Oncolytic Ad Research Articles

Full Remission of CAR-Deficient Tumors by DOTAP-Folate Liposome Encapsulation of Adenovirus.

Adenovirus (Ad)-based vectors have shown considerable promise for gene therapy. However, Ad requires the coxsackievirus and adenovirus receptor (CAR) to enter cells efficiently and low CAR expression is found in many human cancers, which hinder adenoviral gene therapies. Here, cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-folate liposomes (Df) encapsulating replication-deficient Ad were synthesized, which showed improved transfection efficiency in various CAR-deficient cell lines, including epithelial and hematopoietic cell types. When encapsulating replication-competent oncolytic Ad (TAV255) in DOTAP-folate liposome (TAV255-Df), the adenoviral structural protein, hexon, was readily produced in CAR-deficient cells, and the tumor cell killing ability was 5× higher than that of the non-encapsulated Ad. In CAR-deficient CT26 colon carcinoma murine models, replication-competent TAV255-Df treatment of subcutaneous tumors by intratumoral injection resulted in 67% full tumor remission, prolonged survival, and anti-cancer immunity when mice were rechallenged with cancer cells with no further treatment. The preclinical data shows that DOTAP-folate liposomes could significantly enhance the transfection efficiency of Ad in CAR-deficient cells and, therefore, could be a feasible strategy for applications in cancer treatment.

HydrAd: A Helper-Dependent Adenovirus Targeting Multiple Immune Pathways for Cancer Immunotherapy.

Simple SummarySolid tumors are highly immunosuppressive and develop multiple inhibitory mechanisms that must be targeted simultaneously for successful cancer immunotherapy. Adenoviral vectors are promising cancer gene therapy vectors due to their inherent ability to stimulate multiple immune pathways. Adenoviruses are well characterized, and their genomes are easily manipulated, allowing for therapeutic transgene expression. Oncolytic adenoviruses are engineered to replicate specifically in malignant cells, resulting in cancer cell lysis. However, oncolytic adenoviral vectors have limited transgene capacity. Helper-dependent adenoviral vectors have been developed with the capability of expressing multiple transgenes through removal of all viral coding sequences. We have developed a helper-dependent platform for cancer immunotherapy and demonstrate expression of up to four functional transgenes. This platform allows us to target tumors with specific inhibitory pathways using our library of immunomodulatory transgenes in a mix-and-match approach for a synchronized cancer immunotherapy strategy.For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.

Nonhuman Primate Adenoviruses of the Human Adenovirus B Species Are Potent and Broadly Acting Oncolytic Vector Candidates.

The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.

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.

Relaxin-expressing oncolytic adenovirus induces remodeling of physical and immunological aspects of cold tumor to potentiate PD-1 blockade

BackgroundCurrently, several antibody (Ab)-based therapies have shown excellent therapeutic effects in the clinic. Nonetheless, Ab penetration into tumor tissues is limited due to abnormal vasculature, tumor interstitial pressure, and excessive...

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor β Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

AKT and JUN are differentially activated in mesenchymal stem cells after infection with human and canine oncolytic adenoviruses.

There is increasing evidence about the use of oncolytic adenoviruses (Ads) as promising immunotherapy agents. We have previously demonstrated the clinical efficiency of mesenchymal stem cells (MSCs) infected with oncolytic Ads as an antitumoral immunotherapy (called Celyvir) in human and canine patients, using ICOVIR-5 or ICOCAV17 as human and canine oncolytic Ads, respectively. Considering the better clinical outcomes of canine patients, in this study we searched for differences in cellular responses of human and canine MSCs to Ad infection that may help understand the mechanisms leading to higher antitumor immune response. We found that infection of human and canine MSCs with ICOVIR-5 or ICOCAV17 did not activate the NF-κB pathway or the interferon regulatory factors IRF3 and IRF7. However, we observed differences in the profile of cytokines secretion, as infection of canine MSCs with ICOCAV17 resulted in lower secretion of several cytokines. Moreover, we showed that infection of human MSCs with ICOVIR-5 increased the phosphorylation of a number of proteins, including AKT and c-JUN. Finally, we demonstrated that differences in regulation of AKT and c-JUN in human and canine MSCs by ICOVIR-5 or ICOCAV17 are intrinsic to each virus. Our findings suggest that ICOCAV17 induces a more limited host response in canine MSCs, which may be related to a better clinical outcome. This result opens the possibility to develop new human oncolytic Ads with these specific properties. In addition, this improvement could be imitated by selecting specific human MSC on the basis of a limited host response after Ad infection.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/β-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.

