The emergence of combinatorial strategies in the development of RNA oncolytic virus therapies

Oncolytic viruses (OVs) represent an exciting new biological approach to cancer therapy. In particular, RNA viruses have emerged as potent agents for oncolytic virotherapy because of their capacity to specifically target and destroy tumour cells while sparing normal cells and tissues. Several barriers remain in the development of OV therapy, including poor penetration into the tumour mass, inefficient virus replication in primary cancers, and tumour-specific resistance to OV-mediated killing. The combination of OVs with cytotoxic agents, such as small molecule inhibitors of signalling or immunomodulators, as well as stealth delivery of therapeutic viruses have shown promise as novel experimental strategies to overcome resistance to viral oncolysis. These agents complement OV therapy by unblocking host pathways, delivering viruses with greater efficiency and/or increasing virus proliferation at the tumour site. In this review, we summarize recent development of these concepts, the potential obstacles, and future prospects for the clinical utilization of RNA OVs in cancer therapy.     

Oncolytic virotherapy: molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment.     

Oncolytic viruses and histone deacetylase inhibitors—A multi-pronged strategy to target tumor cells

Oncolytic viruses (OVs) have shown promise as cancer therapeutics in pre-clinical and clinical testing; however, it is unlikely that OVs will constitute a stand-alone treatment. Histone deacetylase inhibitors (HDIs) represent a class of anticancer agents known to influence epigenetic modifications of chromatin, alter gene expression and manipulate a variety of signaling pathways, in some cases blunting the cellular antiviral response. Recent studies have shown that combining OV therapy with HDI treatment enhances viral replication and synergistically induces the killing of cancer cells in vitro and in vivo, an effect that has now been demonstrated in variety of virus/HDI combinations. This review discusses the results obtained with the different OV/HDI combinations, the rationale supporting these combinations and the advantages for oncolytic virus therapy.     

Determinants of the efficacy of viro-immunotherapy: A review

Oncolytic virus immunotherapy is rapidly gaining interest in the field of immunotherapy against cancer. The minimal toxicity upon treatment and the dual activity of direct oncolysis and immune activation make therapy with oncolytic viruses (OVs) an interesting treatment modality. The safety and efficacy of several OVs have been assessed in clinical trials and, so far, the Food and Drug Administration (FDA) has approved one OV. Unfortunately, most treatments with OVs have shown suboptimal responses in clinical trials, while they appeared more promising in preclinical studies, with tumours reducing after immune cell influx. In several clinical trials with OVs, parameters such as virus replication, virus-specific antibodies, systemic immune responses, immune cell influx into tumours and tumour-specific antibodies have been studied as predictors or correlates of therapy efficacy. In this review, these studies are summarized to improve our understanding of the determinants of the efficacy of OV therapies in humans and to provide insights for future developments in the viro-immunotherapy treatment field.     

Double trouble for tumours: Exploiting the tumour microenvironment to enhance anticancer effect of oncolytic viruses

Oncolytic viruses (OVs) are selected based on their ability to eliminate malignancies by direct infection and lysis of cancer cells. Originally, OVs were designed to target malignancies by taking advantage of the defects of cancer cells observed in vitro. Subsequent analysis of virus delivery and spread in vivo has demonstrated that the tumour microenvironment can impede the ability of OVs to effectively infect and spread. Despite this limitation, it is becoming increasingly evident that OVs are also able to take advantage of certain features of the tumour microenvironment. Currently, a growing body of the literature is delineating the complex interaction between OVs and the tumour microenvironment that results in an additional therapeutic activity; these viruses are able to target malignancies by rapidly altering the tumour microenvironment into a milieu that potentiates anticancer activity. Herein, we discuss strategies that capitalize on the multifaceted relationship between OVs and host–tumour interactions that enhance the toxicity of OVs to the tumour microenvironment.     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Current landscape and perspective of oncolytic viruses and their combination therapies

Oncolytic virotherapy has become an important strategy in cancer immunotherapy. Oncolytic virus (OV) can reshape the tumor microenvironment (TME) through its replication-mediated oncolysis and transgene-produced anticancer effect, inducing an antitumor immune response and creating favorable conditions for the combination of other therapeutic measures. Extensive preclinical and clinical data have suggested that OV-based combination therapy has definite efficacy and promising prospects. Recently, several clinical trials of oncolytic virotherapy combined with immunotherapy have made breakthroughs. This review comprehensively elaborates the OV types and their targeting mechanisms, the selection of anticancer genes armed in OVs, and the therapeutic modes of action and strategies of OVs to provide a theoretical basis for the better design and construction of OVs and the optimization of OV-based therapeutic strategies.     

