Relaxin-expressing, fiber chimeric oncolytic adenovirus prolongs survival of tumor-bearing mice

Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.     

Use of macrophages to target therapeutic adenovirus to human prostate tumors

New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells.     

Potent antitumor effect of tumor microenvironment‐targeted oncolytic adenovirus against desmoplastic pancreatic cancer

Pancreatic cancer is a leading cause of cancer‐related death. Desmoplastic pancreatic tumors exhibit excessive extracellular matrix (ECM) and are thus highly resistant to anticancer therapeutics, since the ECM restricts drug penetration and dispersion. Here, we designed and generated two hypoxia‐responsive and cancer‐specific hybrid promoters, H(mT)E and H(E)mT. Transgene expression driven by each hybrid promoter was markedly higher under hypoxic conditions than normoxic conditions. Moreover, H(E)mT‐driven transgene expression was highly cancer‐specific and was superior to that of H(mT)E‐driven expression. A decorin‐expressing oncolytic adenovirus (Ad; oH(E)mT‐DCN) replicating under the control of the H(E)mT promoter induced more potent and highly cancer‐specific cell death compared with its cognate control oncolytic Ad, which harbored the endogenous Ad E1A promoter. Moreover, oH(E)mT‐DCN exhibited enhanced antitumor efficacy compared with both the clinically approved oncolytic Ad ONYX‐015 and its cognate control oncolytic Ad lacking DCN. oH(E)mT‐DCN treatment also attenuated the expression of major ECM components, such as collagen I/III, elastin and fibronectin and induced tumor cell apoptosis, leading to extensive viral dispersion within orthotopic pancreatic tumors and pancreatic cancer patient‐derived tumor spheroids. Collectively, these findings demonstrate that oH(E)mT‐DCN exhibits potent antitumor efficacy by degrading the ECM and inducing apoptosis in a multifunctional process. This process facilitates the dispersion and replication of oncolytic Ad, making it an attractive candidate for the treatment of aggressive and desmoplastic pancreatic cancer.     

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)     

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.     

Adenovirus-Mediated Transcriptional Targeting of Colorectal Cancer and Effects on Treatment-Resistant Hypoxic Cells

BackgroundColorectal cancer is the second leading cause of cancer-related mortality and frequently presents with locally advanced or metastatic disease. Adenovirus (Ad) vectors are important gene delivery agents because they offer efficient and broad tissue transduceability. However, their ability to penetrate through multicell layers in colorectal cancers and maintain expression in colon tumor-related hypoxic conditions has yet to be analyzed. Furthermore, their broad tissue tropism presents safety concerns.Materials and MethodsAn ex vivo cultured patient tumor sample model was employed to examine Ad transduction of colorectal tumors.ResultsResults obtained from Ad delivery of the firefly luciferase (FLuc) reporter gene indicated that colon tumor tissue was more amenable to Ad transduction than other tumor histologic types examined (breast and ovary). Ad transduction levels were significantly higher than a range of viral and nonviral methods examined in patient colon tissue. Control of transgene expression using the CXC chemokine receptor 4 (CXCR4) promoter was examined as a strategy to confine expression to tumor cells. An Ad construct carrying FLuc under the control of the human CXCR4 promoter demonstrated low reporter gene expression compared with the ubiquitously expressing cytomegalovirus promoter in normal colon and liver tissue while providing high expression in tumors, demonstrating a ‘tumour-on' and ‘normal-off' phenotype in patient tissue. The effects of changing hypoxia on Ad-related transgene expression were examined in an in vitro model of hypoxic conditions relevant to clinical colorectal tumors. Reporter gene expression varied depending on the level of hypoxia, with significantly reduced levels observed with prolonged hypoxia. However, transgene expression was robust in the cycling hypoxic conditions relevant to colorectal tumors.ConclusionThis study provides novel, clinically relevant data demonstrating the potential for efficient gene delivery to colorectal tumors using Ad.     

