Orca-010, a Novel Potency-enhanced Oncolytic Adenovirus, Exerts Strong Antitumor Activity in Preclinical Models

Improving the antitumor potency of current oncolytic adenoviruses represents one of the major challenges in development of these viruses for clinical use. We have generated an oncolytic adenovirus carrying the safety-enhancing E1A Delta 24 deletion, the potency-enhancing T1 mutation, and the infectivity-enhancing fiber RGD modification. The results of in vitro cytotoxicity assays on 15 human cancer cell lines derived from different tumor types demonstrated that ORCA-010 is more potent than Ad5-Delta 24RGD or ONYX-015. As ORCA-010 will initially be developed for the treatment of prostate cancer, selectivity experiments were performed using primary human prostate cells. ORCA-010 killed cancer cells more effectively than these primary human cells. In both primary prostate fibroblasts and epithelial cells, ORCA-010 was as safe as Ad5-Delta 24RGD. Evaluation of ORCA-010 in in vivo xenograft tumor models in nude mice showed that ORCA-010 significantly inhibited growth of prostate, lung, and ovarian tumors and conferred prolonged survival of tumor-bearing animals. Furthermore, we observed a substantial increase in infectious viral particles in tumors injected with ORCA-010. The number of infectious viral particles increased after treatment and infectious particles remained present up to at least 4 weeks posttreatment. Intratumoral virus replication was associated with substantial necrosis and fibrosis. In conclusion, ORCA-010 is more potent than earlier generation oncolytic adenoviruses, without demonstrating increased toxicity. ORCA-010 exerted strong in vivo antitumor activity and is therefore a suitable candidate for clinical evaluation

Oncolytic Adenovirus ORCA-010 Activates Proinflammatory Myeloid Cells and Facilitates T Cell Recruitment and Activation by PD-1 Blockade in Melanoma

Immune checkpoint inhibitors have advanced the treatment of melanoma. Nevertheless, a majority of patients are resistant, or develop resistance, to immune checkpoint blockade, which may be related to prevailing immune suppression by myeloid regulatory cells in the tumor microenvironment (TME). ORCA-010 is a novel oncolytic adenovirus that selectively replicates in, and lyses, cancer cells. We previously showed that ORCA-010 can activate melanoma-exposed conventional dendritic cells (cDCs). To study the effect of ORCA-010 on melanoma-conditioned macrophage development, we used an in vitro co-culture model of human monocytes with melanoma cell lines. We observed a selective survival and polarization of monocytes into M2-like macrophages (CD14(+)CD80(-)CD163(+)) in co-cultures with cell lines that expressed macrophage colony-stimulating factor. Oncolysis of these melanoma cell lines, effected by ORCA-010, activated the resulting macrophages and converted them to a more proinflammatory state, evidenced by higher levels of PD-L1, CD80, and CD86 and an enhanced capacity to prime allogenic T cells and induce a type-1 T cell response. To assess the effect of ORCA-010 on myeloid subset distribution and activation in vivo, ORCA-010 was intratumorally injected and tested for T cell activation and recruitment in the human adenovirus nonpermissive B16-OVA mouse melanoma model. While systemic PD-1 blockade in this model in itself did not modulate myeloid or T cell subset distribution and activation, when it was preceded by i.t. injection of ORCA-010, this induced an increased rate and activation state of CD8 alpha(+) cDC1, both in the TME and in the spleen. Observed increased rates of activated CD8(+) T cells, expressing CD69 and PD-1, were related to both increased CD8 alpha(+) cDC1 rates and M1/M2 shifts in tumor and spleen. In conclusion, the myeloid modulatory properties of ORCA-010 in melanoma, resulting in recruitment and activation of T cells, could enhance the antitumor efficacy of PD-1 blockade.Peer reviewe

Quantitative Virus-Associated RNA Detection to Monitor Oncolytic Adenovirus Replication

Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.</p

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor

AbstractVirotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer

Adenovirus Armed With TNFa and IL2 Added to aPD-1 Regimen Mediates Antitumor Efficacy in Tumors Refractory to aPD-1

Immune checkpoint inhibitors such as anti-PD-1 have revolutionized the field of oncology over the past decade. Nevertheless, the majority of patients do not benefit from them. Virotherapy is a flexible tool that can be used to stimulate and/or recruit different immune populations. T-cell enabling virotherapy could enhance the efficacy of immune checkpoint inhibitors, even in tumors resistant to these inhibitors. The T-cell potentiating virotherapy used here consisted of adenoviruses engineered to express tumor necrosis factor alpha and interleukin-2 in the tumor microenvironment. To study virus efficacy in checkpoint-inhibitor resistant tumors, we developed an anti-PD-1 resistant melanoma model in vivo. In resistant tumors, adding virotherapy to an anti-PD-1 regimen resulted in increased survival (p=0.0009), when compared to anti-PD-1 monotherapy. Some of the animals receiving virotherapy displayed complete responses, which did not occur in the immune checkpoint-inhibitor monotherapy group. When adenoviruses were delivered into resistant tumors, there were signs of increased CD8 T-cell infiltration and activation, which - together with a reduced presence of M2 macrophages and myeloid-derived suppressor cells - could explain those results. T-cell enabling virotherapy appeared as a valuable tool to counter resistance to immune checkpoint inhibitors. The clinical translation of this approach could increase the number of cancer patients benefiting from immunotherapies.Peer reviewe

Anatomical Differences Determine Distribution of Adenovirus after Convection-Enhanced Delivery to the Rat Brain

Background: Convection-enhanced delivery (CED) of adenoviruses offers the potential of widespread virus distribution in the brain. In CED, the volume of distribution (Vd) should be related to the volume of infusion (Vi) and not to dose, but when using adenoviruses contrasting results have been reported. As the characteristics of the infused tissue can affect convective delivery, this study was performed to determine the effects of the gray and white matter on CED of adenoviruses and similar sized super paramagnetic iron oxide nanoparticles (SPIO). Methodology/Principal Findings: We convected AdGFP, an adenovirus vector expressing Green Fluorescent Protein, a virus sized SPIO or trypan blue in the gray and white matter of the striatum and external capsule of Wistar rats and towards orthotopic infiltrative brain tumors. The resulting Vds were compared to Vi and transgene expression to SPIO distribution. Results show that in the striatum Vd is not determined by the Vi but by the infused virus dose, suggesting diffusion, active transport or receptor saturation rather than convection. Distribution of virus and SPIO in the white matter is partly volume dependent, which is probably caused by preferential fluid pathways from the external capsule to the surrounding gray matter, as demonstrated by co-infusing trypan blue. Distant tumors were reached using the white matter tracts but tumor penetration was limited. Conclusions/Significance: CED of adenoviruses in the rat brain and towards infiltrative tumors is feasible when regional anatomical differences are taken into account while SPIO infusion could be considered to validate proper catheter positioning and predict adenoviral distribution

Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function

Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers

Constitutively active GSK3 beta as a means to bolster dendritic cell functionality in the face of tumor-mediated immune suppression

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3 beta (GSK3 beta) as a pivotal kinase in both DC development and suppression. GSK3 beta inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3 beta induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3 beta activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.Peer reviewe

WEE1 inhibition sensitizes osteosarcoma to radiotherapy

<p>Abstract</p> <p>Background</p> <p>The use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G₂cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G₂arrest and could sensitize OS cells to irradiation induced cell death.</p> <p>Methods</p> <p>WEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.</p> <p>Results</p> <p>WEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G₂arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.</p> <p>Conclusion</p> <p>We show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G₂checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.</p

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