Efficacy Of Oncolytic Adenovirus Research Articles

SOCS3 inhibiting JAK-STAT pathway enhances oncolytic adenovirus efficacy by potentiating viral replication and T-cell activation.

Oncolytic viruses (OVs) are emerging as a potentially useful treatment for malignancies due to the capabilities of direct oncolysis and immune induction. Improving the replication of OVs is an effective approach to enhance the oncolytic effects. Here, we observed that cancer cells with deficiencies in JAK-STAT pathway showed greater sensitivity to oncolytic adenovirus (oAd), and JAK inhibitor could enhance the replication of oAd. Therefore, we constructed a novel oAd expressing SOCS3, a major negative regulator of JAK-STAT pathway, and confirmed that oAd-SOCS3 exhibited a more significant antitumor effect than oAd-Ctrl both in vitro and in vivo. Mechanistically, SOCS3 inhibited the activation of JAK-STAT pathway, resulting in stronger tumor selective replication of oAd and downregulated expression of PD-L1 on cancer cells as well. Both benefits could collectively awaken antitumor immunity. This study highlights the importance of JAK-STAT pathway in viral replication and confirms the treatment of oAd-SOCS3 in potential clinical applications.

IMMU-29. A CHIMERIC ONCOLYTIC ADENOVIRUS CIRCUMVENTS NEUTRALIZING ANTIBODIES DEVELOPED BY PATIENTS WITH RECURRENT GLIOBLASTOMA TREATED WITH DELTA-24-RGD

Abstract Oncolytic virotherapy holds great potential to treat high-grade gliomas by harnessing virus-mediated oncolysis to stimulate anti-tumor immune responses. However, the efficacy of oncolytic adenoviruses can be hampered by neutralizing antibodies. In this study, we examined sera obtained from a phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD to treat patients with recurrent malignant gliomas. We found that a single intratumoral injection of Delta-24-RGD induced the development of neutralizing antibodies in less than half of the patients but demonstrated remarkable outcomes, with 20% of the patients surviving beyond three years. Conversely, multiple injections of the oncolytic adenovirus induced neutralizing antibodies in 80% of patients (P=0.05) but resulted in no long-term survivors. This inverse correlation led us to further investigate the role of neutralizing antibodies during glioma virotherapy. Examination of Delta-24-RGD-treated murine brain tumors using immunofluorescence displayed colocalization of antibodies and viral proteins. To counteract virus neutralization and maximize oncolysis, we engineered a chimeric virus, Delta-24-RGD-H43m, by replacing the hypervariable regions of the hexon protein from the prevalent serotype 5-based Delta-24-RGD with those from the rare serotype 43. The infectivity, replication, and oncolytic capabilities of Delta-24-RGD-H43m were similar to those of the parental virus. Using clinical trial patient sera, we observed that Delta-24-RGD-H43m displayed significant resilience against the inhibitory effects of neutralizing sera from patients (P=0.01). In murine models with pre-existing immunity to adenovirus, Delta-24-RGD-H43m treatment led to a significantly extended median survival and resulted in a higher percentage of long-term survivors than the parental virus (P=0.005). These results underscore the effectiveness of hexon swapping as a strategy to overcome the dominant immune response against oncolytic viruses, provide valuable insights into the interaction between oncolytic viruses and the host immune system, and lay the foundation to translate to the clinical setting immune-evading virotherapies for glioma patients.

Bifidobacterium affects antitumor efficacy of oncolytic adenovirus in a mouse model of melanoma

Gut microbiota plays a key role in modulating responses to cancer immunotherapy in melanoma patients. Oncolytic viruses (OVs) represent emerging tools in cancer therapy, inducing a potent immunogenic cancer cell death (ICD) and recruiting immune cells in tumors, poorly infiltrated by Tcells. We investigated whether the antitumoral activity of oncolytic adenovirus Ad5D24-CpG (Ad-CpG) was gut microbiota-mediated in a syngeneic mouse model of melanoma and observed that ICD was weakened by vancomycin-mediated perturbation of gut microbiota. Ad-CpG efficacy was increased by oral supplementation with Bifidobacterium, reducing melanoma progression and tumor-infiltrating regulatory Tcells. Fecal microbiota was enriched in bacterial species belonging to the Firmicutes phylum in mice treated with both Bifidobacterium and Ad-CpG; furthermore, our data suggest that molecular mimicry between melanoma and Bifidobacterium-derived epitopes may favor activation of cross-reactive Tcells and constitutes one of the mechanisms by which gut microbiota modulates OVs response.

