Species C human adenovirus serotype 5 (HAdV-C5) is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR), HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR)-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.
Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165 000-fold enhanced lung-targeting, compared to Ad5. Additionally, myeloid cell-specific binding is preserved in single cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest endothelial transduction is achieved by leukocyte-mediated “hand-off” of Ad.
adenovirus; targeting; pulmonary; myeloid; endothelial cell; vasculature
New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.
Pancreatic adenocarcinoma is an aggressive malignancy. Oncolytic Ads are genetically modified to target tumor cells while sparing normal cells. We modified the knob domain of the adenovirus (Ad) serotype 5 with a serotype 3 knob domain and incorporated the CXCR4 promoter to regulate Ad E1A gene expression (Ad5/3-CXCR4-E1A). These modifications were made to efficiently infect and lyse pancreatic tumors.
Human pancreatic cancer lines CFPAC-1, PANC-1, AsPC-1, and BxPC-3 were obtained from ATCC. Efficiency of Ad infection in the cells was determined using an Ad construct expressing the green fluorescence protein (GFP) marker in place of the E1A gene (Ad5/3-CXCR4-GFP) and quantified by flow cytometry. Oncolytic activity in the pancreatic cancer cells was determined using the Ad5/3-CXCR4-E1A oncolytic Ad by a crystal violet staining method. To determine the oncolytic effect in vivo, pancreatic cancer cells were implanted on the flanks of 40 SCID mice (4 groups). Tumors were injected intratumorally for 3 days with Ad5/3-CXCR4-E1A, Ad5 wild-type (a positive control), or phosphate buffered saline (a no virus control). Tumor size, overall survival and body condition scale (BCS) were recorded. Statistical analyses included the Kaplan-Meier survival curve, the log-rank test, and ANOVA 1-way.
The serotype 3 fiber-modified Ad with the CXCR4 promoter (Ad5/3-CXCR4-E1A) was most efficient in infecting and lysing pancreatic cancer cells compared with an Ad containing an unmodified fiber knob (Ad5-CXCR4-E1A). Treatment of pancreatic tumor xenografts in vivo with Ad5/3-CXCR4-E1A group resulted in significantly smaller tumors (p = 0.001), higher BCS (p = 0.01), and longer survival time (p = 0.04) than the other treatment groups.
Ad5/3-CXCR4-E1A treatment significantly prolonged survival in SCID mice pancreatic tumor xenografts. This novel construct represents a potential new therapy against pancreatic cancer.
Ad5/3; adenovirus; chemokine receptor; CXCR4; oncolytic virus; fiber chimera; pancreatic adenocarcinoma; serotype; virotherapy
The ability of Dendritic Cells (DC) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DC for clinical administration, their loading with tumor associated antigens (TAA) and their activation, is laborious and expensive, and, due to interindividual variability in the personalized vaccines, poorly standardized. An attractive alternative approach is to load resident DC in vivo by targeted delivery of TAA , using viral vectors and activating them simultaneously. To this end we have constructed genetically modified Adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAA to the CD40 receptor on DC. Preclinical human and murine studies conducted so far have clearly demonstrated the suitability of a “two-component”, i.e. Ad and adaptor molecule, configuration for targeted modification of DC in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in clinical translation of this approach.
Cancer gene therapy approaches have benefited greatly from the utilization of molecular-based therapeutics. Of these, adenovirus-based interventions hold much promise as a platform for targeted therapeutic delivery to tumors. However, a barrier to this progression is the lack of native adenovirus receptor expression on a variety of cancer types. As such, any adenovirus-based cancer therapy must take into consideration retargeting the vector to nonnative cellular surface receptors. Predicated upon the knowledge gained in native adenovirus biology, several strategies to transductionally retarget adenovirus have emerged. Herein, we describe the biological hurdles as well as strategies utilized in adenovirus transductional targeting, covering the progress of both adapter-based and genetic manipulation-based targeting. Additionally, we discuss recent translation of these targeting strategies into a clinical setting.
Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.
Viral gene therapy; Mcl-1 inhibition; apoptosis induction; anti-tumor activity
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G2/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.
