid tumors in nude mice. To use Ad[CgA-E1A] for the treatment of carcinoid liver metastases, it is important that normal hepatocytes do not support virus replication to minimize hepatotoxicity. We therefore evaluated CgA protein expression in normal hepatocytes. We also evaluated CgA gene expression in normal hepatocytes and microdissected tumor cells from carcinoid metastases. RESULTS: We found that Ad[CgA-E1A] replicates similarly to wild-type virus in tumor cells with neuroendocrine features, including the BON carcinoid cell line and the SH-SY-5Y neuroblastoma cell lines, whereas it is attenuated in other cell types. Thus, cells where the CgA promoter is active are selectively killed. We also found that Ad[CgA-E1A] is able to suppress fast-growing human BON carcinoid tumors in nude mice. Furthermore, CgA is highly expressed in microdissected cells from carcinoid metastases, whereas it is not expressed in normal hepatocytes. CONCLUSION: Ad[CgA-E1A] is an interesting agent for the treatment of carcinoid liver metastases in conjunction with standard therapy for these malignancies.