Gene therapy with recombinant adenovirus encoding endostatin encapsulated in cationic liposome in coxsackievirus and adenovirus receptor-deficient colon carcinoma murine models

Adenovirus (Ad)-based antiangiogenesis gene therapy is a promising approach for cancer treatment. Downregulation or loss of coxsackievirus and adenovirus receptor (CAR) is often detected in various human cancers, which hampers adenoviral gene therapy approaches. Cationic liposome-complexed adenoviral vectors have been proven useful in CAR-deficient cells to enhance therapeutic gene transfer in vivo. Here, we investigated the antitumor effects of recombinant adenovirus encoding endostatin (Ad-hE) encapsulated in cationic liposome (Ad-hE/Lipo) on CAR-deficient CT26 colon carcinoma murine models. In vitro, Ad-hE/Lipo enhanced adenovirus transfection in CAR-deficient cells (CT26), and endostatin gene expression was measured by both qualitative and quantitative detection. In addition, an antibody neutralizing assay indicated that neutralizing serum inhibited naked adenovirus 5 (Ad5) at rather higher dilution than the complexes of Ad5 and cationic liposomes (Ad5-CL), which demonstrated that Ad5-CL was more capable of protecting Ad5 from neutralization. In vivo, Ad-hE/Lipo treatment in the murine CT26 tumor model by intratumoral injection resulted in marked suppression of tumor growth and prolonged survival time, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells. In conclusion, recombinant endostatin adenovirus encapsulated with cationic liposome effectively inhibited CAR-deficient tumor growth through an antiangiogenic mechanism in murine models without marked toxicity, thus showing a feasible strategy for clinical applications.     

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice.

AdmATF is a recombinant adenovirus encoding a secreted version of the amino-terminal fragment (ATF) of murine urokinase (uPA). This defective adenovirus was used in three murine models to assess the antitumoral effects associated with local or systemic delivery of ATF, a broad cell invasion inhibitor that antagonizes uPA binding to its cell surface receptor (uPAR). A single intratumoral injection of AdmATF into pre-established MDA-MB-231 human breast xenografts grown in athymic mice, or into pre-established C57/BL6 syngeneic Lewis lung carcinoma resulted in a specific arrest of tumor growth. Neovascularization within and at the vicinity of the injection site was also suppressed, suggesting that AdmATF inhibited primary tumor growth by targeting angiogenesis. AdmATF also interfered with tumor cell establishment at distant sites: (1) lung dissemination of Lewis lung carcinoma cells was significantly reduced following intratumoral injection at the primary site; and (2) systemic administration of AdmATF inhibited subsequent liver metastasis in a LS174T human colon carcinoma xenograft model. These data outline the potential of using a recombinant adenovirus directing the secretion of an antagonist of cell-associated uPA for cancer gene therapy.     

Antitumor effect and cellular immunity activation by murine interferon-beta gene transfer against intracerebral glioma in mouse.

Cationic liposomes containing the human interferon-beta (IFN-beta) gene induce marked growth inhibition in human glioma cells. In vivo experiments using an human glioma implanted into the brains of nude mice have demonstrated a definite growth-inhibitory effect, achieving complete tumor regression with multiple intratumoral injections of the gene. However, nude mouse studies are inadequate to evaluate antitumor effects fully, especially those related to activation of the host immune response. This article aimed to investigate antitumor effects and immune response activation by murine IFN-beta gene transfer in syngeneic mice. In vitro experiments demonstrated a stronger growth-inhibitory effect of liposomes containing the murine IFN-beta gene on a GL261 mouse glioma cell line than exogenously added murine IFN-beta. In in vivo experiments, intratumoral administration of liposomes containing the murine IFN-beta gene resulted in a 16-fold reduction in the mean volume of residual gliomas in the brains of C57BL/6 mice and massive infiltration of cytotoxic T lymphocytes (CTL) within the residual tumor, while few CTL were infiltrated in controls including murine IFN-beta, empty liposomes, naked plasmid expressing murine IFN-beta, and liposomes containing beta-galactosidase gene. In addition, 40% of mice treated with liposomes containing the murine IFN-beta gene were completely cured. These findings indicated that activation of cellular immunity participates in antitumor effects in vivo together with direct effects of the IFN-beta gene.     

