Coxsackie-adenovirus Receptors Research Articles

Enhancing the Antitumor Efficacy of Oncolytic Adenovirus Through Sonodynamic Therapy-Augmented Virus Replication.

The therapeutic efficacy of oncolytic adenoviruses (OAs) relies on efficient viral transduction and replication. However, the limited expression of coxsackie-adenovirus receptors in many tumors, along with the intracellular antiviral signaling, poses significant obstacles to OA infection and oncolysis. Here, we present sonosensitizer-armed OAs (saOAs) that potentiate the antitumor efficacy of oncolytic virotherapy through sonodynamic therapy-augmented virus replication. The saOAs could not only efficiently infect tumor cells via transferrin receptor-mediated endocytosis but also exhibit enhanced viral replication and tumor oncolysis under ultrasound irradiation. We revealed that the sonosensitizer loaded on the viruses induced the generation of ROS within tumor cells, which triggered JNK-mediated autophagy, ultimately leading to the enhanced viral replication. In mouse models of malignant melanoma, the combination of saOAs and sonodynamic therapy elicited a robust antitumor immune response, resulting in significant inhibition of melanoma growth and improved host survival. This work highlights the potential of sonodynamic therapy in enhancing the effectiveness of OAs and provides a promising platform for fully exploiting the antitumor efficacy of oncolytic virotherapy.

D-103 Cancer terminator viruses: A strategy for targeting metastatic cancers

Our laboratory pioneered subtraction hybridization and used this elegant and effective gene identification strategy to clone novel genes of physiological relevance. This methodology defined differentially regulated genes in an unbiased manner that are modified when metastatic human melanoma cells are pharmacologically induced to terminally differentiate and lose irreversibly oncogenic potential following treatment with IFN-band the protein kinase C activator mezerein. This approach, called DISH (differentiation induction subtraction hybridization), identified melanoma differentiation associated (mda) genes relevant to growth control, cancer development, tumor progression, and cancer cell survival. One intriguing gene identified using the DISH scheme was mda-7, which based on chromosomal location, secretory properties and structure has been classified as interleukin-24 (IL-24). mda-7/IL-24 is a novel member of the IL-10 gene family that promotes apoptosis and toxic autophagy in a broad-spectrum of human tumors, exerts potent “bystander” antitumor activity, inhibits tumor angiogenesis, stimulates the immune system and synergizes with conventional therapies (including chemotherapy, radiation, monoclonal antibodies and immunomodulators) to promote selective tumor destruction. mda-7/IL-24 displays profound anti-cancer activity in preclinical animal models (including nude, syngeneic and transgenic mice) and following intratumoral administration using a type 5 adenovirus in a Phase I/II clinical trial in patients with advanced cancers. To enhance the therapeutic efficacy of mda-7/IL-24, we generated a novel class of adenoviruses, called Cancer Terminator Viruses (CTVs), that selectively replicate in cancer cells and simultaneously produce mda-7/IL-24, Ad.5-CTV. A further iteration of this virus employs a chimera between Ad.5 and Ad.3, Ad.5/3, that employs Coxsackie-Adenovirus Receptors (CAR) as well as desmoglien and CD46 to infect cells, thereby increasing its infectivity in cancer cells with reduced CAR. Ad.5/3-CTV displays profound anti-cancer activity in pre-clinical animal models, including GBM, melanoma, and prostate, colorectal and breast cancer. A clinical trial is in preparation to test the effect of the Ad.5/3-CTV in patients with recurrent glioma. To facilitate the delivery of adenoviruses systemically, avoiding immune clearance and liver trapping, we have refined the ultrasound-targeted microbubble-destruction (UTMD) approach. This strategy has allowed us to deliver therapeutic viruses and proteins intravenously to internal target organs, such as the prostate, to evoke pre-clinical antitumor activity. To enhance further the therapeutic efficacy of the UTMD approach, we are employing microbubbles that contain targeting ligands on their surface permitting accumulation in the tumor vasculature, that is, decorated microbubbles. Employing the same cancer-selective promoter used in the original CTV, the rat progression elevated gene-3 promoter (PEG-Prom), we also developed genetic-based imaging approaches for metastases. These approaches use the PEG-Prom (or another cancer-selective promoter) to drive an imaging gene (eg, luciferase or HSV-Tk) linked to a delivery vehicle PEI. These nanoparticle vectors when combined with bioluminescence imaging and SPECT-CT permit non-invasive imaging of tumors and metastases in pre-clinical animal models. As a further iteration of our CTV we have used 2 cancer-selective and a non-selective promoter to generate first in class tripartite Ads that selectively replicate in cancer cells, produce an imaging agent in cancer cells (Lucirerase or HSV-Tk) and simultaneously produce the therapeutic immunogenic cytokine mda-7/IL-24, Ad.Tri-CTV. These “theranostic viruses” permit tumor and metastasis imaging and treatment through application of a single vector. Overall, the novel reagents and methodologies we have developed are providing paradigm shifts in how cancers will be detected and treated in the future.