CD47-retargeted oncolytic adenovirus armed with melanoma differentiation-associated gene-7/interleukin-24 suppresses in vivo leukemia cell growth

Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ leukemia cells though carrying a sCAR-4N1 expression cassette in the viral genome, forming Ad.4N1, in which 4N1 represents the C-terminal CD47-binding domain of thrombospondin-1. The infection and cytotoxicity of Ad.4N1 in leukemia cells were determined to be mediated by the 4N1-CD47 interaction. Ad.4N1 was further engineered to harbor a gene encoding melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), forming Ad.4N1-IL24, which replicated dramatically faster than Ad.4N1, and elicited significantly enhanced antileukemia effect in vitro and in a HL60/Luc xenograft mouse model. Our data suggest that Ad.4N1 could act as a novel oncolytic adenovirus vector for CD47+ leukemia targeting gene transfer, and Ad.4N1 harboring anticancer genes may provide novel antileukemia agents.     

Cancer scene investigation: how a cold virus became a tumor killer

Oncolytic therapy is a novel anticancer treatment with attenuated lytic viruses such as adenovirus (Ad). These viruses kill the host cells through their lytic replication cycle and are thus distinct from classical gene therapy viruses, which serve as gene delivery agents and do not replicate. Oncolytic Ads are genetically engineered so as to replicate only in cancer cells. Their replication cycle leads to viral multiplication, the killing of the host cells and spreading of the infection throughout the tumor. Following success in preclinical studies, their anti-tumor potential is now being evaluated in the clinic. Three oncolytic Ads (dl1520, Ad5-CD/TKrep, and CV706) have completed Phase I and II clinical trials in cancer patients where their administration via multiple routes and in combination with chemo- or radiotherapies, has demonstrated overall safety. These viruses are being re-engineered to arm them with additional therapeutic genes, bolstering their oncolytic activity with a bystander effect. For example, Ad5-CD/TKrep delivers a therapeutic prodrug-activating (suicide) gene. These data indicate that oncolytic Ads are a promising novel cancer treatment approach that can be combined with other modalities, such as gene therapy and classical chemo- and radiotherapies. Further improvements to enhance their specificity, targeting and oncolytic activity are needed however, as these first-generation viruses showed modest anti-tumor activity. To improve their efficacy in the clinic, it will be important to devise and incorporate novel monitoring techniques in the clinical trials, such as analysis of viral replication in biopsies and through the use of creative noninvasive imaging technologies.     

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

Neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing IL-12 effectively treats the parent tumor and protects against recurrence-after resection

The objective of the study was to evaluate the utility of NV1042, a replication competent, oncolytic herpes simplex virus (HSV) containing the interleukin-12 (IL-12) gene, as primary treatment for hepatic tumors and to further assess its ability to reduce tumor recurrence following resection. Resection is the most effective therapy for hepatic malignancies, but is not possible in the majority of the patients. Furthermore, recurrence is common after resection, most often in the remnant liver and likely because of microscopic residual disease in the setting of postoperative host cellular immune dysfunction. We hypothesize that, unlike other gene transfer approaches, direct injection of liver tumors with replication competent, oncolytic HSV expressing IL-12 will not only provide effective control of the parent tumor, but will also elicit an immune response directed at residual tumor cells, thus decreasing the risk of cancer recurrence after resection. Solitary Morris hepatomas, established in Buffalo rat livers, were injected directly with 10(7) particles of NV1042, NV1023, an oncolytic HSV identical to NV1042 but without the IL-12 gene, or with saline. Following tumor injection, the parent tumors were resected and measured and the animals were challenged with an intraportal injection of 10(5) tumor cells, recreating the clinical scenario of residual microscopic cancer. In vitro cytotoxicity against Morris hepatoma cells was similar for both viruses at a multiplicity of infection of 1 (MOI, ratio of viral particles to target cells), with >90% tumor cell kill by day 6. NV1042 induced high-level expression of IL-12 in vitro, peaking after 4 days in culture. Furthermore, a single intratumoral injection of NV1042, but not NV1023, induced marked IL-12 and interferon-gamma (IFN-gamma) expression. Both viruses induced a significant local immune response as evidenced by an increase in the number of intratumoral CD4(+) and CD8(+) lymphocytes, although the peak of CD8(+) infiltration was later with NV1042 compared with NV1023. NV1042 and NV1023 reduced parent tumor volume by 74% (P<.003) and 52% (P<.03), respectively, compared to control animals. Treatment of established tumors with NV1042, but not with NV1023, significantly reduced the number of hepatic tumors after resection of the parent tumor and rechallenge (16.8+/-11 (median=4) vs. 65.9+/-15 (median=66) in control animals, P<.025). In conclusion, oncolytic HSV therapy combined with local immune stimulation with IL-12 offers effective control of parent hepatic tumors and also protects against microscopic residual disease after resection. The ease of use of this combined modality approach, which appears to be superior to either approach alone, suggests that it may have clinical relevance, both as primary treatment for patients with unresectable tumors and also as a neoadjuvant strategy for reducing recurrence after resection.     