Hepatic intra-arterial delivery of a retroviral vector expressing the cytosine deaminase gene, controlled by the CEA promoter and intraperitoneal treatment with 5-fluorocytosine suppresses growth of colorectal liver metastases

Targeting of colorectal liver metastases by regional gene therapy was tested in a clinically relevant syngeneic model. First, the CEA-CD-113 retroviral vector containing the cytosine deaminase gene controlled by the CEA specific tumour cell promoter, was shown in vitro to convert 5-fluorocytosine to 5-fluorouracil, resulting in cancer cell killing with a large bystander effect. Second, 10 days after the establishment of liver metastases, retroviral vectors were delivered to the liver by hepatic artery injection. After 5-fluorocytosine administration for 7 days, most surface metastases disappeared and tumour volumes were suppressed up to 8.2-fold. The results support the development of this approach for patient treatment.     

Cytokine Adjuvanation of Therapeutic Anti‐tumor Immunity Targeted to Cancer Mucosa Antigens

Colorectal cancer immunotherapy is limited by the paucity of available target antigens fulfilling the necessary criteria of tumor‐specificity, sufficient immunogenicity and universal association with disease. A novel class of immune targets, cancer mucosa antigens (CMAs), whose expression normally is confined to mucosae but maintained during neoplastic transformation, promises to overcome these imitations, enjoying the advantage of immune compartmentalization, preventing autoimmune disease, while permitting therapeutic anti‐tumor responses. Indeed, therapeutic immunization against the model CMA guanylyl cyclase c (GCC) extends survival in mouse models of established parenchymal colorectal cancer metastases with antitumor efficacy superior to currently available antigens. Here adjuvanation of therapeutic antitumor immunity to GCC was explored employing the cytokines IL‐2 and GM‐CSF in a mouse model of metastatic colorectal cancer. Combining plasmids expressing murine IL‐2 or GM‐CSF with recombinant viral vector immunization to GCC enhanced antitumor efficacy beyond viral vector immunization alone. These studies support the incorporation of IL‐2 and GM‐CSF in CMA‐targeted immunization regimens for established colorectal cancer metastases.     

Immunocytokines: A Promising Approach to Cancer Immunotherapy

Recombinant antibody-cytokine fusion proteins are immunocytokines that achieve high cytokine concentrations in the tumor microenvironment and thereby effectively stimulate cellular immune responses against malignancies. The activation and expansion of immune effector cells, such as CD8⁺ T lymphocytes, by interleukin-2 immunocytokines resulted in the eradication of established pulmonary and hepatic metastases of murine melanoma and colorectal carcinoma in syngeneic mouse models. These immunocytokines were equally effective in eliminating established bone marrow and liver metastases of murine neuroblastoma by activating natural killer cells. The effective eradication of metastases by immunocytokines resulted in significant prolongation in life span of mice over that of controls receiving equivalent mixtures of antibody and interleukin-2, which failed to reduce the growth of disseminated metastases. Proof of concept was established, indicating that immunocytokine-induced activation and expansion of immune effector cells in the tumor microenvironment can effectively eradicate established tumor metastases. This promising new approach to cancer immunotherapy may lead to clinical applications that improve treatment of cancer patients with minimal residual disease in an adjuvant setting.     

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.     

AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice

TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.     

A phase I/II study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours.

