Pancreatic Ductal Adenocarcinoma (PDAC) may soon become the third-leading cause of cancer-related death in the United States. Aside from curative resection of tumors, there is no highly effective therapy to treat PDAC. Because patients are usually diagnosed in the late stage of the disease, more than 80% of them are not eligible to undergo surgery, which results in a post-diagnosis survival rate of three to four months, and an extremely low five-year survival rate of 6%. Despite discouraging current clinical outcomes, clinical trials treating PDAC patients with adjuvant therapy combined with IFN- (IFN), Fluorouracil (5-FU), Cisplatin (CDDP), and radiation have shown to improve patients' two-year survival rates by 20-41%, and improve their five-year survival rates by 35%. These clinical trials show that IFN-therapy is now one of the few therapeutic regimens that can significantly improve the short- and long-term survival of PDAC patients. Despite its high efficacy, the drawbacks of IFN therapy included high IFN systemic toxicity, which resulted in increased patient drop-out rates in clinical trials, and low levels of the cytokine in tumors, which hampered the chemo-radio-sensitization capability of IFN during therapy. To attempt to improve the efficacy and overcome the drawbacks of IFN therapy, we studied the use of IFN-expressing replication competent oncolytic adenovirus (OAd) vectors in combination with chemoradiation mimicking the aforementioned chemoradiation and IFN-based clinical trials. Because IFN therapy can stimulate a tumor-specific immune response, we first evaluated the anti-tumor effect of oncolytic adenovirus vector expressing hamster IFN (OAd-hamIFN) in a syngeneic immunocompetent PDAC hamster model. To further understand the interaction between IFN-expressing OAd and chemotherapy, radiation, and chemoradiation, and to evaluate the therapeutic effect of the virus used in treatments mimicking the IFN therapy, we also tested the effect of a human IFN-expressing oncolytic adenovirus (OAd-IFN) in immunodeficient mice bearing human PDAC xenografts. To increase infectivity of OAd-hamIFN in hamster pancreatic cancer cell lines, we included the RGD-4C (Arginive-Glycine-Aspartic) motif in the HI loop of OAd-hamIFN fiber. This modification is known to shift viral tropism from the Coxsackie and Adenovirus Receptor (CAR) to integrins αvβ3 and αvβ5, which are widely expressed in hamster pancreatic cancer cell lines. To increase infectivity of OAd-IFN in human pancreatic cancer cells lines, we replaced the adenovirus type 5 fiber with the chimeric adenovirus 5/3 fiber, which consisted of the fiber of adenovirus type 5 with the knob of adenovirus type 3. This modification has been proven to shift viral tropism from CAR receptor, which shows low expression in human pancreatic cancer cells, to Desmoglein type-2 and CD46 proteins, which are widely expressed in human pancreatic cancer cell lines. To improve spreading in tumors and oncolytic effect of both OAd-hamIFN and OAd-IFN in PDAC cells, and to achieve replication-dependent expression of IFN, we included the Adenovirus Death Protein (ADP) and respective IFN genes in the adenovirus (Ad) E3 region. To restrict the replication of human IFN expressing OAd vector (OAd-IFN) in human PDAC cells, we added the cyclooxygenase-2 (Cox-2) promoter upstream of the Ad E1 region, which is the region responsible for initiating viral replication. Combinations of 5-FU, radiation, and 5-FU + Radiation with OAd-hamIFN in hamster PDAC cells, or with OAd-IFN in human PDAC cells, resulted in highly synergistic and cytotoxic combinations in vitro. Studies in the syngeneic hamster PDAC model showed that including OAd-hamIFN in combination with therapeutics used in IFN therapy improved treatment efficacy, and that using the virus in treatment mimicking the IFN therapy (OAd-hamIFN + 5-FU + Radiation) was the most effective treatment strategy in the study. When we analyzed the effect of OAd-IFN in combination with 5-FU + CDDP in human PDAC cells, the combinations were antagonistic and weakly cytotoxic. But adding radiation to the treatment (OAd-IFN + (5-FU + CDDP) + Radiation) overcame the chemotherapy antagonism, which resulted in highly synergistic and extremely potent treatments in vitro and in mice bearing PDAC xenografts. Because radiation eliminated the antagonism of chemotherapy to the virus in vivo, we tested different radiation protocols in combination with OAd-IFN in mice bearing PDAC tumors. We concluded that administering radiation before infecting tumors with OAd-IFN improved treatment efficacy and that using radiation before OAd-IFN infection should potentiate the effect of Cox-2 controlled IFN-expressing OAds in combination with chemoradiation. In summary, our data strongly support including an IFN expressing OAd in treatments mimicking IFN therapy. As this therapy is one of the few therapeutic regimens shown to improve patients' short- and long-term survival rates, developing a virus-based IFN treatment protocol may result in a highly effective means to treat PDAC.
University of Minnesota Ph.D. dissertation. February 2018. Major: Pharmacology. Advisor: Masato Yamamoto. 1 computer file (PDF); ix, 129 pages.
Use of Oncolytic Adenovirus expressing Interferon (IFN) Alpha as a tool to improve IFN-based chemoradiation regimen to treat pancreatic cancer.
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