Oncolytic adenovirus H101 ameliorate the efficacy of anti-PD-1 monotherapy in colorectal cancer.

Immune checkpoint blockade therapy with anti-programmed cell death (PD)-1 antibodies provides therapeutic effect for many patients of various cancers but remains inadequate in colorectal cancer (CRC) patients. The present study aims to assess the efficacy of oncolytic adenovirus (OncoAd ) in enhancing the anti-PD-1 treatment of CRC. The estimating relative subsets of RNA transcripts algorithm was used for estimating the infiltrated immune cells in melanoma and CRC tissues. The efficacy of OncoAd with anti-PD-1 monotherapy was performed in a CT26 CRC mouse model in vivo. Flow cytometric analysis of peripheral blood and tumor tissues determined the difference anti-tumor immune efficacy of OncoAd with anti-PD-1 monotherapy. The Cancer Genome Atlas database indicated that CD8+ T cells and regulatory T cells were significantly elevated in melanoma compared to CRC cohorts. Moreover, intratumor injection of oncolytic adenovirus enhanced T cell infiltration and decreased Treg percentages in the CT26 CRC colorectal cancer mouse model. Combinatorial OncoAd with anti-PD-1 antibody treatment markedly enhanced the anti-tumor efficacy of anti-PD-1 by significantly decreasing the tumor volume and reducing tumor growth in a CRC mouse model. To the end, OncoAd treatment increased the CD8/Treg ratio, indicating that OncoAd intratumor injection ameliorate the anti-tumor immune response of anti-PD-1 therapy. The present study elucidates that OncoAd promotes intratumor T cell infiltration and improves anti-PD-1 immunotherapy, thereby providing a potent combinatorial therapeutic strategy for CRC.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

An Oncolytic Adenovirus Armed with a Nanobody Against CD47 Reprograms Tumor Immune Microenvironment and Drives Durable Antitumor Immunity

Background: Oncolytic virotherapy utilizes natural or engineered viruses to kill tumors selectively, representing a promising cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses (OVs) elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in don't eat me signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As ideal gene delivery vectors in cancer therapy, OVs can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Importantly, OVs can intratumorally deliver anti-CD47 antibodies without causing uncontrolled off-tumor toxicity. Methods: We developed a recombinant oncolytic adenovirus loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. An oncolytic adenovirus without transgene of interest was used as control. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer mouse models were established to evaluated in vivo antitumor efficacy of oncolytic adenoviruses. Findings: The recombinant oncolytic adenovirus armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice. Additionally, the recombinant oncolytic adenovirus elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in mice treated with the anti-CD47 oncolytic adenovirus; tumorigenesis was completely inhibited in these mice after tumor re-challenge. Interpretation: The recombinant anti-CD47 oncolytic adenovirus has an effectual antitumor activity and may be a promising antitumor agent. Funding Statement: This work was supported by the National Science and Technology Major Projects of New Drugs (2018ZX09201018-013), the National Science and Technology Major Project for Infectious Diseases Control (2017ZX10203206-004), the National Natural Science Foundation of China (81101728), Sichuan Regional Innovation Cooperation Project (20QYCX0100), the Innovation Spark Project of Sichuan University (2018SCUH0084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu, China.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas

BackgroundPediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved...