MDA-7/IL-24; soluble clusterin; nuclear clusterin; G2/M arrest; apoptosis
Microsomal triglyceride transfer protein (MTP), essential for apoB-lipoprotein biosynthesis, evolved as a phospholipid transfer protein and acquired triglyceride transfer activity during a transition from invertebrates to vertebrates. But it is unknown whether MTP directly transfers lipids onto apoB in vivo and, if it does, whether both neutral and polar lipid transfer activities of MTP are critical for lipoprotein assembly. The molecular bases for differences in lipid transfer activities with respect to distinct domains in dMTP and hMTP are not obvious because both proteins have very similar primary, secondary and tertiary structures. We used an in vivo approach to delineate physiological significance of these distinct lipid transfer activities by expressing Drosophila (transfers phospholipids) and human MTP (transfers phospholipids and triglycerides) orthologs using adenoviruses in liver-specific MTP deficient (L-MTP−/−) mice that have low plasma and high hepatic lipids. Both orthologs improved plasma lipids but plasma triglycerides were lower in dMTP mice due to lower hepatic triglyceride and apoB production. Hepatosteatosis in L-MTP−/− mice was ameliorated to similar levels by both. Attenuation of hepatosteatosis upon dMTP expression pertained to enhanced β-oxidation with no changes in lipogenesis. Phospholipid transfer activity of MTP promoted biogenesis of both apoB48 and apoB100-containing VLDL in addition to a phospholipid-rich apoB48-containing HDL-size particle. Triglyceride transfer activity augmented the biosynthesis of triglyceride-rich lipoproteins by increasing the formation of these particles in the lumen of the endoplasmic reticulum. Based on these findings, we posit that the selective inhibition of MTP triglyceride transfer activity might reduce hyperlipidemia while protecting liver from excess lipid accumulation.
The editors of Journal of Ovarian Research would like to thank all of our reviewers who have contributed to the journal in volume 5 (2012).
Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.
adenovirus; adenoviral assembly; cancer gene therapy; nucleophosmin 1; protein V
Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.
As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.
These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.
Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.
Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.
Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.
Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.
We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
Conditionally replicative adenoviral (CRAd) virotherapy represents a promising therapeutic approach for cancer. We have demonstrated that a serotype chimeric adenoviral 5/3 fiber-knob modification achieves enhanced ovarian cancer infectivity, conditional replication, and oncolytic activity. This study evaluated the safety of intraperitoneal (IP) Ad5/3-Δ24 in advance of a phase I clinical trial in gynecologic cancers. Syrian hamster cohorts were treated with IP Ad5/3-Δ24 or control buffer for 3 consecutive days and euthanized on study days 8, 17, 57, and 89. Blood and tissue samples were harvested from each animal. For biodistribution studies, presence and quantitation of viral levels within samples were determined via quantitative polymerase chain reaction. For safety studies, animals were assessed for adverse vector-related tissue or laboratory effects. In the biodistribution study, low levels of Ad5/3-Δ24 DNA were noted outside of the abdominal cavity. Viral DNA levels in tissues obtained from the peritoneal cavity peaked at day 8 and declined thereafter. In the safety study, no specific histopathologic changes were attributable to virus administration. Hematologic findings noted in the 1 × 1011 viral particles (vp)/dose group on Days 4 and/or 8 were indicative of an Ad5/3-Δ24–specific generalized inflammatory response; these findings resolved by day 56. The no observable adverse effect level was determined to be 1 × 1010 vp/dose. This study elucidates the safety profile of IP administration of the serotype chimeric infectivity-enhanced CRAd, Ad5/3-Δ24, and provides guidance for a planned phase I trial for patients with recurrent gynecologic cancers.
In this study, Kim and colleagues evaluate the biodistribution, safety, and immunological response associated with intraperitoneal administration of a novel adenoviral vector with a 5/3 fiber-knob modification in a hamster model. These studies were done in advance of a planned phase I human clinical trial in ovarian and other select gynecological cancers.
The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5). Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR), with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP), with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease.
Recombinant adenovirus vectors (Ad) have been recognized as effective in vivo gene delivery vehicles and utilized as gene therapy agents for a number of cancers. The elucidation of viral entry mechanisms has allowed the development of recombinant vectors that exploit existing cell surface receptors to achieve entry into the cell. B lymphocytes are normally resistant to infection by adenovirus 5, likely due to the lack of the Coxsackie and Adenovirus receptor (CAR). Using reverse-transcriptase PCR and flow cytometry, the CD40 receptor has been shown to be expressed on many lymphoma cells. We exploited this finding to develop a gene therapy strategy for treatment of canine B-cell lymphoma. Ad5 was targeted to cells expressing CD40 via CD40 ligand (CD40L) and was effective in infecting CD40-expressing control cells; however, both primary canine lymphoma cells and cell lines demonstrated limited evidence of transduction. Following receptor binding, adenovirus entry into cells may require interaction with αvβ3/5 integrins; we demonstrate that canine lymphoma cells are deficient in these integrins. Reduced αvβ3 integrin expression may render these cells incapable of internalizing Ad vectors. Thus, any viral targeting approaches for treatment of canine lymphoma must also take into account the potential lack of internalization signals.