Oncolytic Immunotherapy: Can’t Start a Fire Without a Spark

Recent advances in cancer immunotherapy have renewed interest in oncolytic viruses (OVs) as a synergistic platform for the development of novel antitumor strategies. Cancer cells adopt multiple mechanisms to evade and suppress antitumor immune responses, essentially establishing a non-immunogenic (‘cold’) tumor microenvironment (TME), with poor T-cell infiltration and low mutational burden. Limitations to the efficacy of immunotherapy still exist, especially for a variety of solid tumors, where new approaches are necessary to overcome physical barriers in the TME and to mitigate adverse effects associated with current immunotherapeutics. OVs offer an attractive alternative by inducing direct oncolysis, immunogenic cell death, and immune stimulation. These multimodal mechanisms make OVs well suited to reprogram non-immunogenic tumors and TME into inflamed, immunogenic (‘hot’) tumors; enhanced release of tumor antigens by dying cancer cells is expected to augment T-cell infiltration, thereby eliciting potent antitumor immunity. Advances in virus engineering and understanding of tumor biology have allowed the optimization of OV-tumor selectivity, oncolytic potency, and immune stimulation. However, OV antitumor activity is likely to achieve its greatest potential as part of combinatorial strategies with other immune or cancer therapeutics.     

Mechanical effects of the surface ectoderm on optic vesicle morphogenesis in the chick embryo

Precise shaping of the eye is crucial for proper vision. Here, we use experiments on chick embryos along with computational models to examine the mechanical factors involved in the formation of the optic vesicles (OVs), which grow outward from the forebrain of the early embryo. First, mechanical dissections were used to remove the surface ectoderm (SE), a membrane that contacts the outer surfaces of the OVs. Principal components analysis of OV shapes suggests that the SE exerts asymmetric loads that cause the OVs to flatten and shear caudally during the earliest stages of eye development and later to bend in the caudal and dorsal directions. These deformations cause the initially spherical OVs to become pear-shaped. Exposure to the myosin II inhibitor blebbistatin reduced these effects, suggesting that cytoskeletal contraction controls OV shape by regulating tension in the SE. To test the physical plausibility of these interpretations, we developed 2-D finite-element models for frontal and transverse cross-sections of the forebrain, including frictionless contact between the SE and OVs. With geometric data used to specify differential growth in the OVs, these models were used to simulate each experiment (control, SE removed, no contraction). For each case, the predicted shape of the OV agrees reasonably well with experiments. The results of this study indicate that differential growth in the OV and external pressure exerted by the SE are sufficient to cause the global changes in OV shape observed during the earliest stages of eye development.     

The viral approach to breast cancer immunotherapy

Despite years of intensive research, breast cancer remains the leading cause of death in women worldwide. New technologies including oncolytic virus therapies, virus, and phage display are among the most powerful and advanced methods that have emerged in recent years with potential applications in cancer prevention and treatment. Oncolytic virus therapy is an interesting strategy for cancer treatment. Presently, a number of viruses from different virus families are under laboratory and clinical investigation as oncolytic therapeutics. Oncolytic viruses (OVs) have been shown to be able to induce and initiate a systemic antitumor immune response. The possibility of application of a multimodal therapy using a combination of the OV therapy with immune checkpoint inhibitors and cancer antigen vaccination holds a great promise in the future of cancer immunotherapy. Display of immunologic peptides on bacterial viruses (bacteriophages) is also increasingly being considered as a new and strong cancer vaccine delivery strategy. In phage display immunotherapy, a peptide or protein antigen is presented by genetic fusions to the phage coat proteins, and the phage construct formulation acts as a protective or preventive vaccine against cancer. In our laboratory, we have recently tested a few peptides (E75, AE37, and GP2) derived from HER2/neu proto-oncogene as vaccine delivery modalities for the treatment of TUBO breast cancer xenograft tumors of BALB/c mice. Here, in this paper, we discuss the latest advancements in the applications of OVs and bacterial viruses display systems as new and advanced modalities in cancer immune therapeutics.     

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a “push and pull” manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.     

Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.     

The pros and cons of interferons for oncolytic virotherapy

Interferons (IFN) are potent immune stimulators that play key roles in both innate and adaptive immune responses. They are considered the first line of defense against viral pathogens and can even be used as treatments to boost the immune system. While viruses are usually seen as a threat to the host, an emerging class of cancer therapeutics exploits the natural capacity of some viruses to directly infect and kill cancer cells. The cancer-specificity of these bio-therapeutics, called oncolytic viruses (OVs), often relies on defective IFN responses that are frequently observed in cancer cells, therefore increasing their vulnerability to viruses compared to healthy cells. To ensure the safety of the therapy, many OVs have been engineered to further activate the IFN response. As a consequence of this IFN over-stimulation, the virus is cleared faster by the immune system, which limits direct oncolysis. Importantly, the therapeutic activity of OVs also relies on their capacity to trigger anti-tumor immunity and IFNs are key players in this aspect. Here, we review the complex cancer–virus–anti-tumor immunity interplay and discuss the diverse functions of IFNs for each of these processes.     