Colorectal cancer (CRC) is the second commonest cause of cancer death in the UK, with greater than 40% of these patients destined to die of the disease despite current medical management. Death is commonly due to liver metastases with sequelae including progressive liver dysfunction. Most patients with liver metastases present with tumours that are unresectable and incurable with existing therapies. The median survival for CRC patients after diagnosis with liver metastases is approximately 6 months or less. The human p53 gene is a tumour suppressor gene involved in the control of cell proliferation. Loss of wild-type p53 function is associated with the uncontrolled growth of many types of human cancers. The reintroduction and expression of wild-type p53 into p53 altered tumour cells has been shown to suppress tumour growth or induce apoptosis in both in vitro and in vivo models. In our experience greater than 50% of CRC tumours have p53 alterations. This study seeks to evaluate the safety, biological efficacy and the effectiveness of wtp53-CMV-Ad treatment which is a recombinant adenoviral vector containing the wild-type human p53 gene. It will be administered by infusion via the hepatic artery, for the regional gene therapy of malignant liver tumours. Study patients will have incurable metastatic (CRC) malignant tumours of the liver with evidence of p53 alteration in their liver tumours. In vitro studies have demonstrated p53-specific antiproliferative effects of wtp53-CMV-Ad on human liver tumour cells and in vivo studies have demonstrated p53-specific antiproliferative effects on human liver tumour cells. The vector Ad-p53 is a recombinant, replication-defective adenovirus based on adenovirus serotype 5. It contains a sequence encoding wild-type p53 whose expression is under the control of the human cytomegalovirus immediate early promoter-enhancer. This construct will be growth in 293 cells which contain the adenoviral E1A and E1B coding sequences which have been removed from the vector to render it replication defective. The study design is an open-label, non-randomised, single-dose, dose escalation Phase I/II clinical trial anticipated to involve a maximum of 19 patients. wtp53-CMV-Ad will be administered by infusion in a reservoir connected to the hepatic artery, for regional gene therapy (surgically implanted pump) in 3 escalating doses to successive cohorts of 3 patients each until the maximum tolerated dose is determined. Subsequently, 10 patients will be treated with this dose. Regional wtp53-CMV-Ad therapy will be administered as a single bolus infusion via hepatic artery catheter. The route of administration of wtp53-CMV-Ad via hepatic artery infusion is designed to maximise gene therapy exposure to the malignant tumours while minimising exposure to normal tissues outside the liver. The clinical protocol is designed to monitor treatment toxicity. Another objective is to evaluate the biological efficacy, including efficiency and stability of gene transfer by analysis of tumour tissues following therapy. As an important part of this objective the pharmacokinetics of wtp53-CMV-Ad will be studied. Clinical evidence of anti-tumour efficacy will also be collected. In addition, the safety and efficacy of different doses levels of wtp53-CMV-Ad will be studied.     

Gene-based intramuscular interferon-beta therapy for experimental autoimmune encephalomyelitis.

In contrast to serial injections of recombinant interferon-beta (IFN-beta) for long-term therapy of multiple sclerosis (MS), prolonged systemic delivery of proteins derived through in vivo gene transfer may provide a more clinically relevant alternative. Here we compare the therapeutic efficacies of electroporation (EP)-mediated intramuscular IFN-beta gene transfer with repeated alternate-day injections of recombinant IFN-beta after the onset of relapsing-remitting experimental autoimmune encephalomyelitis (EAE), an animal model widely used in MS research. We show for the first time that a single EP-mediated intramuscular administration of 20 microg of an IFN-beta-expressing plasmid provides long-term expression of interferon-inducible genes and is therapeutic in ongoing established EAE. The achieved therapeutic effects of IFN-beta gene delivery were comparable to an 8-week regimen of 10,000 IU rIFN-beta injected every other day and involved a significant inhibition of disease progression and a significant reduction of EAE relapses compared to untreated or null-vector-treated mice. Our results indicate the viability of a convenient and effective gene-based alternative for long-term IFN-beta protein therapy in MS.     

PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis

Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.     

Anti-tumor immunity induced by in vivo adenovirus vector-mediated expression of CD40 ligand in tumor cells.