Menstrual Blood-Derived Stem Cells as Delivery Vehicles for Oncolytic Adenovirus Virotherapy for Colorectal Cancer

Oncolytic adenoviruses (Ads) have potential applications in cancer therapy due to their ability to replicate and induce tumor cell death. However, their clinical application has been limited by the lack of efficient cell-based delivery systems that can provide protection from immune attack and prevent virus clearance by neutralizing antibodies. We previously demonstrated that menstrual blood-derived mesenchymal stem cells (MenSCs) can specifically target tumor cells and serve as a novel drug delivery platform. We engineered CRAd5/F11 chimeric oncolytic Ads that can infect MenSCs and preserve their tumor targeting ability in vitro. MenSCs loaded with these Ads were transplanted in a mouse tumor model. We found that a large number of the CRAd5/F11 viruses were accumulated in tumor site and mediated marked inhibitory effects against colorectal cancer (CRC). Thus, we concluded that MenSC-cloaked oncolytic Ads hold great potential as a novel virus-delivery platform for the therapy of various cancers, including CRC.

Systemic administration of human mesenchymal stromal cells infected with polymer-coated oncolytic adenovirus induces efficient pancreatic tumor homing and infiltration

Oncolytic adenovirus (oAd)-mediated gene therapy is a promising approach for cancer treatment because of its cancer cell-restricted replication and therapeutic gene expression. However, systemic administration of oAd is severely restricted by their immunogenic nature and poor tumor homing ability, thus oAd cannot be utilized to treat disseminated metastases. In this study, human bone marrow-derived mesenchymal stromal cell (hMSCs) was used as a viral replication-permissive carrier for oAd with an aim to improve the systemic delivery of the virus to tumor tissues. To overcome the poor delivery of oAd into hMSCs, a relaxin (RLX)-expressing oncolytic Ad (oAd/RLX), which degrades dense tumor extracellular matrix of highly desmoplastic pancreatic cancer, was complexed with biodegradable polymer (poly (ethyleneimine)-conjugated poly(CBA-DAH); PCDP), generating oAd/RLX-PCDP complex. oAd/RLX-PCDP complex enhanced the internalization of oAd into hMSC, leading to superior viral production and release from hMSCs, along with high RLX expression. Furthermore, systemic administration of oAd/RLX-PCDP-treated hMSCs elicited more potent antitumor effect compared to naked oAd/RLX or oAd/RLX-treated hMSC in pancreatic tumor model. This potent antitumor effect of systemically administered oAd/RLX-PCDP-treated hMSCs was achieved by superior viral replication in tumor tissues than any other treatment group. In conclusion, these results demonstrate that hMSCs are effective carriers for the systemic delivery of oAd to tumor sites and treatment of pancreatic cancer.

A MicroRNA Derived from Adenovirus Virus-Associated RNAII Promotes Virus Infection via Posttranscriptional Gene Silencing.

The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection.IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.

The practical self-targeted oncolytic adenoviral nanosphere based on immuno-obstruction method via polyprotein surface precipitation technique enhances transfection efficiency for virotherapy

Recent developments in tumour treatment had focused on virotherapies that were currently revolutionising new innovated treatment pathways. This study focused on the fabrication of oncolytic adenoviral vector (Ad) nanosphere that self-targeted at lung tumour cells (A549), utilising the immune response for upper respiratory tract infection, caused by the Ad infection. This system was dependent upon T-cell immune response, surface charge and blood metabolism. Oncolytic Ad attacked lung A549 tumour cells by incorporated its own DNA to replace A549's, the triggered immune response generated T-cells also further attack A549. Direct Ad injection was demonstrated to be lethal and prohibited in vivo. In this research a multifunctional principal using polyprotein surface precipitation technique (PSP) whist maintaining biological controls for self-assembly polyprotein Ad nanosphere both biocompatible and reproducible, was demonstrated as a result of the enhanced transfection efficiency and a successful multifunctional drug delivery system for virotherapy.

Antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration

Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy

ABSTRACT Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX.

