Neutralizing Antibodies Impair the Oncolytic Efficacy of Reovirus but Permit Effective Combination with T cell-Based Immunotherapies.

Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell-deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell-based immunotherapeutic strategies.

Intertumoral Differences Dictate the Outcome of TGF-ß Blockade on the Efficacy of Viro-Immunotherapy.

The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8+ T cells to the tumor and thereby enhance the efficacy of immunotherapeutic strategies that depend on high T-cell density, such as CD3-bispecific antibody (bsAb) therapy. TGF-ß signaling might represent another barrier to effective Reo&CD3-bsAb therapy due to its immunoinhibitory characteristics. Here, we investigated the effect of TGF-ß blockade on the antitumor efficacy of Reo&CD3-bsAb therapy in the preclinical pancreatic KPC3 and colon MC38 tumor models, where TGF-ß signaling is active. TGF-ß blockade impaired tumor growth in both KPC3 and MC38 tumors. Furthermore, TGF-ß blockade did not affect reovirus replication in both models and significantly enhanced the Reo-induced T-cell influx in MC38 colon tumors. Reo administration decreased TGF-ß signaling in MC38 tumors but instead increased TGF-ß activity in KPC3 tumors, resulting in the accumulation of α-smooth muscle actin (αSMA+) fibroblasts. In KPC3 tumors, TGF-ß blockade antagonized the antitumor effect of Reo&CD3-bsAb therapy, even though T-cell influx and activity were not impaired. Moreover, genetic loss of TGF-ß signaling in CD8+ T cells had no effect on therapeutic responses. In contrast, TGF-ß blockade significantly improved therapeutic efficacy of Reo&CD3-bsAb in mice bearing MC38 colon tumors, resulting in a 100% complete response. Further understanding of the factors that determine this intertumor dichotomy is required before TGF-ß inhibition can be exploited as part of viroimmunotherapeutic combination strategies to improve their clinical benefit. Significance: Blockade of the pleiotropic molecule TGF-ß can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-ß blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.

Immunostimulatory Profile of Cancer Cell Death by the AdV-Lumc007-Derived Oncolytic Virus 'GoraVir' in Cultured Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus' therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.

Reovirus Type 3 Dearing Variants Do Not Induce Necroptosis in RIPK3-Expressing Human Tumor Cell Lines.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

Preinduced reovirus-specific T-cell immunity enhances the anticancer efficacy of reovirus therapy.

BACKGROUND: Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells. METHODS: We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells. RESULTS: Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth. CONCLUSIONS: These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.

Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

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.

Induction of Robust Type I Interferon Levels by Oncolytic Reovirus Requires Both Viral Replication and Interferon-α/ß Receptor Signaling.

Oncolytic viruses are promising agents for cancer therapy because they selectively infect and kill tumor cells, and because they trigger immune responses that can boost anticancer immunity. Key to the latter process is the production of type I interferons (IFN-Is) that can turn noninflamed "cold" tumors into "hot" ones. Besides this desired anticancer effect, IFN-Is are antiviral and successful oncolytic virotherapy thus relies on tightly controlled IFN-I levels. This requires a profound understanding of when and how tumor cells induce IFN-I in response to specific viruses. In this study, we uncovered two key factors that augment IFN-I production in transformed human myeloid cells infected with a tumor-selective reovirus. Viral replication and IFN-α/ß receptor (IFNAR) signaling progressively reinforced the levels of IFN-I expressed by infected cells. Mechanistically, both augmented the activation of interferon regulatory factor 3, a key transcription factor for IFNß expression. Our findings imply that reovirus-permissive tumor cells themselves are a major source of IFN-I expression. As tumors can perturb the IFNAR pathway for their own survival, reovirus-exposed IFNAR-unresponsive tumors may need additional therapeutic intervention to promote the secretion of sufficient IFN-I into the tumor microenvironment. Our increased understanding of the parameters that affect reovirus-induced IFN-I levels could aid in the design of tailored virus-based cancer therapies.