Modeling the Efficacy of Oncolytic Adenoviruses In Vitro and In Vivo: Current and Future Perspectives

Oncolytic adenoviruses (OAd) selectively target and lyse tumor cells and enhance anti- tumor immune responses. OAds have been used as promising cancer gene therapies for many years and there are a multitude of encouraging pre-clinical studies. However, translating OAd therapies to the clinic has had limited success, in part due to the lack of realistic pre-clinical models to rigorously test the efficacy of OAds. Solid tumors have a heterogenous and hostile microenvironment that provides many barriers to OAd treatment, including structural and immunosuppressive components that cannot be modeled in two-dimensional tissue culture. To replicate these characteristics and bridge the gap between pre-clinical and clinical success, studies must test OAd therapy in three-dimensional culture and animal models. This review focuses on current methods to test OAd efficacy in vitro and in vivo and the development of new model systems to test both oncolysis and immune stimulatory components of oncolytic adenovirotherapy.

EXTH-11. TREATMENT WITH DELTA-24-RGDOX OF SUBCUTANEOUS TUMORS RESULTS IN ABSCOPAL EFFECT ERADICATING INTRACRANIAL MELANOMAS

Abstract Immune checkpoint blockade has revolutionized cancer therapy; however the therapeutic benefit is limited to only a subset of patients with immunogenic (“hot”) tumors and is compromised by immune-related adverse events. We have reported the efficacy of oncolytic adenovirus Delta-24-RGDOX (DNX-2440) in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas, including intracranial melanomas. We tested the hypothesis in the subcutaneous (s.c.)/s.c. and s.c./intracranial (i.c.) melanoma models derived from luciferase-expressing B16-Red-FLuc cells in C57BL/6 mice. First, through monitoring tumor growth with bioluminescence imaging, we found that, in both s.c./s.c. and s.c./i.c. models, three injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. or i.c. tumor, thereby prolonging survival. Next, through cell profiling with flow cytometry, we observed that the virus increased the presence of T cells and effector T cell frequency in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, tumor-draining lymph nodes (TDLNs), spleens, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the frequency of exhausted T cells and regulatory T cells in the virus-injected and untreated i.c. tumors. Consequently, the virus promoted recruitment and/or in situ expansion of antigen-specific T cells in tumors expressing the target antigen. Therefore, we concluded that local intratumoral injection of Delta-24-RGDOX resulted in systemic immune activity against the disseminated tumors. Furthermore, we speculate that given the immunogenicity, cancer-selectivity and intratumoral administration of the virus, Delta-24-RGDOX is expected to have an improved safety profile when compared to immune checkpoint blockade treatment strategies. This is the first report demonstrating that local administration of oncolytic adenovirus results in eradication of intracranial tumors, suggesting Delta-24-RGDOX could be used to manage brain metastases of melanoma.

Oncolytic Adenovirus—A Nova for Gene-Targeted Oncolytic Viral Therapy in HCC

Hepatocellular carcinoma (HCC) is one of the most frequent cancers worldwide, particularly in China. Despite the development of HCC treatment strategies, the survival rate remains unpleasant. Gene-targeted oncolytic viral therapy (GTOVT) is an emerging treatment modality—a kind of cancer-targeted therapy—which creates viral vectors armed with anti-cancer genes. The adenovirus is a promising agent for GAOVT due to its many advantages. In spite of the oncolytic adenovirus itself, the host immune response is the determining factor for the anti-cancer efficacy. In this review, we have summarized recent developments in oncolytic adenovirus engineering and the development of novel therapeutic genes utilized in HCC treatment. Furthermore, the diversified roles the immune response plays in oncolytic adenovirus therapy and recent attempts to modulate immune responses to enhance the anti-cancer efficacy of oncolytic adenovirus have been discussed.

Enhanced Antitumor Efficacy of Oncolytic Adenovirus-loaded Menstrual Blood-derived Mesenchymal Stem Cells in Combination with Peripheral Blood Mononuclear Cells.