溶瘤病毒载体研究进展

溶瘤病毒(oncolytic virus,OVs)疗法是治疗肿瘤的一种新方法,它可通过直接杀死肿瘤细胞并引起机体的免疫反应发挥作用.然而天然溶瘤病毒有一定的局限性,因此需将各种不同的病毒作为载体,通过基因修饰的方法增强或减弱病毒毒力并导入新的功能性基因,以提高其溶瘤作用.目前可以作为溶瘤病毒载体的有单纯疱疹病毒、腺病毒、牛痘病毒、水泡性口炎病毒、麻疹病毒、腮腺炎病毒、脊髓灰质炎病毒等.这些病毒载体均可通过不同的基因修饰方法,靶向性感染并杀死不同的肿瘤细胞,获得较好的溶瘤作用.本文综述了几种溶瘤病毒载体的基因修饰方法,以及修饰后的溶瘤效果.     

A homolog of interleukin-10 is encoded by the poxvirus orf virus.

A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function.     

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.     

Redirecting oncolytic viruses: Engineering opportunists to take control of the tumour microenvironment

The transformation of healthy cells to malignant often drives them to become inherently susceptible to viral infection as a trade-off to achieve uninhibited growth and immune escape. Enter oncolytic viruses (OVs), an exciting class of viruses that specifically infect cancer cells, leaving healthy tissue unharmed. Unfortunately, there is more to this story. Tumours are much more than a group of cancer cells, the surrounding tumour microenvironment (TME) comprises a collection of cells which influence and nourish the development and spread of the tumour. While initially quite promising, OV therapy has been met with a myriad of barriers due to the unwelcoming nature of the TME. Riddled with immunosuppressive factors and physical barriers, many tumours have proven impenetrable by OVs. Herein, we review the diverse array of approaches being used to target each component of the TME from enhancing entry into specific tumour types, breaking through the dense tumour stroma, eliminating cancer stem cells, and activating the immune system. We highlight the value of combination approaches which have led to complete successes in several in vivo models, some of which have entered clinical development.     

The two-faces of NK cells in oncolytic virotherapy

Oncolytic viruses (OVs) are immunotherapeutics capable of directly killing cancer cells and with potent immunostimulatory properties. OVs exert their antitumor effect, at least partially, by activating the antitumor immune response, of which NK cells are an important component. However, if on the one hand increasing evidence revealed that NK cells are important mediators of oncolytic virotherapy, on the other hand, NK cells have evolved to fight viral infections, and therefore they can have a detrimental effect for the efficacy of OVs. In this review, we will discuss the dichotomy between the antitumor and antiviral functions of NK cells related to oncolytic virotherapy. We will also review NK cell-based and OV-based therapies, engineered OVs aimed at enhancing immune stimulation, and combination therapies involving OVs and NK cells currently used in cancer immunotherapy.     

A novel virus carrier state to evaluate immunotherapeutic regimens: regulatory T cells modulate the pathogenicity of antiviral memory cells

Restrictions in the diversity of an adaptive immune repertoire can facilitate viral persistence. Because a host afflicted with an immune deficiency is not likely to purge a persistent infection using endogenous mechanisms, it is important to explore adoptive therapies to supplement the host with a functional immune defense. In this study, we describe a virus carrier state that results from introducing lymphocytic choriomeningitis virus (LCMV) into adult mice possessing a restricted T cell repertoire. On infection of these mice, LCMV establishes systemic persistence, and within the CNS the virus infects astrocytes (and later oligodendrocytes) rather than its traditional parenchymal target neurons. To determine whether LCMV could be purged from a novel target selection in the absence of an endogenous immune repertoire, we adoptively transferred virus-specific memory cells into adult carrier mice. The memory cells purged virus from the periphery as well as the CNS, but they induced fatalities not typically associated with adoptive immunotherapy. When the repertoire of the recipient mice was examined, a deficiency in natural regulatory T cells was noted. We therefore supplemented carrier mice with regulatory T cells and simultaneously performed adoptive immunotherapy. Cotransfer of regulatory T cells significantly reduced mortality while still permitting the antiviral memory cells to purge the persistent infection. These data indicate that regulatory T cells can be used therapeutically to lessen the pathogenicity of virus-specific immune cells in an immunodeficient host. We also propose that the novel carrier state described herein will facilitate the study of immunotherapeutic regimens.     

Tumour vasculature: Friend or foe of oncolytic viruses?

In the past two decades there have been substantial advances in understanding the anti-cancer mechanisms of oncolytic viruses (OVs). OVs can mediate their effects directly, by preferentially infecting and killing tumour cells. Additionally, OVs can indirectly generate anti-tumour immune responses. These differing mechanisms have led to a paradoxical divergence in strategies employed to further increase the potency of oncolytic virotherapies. On one hand, the tumour neovasculature is seen as a vital lifeline to the survival of the tumour, leading some to use OVs to target the tumour vasculature in hopes to starve cancers. Therapeutics causing vascular collapse can potentiate tumour hypoxia, nutrient restriction and pro-inflammatory cytokine release, which has shown promise in oncological studies. On the other hand, the same vasculature plays an important role for the dissemination of OVs, trafficking of effector cells and other therapeutics, which has prompted researchers to find ways of normalizing the vasculature to enhance infiltration of leukocytes and delivery of therapeutic agents. This article describes the recent developments of therapies aimed to shut down versus normalize tumour vasculature in order to inform researchers striving to optimize OV-based therapies.