Preconditioning of the tumor microenvironment with oncolytic reovirus converts CD3-bispecific antibody treatment into effective immunotherapy.

BACKGROUND: T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. METHODS: The mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. RESULTS: Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. CONCLUSIONS: Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

Pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer.

The use of oncolytic viruses (OVs) for cancer treatment is emerging as a successful strategy that combines the direct, targeted killing of the cancer with the induction of a long-lasting anti-tumor immune response. Using multiple aggressive murine models of triple-negative breast cancer, we have recently demonstrated that the early administration of oncolytic Maraba virus (MRB) prior to surgical resection of the primary tumor is sufficient to minimize the metastatic burden, protect against tumor rechallenge, cure a fraction of the mice and sensitize refractory tumors to immune checkpoint blockade without the need for further treatment. Here, we apply our surgical model to other OVs: Vesicular stomatitis virus (VSV), Adenovirus (Ad), Reovirus (Reo) and Herpes simplex virus (HSV) and show that all of the tested OVs could positively change the outcome of the treated animals. The growth of the primary and secondary tumors was differently affected by the various OVs and most of the viruses conferred survival benefits in this neoadjuvant setting despite the absence of direct treatment following rechallenge. This study establishes that OV-therapy confers long-term protection when administered in the pre-operative window of opportunity.

Arming oncolytic reovirus with GM-CSF gene to enhance immunity.

Oncolytic reovirus administration has been well tolerated by cancer patients in clinical trials. However, its anti-cancer efficacy as a monotherapy remains to be augmented. We and others have previously demonstrated the feasibility of producing replication-competent reoviruses expressing a heterologous transgene. Here, we describe the production of recombinant reoviruses expressing murine (mm) or human (hs) GM-CSF (rS1-mmGMCSF and rS1-hsGMCSF, respectively). The viruses could be propagated up to 10 passages while deletion mutants occurred only occasionally. In infected cell cultures, the secretion of GM-CSF protein (up to 481 ng/106 cells per day) was demonstrated by ELISA. The secreted mmGM-CSF protein was functional in cell culture, as demonstrated by the capacity to stimulate the survival and proliferation of the GM-CSF-dependent dendritic cell (DC) line D1, and by its ability to generate DCs from murine bone marrow cells. Importantly, in a murine model of pancreatic cancer we found a systemic increase in DC and T-cell activation upon intratumoral administration of rS1-mmGMCSF. These data demonstrate that reoviruses expressing functional GM-CSF can be generated and have the potential to enhance anti-tumor immune responses. The GM-CSF reoviruses represent a promising new agent for use in oncolytic virotherapy strategies.

Antibody-Neutralized Reovirus Is Effective in Oncolytic Virotherapy.

Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus-antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161-73. ©2018 AACR.

Characterization of a replicating expanded tropism oncolytic reovirus carrying the adenovirus E4orf4 gene.

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.

Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells.

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus' tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two- and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus' capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.

Oncolytic Reovirus Infection Is Facilitated by the Autophagic Machinery.

Mammalian reovirus is a double-stranded RNA virus that selectively infects and lyses transformed cells, making it an attractive oncolytic agent. Despite clinical evidence for anti-tumor activity, its efficacy as a stand-alone therapy remains to be improved. The success of future trials can be greatly influenced by the identification and the regulation of the cellular pathways that are important for reovirus replication and oncolysis. Here, we demonstrate that reovirus induces autophagy in several cell lines, evident from the formation of Atg5-Atg12 complexes, microtubule-associated protein 1 light chain 3 (LC3) lipidation, p62 degradation, the appearance of acidic vesicular organelles, and LC3 puncta. Furthermore, in electron microscopic images of reovirus-infected cells, autophagosomes were observed without evident association with viral factories. Using UV-inactivated reovirus, we demonstrate that a productive reovirus infection facilitates the induction of autophagy. Importantly, knock-out cell lines for specific autophagy-related genes revealed that the expression of Atg3 and Atg5 but not Atg13 facilitates reovirus replication. These findings highlight a central and Atg13-independent role for the autophagy machinery in facilitating reovirus infection and contribute to a better understanding of reovirus-host interactions.