Several studies have evaluated the efficacy of using human oncolytic adenovirus (OAdv)-loaded mesenchymal stem cells (MSC) for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow-mesenchymal stem cells infected with the OAdv ICOVIR-5, for treatment of patients with neuroblastoma. Results from this clinical trial point out the role of the immune system in the clinical outcome. In this context, a better understanding of the immunophenotypic changes of human MSCs upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMC) could guide strategies to improve the antitumor efficacy of infected MSCs. In this work, we show how infection by an OAdv induces toll-like receptor 9 overexpression and activation of the NFĸB pathway in menstrual blood-derived MSCs, leading to a specific cytokine secretion profile. Moreover, a proinflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T cells and natural killer cells (NK cell), is generated when OAdv-loaded MSCs are cocultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated by monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the OAdv-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy.

Unleashing the Full Potential of Oncolytic Adenoviruses against Cancer by Applying RNA Interference: The Force Awakens

Oncolytic virus therapy of cancer is an actively pursued field of research. Viruses that were once considered as pathogens threatening the wellbeing of humans and animals alike are with every passing decade more prominently regarded as vehicles for genetic and oncolytic therapies. Oncolytic viruses kill cancer cells, sparing healthy tissues, and provoke an anticancer immune response. Among these viruses, recombinant adenoviruses are particularly attractive agents for oncolytic immunotherapy of cancer. Different approaches are currently examined to maximize their therapeutic effect. Here, knowledge of virus–host interactions may lead the way. In this regard, viral and host microRNAs are of particular interest. In addition, cellular factors inhibiting viral replication or dampening immune responses are being discovered. Therefore, applying RNA interference is an attractive approach to strengthen the anticancer efficacy of oncolytic viruses gaining attention in recent years. RNA interference can be used to fortify the virus’ cancer cell-killing and immune-stimulating properties and to suppress cellular pathways to cripple the tumor. In this review, we discuss different ways of how RNA interference may be utilized to increase the efficacy of oncolytic adenoviruses, to reveal their full potential.

Correction for Spurrell et al., "STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus".

Correction for Spurrell et al., "STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus".

Mesenchymal stem cell carriers enhance antitumor efficacy of oncolytic adenoviruses in an immunocompetent mouse model

Oncolytic virotherapy represents a promising alternative for cancer treatment; however, viral delivery to the tumor represents a major challenge. Mesenchymal stem cells (MSCs) chemotax to tumors, and can serve as a viral delivery tool. Previously, we demonstrated antitumor therapeutic efficacy for mesenchymal stem cells (MSCs) infected with the oncolytic human adenovirus ICOVIR5 (Celyvir) for treatment of neuroblastoma patients. Given the lack of suitable immunocompetent preclinical models, the mechanism underlying Celyvir antitumor activity remains unknown. In this study, we used the syngeneic murine CMT64 cell line as a human adenovirus-semi-permissive tumor model and demonstrate the homing capacity of mouse Celyvir (mCelyvir) to CMT64 tumors. We found that the combined treatment of mCelyvir and intratumoral injections (i.t.) of ICOVIR5 was more effective than treatment with i.t. ICOVIR5 alone. Interestingly, the superior therapeutic effect of the combined therapy was associated with a higher tumor infiltration of CD8+ and CD4+ T cells. Our findings suggest that the use of MSCs as carriers of oncolytic adenovirus can improve the clinical efficacy of anti-cancer virotherapy, not only by driving the adenovirus to tumors, but also through their potential to recruit T cells.