Human Islets- Like Cell Aggregates (ICAs) express Coxsackie- Adenovirus receptors (CAR) and Decay Accelerating Factor (DAF or CD55) and reveal a distinctive microRNA diabetes-associated signature

Human Islets- Like Cell Aggregates (ICAs) express Coxsackie- Adenovirus receptors (CAR) and Decay Accelerating Factor (DAF or CD55) and reveal a distinctive microRNA diabetes-associated signature

Enhanced prostate cancer gene transfer and therapy using a novel serotype chimera cancer terminator virus (Ad.5/3-CTV)

Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting.

Abstract 61: A new serotype chimera Cancer Terminator Virus (Ad.5/3-CTV) expands the efficiency and specificity of prostate cancer gene transfer and therapy

Abstract Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating prostate cancer (PC). We previously constructed a unique CRCA, Cancer Terminator Virus (CTV), in which the E1A gene, necessary for adenoviral replication, is controlled by the cancer-selective Progression Elevated Gene-3 (PEG-3) promoter and which simultaneously expresses melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) from the E3 region of the adenovirus. mda-7/IL-24, a novel member of the IL-10-related cytokine gene family, displays almost ubiquitous antitumor properties in vitro and in vivo, leading to its rapid entry into the clinic, where its safety and clinical efficacy, when administered by adenovirus (Ad.mda-7; INGN 241), was observed in a phase I clinical trial in humans with advanced carcinomas and melanomas. mda-7/IL-24 preferentially induces apoptosis in cancer cells while exerting no discernible toxic effects toward normal cells and it also elicits potent “antitumor bystander activity” in distant cancer cells as a consequence of autocrine and paracrine secretion of MDA-7/IL-24. The CTV was generated on a serotype 5 background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC in Hi-myc transgenic mice. Ad.5/3.CTV exerted a marked anti-tumor ‘bystander’ effect in vivo confirming therapeutic utility. In these contexts, and considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7, the use of Ad.5/3-CTV to administer therapeutic and cytotoxic mda-7/IL24 to selectively induce cytolysis and apoptosis in prostate tumor cells represents a viable treatment options. (Supported by R01 CA097318 and P01 CA104177) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 61. doi:1538-7445.AM2012-61

Enhanced delivery of mda‐7/IL‐24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the apogossypol derivative BI‐97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells

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.

Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells

Gene therapy is being examined as a potential strategy for treating prostate cancer. Serotype 5 adenovirus (Ad.5) is routinely used as a vector for transgene delivery. However, the infectivity of Ad.5 is dependent on Coxsackie-adenovirus receptors (CARs); many tumor types show a reduction in this receptor in vivo, thereby limiting therapeutic gene transduction. Serotype chimerism is one approach to circumvent CAR deficiency; this strategy is used to generate an Ad.5/3-recombinant Ad that infects cancer cells through Ad.3 receptors in a CAR-independent manner. In this report, the enhanced transgene delivery and efficacy of Ad.5/3-recombinant virus was evaluated using an effective wide-spectrum anticancer therapeutic melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24). Our data show that in low CAR human prostate cancer cells (PC-3), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than an Ad.5 virus encoding mda-7/IL-24 (Ad.5-mda-7) in infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor 'bystander' effect in a nude mouse xenograft model. Considering the fact that Ad.5-mda-7 has shown significant objective responses in a phase I clinical trial for solid tumors, Ad.5/3-mda-7 is predicted to exert enhanced therapeutic benefit in patients with prostate cancer.