Exploring Reovirus Plasticity for Improving Its Use as Oncolytic Virus.

Reoviruses are non-enveloped viruses with a segmented double stranded RNA genome. In humans, they are not associated with serious disease. Human reoviruses exhibit an inherent preference to replicate in tumor cells, which makes them ideally suited for use in oncolytic virotherapies. Their use as anti-cancer agent has been evaluated in several clinical trials, which revealed that intra-tumoral and systemic delivery of reoviruses are well tolerated. Despite evidence of anti-tumor effects, the efficacy of reovirus in anti-cancer monotherapy needs to be further enhanced. The opportunity to treat both the primary tumor as well as metastases makes systemic delivery a preferred administration route. Several pre-clinical studies have been conducted to address the various hurdles connected to systemic delivery of reoviruses. The majority of those studies have been done in tumor-bearing immune-deficient murine models. This thwarts studies on the impact of the contribution of the immune system to the tumor cell eradication. This review focuses on key aspects of the reovirus/host-cell interactions and the methods that are available to modify the virus to alter these interactions. These aspects are discussed with a focus on improving the reovirus' antitumor efficacy.

Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

Modification of mammalian reoviruses for use as oncolytic agents.

The Reoviridae are a family of viruses with a non-enveloped icosahedral capsid and a segmented double-stranded RNA genome. Prototypes of the mammalian Orthoreoviruses have been isolated from human respiratory and enteric tracts and are not associated with human disease. One of these, human reovirus type 3 Dearing (T3D), usually serves as a model for the family. In the last decade the mammalian Orthoreoviruses, especially T3D, have been evaluated as oncolytic agents in experimental cancer therapy. This is based on the observation that reoviruses induce cell death and apoptosis in tumor cells, but not in healthy non-transformed cells. Several clinical trials have been initiated in Canada, the USA, and the UK, to study the feasibility and safety of this approach. Due to the segmented structure of their double-stranded RNA genomes genetic modification of Reoviridae has been notoriously difficult. Several techniques have been described recently that facilitate the genetic modification of reovirus genomes. The basis for reverse genetics of reovirus is the discovery in 1990 that reovirus RNA is infectious. Subsequently, it took ten years before a foreign gene was introduced into the reovirus genome. Here we review the methods for reovirus modification and their use for generating new reovirus-derived oncolytic agents.

KRAS(D13) Promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.

Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRAS(D13). However, deletion of KRAS(D13) or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of beta-catenin(DeltaS45) had no effect. Likewise, KRAS(D13)- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRAS(D13) increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication.

Relaxed template specificity in fowl adenovirus 1 DNA replication initiation.

The fowl adenovirus 1 (FAdV-1) isolates PHELPS and OTE are highly similar, but have striking differences in the repeat region of the inverted terminal repeat (ITR). Whilst the repeat region in OTE conforms to the conventional human adenovirus repeat region (5'-CATCATC), that of PHELPS contains guanidine residues at positions 1, 4 and 7 (5'-GATGATG). This implies that the FAdV-1 isolates PHELPS and OTE have either distinct template specificity at replication initiation or, alternatively, a relaxed specificity for replication initiation. In this study, the distinct sequence variation at the origin of DNA replication in the ITRs of the FAdV-1 PHELPS and OTE isolates was confirmed. Sequence analyses of the pTP and Pol genes of both PHELPS and OTE did not reveal differences that could explain the distinct template specificity. Replication assays demonstrated that linear DNA fragments flanked by either 5'-CATCATC or 5'-GATGATG termini replicated in cells upon infection with FAdV-1 OTE and FAdV-1 PHELPS. This was evident from the appearance of DpnI-resistant fragments in a minireplicon assay. From these data, it is concluded that FAdV-1 has relaxed, rather than changed, its template specificity at replication initiation.