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.

Abstract 4640: Sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF using biodegradable polymer matrix for cancer immunotherapy

Abstract Administration of dendritic cells (DCs) combined with oncolytic adenovirus (Ad) expressing antitumor cytokines induces a potent antitumor effect and antitumor immunity by ameliorating the immunosuppressive tumor microenvironment. However, this combination therapy has significant limitations due to rapid dissemination and inactivation of the therapeutics at the tumor site, necessitating multiple injections of both therapeutics. In this study, we investigated injectable and biodegradable gelatin-based hydrogel as a carrier for oncolytic Ad co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (oAd) and DCs to elicit a potent antitumor effect with single administration. Gel matrix enabled sustained release of both oAd and DCs while preserving their biological activity over a considerable time period, leading to efficient retention of both therapeutics in tumor tissue. Further, tumors treated with oAd- and DC-loaded gel (oAd+DC/gel) showed a significantly greater expression level of IL-12, GM-CSF, and interferon-γ (IFN-γ) than either single treatment (oAd or DC) or oAd in combination with DC (oAd+DC), resulting in efficient activation of both endogenous and exogenous DCs, migration of DCs to draining lymph nodes, and tumor infiltration of CD4+ and CD8+ T cells. Moreover, oAd+DC/gel resulted in a significantly higher number of tumor-specific IFN-γ-secreting immune cells compared with oAd+DC. Lastly, oAd+DC/gel significantly attenuated tumor-mediated thymic atrophy, which is associated with immunosuppression in the tumor microenvironment, compared with oAd+DC. Taken together, these results demonstrate that gelatin gel-mediated co-delivery of oncolytic Ad and DCs might be a promising strategy to efficiently retain both therapeutics in tumor tissue and induce a potent antitumor immune response for an extended time period via a single administration. Citation Format: Eonju Oh, Jung-Eun Oh, JinWoo Hong, YoonHo Chung, Yunki Lee, Ki Dong Park, Sung Wan Kim, Chae-Ok Yun. Sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF using biodegradable polymer matrix for cancer immunotherapy [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 4640.

Histone deacetylase inhibitor trichostatin A sensitises cisplatin-resistant ovarian cancer cells to oncolytic adenovirus.

Ovarian cancer is often termed a silent killer due to the late onset of symptoms. Whilst patients initially respond to chemotherapy, they rapidly develop chemo-resistance. Oncolytic adenoviruses (OAds) are promising anti-cancer agents engineered to “hijack” the unique molecular machinery of cancer cells enabling tumour-selective viral replication. This allows spread to adjacent cells and amplification of oncolysis within the tumour. OAds represent an excellent opportunity for ovarian cancer therapy via intra-peritoneal delivery, however the efficacy of OAds thus far is limited. Here, we evaluate chromatin (histone) modification in chemo-resistant cells and its relationship to Ad efficacy (wild-type or oncolytic Ad). In contrast to cisplatin-sensitive A2780 cells that show an efficient reduction of cell viability by Ad in the presence of cisplatin, cisplatin-resistant A2780/cp70 cells show diminishing Ad-mediated reduction of cell viability with escalating doses of cisplatin. Histone deacetylase (HDAC)-2 and to a lesser extent HDAC1 were up-regulated in cisplatin-resistant but not cisplatin-sensitive cells. Cisplatin-resistant cells treated with a pan-HDAC inhibitor trichostatin A (TsA) significantly enhanced Ad-mediated reduction of cell viability in the presence of cisplatin. Cells treated with TsA alone did not reduce cell viability suggesting these findings are Ad-dependent. Thus, we identify HDAC inhibition as a potential means to sensitise cisplatin-resistant ovarian cancer cells to virotherapies, an observation that may offer improved outcomes for patients with late stage, chemotherapy-resistant ovarian cancer.