E1B-55kD-deleted oncolytic adenovirus armed with canstatin gene yields an enhanced anti-tumor efficacy on pancreatic cancer

Conditionally-replicating adenovirus (CRAd) therapy is currently being tested against pancreatic cancer and has shown some promise. To improve the efficacy, a novel virus CRAd-Cans was designed by deletion of E1B-55 kDa gene for selective replication in tumor cells, as well as carrying a new angiogenesis inhibitor gene, canstatin. CRAd-Cans mediated higher expression of canstatin in BxPC-3 pancreatic cancer cell line compared to the replication-deficient adenovirus Ad5-Cans. The modified CRAd-Cans manifested the same selective replication and cytocidal effects in pancreatic cancer cells as ONYX-015 in vitro, yet showed greater reduction of tumor growth in nude mice with markedly prolonged survival rate in vivo (P < 0.05), compared to that of either ONYX-015 or Ad5-Cans. Pathological examination revealed viral replication, decreased microvessel density and increased cancer cell apoptosis in CRAd-Cans-treated xenografts. The results suggest that the novel oncolytic virus CRAd-Cans, showing synergistic effects of oncolytic therapy and anti-angiogenesis therapy, is a new promising therapeutics for pancreatic cancer.     

Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.     

Deficiency of caspase 3 in tumor xenograft impairs therapeutic effect of measles virus Edmoston strain

The oncolytic measles virus Edmonston (MV-Edm) strain shows considerable oncolytic activity against a variety of human tumors. In this study, we report MV-Edm is able to trigger apoptosis pathways in infected tumor cells and elucidate the roles of cellular apoptosis in the whole oncolytic process. We also show that activated caspase 3, a key executioner of apoptosis, plays key roles in the oncolytic virotherapy. Activated caspase 3 can accelerate viral replication in cervical cancer cells and enhance the killing effects of the virus. Deficiency of caspase 3 either in tumor cells or in tumor xenograft significantly desensitized tumor to oncolysis with MV-Edm. In the infected cells, caspase 3 regulates interferon α release, which can inhibit viral replication in neighboring tumor cells. We propose that caspase-3 activation enhances the oncolytic effects of MV-Edm, thus inhibiting tumor growth in mice.     

Cotargeting tumor and tumor endothelium effectively inhibits the growth of human prostate cancer in adenovirus-mediated antiangiogenesis and oncolysis combination therapy

Tumor-endothelial interaction contributes to local prostate tumor growth and distant metastasis. In this communication, we designed a novel approach to target both cancer cells and their "crosstalk" with surrounding microvascular endothelium in an experimental hormone refractory human prostate cancer model. We evaluated the in vitro and in vivo synergistic and/or additive effects of a combination of conditional oncolytic adenovirus plus an adenoviral-mediated antiangiogenic therapy. In the in vitro study, we demonstrated that human umbilical vein endothelial cells (HUVEC) and human C4-2 androgen-independent (AI) prostate cancer cells, when infected with an antiangiogenic adenoviral (Ad)-Flk1-Fc vector secreting a soluble form of Flk1, showed dramatically inhibited proliferation, migration and tubular formation of HUVEC endothelial cells. C4-2 cells showed maximal growth inhibition when coinfected with Ad-Flk1-Fc and Ad-hOC-E1, a conditional replication-competent Ad vector with viral replication driven by a human osteocalcin (hOC) promoter targeting both prostate cancer epithelial and stromal cells. Using a three-dimensional (3D) coculture model, we found that targeting C4-2 cells with Ad-hOC-E1 markedly decreased tubular formation in HUVEC, as visualized by confocal microscopy. In a subcutaneous C4-2 tumor xenograft model, tumor volume was decreased by 40-60% in animals treated with Ad-Flk1-Fc or Ad-hOC-E1 plus vitamin D3 alone and by 90% in a combined treatment group, compared to untreated animals in an 8-week treatment period. Moreover, three of 10 (30%) pre-established tumors completely regressed when animals received combination therapy. Cotargeting tumor and tumor endothelium could be a promising gene therapy strategy for the treatment of both localized and metastatic human prostate cancer.