Abstract A28: Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy

Abstract Oncolytic viruses (OVs) are a new class of anti-cancer agent. They infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs disrupt chemokine and cytokine networks, which may influence clinical efficacy. This study investigates the role of innate immune responses, in particular IL17F and Natural Killer cells (NK), on the efficacy of oncolytic adenoviruses. A transcriptional array (Qiagen RT2 Profiler) of 84 human chemokines and cytokines revealed highly consistent changes (r=0.623, p<0.0001) following infection of ovarian cancer lines (TOV21G, OVCAR4) by the E1A CR2-deleted oncolytic adenovirus dl922-947. The most highly upregulated cytokine in both cells was IL17F (fold change=166.9 in TOV21G, p<0.0001, and 97.6 in OVCAR4, p<0.05), whilst CXCL1 and CCL2 were the most down-regulated. Similarly, an increase in IL17F transcription was seen after dl922-947 infection in HeLa cells (fold change=4.3, p<0.05) and after Ad5 wild-type infection in OVCAR4 (fold change=18.3, p<0.01). By ELISA, an increase in IL17F production was also observed after dl922-947 infection in TOV21G (mean IL17F was 4930pg/106 cells for dl922-947 versus 1167pg/106 for mock infection, n=2 in triplicates, p<0.05). We also observed increased transcription (median fold-change=91.5, p<0.05) and translation (median 2086pg/106 for dl922-947 vs 1381pg/106 for mock infection, p<0.05) of IL17F in 6 primary lines derived from ascites of patient with ovarian cancer, suggesting immediate clinical relevance of our findings. As human adenoviruses do not replicate in mice, HeLa subcutaneous xenografts in CD1 nu/nu mice were used to evaluate the infiltration of immune cells after dl922-947 infection. Tumors were harvested 48 hours following a single intra-tumoral injection of dl922-947 (1010 particles). IL17F transcription increased significantly in virus-treated tumors (mean fold-change=1 for mock-infected, n=5, vs 10.7 for dl922-947-infected, n=7), accompanied by a significant neutrophil infiltration (as detected by NIMP-R14, which binds Ly6G and Ly6C; median histoscores 2 and 16.5 for mock and dl922-947 infected tumors, respectively, p<0.05). We are currently generating TOV21G IL17F-/- cells using CRISPR-Cas9 technology, which will be used to evaluate potential changes in dl922-947 efficacy in vitro and in vivo. NK have potential impact on oncolytic virus efficacy (1). The interaction between NK and dl922-947 was evaluated in vitro using peripheral blood NK from health donors. Significant increases in CD107a+ NK, a degranulation marker, and interferon-Υ production were observed after co-culture with dl922-947-infected TOV21G, compared to co-culture with mock-infected TOV21G, by flow cytometry and ELISA, respectively (CD107a in 9.0% vs 1.8% of total NK, p<0.01 and interferon-Υ were 8559 +/-356pg/mL vs 4 +/-1pg/mL, respectively). Furthermore, Ad-CMV-GFP, an E1-deleted Ad5 vector, infects ovarian cancer lines but not NK-92 cells (92% vs 0% infected, respectively, p<0.001), suggesting the activation observed was not due to direct infection of NK by Ad5. We will investigate alteration of NK cytotoxicity against ovarian cancer cells after exposure to dl922-947 infection by calcein cytotoxicity assays, using both peripheral blood NK and NK from ascites of women with ovarian cancer. Any change in efficacy of dl922-947 with or without NK depletion in vivo will be assessed using ovarian cancer (TOV21G) intraperitoneal xenografts in CD1 nu/nu mice. To conclude, adenovirus infection leads to distinctive changes in chemokines and cytokines, as well as activation of NK. In particular, IL17F, but not IL17A, was up-regulated after adenovirus infection. The influence of these observations on oncolytic adenovirus efficacy is currently being evaluated. 1. Alvarez-Breckenridge CA, et al. Nat Med. 2012 Dec;18(12):1827-34. Citation Format: Elaine YL Leung, Melanie Weigert, Josephine Walton, Darren Ennis, Dimitris Athineos, Suzanne Dowson, Chris Hansell, Karen Blyth, Gerard Graham, Iain McNeish. Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A28.

The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model.