Hepatocellular carcinoma-targeting oncolytic adenovirus overcomes hypoxic tumor microenvironment and effectively disperses through both central and peripheral tumor regions

Cancer-specific promoter driven replication of oncolytic adenovirus (Ad) is cancer-specific, but shows low transcriptional activity. Thus, we generated several chimeric α-fetoprotein (AFP) promoter variants, containing reconstituted enhancer and silencer regions, to preferentially drive Ad replication in hepatocellular carcinoma (HCC). Modified AFP promoter, containing 2 enhancer A regions and a single enhancer B region (a2bm), showed strong and HCC-specific transcription. In AFP-positive HCCs, gene expression was 43- to 456-fold higher than those of control AFP promoter lacking enhancers. a2bm promoter was further modified by inserting multiple hypoxia-responsive elements (HRE) to generate Ha2bm promoter, which showed stronger transcriptional activity than a2bm promoter under hypoxic conditions. Ha2bm promoter-regulated oncolytic Ad (Ha2bm-d19) showed a stronger antitumor and proapoptotic effect than did a2bm promoter-regulated oncolytic Ad (a2bm-d19) in HCC xenograft tumors. Systemically administered Ha2bm-d19 caused no observable hepatotoxicity, whereas control replication-competent Ad, lacking cancer specificity (d19), induced significant hepatic damage. Ha2bm-d19 caused significantly lower expression of interleukin-6 than d19, showing that HCC-targeted delivery of Ad attenuates induction of the innate immune response against Ad. This chimeric AFP promoter enabled Ad to overcome the hypoxic tumor microenvironment and target HCC with high specificity, rendering it a promising candidate for the treatment of aggressive HCCs.

Sensitisation to mitoxantrone-induced apoptosis by the oncolytic adenovirus Ad\u2206\u2206 through Bcl-2-dependent attenuation of autophagy

Anti-apoptotic Bcl-2 is frequently activated in human malignant cells to promote cell survival and inhibit cell death. Replication-selective oncolytic adenoviruses deleted in the functional Bcl-2 homologue E1B19K potently synergise with apoptosis-inducing chemotherapeutic drugs, including mitoxantrone for prostate cancer. Here, we demonstrate that our previously generated oncolytic mutant Ad∆∆ (E1B19K- and E1ACR2-deleted) caused potent synergistic apoptotic cell death in both drug-sensitive 22Rv1, and drug-insensitive PC3 and PC3M prostate cancer cells. The synergistic cell killing was dependent on Bcl-2 expression and was prevented by Bcl-2 knockdown, which led to activation of the autophagy pathway. Mitoxantrone-induced autophagy, which was decreased in combination with Ad∆∆-infection resulting in increased apoptosis. Expression of the viral E1A12S protein alone mimicked the synergistic effects with Ad∆∆ in combination with mitoxantrone while intact wild-type virus (Ad5) had no effect. Early and late-stage inhibition of autophagy by Atg7 knockdown and chloroquine respectively, promoted apoptotic cell killing with mitoxantrone similar to Ad∆∆. These findings revealed currently unexplored actions of E1B19K-deleted oncolytic adenoviruses and the central role of Bcl-2 in the synergistic cell killing. This study suggests that cancers with functional Bcl-2 expression may be selectively re-sensitised to drugs by Ad∆∆.

Suppression of Oncolytic Adenovirus-Mediated Hepatotoxicity by Liver-Specific Inhibition of NF-κB

Telomerase-specific replication-competent adenoviruses (Ads), i.e., TRADs, which possess an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, are promising agents for cancer treatment. However, even though oncolytic Ads, including TRAD, are intratumorally administered, they are disseminated from the tumor to systemic circulation, causing concern about oncolytic Ad-mediated hepatotoxicity (due mainly to leaky expression of Ad genes in liver). We reported that inhibition of nuclear factor-κB (NF-κB) leads to the suppression of replication-incompetent Ad vector-mediated hepatotoxicity via reduction of the leaky expression of Ad genes in liver. Here, to develop a TRAD with an improved safety profile, we designed a TRAD that carries a liver-specific promoter-driven dominant-negative IκBα (DNIκBα) expression cassette (TRAD-DNIκBα). Compared with a conventional TRAD, TRAD-DNIκBα showed hepatocyte-specific inhibition of NF-κB signaling and significantly reduced Ad gene expression and replication in the normal human hepatocyte cell line. TRAD-induced hepatotoxicity was largely suppressed in mice following intravenous administration of TRAD-DNIκBα. However, the replication profiles and oncolytic activities of TRAD-DNIκBα were comparable with those of the conventional TRAD in human non-hepatic tumor cells. These results indicate that oncolytic Ads containing the liver-specific DNIκBα expression cassette have improved safety profiles without inhibiting oncolytic activities.