CD40 ligand (CD40L), the ligand for CD40 on antigen-presenting cells, is essential for the initiation of antigen-specific T cell responses, an important component of the immune response to tumors. This study is based on the hypothesis that in vivo genetic modification of tumor cells to express CD40L will trigger CD40 on local antigen-presenting cells to present tumor antigen to the cellular immune systems, thus eliciting anti-tumor immunity to suppress growth of the tumor. To examine this concept, subcutaneous tumors of three different murine tumor models in two strains of mice were infected with a recombinant adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In the B16 (H-2b, melanoma) and CT26 (H-2d, colon cancer) murine models, injection of AdmCD40L into established subcutaneous tumors resulted in sustained tumor regression and tumor-free status in >60% of animals. Intratumoral injection of AdmCD40L also significantly suppressed the growth of established, weakly immunogenic Lewis lung carcinoma (H-2b) tumors, but to a lesser extent. Ex vivo AdmCD40L-transduced tumor cells implanted in syngeneic hosts induced significant antitumor response against preexisting identical tumors at a distant site. Both in vivo and in vitro AdmCD40L modification of tumors to express CD40L elicited tumor-specific cytolytic T lymphocytes responses, and the transfer of spleen cells from treated mice efficiently protected naive mice against a subsequent tumor challenge. These results support the concept that transduction of tumors with a recombinant CD40L adenovirus vector may be a useful strategy for cancer immunotherapy.     

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

To improve adenovirus-mediated gene delivery to skeletal muscle, we have used a recombinant adenovirus vector encoding the human Coxsackievirus and adenovirus receptor (hCAR). Because CAR is expressed at a lower level in rodent myoblasts and muscle fibers than in other tissues, we expected that elevated expression of CAR in skeletal muscle would improve the efficacy of adenovirus-mediated gene transfer. Since the mouse myoblasts, C2C12 cells, showed low sensitivity to infection by recombinant adenovirus 5, we initially infected these cells at a high multiplicity of infection (MOI) of 250 with the recombinant adenovirus containing hCAR cDNA and LacZ gene. Subsequent infection by recombinant adenovirus containing the marker gene, green fluorescence protein, became efficient even at a low MOI of 25. Thus, elevated hCAR expression in mouse muscle fibers made a second virus inoculation at low doses possible. We also demonstrated that the elevated hCAR expression did not influence muscle membrane integrity. Our results suggest that co-expression of CAR and a therapeutic gene by adenovirus vector constitutes a novel strategy to advance gene therapy for hereditary muscle diseases.     

Syngeneic hematopoietic stem cell transplantation enhances the antitumor immunity of intratumoral type I interferon gene transfer for sarcoma

Sarcoma at advanced stages remains a clinically challenging disease. Interferons (IFNs) can target cancer cells by multiple antitumor activities, including the induction of cancer cell death and enhancement of immune response. However, the development of an effective cancer immunotherapy is often difficult, because cancer generates an immunotolerant microenvironment against the host immune system. An autologous hematopoietic stem cell transplantation (HSCT) is expected to reconstitute a fresh immune system, and expand tumor-specific T cells through the process of homeostatic proliferation. Here we examined whether a combination of autologous HSCT and IFNs could induce an effective tumor-specific immune response against sarcoma. First, we found that a type I IFN gene transfer significantly suppressed the cell growth of various sarcoma cell lines, and that IFN-β gene transfer was more effective in inducing cell death than was IFN-α in sarcoma cells. Then, to examine the antitumor effect in vivo, human sarcoma cells were inoculated in immune-deficient mice, and a lipofection of an IFN-β-expressing plasmid was found to suppress the growth of subcutaneous tumors significantly. Finally, the IFN gene transfer was combined with syngeneic HSCT in murine osteosarcoma models. Intratumoral IFN-β gene transfer markedly suppressed the growth of vector-injected tumors and inhibited formation of spontaneous lung and liver metastases in syngeneic HSCT mice, and an infiltration of many immune cells was recognized in metastatic tumors of the treated mice. The treated mice showed no significant adverse events. A combination of intratumoral IFN gene transfer with autologous HSCT could be a promising therapeutic strategy for patients with sarcoma.     

Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.

The improvement of initial tumor cell transduction with viral vectors is a major task in tumor gene therapy. We have developed mouse tumor models with hepatic metastases to study transduction of tumor cells after systemic adenovirus vector application. The tumor models were established by intraportal transplantation of human tumor cell lines into immunodeficient mice. Liver metastases derived from cervix, colon, breast, and liver cancer lines were analyzed for distribution of extracellular matrix, vascularization, and transgene expression after tail vein injection of adenovirus vectors. Overall, xenografts resembled the morphology of corresponding tumors in cancer patients. Adenovirus-mediated gene delivery depended on tumor vascularization and direct contact between blood vessels and tumor cells. These models represent important tools for studying and improving tumor gene therapy approaches.     

A therapeutic cancer vaccine targeting carcinoembryonic antigen in intestinal carcinomas

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.     

Enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model

For cancer gene therapy, replicative adenovirus is a promising vector to overcome low infectivity and poor gene delivery of nonreplicative adenovirus in vivo, but its therapeutic efficacy is still unsatisfactory because of the limited spread of replicative virus in a solid tumor. Therefore, the combined therapy with other antitumor agents may be necessary. Nonreplicative adenovirus expressing a therapeutic gene may be a promising candidate because E1 proteins expressed by replicative adenovirus would render nonreplicative adenovirus replicative, augmenting a transgene expression. In this study, we first found that mouse hepatoma Hepa 1-6 cells were permissive for the replication and cytopathic effect of human adenovirus, which enabled us to examine the potential of combined replicative adenovirus and nonreplicative adenovirus expressing an immunostimulator in an immunocompetent mouse-syngeneic Hepa 1-6 tumor model. Nonreplicative adenovirus expressing interleukin-12 (AdIL-12) was used as a model. In vitro coinfection of two adenoviruses produced higher concentrations of IL-12 than infection of AdIL-12 alone in this cell line. In vivo experiments with Hepa 1-6 tumors in syngeneic immunocompetent C57BL/6 mice showed higher concentrations of serum IL-12 and greater therapeutic efficacy in the combination therapy than infection of either adenovirus. These data indicate that the combination of replicative adenovirus and nonreplicative adenovirus expressing an immunostimulator appears to be very efficacious for cancer gene therapy.     

Intratumoral delivery of adenovirus-mediated interleukin-12 gene in mice with metastatic cancer in the liver

Clinical trials of recombinant human interleukin-12 (rhIL-12) delivered by intravenous administration have shown dose-limiting toxicities with limited tumor responses at the doses tested. We have previously reported that intratumoral injection of an adenovirus vector expressing murine interleukin-12 (Adv.RSV-mIL-12) was effective in inducing antitumor immune responses, tumor regression, and long-term survival in mice with established metastatic cancer in the liver. We now report additional studies in the same murine tumor model to assess the safety of intratumoral Adv.RSV-mIL-12 injection. At vector doses that were previously shown to be therapeutically effective, no inflammation in the liver or lungs, and no significant elevations in serum creatinine and aminotransferases were seen after vector injection. Serum elevations of IL-12 and interferon-gamma (IFN-gamma) were 17- and 19-fold lower than peak levels after intravenous recombinant IL-12 at the maximal tolerated dose in clinical trials. No elevations in serum proinflammatory cytokines (interleukin-6, tumor necrosis factor-alpha) were noted up to 2 weeks after vector injection. No systemic dissemination of the vector was detected on polymerase chain reaction (PCR) assays at therapeutically effective vector doses. At higher supratherapeutic vector doses of Adv.RSV-mIL-12, however, inflammation in the liver and lungs with elevation in serum aminotransferases were seen, but not in controls injected with the equivalent particle number of an empty adenoviral vector. These results support the cautious testing in patients with hepatic metastases of adenovirus mediated IL-12 gene delivery by intratumoral injection.