SnapShot: Cancer Immunotherapy with Oncolytic Viruses.

Oncolytic viruses (OVs) preferentially infect and kill cancer cells without harming normal cells. OVs can revert cancer-associated immune suppression and initiate clinically meaningful antitumor immune responses. OVs and their resultant immunological events can act at both primary and metastatic sites. Thus, OVs can be exploited for cancer gene therapies and immunotherapies alone or in combination with other interventions, including immune checkpoint blockade.

Optimal delivery of RNA interference by viral vectors for cancer therapy.

In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.

Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, sensitizes cancer cells to VSVΔ51 oncolytic virotherapy.

The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Safety and efficacy of autologous whole cell vaccines in hematologic malignancies: A systematic review and meta-analysis.

Autologous cell vaccines use a patient's tumor cells to stimulate a broad antitumor response in vivo. This approach shows promise for treating hematologic cancers in early phase clinical trials, but overall safety and efficacy remain poorly described. We conducted a systematic review assessing the use of autologous cell vaccination in treating hematologic cancers. Primary outcomes of interest were safety and clinical response, with secondary outcomes including survival, relapse rate, correlative immune assays and health-quality related metrics. We performed a search of MEDLINE, Embase and the Cochrane Register of Controlled Trials including any interventional trial employing an autologous, whole cell product in any hematologic malignancy. Risk of bias was assessed using a modified Institute of Health Economics tool. Across 20 single arm studies, only 341 of 592 enrolled participants received one or more vaccinations. Primary reasons for not receiving vaccination included rapid disease progression/death and manufacturing challenges. Overall, few high-grade adverse events were observed. One death was reported and attributed to a GM-CSF producing allogeneic cell line co-administered with the autologous vaccine. Of 58 evaluable patients, the complete response rate was 21.0% [95% CI, 10.4%-37.8%)] and overall response rate was 35.8% (95% CI, 24.4%-49.0%). Of 97 evaluable patients for survival, the 5-years overall survival rate was 64.9% (95% CI, 52.6%-77.2%) and disease-free survival was 59.7% (95% CI, 47.7%-71.7%). We conclude that, in hematologic malignancies, based on limited available data, autologous cell vaccines are safe and display a trend towards efficacy but that challenges exist in vaccine manufacture and administration.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis.

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.

Enhancement of oncolytic virotherapy by vanadium(V) dipicolinates.

Oncolytic viruses rewire the immune system and can lead to long-lasting antitumor defenses against primary and metastatic tumors. However, results from clinical studies have shown heterogeneity in responses suggesting that multiplexed approaches may be necessary to consistently generate positive outcomes in patients. To this end, we explored the combination of oncolytic rhabdovirus VSV∆51 with vanadium(V) dipicolinate derivatives, which have already been explored for their antidiabetic properties in animal models. The combination of vanadium-based dipicolinate compounds with VSV∆51 significantly increased viral replication and cytotoxicity in the human renal cell carcinoma cell line 786-0. The effects of three vanadium(V)-dipicolinate coordination complexes ([VO2dipic]-, [VO2dipic-OH]- and [VO2dipic-Cl]- with -OH or -Cl in the para position) were compared to that of the simple salts using spectroscopy and speciation profiles. Like the vanadate salts and the vanadyl cation, all dioxovanadium(V) dipicolinate complexes tested were found to increase viral infection and cytotoxicity when used in combination with VSV∆51. Viral sensitization is dependent on the vanadium since free dipicolinate ligands exerted no effect on viral infection and viability. The ability of these complexes to interact with interfaces and the stability of the complexes were evaluated under physiological conditions. Results indicate that these complexes undergo hydrolysis in cell culture media thereby generating vanadate. The vanadium dipicolinate derivatives in the context of immunovirotherapy shares similarities with previous studies exploring the antidiabetic properties of the compounds. The synergy between vanadium compounds and the oncolytic virus suggests that these compounds may be valuable in the development of novel and effective pharmaco-viral therapies.

Multi-modal Potentiation of Oncolytic Virotherapy by Vanadium Compounds.

Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in tumor cells. However, the efficacy of OVs as single agents remains limited. We introduce a strategy that boosts the therapeutic efficacy of OVs by combining their activity with immuno-modulating, small molecule protein tyrosine phosphatase inhibitors. We report that vanadium-based phosphatase inhibitors enhance OV infection in vitro and ex vivo, in resistant tumor cell lines. Furthermore, vanadium compounds increase antitumor efficacy in combination with OV in several syngeneic tumor models, leading to systemic and durable responses, even in models otherwise refractory to OV and drug alone. Mechanistically, this involves subverting the antiviral type I IFN response toward a death-inducing and pro-inflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced antitumor immune stimulation. Overall, we showcase a new ability of vanadium compounds to simultaneously maximize viral oncolysis and systemic anticancer immunity, offering new avenues for the development of improved immunotherapy strategies.