Neuroblastomas and medulloblastomas exhibit more Coxsackie adenovirus receptor expression than gliomas and other brain tumors

Adenoviral vector-mediated treatment is a potential therapy for tumors of the central nervous system. To obtain a significant therapeutic effect by adenoviral vectors, a sufficient infection is required, the power of which depends predominantly on the level of Coxsackie adenovirus receptors. We stained surgical biopsies of central nervous system tumors and neuroblastomas for Coxsackie adenovirus receptors. For gliomas, the level of the receptor was low and markedly variable among individual tumors. By contrast, neuroblastomas and medulloblastomas exhibited a higher degree of Coxsackie adenovirus receptor expression than gliomas and other brain tumors. We conclude that neuroblastomas and medulloblastomas could be suitable for adenovirus-mediated gene therapy. Adverse effects of the treatment, however, must be considered because neurons and reactive astrocytes also express a significant amount of the receptor.

P53 therapy in a patient with Li-Fraumeni syndrome

Li-Fraumeni syndrome is an autosomal dominant disorder that greatly increases the risk of developing multiple types of cancer. The majority of Li-Fraumeni syndrome families contain germ-line mutations in the p53 tumor suppressor gene. We describe treatment of a refractory, progressive Li-Fraumeni syndrome embryonal carcinoma with a p53 therapy (Advexin) targeted to the underlying molecular defect of this syndrome. p53 treatment resulted in complete and durable remission of the injected lesion by fluorodeoxyglucose-positron emission tomography scans with improvement of tumor-related symptoms. With respect to molecular markers, the patient's tumor had abnormal p53 and expressed coxsackie adenovirus receptors with a low HDM2 and bcl-2 profile conducive for adenoviral p53 activity. p53 treatment resulted in the induction of cell cycle arrest and apoptosis documented by p21 and cleaved caspase-3 detection. Increased adenoviral antibody titers after repeated therapy did not inhibit adenoviral p53 activity or result in pathologic sequelae. Relationships between these clinical, radiographic, and molecular markers may prove useful in guiding future application of p53 tumor suppressor therapy.

879. Transduction Enhancement by the Adenovirus/Polymer Complexes Derivatized with Protein Transduction Domain Peptides

Top of pageAbstract Background: Cellular uptake of group C adenoviruses critically depends on the availability of the cognate Coxsackie Adenovirus Receptors (CARs) on the cell surface. In cardiovascular tissues paucity of CARs is a limiting factor for the successful application of adenoviral vectors. Additionally, immune responses elicited by Ad infection diminish transgene expression due to the elimination of free Ad and transduced cells. Polymer modification of adenoviral surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties. We report here that a covalent modification of Ad surface with a photoactivatable polymer derivatized with protein transduction domains (PTD), TAT and Antp, increases transduction efficiency. Methods: A positively charged, thiol-reactive, photoactivatable polyallylamine (PAA) derivative was coupled with TAT-Cys and Antp-Cys. Ad samples suspended in water or PBS of different ionic strength (x1 and x5) were admixed with PTD-derivatized and unmodified polymers to obtain final polymer/Ad ratios ranging from 1:100 to 1:5 (w/w). Initial charge-based polymer/virus attachment was rendered covalent by 15 sec UV light (350 nm) illumination. Surface charge and size of the Ad/polymer complexes were measured by zeta-potentiometry and dynamic light scattering, respectively. Ad surface modification was examined by transmission electron microscopy (TEM) employing colloidal anionic gold. Transduction of a rat aortic smooth muscle cell line (A10) and primary bovine aortic endothelial cells (BAEC) was assessed by the fluorescence microscopy and the fluorimetry of cell lysates. In some experiments the cells were pretreated with the recombinant soluble knob protein (5 |[mu]|g/ml) prior to the Ad infection. Results: Association of Ad with unmodified polymer increased surface charge of the vector from |[minus]|31.67 mv to |[minus]|11.89 mv, while the interaction with the Antp- and TAT-modified polymers inverted the surface charge to +3.75 mv and +12.09 mv, respectively. Recharging of the Ad surface was confirmed by the TEM demonstrating the attraction of 5 nm anionic gold particles to the surface of the Ad modified with TAT-derivatized polymer, but not to the surface of naive Ad. The association of Ad-GFP with PTD-derivatized polymers resulted in 3-7-fold increase of GFPexpression in cells, whereas unmodified polymer-derivatized virus demonstrated the same efficiency as the naive Ad. Pretreatment of A10 cells with knob protein caused 86% transduction inhibition with free and unmodified polymer-derivatized Ad, while the transgene expression of the PTD/polymer-modified Ad was not affected. Optimization of the transduction protocol revealed maximal enhancement of transgene expression at a polymer/Ad ratio of 1:10. Also, complexes prepared in x5 PBS achieved higher GFP expression than those prepared in water or x1 PBS, which correlated with smaller size of complexes (330 nm vs 1360 and 1060 nm diameter, respectively). Conclusion: Covalent attachment of PDT-decorated polymers to the Ad surface increases transduction in vitro.

