Joohee Han

Generation of a novel, cyclooxygenase-2-targeted, interferon-expressing, conditionally replicative adenovirus for pancreatic cancer therapy

BACKGROUND Oncolytic adenoviruses provide a promising alternative for cancer treatment. Recently, adjuvant interferon (IFN)-alfa has shown significant survival benefits for pancreatic cancer, yet was impeded by systemic toxicity. To circumvent these problems adenovirus with high-level targeted IFN-alfa expression can be generated. METHODS Conditionally replicative adenoviruses (CRAds) with improved virulence and selectivity for pancreatic cancer were generated. The vectors were tested in vitro, in vivo, and in human pancreatic cancer and normal tissue specimens. RESULTS Adenoviral death protein and fiber modifications significantly improved oncolysis. CRAds selectively replicated in vitro, in vivo and showed persistent spread in cancer xenografts. They showed high-level replication in human pancreatic cancer specimens, but not in normal tissues. Improved IFN-CRAd oncolytic efficiency was shown. CONCLUSIONS Optimized cyclooxygenase-2 CRAds show highly favorable effects in vitro and in vivo. We report a pancreatic cancer-specific, highly virulent, IFN-expressing CRAd, and we believe that adenovirus-based IFN therapy offers a new treatment opportunity for pancreatic cancer patients.

Oncolytic adenovirus expressing interferon alpha in a syngeneic Syrian hamster model for the treatment of pancreatic cancer

BACKGROUND The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. METHODS Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. RESULTS All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. CONCLUSION The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

Oncolytic adenoviruses targeted to Human Papilloma Virus-positive head and neck squamous cell carcinomas

OBJECTIVES In recent years, the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has markedly increased. Our aim was to design a novel therapeutic agent through the use of conditionally replicative adenoviruses (CRAds) that are targeted to the HPV E6 and E7 oncoproteins. METHODS Each adenovirus included small deletion(s) in the E1a region of the genome (Δ24 or CB016) intended to allow for selective replication in HPV-positive cells. In vitro assays were performed to analyze the transduction efficiency of the vectors and the cell viability following viral infection. Then, the UPCI SCC090 cell line (HPV-positive) was used to establish subcutaneous tumors in the flanks of nude mice. The tumors were then treated with either one dose of the virus or four doses (injected every fourth day). RESULTS The transduction analysis with luciferase-expressing viruses demonstrated that the 5/3 fiber modification maximized virus infectivity. In vitro, both viruses (5/3Δ24 and 5/3CB016) demonstrated profound oncolytic effects. The 5/3CB016 virus was more selective for HPV-positive HNSCC cells, whereas the 5/3Δ24 virus killed HNSCC cells regardless of HPV status. In vivo, single injections of both viruses demonstrated anti-tumor effects for only a few days following viral inoculation. However, after four viral injections, there was statistically significant reductions in tumor growth when compared to the control group (p<0.05). CONCLUSION CRAds targeted to HPV-positive HNSCCs demonstrated excellent in vitro and in vivo therapeutic effects, and they have the potential to be clinically translated as a novel treatment modality for this emerging disease.

Combination of interferon-expressing oncolytic adenovirus with chemotherapy and radiation is highly synergistic in hamster model of pancreatic cancer

Recent clinical trials utilizing Interferon-alpha (IFN) in combination with chemoradiation have demonstrated significant improvements in the survival of patients with pancreatic cancer. However, efficacy was limited by the systemic toxicity of IFN and low intratumoral levels of the cytokine. We sought to address these drawbacks by using an Oncolytic Adenovirus expressing IFN (OAd-hamIFN) in combination with chemotherapy and/or radiation in regimens mimicking the IFN-based therapies used in clinical trials. IFN expressed from OAd-hamIFN potentiated the cytotoxicity of radiation and chemotherapy (5-FU, Gemcitabine, and Cisplatin), and enhanced pancreatic cancer cell death in both in vitro and in vivo experimental settings. Notably, synergism was demonstrated in therapeutic groups that combined the interferon-expressing oncolytic virus with chemotherapy and radiation. In an in vivo immunocompetent hamster model, treatment regimens combining oncolytic virus therapy with 5-FU and radiation demonstrated significant tumor growth inhibition and enhanced survival. This is the first study to report synergism between an IFN-expressing oncolytic adenovirus and chemoradiation-based therapies. When combined with an IFN-expressing OAd, there is a significant enhancement of radiation and especially chemoradiation, which may broaden the application of this new therapeutic approach to the pancreatic cancer patients who cannot tolerate existing chemotherapy regimens.