Oncolytic Maraba Virus MG1 as a Treatment for Sarcoma.

The poor prognosis of patients with advanced bone and soft-tissue sarcoma has not changed in the past several decades, highlighting the necessity for new therapeutic approaches. Immunotherapies, including oncolytic viral (OV) therapy, have shown great promise in a number of clinical trials for a variety of tumor types. However, the effective application of OV in treating sarcoma still remains to be demonstrated. Although few pre-clinical studies using distinct OVs have been performed and demonstrated therapeutic benefit in sarcoma models, a side-by-side comparison of clinically relevant OV platforms has not been performed. Four clinically relevant OV platforms (Reovirus, Vaccinia virus, Herpes-simplex virus and Rhabdovirus) were screened for their ability to infect and kill human and canine sarcoma cell lines in vitro, and human sarcoma specimens ex vivo. In vivo treatment efficacy was tested in a murine model. The rhabdovirus MG1 demonstrated the highest potency in vitro. Ex vivo, MG1 productively infected more than 80% of human sarcoma tissues tested, and treatment in vivo led to a significant increase in long-lasting cures in sarcoma-bearing mice. Importantly, MG1 treatment induced the generation of memory immune response that provided protection against a subsequent tumor challenge. This study opens the door for the use of MG1-based oncolytic immunotherapy strategies as treatment for sarcoma or as a component of a combined therapy.

Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans.

The efficacy and safety of biological molecules in cancer therapy, such as peptides and small interfering RNAs (siRNAs), could be markedly increased if high concentrations could be achieved and amplified selectively in tumour tissues versus normal tissues after intravenous administration. This has not been achievable so far in humans. We hypothesized that a poxvirus, which evolved for blood-borne systemic spread in mammals, could be engineered for cancer-selective replication and used as a vehicle for the intravenous delivery and expression of transgenes in tumours. JX-594 is an oncolytic poxvirus engineered for replication, transgene expression and amplification in cancer cells harbouring activation of the epidermal growth factor receptor (EGFR)/Ras pathway, followed by cell lysis and anticancer immunity. Here we show in a clinical trial that JX-594 selectively infects, replicates and expresses transgene products in cancer tissue after intravenous infusion, in a dose-related fashion. Normal tissues were not affected clinically. This platform technology opens up the possibility of multifunctional products that selectively express high concentrations of several complementary therapeutic and imaging molecules in metastatic solid tumours in humans.

Combination of Paclitaxel and MG1 oncolytic virus as a successful strategy for breast cancer treatment.

BACKGROUND: Breast cancer is the most common malignant disease amongst Western women. The lack of treatment options for patients with chemotherapy-resistant or recurrent cancers is pushing the field toward the rapid development of novel therapies. The use of oncolytic viruses is a promising approach for the treatment of disseminated diseases like breast cancer, with the first candidate recently approved by the Food and Drug Administration for use in patients. In this report, we demonstrate the compatibility of oncolytic virotherapy and chemotherapy using various murine breast cancer models. This one-two punch has been explored in the past by several groups with different viruses and drugs and was shown to be a successful approach. Our strategy is to combine Paclitaxel, one of the most common drugs used to treat patients with breast cancer, and the oncolytic Rhabdovirus Maraba-MG1, a clinical trial candidate in a study currently recruiting patients with late-stage metastatic cancer. METHODS: We used the EMT6, 4 T1 and E0771 murine breast cancer models to evaluate in vitro and in vivo the effects of co-treatment with MG1 and Paclitaxel. Treatment-induced cytotoxicity was assessed and plaque assays, flow cytometry, microscopy and immunocytochemistry analysis were performed to quantify virus production and transgene expression. Orthotopically implanted tumors were measured during and after treatment to evaluate efficacy and Kaplan-Meier survival curves were generated. RESULTS: Our data demonstrate not only the compatibility of the treatments, but also their synergistic cytopathic activity. With Paclitaxel, EMT6 and 4 T1 tumors demonstrated increased virus production both in vitro and in vivo. Our results also show that Paclitaxel does not impair the safety profile of the virus treatment. Importantly, when combined, MG1 and the drug controlled tumor growth and prolonged survival. CONCLUSIONS: The combination of MG1 and Paclitaxel improved efficacy in all of the breast cancer models we tested and thus is a promising alternative approach for the treatment of patients with refractory breast cancer. Our strategy has potential for rapid translation to the clinic, given the current clinical status of both agents.

From scourge to cure: tumour-selective viral pathogenesis as a new strategy against cancer.