Oncolytic adenoviruses (Ad) represent an innovative approach to cancer therapy. Its efficacy depends on multiple actions, including direct tumor lysis and stimulation of antiviral and antitumor immune responses. In this study, we investigated the roles of T-cell responses in oncolytic adenoviral therapy. An immunocompetent and viral replication-permissive Syrian hamster tumor model was used. The therapeutic mechanisms of oncolytic Ad were investigated by T-cell deletion, immunohistochemical staining, and CTL assay. Deletion of T cells with an anti-CD3 antibody completely demolished the antitumor efficacy of oncolytic Ad. Intratumoral injection of Ad induced strong virus- and tumor-specific T-cell responses, as well as antiviral antibody response. Both antiviral and antitumor T-cell responses contributed to the efficacy of oncolytic Ad. Deletion of T cells increased viral replication and extended the persistence of infectious virus within tumors but almost abrogated the antitumor efficacy. Preexisting antiviral immunity promoted the clearance of injected oncolytic Ad from tumors but had no effect on antitumor efficacy. Strikingly, the repeated treatment with oncolytic Ad has strong therapeutic effect on relapsed tumors or tumors insensitive to the primary viral therapy. These results demonstrate that T cell-mediated immune responses outweigh the direct oncolysis in mediating antitumor efficacy of oncolytic Ad. Our data have a high impact on redesigning the regimen of oncolytic Ad for cancer treatment. Clin Cancer Res; 23(1); 239-49. ©2016 AACR.

Lentivirus-mediated p21/Waf-1 short hairpin RNA enhances the cytotoxic effects and replicative potential of a bladder cancer-specific oncolytic adenovirus in vitro

Our previous work confirmed that the bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A could selectively replicate in bladder cancer cells, thus causing specific tumor cell lysis. The replicative potential is a crucial factor in determining the therapeutic efficacy of oncolytic adenoviruses. However, viral replication is attenuated by the low-activity promoter that we used, thus compromising viral cytotoxicity. In this study, we investigated the effect of the cell cycle-dependent kinase inhibitor p21/Waf-1 on an adenovirus. We used lentivirus-mediated short hairpin RNA to knock down p21/Waf-1 in two bladder cancer cell lines EJ and 5637. The p21/Waf-1 knockdown not only induced stronger cytopathic effects but also augmented apoptosis, which was closely associated with the enhancement of Fas and the subsequent significant activation of caspase-3. A replicative assay showed that p21/Waf-1 knockdown increased the viral particle production. Western blot analysis confirmed that p21/Waf-1 knockdown upregulated the expression of androgen receptor (AR) and two adenovirus replication indicators E1A and hexon. A luciferase activity assay indicated higher transcriptional activity of the uroplakin II (UPII) promoter in the p21/Waf-1 knockdown cells, and one possible mechanism could be that the increased expression of AR induced the UPII promoter through the AR-binding sites of the prostate stem cell antigen enhancer. These findings indicating that p21/Waf-1 knockdown could enhance cell killing and viral replication have significant implications for the development of bladder cancer-specific oncolytic adenovirus therapies.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922-947 (E1A CR2-deleted), dl1520 (E1B-55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel-resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non-CAR receptors to infect cells, are also significantly more effective in paclitaxel-resistant ovarian cell models. Inhibition of CDK4/6 using PD-0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

Association of oncolytic adenoviruses with chemotherapies: An overview and future directions

Oncolytic adenoviruses have been used in different preclinical and clinical studies, showing their capacity to kill tumor cells without major adverse events. However, these studies also underline the limitations of this approach. The efficacy of oncolytic adenoviruses is hampered by their limited ability to transduce some tumor types, their lack of selectivity, and their poor dissemination within tumors. In addition, the host immune response may limit oncolytic adenovirus efficacy. Combining oncolytic adenoviruses with chemotherapeutics constitutes an appealing strategy to increase their potency. The first part of this review describes the molecular basis of oncolytic adenoviruses, their use in preclinical studies and clinical trials, their limitations, and strategies to circumvent these limitations. The second part will focus on studies combining oncolytic adenoviruses with chemotherapeutic drugs, including standard chemotherapeutic drugs, molecularly targeted drugs, and other drugs that have been combined with oncolytic adenoviruses. Finally, based on these studies, we describe future directions and general rules that could be followed to identify chemotherapeutic drugs displaying additive/synergistic effects when combined with oncolytic adenoviruses.