gene therapy; adenovirus; canine; lymphoma; transduction; CD40; integrins
Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes.
adenovirus; administration route; melanoma
The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
ROS; caspase; ER stress; CD95; cell death
The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
MDA-7; IL-24; Apoptosis; Autophagy; Ceramide; ROS; Ca2+; Clinical trial; Signal transduction; PERK; ER stress; MCL-1
To determine the maximum tolerated dose (MTD), toxicity spectrum, clinical activity, and biological effects of the tropism-modified infectivity-enhanced CRAd, Ad5-Δ24-RGD, in patients with gynecologic malignancies.
Cohorts of eligible patients were treated daily for 3 days via intraperitoneal catheter. Vector doses ranged from 1×109 to 1×1012 viral particles/day. Toxicity was evaluated utilizing CTCv3.0. CA-125 and RECIST criteria were utilized to determine clinical efficacy. Corollary biologic studies included assessment of CRAd replication, wild type virus generation, viral shedding, and neutralizing antibody response.
Twenty-one patients were enrolled. Adverse clinical effects were limited to G1/2 fever, fatigue, or abdominal pain. No vector related grade 3/4 toxicities were noted. No clinically significant laboratory abnormalities were noted. The MTD was not reached. Over a 1 month follow up, 15 (71%) patients had stable disease and six (29%) had progressive disease. No partial or complete responses were noted. Seven patients had a decrease in CA-125; 4 had a >20% drop. RGD-specific-PCR demonstrated the presence of study vector in ascites of 16 patients. Seven revealed an increase in virus after day 3, suggesting replication of Ad5-Δ24-RGD. Minimal wild type virus generation was detected. Viral shedding studies demonstrated insignificant shedding in the serum, saliva, and urine. Anti-adenoviral neutralizing antibody effects were prevalent.
This study, the first to evaluate an infectivity enhanced CRAd in human cancer, demonstrates the feasibility, safety, potential antitumor response, and biologic activity of this approach in ovarian cancer. Further evaluation of infectivity enhanced virotherapy approaches for gynecologic malignancies is warranted.
Ovarian cancer; Gene therapy; Virotherapy; Replicative Adenovirus; CRAd
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R–like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK−/− cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B–dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methylade-nine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.
Due to advances in viral design, oncolytic adenoviruses have emerged as a promising approach for treatment of breast cancer. Tumor tissue slices offer a stringent model system for preclinical evaluation of adenovirus therapies, since the slices retain a morphology and phenotype that more closely resembles the in vivo setting than cell line cultures, and it has been shown to have utility in the evaluation of viral infectivity and replication. In this study, we evaluated the efficacy of viral infection and replication using a tropism-modified oncolytic adenovirus.
Breast tumor tissue slices were infected with a tropism-modified oncolytic adenovirus, and a wild-type adenovirus for comparison. Efficiency of infection was evaluated using fluorescent microscopy, as the viruses used have been modified to express red fluorescent protein. Replication of the viruses was evaluated with quantitative real-time PCR to assay viral E4 genome copy number, a surrogate indicator for the number of virions. The breast tumor tissue slices were evaluated for the expression of CD46 expression by immunohistochemistry.
Infection and replication of our tropism modified oncolytic virus has been observed in breast cancer tissue slice model system and is comparative to wild-type virus. A qualitative increase in the number of cells showing RFP expression was observed correlating with increasing multiplicity of infection. Higher relative infectivity of the virus was observed in tumor tissue compared with normal breast tissue. Replication of the virus was demonstrated through increases in E4 copy number at 48 and 72 hours after infection in human breast tumor slices.
We have shown that a tropism modified oncolytic oncolytic adenovirus can infect and replicate in breast cancer tissue slices, which may be an important preclinical indicator for its therapeutic utility.
adenovirus; breast cancer; CD46; CAR; oncolytic; tissue slice model
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect anti-tumor activity through inhibition of angiogenesis, stimulation of an anti-tumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.
mda-7/IL-24; apoptosis; autophagy; bystander antitumor activity; cancer terminator virus