Tumour mutations corrupt cellular pathways, and accumulate to disrupt, dysregulate, and ultimately avoid mechanisms of cellular control. Yet the very changes that tumour cells undergo to secure their own growth success also render them susceptible to viral infection. Enhanced availability of surface receptors, disruption of antiviral sensing, elevated metabolic activity, disengagement of cell cycle controls, hyperactivation of mitogenic pathways, and apoptotic avoidance all render the malignant cell environment highly supportive to viral replication. The therapeutic use of oncolytic viruses (OVs) with a natural tropism for infecting and subsequently lysing tumour cells is a rapidly progressing area of cancer research. While many OVs exhibit an inherent degree of tropism for transformed cells, this can be further promoted through pharmacological interventions and/or the introduction of viral mutations that generate recombinant oncolytic viruses adapted to successfully replicate only in a malignant cellular environment. Such adaptations that augment OV tumour selectivity are already improving the therapeutic outlook for cancer, and there remains tremendous untapped potential for further innovation.

Resistance to two heterologous neurotropic oncolytic viruses, Semliki Forest virus and vaccinia virus, in experimental glioma.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference.

HDAC inhibition suppresses primary immune responses, enhances secondary immune responses, and abrogates autoimmunity during tumor immunotherapy.

Histone deacetylase inhibitors (HDACi) can modulate innate antiviral responses and render tumors more susceptible to oncolytic viruses (OVs); however, their effects on adaptive immunity in this context are largely unknown. Our present study reveals an unexpected property of the HDACi MS-275 that enhances viral vector-induced lymphopenia leading to selective depletion of bystander lymphocytes and regulatory T cells while allowing expansion of antigen-specific secondary responses. Coadministration of vaccine plus drug during the boosting phase focuses the immune response on the tumor by suppressing the primary immune response against the vaccine vector and enhancing the secondary response against the tumor antigen. Furthermore, improvement of T cell functionality was evident suggesting that MS-275 can orchestrate a complex array of effects that synergize immunotherapy and viral oncolysis. Surprisingly, while MS-275 dramatically enhanced efficacy, it suppressed autoimmune pathology, profoundly improving the therapeutic index.

Immune profiling of dedifferentiated liposarcoma and identification of novel antigens for targeted immunotherapy.

Dedifferentiated liposarcoma (DDLS) is an aggressive, recurring sarcoma with limited treatments. T-cell immunotherapies selectively target malignant cells, holding promise against DDLS. The development of successful immunotherapy for DDLS requires a thorough evaluation of the tumor immune microenvironment and the identification and characterization of targetable immunogenic tumor antigens. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens, we used the nCounter NanoString platform, analyzing gene expression profiles across 29 DDLS and 10 healthy adipose tissue samples. Hierarchical clustering of tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumors and 14 non-inflamed tumors, demonstrating tumor heterogeneity within this sarcoma subtype. Among the identified antigens, PBK and TTK exhibited substantial upregulation in mRNA expression compared to healthy adipose tissue controls, further corroborated by positive protein expression by IHC. This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS, and provides a novel targetable antigen in DDLS. The results of this study lay the groundwork for the development of a novel immunotherapy for this highly aggressive sarcoma.

Tepilamide Fumarate as a Novel Potentiator of Virus-Based Therapy.

Oncolytic virotherapy, using viruses such as vesicular stomatitis virus (VSVΔ51) and Herpes Simplex Virus-1 (HSV-1) to selectively attack cancer cells, faces challenges such as cellular resistance mediated by the interferon (IFN) response. Dimethyl fumarate (DMF) is used in the treatment of multiple sclerosis and psoriasis and is recognized for its anti-cancer properties and has been shown to enhance both VSVΔ51 and HSV-1 oncolytic activity. Tepilamide fumarate (TPF) is a DMF analog currently undergoing clinical trials for the treatment of moderate-to-severe plaque psoriasis. The aim of this study was to evaluate the potential of TPF in enhancing the effectiveness of oncolytic viruses. In vitro, TPF treatment rendered 786-0 carcinoma cells more susceptible to VSVΔ51 infection, leading to increased viral replication. It outperformed DMF in both increasing viral infection and increasing the killing of these resistant cancer cells and other cancer cell lines tested. Ex vivo studies demonstrated TPF's selective boosting of oncolytic virus infection in cancer cells without affecting healthy tissues. Effectiveness was notably high in pancreatic and ovarian tumor samples. Our study further indicates that TPF can downregulate the IFN pathway through a similar mechanism to DMF, making resistant cancer cells more vulnerable to viral infection. Furthermore, TPF's impact on gene therapy was assessed, revealing its ability to enhance the transduction efficiency of vectors such as lentivirus, adenovirus type 5, and adeno-associated virus type 2 across various cell lines. This data underscore TPF's potential role in not only oncolytic virotherapy but also in the broader application of gene therapy. Collectively, these findings position TPF as a promising agent in oncolytic virotherapy, warranting further exploration of its therapeutic potential.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.