639. Oncolytic Adenovirus Expressing IFN Alpha Works Synergistically with Chemoradiation

Aside from curative resection, there is no curative treatment against pancreatic adenocarcinoma (PDAC) at present. Late diagnosis and high recurrence results in five-year survival of 6%. Notably, Phase II trials based on adjuvant therapy combining systemic IFN Alpha (IFN) and chemoradiation reported 30-50% increase in two-year survival and impressive 35% increase in five-year survival of PDAC patients. Despite promising results, trial drawbacks included high patient dropout due to IFN systemic toxicity and low IFN levels in tumors. Low intratumoral IFN hampered the full potential of the therapy while IFN is known to induce tumor apoptosis, chemoradio sensitization, and decreased tumor neo-vasculatization.Aiming to improve efficacy and tolerability of IFN therapy, we have developed an oncolytic adenovirus expressing human IFN (OAd-IFN). Vector has Ad5/3 fiber modification and overexpresses Adenoviral Death Protein respectively contributing to increased infectivity and oncolysis. Taking advantage of Cox-2 up-regulation in PDAC, the Cox-2 promoter was included in the upstream of Adenovirus E1 region, restricting vector replication to cancer cells. Human IFN-alpha gene was placed in the Adenovirus E3 region in the way that its expression is controlled by the adenovirus major late promoter. Therefore, IFN expression in this vector is replication dependent. To test the vector in an immunocompetent syngeneic hamster model of pancreatic cancer, OAd-IFN expressing hamster IFN was generated. Vector contains RGD fiber modification enhancing its infectivity in hamster cells.MTS and crystal violet assays demonstrated sensitization of PDAC cells to chemotherapy (5-FU, Cisplatin, and Gemcitabine), and radiation (4 and 8Gy) by OAd-IFN. Comparison between OAd-IFN and control vector not expressing IFN (OAd-LUC) indicated that IFN expressed by OAd-IFN is functional in combination therapy sensitizing PDAC cells to chemoradiation. Colony formation assay showed that combinations of OAd-IFN with chemotherapy, radiation, or chemoradiation are synergistic and exhibit superior killing effect compared to groups without OAd-IFN.In vivo studies using immunocompetent syngeneic hamster model of pancreatic cancer showed that combinations including OAd-IFN resulted in augmented tumor shrinkage and survival compared to groups treated with chemotherapy, radiation, chemoradiation, or OAd-LUC + radiation. Hexon staining and viral DNA quantification by qPCR show OAd-IFN effectively replicates and spreads in tumors.Our data suggests OAd-IFN synergistically improves chemoradioation in PDAC cells, and shows superior therapeutic effect when treating immunocompetent model of pancreatic cancer. Vector capacity to express high levels of IFN intratumorally and the strong synergism between OAd-IFN and radiation, chemotherapy, and chemoradiation indicates OAd-IFN will contribute to more olerable and effective IFN therapy. Strong synergism between OAd-IFN and chemoradiation combined with focal expression of IFN can help to reduce not only IFN toxicity, but also dose-dependent toxicity of chemoradiation. Considering systemic IFN injection combined with chemoradiation is one of the few therapies to effectively treat pancreatic cancer, usage of OAd-IFN in combination with chemoradiation has great potential to result in more effective and tolerable therapy agaisnt pancreatic cancer.

632. Oncolytic Adenoviruses Targeted to the HPV E6 and E7 Oncoproteins as a Novel Treatment for Head & Neck Squamous Cell Carcinomas