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells.

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and alphaV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that alphaVbeta3 and alphaVbeta5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.

Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway.

Oncolytic adenoviruses are promising therapies for the treatment of gliomas. However, untargeted viral replication and the paucity of coxsackie-adenovirus receptors (CARs) on tumor cells are major stumbling blocks for adenovirus-based treatment. We studied the antiglioma activity of the tumor-selective Delta-24 adenovirus, which encompasses an early 1 A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, and of the Delta-24-RGD adenovirus. Delta-24-RGD has an RGD-4C peptide motif inserted into the adenoviral fiber, which allows the adenovirus to anchor directly to integrins. CAR and integrin expression were examined by flow cytometry in six glioma cell lines and in normal human astrocytes (NHAs). Adenoviral vectors containing green fluorescent protein (GFP) (AdGFP and AdGFP-RGD) were used to infect glioma cell lines with high or low CAR expression. Viability of glioma cells infected with different adenoviruses was assessed by trypan blue staining. Adenovirus replication was quantified with the infection-dose replication assay. Athymic mice carrying glioma xenografts received intratumoral injections of Delta-24-RGD or Delta-24 and were followed for survival, which was analyzed by the Kaplan-Meier method and the log-rank test. All statistical tests were two-sided. Half the glioma cell lines expressed low levels of CAR (defined as <50% of cells expressing detectable CAR); all lines expressed integrins in more than 50% of cells. Infection of U-87 MG cells (a low-CAR-expressing line) with AdGFP-RGD resulted in approximately six times more GFP-positive cells than infection with AdGFP. Delta-24-RGD was more cytopathic to both low- and high-CAR-expressing glioma lines than Delta-24, and it replicated more efficiently in both cell lines. In the xenografted mice, intratumoral injection of Delta-24-RGD was associated with longer survival than intratumoral injection of Delta-24 (P<.001, log-rank test). Furthermore, 60% of Delta-24-RGD-treated mice but only 15% of Delta-24-treated mice survived more than 4 months (difference = 45%, 95% CI = 21% to 68%). The antitumor activity of Delta-24-RGD suggests that it has the potential to be an effective agent in the treatment of gliomas.

Syn3 provides high levels of intravesical adenoviral-mediated gene transfer for gene therapy of genetically altered urothelium and superficial bladder cancer.

Using our model to grow superficial human bladder cancer in the mouse bladder, we have found that the polyamide compound, Syn3, when injected intravesically for 1 hour at 1 mg/mL on two consecutive days, markedly increases rAd-beta-gal intravesical gene transfer and expression. This enhanced transgene expression was much greater than obtain by the use of 22% ethanol, which had previously been shown to increase intravesical adenoviral gene transfer, whereas little or no gene expression was seen with exposure to only rAd-beta-gal. beta-Galactosidase staining was seen in virtually every normal urothelial and superficial tumor cell present, including tumors that express little or no coxsackie-adenovirus receptors when Syn3 was present. High adenoviral-mediated gene transfer was also documented in the pig bladder using Syn3 in a similar protocol. Therefore, Syn3 may overcome the limitations of adequate intravesical adenoviral-mediated gene transfer and, when combined with an appropriate adenoviral-mediated gene, could offer an effective approach to the treatment of superficial bladder cancer and perhaps even genetically altered precursor lesions.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases.