Adenovirus Death Protein Research Articles

Oncolytic Adenovirus for the Targeting of Paclitaxel-Resistant Breast Cancer Stem Cells.

Adjuvant systemic therapies effectively reduce the risk of breast cancer recurrence and metastasis, but therapy resistance can develop in some patients due to breast cancer stem cells (BCSCs). Oncolytic adenovirus (OAd) represents a promising therapeutic approach as it can specifically target cancer cells. However, its potential to target BCSCs remains unclear. Here, we evaluated a Cox-2 promoter-controlled, Ad5/3 fiber-modified OAd designed to encode the human sodium iodide symporter (hNIS) in breast cancer models. To confirm the potential of OAds to target BCSCs, we employed BCSC-enriched estrogen receptor-positive (ER+) paclitaxel-resistant (TaxR) cells and tumorsphere assays. OAd-hNIS demonstrated significantly enhanced binding and superior oncolysis in breast cancer cells, including ER+ cells, while exhibiting no activity in normal mammary epithelial cells. We observed improved NIS expression as the result of adenovirus death protein deletion. OAd-hNIS demonstrated efficacy in targeting TaxR BCSCs, exhibiting superior killing and hNIS expression compared to the parental cells. Our vector was capable of inhibiting tumorsphere formation upon early infection and reversing paclitaxel resistance in TaxR cells. Importantly, OAd-hNIS also destroyed already formed tumorspheres seven days after their initiation. Overall, our findings highlight the promise of OAd-hNIS as a potential tool for studying and targeting ER+ breast cancer recurrence and metastasis.

Novel conditionally replicating adenovirus-mediated efficient detection of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads.

Adenovirus Vector With ADP Gene Induces Cytopathic Effects in HEK293 Cells Without Significant Elevation of Virus Titers.

Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome. We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined. The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers. The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells.

Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein.

The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.

Cancer imaging and therapy utilizing a novel NIS-expressing adenovirus: The role of adenovirus death protein deletion

Encoding the sodium iodide symporter (NIS) by an adenovirus (Ad) is a promising strategy to facilitate non-invasive imaging and radiotherapy of pancreatic cancer. However, insufficient levels of NIS expression in tumor cells have limited its clinical translation. To optimize Ad-based radiotherapy and imaging, we investigated the effect of Ad death protein (ADP) deletion on NIS expression. We cloned two sets of oncolytic NIS-expressing Ads that differed only in the presence or absence of ADP. We found that ADP expression negatively affected NIS membrane localization and inhibited radiotracer uptake. ADP deletion significantly improved NIS-based imaging in pancreatic cancer models including patient-derived xenografts, where effective imaging was possible for up to 6 weeks after a single virus injection. This study demonstrates that improved oncolysis may hinder the therapeutic effect of oncolytic viruses designed to express NIS. In vivo studies in combination with 131I showed potential for effective radiotherapy. This also highlights the need for further investigation into optimal timing of 131I administration and suggests that repeated doses of 131I should be considered to improve efficacy in clinical trials. We conclude that ADP deletion is essential for effective NIS-based theranostics in cancer.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.

The Adenovirus Death Protein - a small membrane protein controls cell lysis and disease.

Human adenoviruses (HAdVs) cause widespread acute and persistent infections. Infections are usually mild and controlled by humoral and cell-based immunity. Reactivation of persistently infected immune cells can lead to a life-threatening disease in immunocompromised individuals, especially children and transplant recipients. To date, no effective therapy or vaccine against HAdV disease is available to the public. HAdV-C2 and C5 are the best-studied of more than 100 HAdV types. They persist in infected cells and release their progeny by host cell lysis to neighbouring cells and fluids, a process facilitated by the adenovirus death protein (ADP). ADP consists of about 100 amino acids and harbours a single membrane-spanning domain. It undergoes post-translational processing in endoplasmic reticulum and Golgi compartments, before localizing to the inner nuclear membrane. Here, we discuss the current knowledge on how ADP induces membrane rupture. Membrane rupture is essential for both progression of disease and efficacy of therapeutic viruses in clinical applications, in particular oncolytic therapy.

IFN Expressing Oncolytic Adenovirus as a Tool to Improve Pancreatic Cancer Therapy

Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd cause of cancer related death in the US. Aside from surgery, which is not applicable to 80% of patients, there is no highly effective therapy to treat the disease. Due to late diagnosis and advanced disease, PDAC patients live 3–6 months post‐diagnosis and 5‐year survival is 6%. Despite this discouraging scenario, clinical trials testing use of adjuvant therapy combined with IFN‐α (IFN), 5‐FU, CDDP, and radiation improved the 2‐year survival of patients by 20–41%, and the 5‐year survival by 35%. While efficacious, IFN‐therapy drawbacks included high IFN systemic toxicity, increasing patient drop‐out from trials, and low levels of the cytokine in tumors, reducing chemo‐radio sensitization of IFN in tumors. Aiming to perfect this promising therapy, we studied the use of IFN‐expressing oncolytic adenovirus (OAd) vectors in combination with chemoradiation mimicking the aforementioned IFN‐chemoradiation based clinical trials. As IFN can stimulate tumor specific immune response, we first evaluated the effect of a hamster IFN expressing vector (OAd‐hamIFN) in a syngeneic immunocompetent hamster model of PDAC. To analyse the interaction of OAd with chemotherapy and radiation without the interference of the immune system, we later accessed the effect of a human IFN‐expressing vector (OAd‐IFN) in immunodeficient mice bearing human PDAC xenografts. In both studies, OAd vectors included 5/3 fiber modification to enhance infectivity in PDAC cells, over expressed Adenovirus Death Protein (ADP) to potentiate cell oncolysis, and expressed IFN in a replication dependent manner. As PDAC cells upregulate Cox‐2, we used the Cox‐2 promoter in the OAd‐IFN to restrict viral replication to human PDAC cells. Data showed that combinations of 5‐FU, radiation, and 5‐FU + radiation with either OAd‐hamIFN in hamster PDAC cells, or OAd‐IFN in human PDAC cells resulted in highly synergistic and cytotoxic combinations in vitro. Studies in immunocompetent hamsters showed that use of OAd‐hamIFN in combination with therapeutics used in IFN‐therapy improved treatment efficacy, and that use of the virus mimicking the IFN‐therapy was the most efficacious treatment. When analyzing effect of OAd‐IFN in combination with 5‐FU + CDDP in human PDAC cells, combinations were shown to be antagonistic and weakly cytotoxic. However, addition of radiation to treatment (OAd‐IFN + (5‐FU + CDDP) + radiation) overruled chemotherapy antagonism, resulting in highly synergistic and extremely potent treatments in vitro and in mice bearing PDAC xenografts. As radiation was shown to abolish antagonism of chemotherapy to virus in vivo, we tested different radiation protocols in combination with virus. We concluded that the best radiation protocol comprised of infecting tumors before irradiation. In summary, our data strongly supported inclusion of an IFN expressing OAd in treatments mimicking IFN‐therapy. As this therapy is one of the few showing an impact in patient survival, improvement of therapy with the virus might finally result in an potent treatment against PDAC.Support or Funding InformationNIH/NCIP50CA101955, NIH/NCIR01CA094084This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An oncolytic adenovirus 11p vector expressing adenovirus death protein in the E1 region showed significant apoptosis and tumour-killing ability in metastatic prostate cells.

The usefulness for cancer therapy of replication-competent adenoviral vectors expressing therapeutic genes from the E3 region has been evaluated, but few reports have described replication-competent adenoviruses with insertions at the E1 region in the full viral genome. We investigated in different prostate cancer cells the oncolytic efficacy of the replication-competent adenovirus 11p vectors expressing adenovirus death (RCAd11pADP) and red fluorescence (RCAd11pRFP) proteins from the upstream E1 region. ADP/RFP gene expression was 2-3 logs higher in PC3 and DU145 cells than in LNCaP and RWPE-1 cells. E1A protein expression in PC3 and DU145 cells was notably increased after infection with the RCAd11pADP or RCAd11pRFP vector compared with the Ad11pwt virus. Toxicity assays revealed 2-5-fold greater oncolytic effects of RCAd11pADP compared to Ad11pwt. Although all three viruses suppressed subcutaneous PC3 tumour growth in nude mice, RCAd11pRFP had greater oncolytic effects than did the Ad11pwt virus, and RCAd11pADP exhibited significant anti-tumour effects via apoptosis in a xenograft model. Interestingly, the apoptosis triggered by RCAd11pADP was markedly enhanced in comparison to that by the vector expressing ADP from E3 region. Taken together, our findings suggest that RCAd11pADP can potentially be used for the treatment of prostate metastases in clinical settings.

Abstract 5925: A novel oncolytic adenovirus for radioiodine therapy and imaging

Abstract Recently, the use of oncolytic viruses encoding the sodium-iodide symporter (NIS) has become an attractive approach to achieve radionuclide imaging of cancer. However, the potential of virus-induced NIS expression to facilitate therapy with radioactive 131I has not been fully explored. We designed a tumor-selective, infectivity-improved oncolytic adenovirus (Ad5/3-Cox2-NIS-ADP) modified to express high quantities of NIS. This virus induced radioisotope uptake in prostate and lung cancer cells and facilitated SPECT/CT imaging, but more importantly, supported the effective therapy with 131I. In our original vector, enhanced oncolysis was mediated by overexpression of adenoviral death protein (ADP). Although this structure was operative in detection and therapeutic regimens, we were concerned that the cytolytic effect of ADP may affect NIS membrane localization, and diminish its effectiveness as a theranostic tool. We therefore designed an identical Ad5/3-Cox2-NIS-ADP-deleted virus {ADP(-)} and assessed the impact of ADP on NIS expression and radioisotope uptake. Western blot and immunocytochemical analyses of pancreatic cancer cells infected with ADP(-) demonstrated higher NIS levels when compared to the ADP(+) counterpart. This correlated with an improved 125I uptake in vitro. SPECT/CT imaging studies assessing 99mTcO4- accumulation in mice to visualize pancreatic adenocarcinoma (PDAC) using patient-derived xenografts (PDX) showed that a single dose of ADP(-) accumulated far greater levels of 99mTcO4- when compared to ADP(+). Remarkably, ADP(-) produced stronger signal that was maintained up until day 32, and this outlasted ADP(+) and the control vector, replication-deficient AdCMV-NIS, currently employed in a clinical trial. Notably, within the PDAC, ADP(-) showed a distinct NIS cell membrane distribution pattern as it co-localized with cell membrane bound-cytokeratin-4. Unlike ADP(-), ADP(+) produced a punctate NIS staining pattern, with little to no cell membrane localization. These results support our hypothesis that ADP-cytolytic effect reduces NIS membrane localization, subsequently affecting radionuclide uptake. To evaluate the therapeutic potential of NIS-based radiotherapy, we treated mice bearing PDAC tumors with virotherapy alone or in combination with 131I. NIS-expressing OAds in combination with 131I significantly reduced tumor growth when compared to viro- or radiotherapy alone. The therapeutic effect in mice treated with ADP(-) combined with 131I outperformed that with ADP(+) or AdCMV-NIS vectors. Quantitative analyses of 131I uptake in tumor tissues with a gamma counter showed a clear trend where ADP(-) retained higher 131I uptake than ADP(+). These findings support the clinical applicability of the ADP-deleted OAd as the more sensitive tool for NIS-based cancer diagnosis and therapy. We are currently investigating the biodistributon and toxicity of our vectors in a pig model. Citation Format: Lisa Koodie, Eriko Iguchi Kawakami, Kari Jacobsen, Zuzan Cayci, Andrew Taylor, Edward W. Greeno, Robert J. Schumacher, John C. Morris, Martin E. Fernandez-Zapico, Julia Davydova. A novel oncolytic adenovirus for radioiodine therapy and imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5925.

Effect of adenoviral death protein on NIS-based iodine therapy and imaging for pancreatic cancer

Effect of adenoviral death protein on NIS-based iodine therapy and imaging for pancreatic cancer

413. Effect of Adenoviral Death Protein on NIS-Based Iodine Therapy and Imaging for Pancreatic Cancer

In this study, we assess our novel Oncolytic Adenovirus (OAd) expressing a dual function therapeutic and imaging transgene, the sodium-iodide symporter (NIS) as a promising alternative for pancreatic cancer treatment. We hypothesized that NIS expression in pancreatic cancer will induce uptake of radioiodine and allow noninvasive SPECT/CT imaging with 123I. We designed our OAd-NIS to overcome the low efficacy of previous vectors. These viruses have a modified Ad5/Ad3 fiber-knob shown to improve the poor transduction of pancreatic cancer cells. Viral replication is controlled under the Cox2 promoter allowing specific delivery of viral genes, and most of adenoviral E3 genes are deleted and replaced with NIS and Adenovirus Death Protein (ADP) genes. We have previously demonstrated significantly improved ability of ADP-overexpressing OAds to enhance viral release and cytolytic activity. Although improved oncolysis enhances viral release and improve the therapeutic effect in solid tumors, it can potentially reduce radioiodide tracer uptake. To test this possibility we designed and compared identical vectors with ADP (OAd-NIS-ADP) and without ADP (OAd-NIS-noADP) in vitro and in vivo. We first compared virus killing ability in pancreatic cancer cell lines. The ADP+ vector was significantly more cytolytic that no-ADP counterpart and showed improved viral replication and spread. Cox2-controlled OAd did not produce cell death in Cox2-negative control, confirming selectivity. We next assessed OAds ability to induce NIS protein expression and radioiodine uptake. NIS-OAds efficiently produce glycosylated NIS multimers as early as 2-days post infection. Infection with no-ADP vectors resulted in a significantly greater radioiodine uptake (125I) compared to ADP+ viruses. Importantly, ex vivo uptake test in human patient tissues confirmed the high level of NIS in pancreatic adenocarcinoma samples, and revealed no 125I uptake in normal pancreas. We further assessed the ability of our vectors to visualize human pancreatic cancer xenografts in a mouse model by monitoring 99mTcO4− accumulation with SPECT-CT. The OAd-NIS-noADP showed an earlier and more sustained radioisotope uptake when compared to ADP+ supporting its use as a more sensitive diagnostic tool for pancreatic cancer. Of contrary, the ADP+ vector significantly outperformed OAd-NIS-noADP in tumor shrinkage. These results suggest that while OAd-NIS-noADP produces a greater radiotracer uptake and can be used as a diagnostic tool, its ADP+ counterpart results in a better therapeutic effect when applied as a monotherapy. It is now essential to estimate the therapeutic ability of both vectors upon combination with I-131 (ongoing study). Further investigation will include the imaging and therapeutic studies in patient-derived xenografts. Ultimately, the goal of our research is to design a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

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.

421. Evaluating the Efficacy of Combination Cancer Treatment: Oncolytic Adenovirus and 5FU Chemotherapy

Previous studies have shown that Interferon(IFN)-based chemoradiation therapy can improve survival after resection of pancreas cancer. However, its clinical utility to this point has been limited due to the severe toxicity related to its use systemically. Gene Therapy represents a promising approach to deliver localized treatments that are effective and minimally toxic. Our aim in this study is to evaluate our group's novel oncolytic adenovirus (OAd) which allows targeting IFN treatment to cancer cells while sparing healthy tissue. The OAd, 5/3Cox2DE3ADPIFN, is selectively replicative in Cox2 (+) cell lines, allowing for specificity in therapy. It has also been modified to with a 5/3 chimeric knob to facilitate transduction, an overexpression of the adenoviral death protein, and expresses our IFN gene of interest. This study was conducted to analyze the combination of 5FU chemotherapy and our OAd in vitro in order to assess the interaction of treatments and determine the optimum combined treatment regimen. Treatments were analyzed in pancreatic cancer and esophageal adenocarcinoma cell lines S2013 and OE19. Recombinant OAds expressing luciferase rather than IFN were used in this study to isolate the combination of 5FU and the virus. Two viral models were evaluated: our selective therapeutic virus (Cox2) and a nearly identical but universally replicative virus (wild type). Cells were treated with 0, 1 or 10 viral particles per cell and 0, 5, 10, or 20 uM 5FU. Three timing regimens were used: simultaneous administration, 5FU 4 hours before virus, and virus 48 hours before 5FU. Crystal Violet and MTS Assays were used to measure cell death. Viral Copy number was used to assess viral replication using qPCR with a viral E4 primer. Cell death analysis showed an earlier killing effect from the wild type virus but otherwise similar patterns. 5FU and each virus produced dose dependent cell death independently. There was a significant additive effect seen in cell death from combining treatments when using 5FU before virus in both S2013 and OE19. Simultaneous treatment showed an additive killing effect with the combination in S2013, but a reduced killing by the combination compared to virus alone in OE19. There was also reduced combination killing when using virus before 5FU in S2013. Viral copy experiments in S2013 using simultaneous treatment showed dose dependent inhibition of viral replication, with only the 20uM dose significantly limiting viral replication over time. Our Cox2 OAd shows a killing effect similar to wild type in multiple cancer cell lines. When combined with 5FU treatment the expected summation in overall cell death from the independent treatments varies by timing of administrations as well as by type of cancer cell line. The reduced killing of the combination treatment in the simultaneous and virus before 5FU regimens, as well as the replication inhibition shown by viral copy number, may suggest an inhibition of the virus by 5FU under certain conditions. This also suggests the possibility of a gene therapy treatment regimen with optimal therapeutic effect, which appears to be 5FU before virus based on the results collected. Further studies investigating different chemotherapeutic drugs and regimens should be conducted to examine these trends.

Abstract 1816: Impact of 5FU and radiation on replication of interferon-expressing adenovirus

Abstract Pancreatic Cancer is a disease for which there are very limited efficacious treatments, resulting in a 5-year survival rate for affected patients around only 6%. A recombinant oncolytic adenovirus (OAd-IFN) developed by our group to target pancreatic cancer utilizes enhanced oncolysis with an overexpression of the adenoviral death protein, enhanced infectivity with a genetically modified fiber, conditional replication targeted to pancreatic cancer using the Cox-2 promoter, and an expression of human interferonαto enhance the therapeutic effects of chemotherapy and radiation through the antitumor effects of the cytokine. Our previous work analyzing the therapeutic abilities of IFN expressing vectors in human pancreatic cancer xenografts in immonudeficient nude mice and in syngeneic hamster pancreatic cancer nodules in immunocompetent Syrian hamsters, showed a more evident antitumor effect of OAd-IFN in the latter models. This suggests the success of OAd-IFN as an immune system stimulator, making it a promising candidate for combination therapy approaches. The goal of this study is to investigate the possibility for use of OAd-IFN as a therapy for pancreatic cancer in conjunction with 5FU and radiation. Specifically, the adenoviral replicative ability was measured within pancreatic cancer Mia-Paca 2 cells under various conditions of 5FU and radiation exposure. Replication was analyzed by a correlation to the increase in amount of viral DNA extracted from treated cells at two time points post infection. In two preliminary trials, cells were infected with the virus two days prior to receiving 5FU and radiation, and another group of cells was treated with 5FU before viral infection. Based on the initial data collected, radiation seemed to have no effect on viral replication. 5FU treatment prior to viral infection resulted in an increase in replication at low dose, and a decrease in replication with high dose, however, this effect was only seen at the first time point 3 days after infection. Similarly, 5FU treatment post infection showed a minor dose dependent inhibition of viral replication at high doses, with little to no effect observed in the low dose. Combining viral infection with 5FU appears to have an impact on the replication of OAd-IFN in vitro; however, further trials need to be done to explore the effect of various timing among viral infection and therapy treatments as well as different therapy concentrations and viral titers to further analyze the potential combination therapies. Citation Format: Jordan Sell. Impact of 5FU and radiation on replication of interferon-expressing adenovirus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2015-1816

320. Adenovirus Expansion of Radioiodine Therapy to Non-Thyroid Cancer Treatment and Diagnostics

Radioiodine therapy has successfully treated and diagnosed cancers over the last 50 years. The clinical scope of radioiodine is limited in thyroid cancers because of restricted iodine uptake. Our lab proposes to expand the clinical use of radioiodine into non-thyroid cancer theranostics by targeting cancers (e.g. pancreatic and prostate) with oncolytic adenovirus to over express the human sodium iodine symporter (NIS). The adenovirus expression of the symporter allows iodine to concentrate into the targeted cancer cells providing diagnostic imaging with I123 and therapy with I131.We designed infectivity-enhanced oncolytic Adenoviruses (NIS-OAd), Ad5/3_TSP_E3_ADP_NIS, to express NIS and adenovirus death protein (ADP) from the E3 region. The Ad5/3 fiber modification enables the infection of CAR negative cells. Vector replication is controlled through a tumor specific promoter (TSP, e.g. COX2 in pancreatic and Pb in prostate cancer). We have shown the specificity of our viruses with TSPs in vitro with multiple pancreatic and prostate cancer cell lines. The expression of ADP resulted in an increase in oncolytic activity and transgene expression. The functionality of NIS-OAds was tested with in vitro radioiodine uptake assay and showed up to a 10-fold increase in radioiodine uptake in prostate and pancreatic cancer cell lines. This endorses that oncolytic viruses increase functional NIS expression in tumors.Our animal model experiments included nude mice xenographs to evaluate therapeutic and imaging affects with i.v. injections of NIS-OAds in A549 lung and LnCap prostate cancer cells. In A549 models, the combination of NIS-OAds and I131 had an increase in therapeutic effect over virus alone signifying functional NIS expression in solid tumors. LnCap xenographs had SPECT/CT images taken after NIS-OAds and non-replicating virus administration with I123. NIS-OAds had the highest tumor specific signal confirming that NIS-OAds can be diagnostic in tumors.Viral functionality in human tissue was evaluated by PCR in ex vivo tissue slices showing that virus replication was specific to cancerous tissues and did not occur in normal tissue. This study supports the high-level replication control of NIS-OAds in non-thyroid cancer tissues. A related non-replicating NIS expressing Ad is also being evaluated in prostate cancer by Dr. Morris, our collaborator, in a stage I clinical trial. Together the virus specificity, imaging, and therapeutic data support the potential of NIS-OAds as cancer theranostics.In sum, this data provides key functionality and safety support of NIS expressing Ad use in humans. Current projects include therapeutic and imaging animal studies with radioiodine in pancreatic cancer immuno-competent Syrian hamsters, and human xenographs in nude mice. Further studies will evaluate the distribution of NIS expression in vivo and cytotoxicity of virus with radiation. Ultimately the goal of our research is to form a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

435. Effects of Chemotherapy and Radiation on Replication of Interferon-Expressing Adenovirus in Pancreatic Cancer Cells

Pancreatic Cancer is a disease for which there are very limited efficacious treatments, resulting in a 5-year survival rate for affected patients around only 6%. Our group has developed a recombinant oncolytic adenovirus (OAd-IFN) aimed for use as an additional treatment for this disease. Manipulations in the viral genome include enhanced oncolysis with an overexpression of the adenoviral death protein, enhanced infectivity with a genetically modified fiber that has replaced the serotype 3 Ad fiber with that of a serotype 5 Ad, conditional replication targeted to pancreatic cancer cells by linking replication with the Cox-2 promoter, and an expression of the gene for human interferon α within the E3 region to enhance the therapeutic effects of chemotherapy and radiation through the antitumor effects of the cytokine.Our previous work analyzing the therapeutic abilities of IFN expressing vectors in human pancreatic cancer xenografts in immonudeficient nude mice and in syngeneic hamster pancreatic cancer nodules in immunocompetent Syrian hamsters, showed a more evident antitumor effect of OAd-IFN in the latter models. This suggests the success of OAd-IFN as an immune system stimulator, making it a promising candidate for combination therapy approaches. In vitro studies in hamster pancreatic cancer cell lines showed significantly improved killing effects when OAd-IFN was added to conventional chemotherapy and radiation treatments, as well as greater killing ability with triple therapies using OAd-IFN versus an OAd without IFN. Moreover, killing effects became more pronounced over time, showing that the oncolytic impact of the IFN depends on replication of the virus to express the IFN gene.The goal of this study is to investigate the effects of chemotherapy and radiation on the replication of our OAd-IFN, since understanding these interactions is necessary to evaluate the possibility of using this virus as part of a combined therapeutic regimen. Specifically, the adenoviral replicative ability was measured within pancreatic cancer Mia-Paca 2 cells under various conditions of 5FU and radiation exposure. Replication was analyzed by quantifying viral DNA extracted from treated cells at two time points post infection. In preliminary trials, cells were infected with the virus either two days prior to receiving 5FU and radiation or one day post 5FU treatment. Based on the initial data collected, radiation seemed to have no effect on viral replication. 5FU treatment prior to viral infection resulted in an increase in replication at low dose, and a decrease in replication at high dose, however, this effect was only seen at the first time point. Similarly, 5FU treatment post infection showed minor inhibition of viral replication at high doses, with little to no effect observed in the low dose. Further trials including a simultaneous treatment with 5FU and virus are being done to better explore these observed effects, and future trials will be conducted exploring for similar patterns using Gemcitabine and Cisplatin in place of 5FU.

Development of replication-competent adenovirus for bladder cancer by controlling adenovirus E1a and E4 gene expression with the survivin promoter.

Survivin is a member of the inhibitors of apoptosis protein family. Here, we examined survivin expression and confirmed abundant survivin expression in bladder cancer cells. This expression pattern indicated that the transcriptional regulatory elements that control survivin expression could be utilized to discriminate cancer from normal cells. We therefore generated a novel adenovirus termed Ad5/35E1apsurvivinE4 with the following characteristics: 1) E1A and E4 protein expression was dependent on survivin promoter activity; 2) the green fluorescence protein gene was inserted into the genome under the control of the CMV promoter; 3) most of the E3 sequences were deleted, but the construct was still capable of expressing the adenovirus death protein with potent cytotoxic effects; and 4) the fiber knob was from serotype 35 adenovirus. As expected from the abundant survivin expression observed in bladder cancer cells, Ad5/35E1apsurvivinE4 replicated better in cancer cells than in normal cells by a factor of 106 to 102. Likewise, Ad5/35E1apsurvivinE4 exerted greater cytotoxic effects on all bladder cancer cell lines tested. Importantly, Ad5/35E1apsurvivinE4 inhibited the growth of Ku7-Luc orthotopic xenografts in nude mice. Taken together, Ad5/35E1apsurvivinE4 indicates that the survivin promoter may be utilized for the development of a replication-competent adenovirus to target bladder cancers.

Tu2022 Optimizing Outcomes of Complex Ventral Hernia Repairs Using a Sandwich Reconstruction Technique With Novel Hybrid-VAC Closure

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.

Tu2021 Interferon Expressing Oncolytic Adenovirus for Esophageal Cancer

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.