Introduction:Recent reports have shown that the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has been steadily increasing. HNSCCs are known to have a high recurrence rate and compared to their HPV-negative counterparts, these tumors have a unique biology which necessitates the development of novel treatment modalities. The HPV E6 and E7 oncoproteins are attractive therapeutic targets as they interact with key cell cycle regulatory components, namely p53 and pRb. Our research group has applied conditionally replicating oncolytic adenoviruses (CRAd) with modifications in the E1a region of the genome allowing for selective replication in E6 and E7 expressing HNSCCs.Methods:The in vitro transduction efficiency of E1-deleted luciferase expressing vectors with fiber modifications was assessed in multiple HPV-positive and negative cell lines. The CRAds were designed with a distinct deletion in the E1a region of the adenoviral genome (D24 and CB016) intended to allow for selective replication in HPV-positive HNSCC cells. Additionally, the CRAds have a luciferase transgene in the E3 region that is expressed in a replication dependent manner. By using a luciferase assay, the degree of viral replication was analyzed in numerous HNSCC cell lines. In vitro cell viability following viral infection was analyzed with crystal violet and MTS assays. A HPV-positive cell line (UPCI SCC 090) was used to establish subcutaneous tumors in female nude mice. They were subsequently treated with either one intratumoral viral injection or four injections (given every fourth day).Results:The 5/3 fiber modification significantly increased viral infectivity in all tested HNSCC cell lines. The 5/3 CB016 vector replicated selectively in HPV-positive cell lines, while the 5/3 D24 virus replicated in all cell lines regardless of HPV status. Both of the vectors demonstrated profound cytocidal effects in the crystal violet and MTS assays. For the in vivo experiments, a single intratumoral injection of virus demonstrated an anti-tumor effect for only one week following injection. Given this limited effect, an additional in vivo experiment was performed to analyze the efficacy of multiple intratumoral injections (3.5 x1011 vp/injection). This setup resulted in statistically significant tumor growth suppression at day 26 when compared to the saline control group (p<0.05 for 5/3 CB016, p<0.01 for 5/3 D24).Conclusion:CRAds designed to target HPV-positive HNSCCs demonstrated tremendous, yet selective in vitro cytocidal effects. The profound tumor growth suppression in a mouse model (particularly by multiple injections) shows the excellent potential of our viruses to be clinically translated as a new therapeutic entity for this emerging disease process.

Abstract 3308: Systemic administration of fiber redesigned infectivity-selective oncolytic adenovirus for pancreatic cancer.

Adenovirus (Ad) has frequently been used as a backbone of oncolytic viral agents, while lack of selectivity to cancer cells at the stage of infection has greatly hampered in vivo efficacy. Unlike loco-regional therapy, systemic application of cancer gene therapy mandates different level of selectivity of gene delivery. The controlled distribution and selective transduction of the vector would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer. We have recently developed transductionally-targeted Ads by high-throughput screening of high-diversity (10ˆ9-level) Ad-library. Mesothelin (MSLN) is highly over-expressed in pancreatic cancers. Ad with redesigned AB-loop targeting MSLN, AdML-VTIN, was successfully isolated from the high throughput screening of Ad library by infectivity for MSLN-expressing 293 cells (293-MSLN). The in vitro binding of AdML-VTIN corresponded to the level of MSLN-expression and the suppression of MSLN with siRNA or antibody significantly reduced the binding to MSLN-expressing cells, which indicates that mesothelin is a receptor moiety for the isolated targeting ligand. The binding ability of this virus in Panc-1 (MSLN-positive) was higher than that of Ad with 5/3 modified fiber which is known to exhibit highest infectivity in pancreatic cancer cells. The in vivo antitumor effect was compared in Panc-1 and MiaPaCa-2 (MSLN-negative) subcutaneous xenografts. The injection of the MSLN-retargeted oncolytic Ad showed significant antitumor effect against Panc-1 tumors, and about half of the tumor disappeared. Contrarily, the same virus showed no antitumor effect in MiaPaCa-2. Viral DNA quantitation also supported the selective viral replication in Panc-1. Upon systemic administration in the nude mice with Panc-1 subcutaneous xenografts, the sequestration of AdML-VTIN to the liver was significantly lower than that of wild-type Ad5, and the copy number of this retargeted virus was significantly (1000 fold) higher in the tumor. These data indicates the systemic delivery of this vector is efficient for MSLN-expression pancreatic cancer xenografts. In this study, our MSLN-targeted Ad vector exhibited significant infectivity to MSLN-positive pancreatic cancer cells in vitro and in vivo. Moreover, this virus showed significantly reduced liver sequestration as well as dramatically augmented delivery to the tumors upon systemic administration. This new genetically modified Ad transductionally retargeted to pancreatic cancer may embody a next generation targeting for this devastating disease in clinical settings. Citation Format: Yoshiaki Miura, Joohee Han, Julia Davydova, Masato Yamamoto. Systemic administration of fiber redesigned infectivity-selective oncolytic adenovirus for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3308. doi:10.1158/1538-7445.AM2013-3308 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.