Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 de novo. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. IMPORTANCE As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.

Radiofrequency Hyperthermia Enhances Locally Delivered Oncolytic Immuno-Virotherapy for Pancreatic Adenocarcinoma.

PURPOSE: To investigate the effect of radiofrequency hyperthermia (RFH)-enhanced oncolytic immuno-virotherapy on in vitro pancreatic adenocarcinoma cell line and in vivo rat pancreatic cancer model. MATERIALS AND METHODS: Rat pancreatic adenocarcinoma cell line and 24 Lewis rats with orthotopic pancreatic adenocarcinomas underwent treatment with either (1) oncolytic virotherapy (talimogene laherparepvec [T-VEC]) plus RFH at 42 °C for 30 min; (2) oncolytic virotherapy-only; (3) RFH-only; or (4) saline (control). MTS assays and flow cytometry were used to analyze tumor cell viability and apoptosis levels 24 h after treatment. In the in vivo studies, bioluminescence optical/x-ray imaging and ultrasound imaging was used to assess tumor viability and size 7 and 14 days after treatment. Histopathologic analysis was performed after hematoxylin and eosin staining, TUNEL, Ki-67, and immunohistochemical staining with CD8 and ANK61. RESULTS: Combination therapy (T-VEC + RFH) induced decreased cell viability and increased cell apoptosis compared to T-VEC alone, RFH alone, or control. Optical/x-ray imaging and ultrasound imaging demonstrated decreased tumor bioluminescent signal and tumor volume relative to baseline after combination therapy compared to T-VEC alone, RFH alone, or control. Histopathology demonstrated decreased tumor volume and cell proliferation, increased CD8+ T cell and NK cell infiltration in tumors treated with the combination therapy compared to other three groups. CONCLUSION: RFH enhances locally delivered oncolytic immuno-virotherapy for pancreatic adenocarcinoma, with decreased cell viability and increased apoptosis observed after combination therapy in vitro, and decreased cell viability and tumor volume and increased immune cell infiltrate observed after combination therapy in vivo.

Oncolytic Virotherapy Treatment of Breast Cancer: Barriers and Recent Advances.

Oncolytic virotherapy (OV) is an emerging class of immunotherapeutic drugs. Their mechanism of action is two-fold: direct cell lysis and unmasking of the cancer through immunogenic cell death, which allows the immune system to recognize and eradicate tumours. Breast cancer is the most common cancer in women and is challenging to treat with immunotherapy modalities because it is classically an immunogenically "cold" tumour type. This provides an attractive niche for OV, given viruses have been shown to turn "cold" tumours "hot," thereby opening a plethora of treatment opportunities. There has been a number of pre-clinical attempts to explore the use of OV in breast cancer; however, these have not led to any meaningful clinical trials. This review considers both the potential and the barriers to OV in breast cancer, namely, the limitations of monotherapy and the scope for combination therapy, improving viral delivery and challenges specific to the breast cancer population (e.g., tumour subtype, menopausal status, age).

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

The role of telomerase and viruses interaction in cancer development, and telomerase-dependent therapeutic approaches.

Human telomerase reverse transcriptase (hTERT) is an enzyme that is critically involved in elongating and maintaining telomeres length to control cell life span and replicative potential. Telomerase activity is continuously expressed in human germ-line cells and most cancer cells, whereas it is suppressed in most somatic cells. In normal cells, by reducing telomerase activity and progressively shortening the telomeres, the cells progress to the senescence or apoptosis process. However, in cancer cells, telomere lengths remain constant due to telomerase's reactivation, and cells continue to proliferate and inhibit apoptosis, and ultimately lead to cancer development and human death due to metastasis. Studies demonstrated that several DNA and RNA oncoviruses could interact with telomerase by integrating their genome sequence within the host cell telomeres specifically. Through the activation of the hTERT promoter and lengthening the telomere, these cells contributes to cancer development. Since oncoviruses can activate telomerase and increase hTERT expression, there are several therapeutic strategies based on targeting the telomerase of cancer cells like telomerase-targeted peptide vaccines, hTERT-targeting dendritic cells (DCs), hTERT-targeting gene therapy, and hTERT-targeting CRISPR/Cas9 system that can overcome tumor-mediated toleration mechanisms and specifically apoptosis in cancer cells. This study reviews available data on the molecular structure of telomerase and the role of oncoviruses and telomerase interaction in cancer development and telomerase-dependent therapeutic approaches to conquest the cancer cells.

Oncolytic Viruses as a Platform for the Treatment of Malignant Brain Tumors.

Malignant brain tumors remain incurable diseases. Although much effort has been devoted to improving patient outcome, multiple factors such as the high tumor heterogeneity, the strong tumor-induced immunosuppressive microenvironment, and the low mutational burden make the treatment of these tumors especially challenging. Thus, novel therapeutic strategies are urgent. Oncolytic viruses (OVs) are biotherapeutics that have been selected or engineered to infect and selectively kill cancer cells. Increasingly, preclinical and clinical studies demonstrate the ability of OVs to recruit T cells and induce durable immune responses against both virus and tumor, transforming a "cold" tumor microenvironment into a "hot" environment. Besides promising clinical results as a monotherapy, OVs can be powerfully combined with other cancer therapies, helping to overcome critical barriers through the creation of synergistic effects in the fight against brain cancer. Although many questions remain to be answered to fully exploit the therapeutic potential of OVs, oncolytic virotherapy will clearly be part of future treatments for patients with malignant brain tumors.

Immunomodulatory effects of ablation therapy on tumors: Potentials for combination with immunotherapy.

As a promising area of tumor treatment, immunotherapies, such as immune checkpoint inhibitors, have been applied to various types of cancer. However, many patients do not respond to such therapies. Increasing application of tumor ablation therapy, a minimally invasive treatment, has been observed in the clinic. Although it can boost the anti-tumor immune response of patients in many ways, ablation alone is not sufficient to remove the tumor completely or stop tumor recurrence in the long term. Currently, there is emerging research focusing on ablation in combination with immunotherapy, aiming to confirm the therapeutic value of this treatment for cancer patients. Hence, in this article, we review the classification, guideline recommendations, and immunomodulatory effects of ablation therapy, as well as the pre-clinical and clinical research of this combination therapy.

Virotherapy: From single agents to combinatorial treatments.

Virotherpay is emerging as a promising strategy against cancer, and three oncolytic viruses (OVs) have gained approval in different countries for the treatment of several cancer types. Beyond the capability to selectively infect, replicate and lyse cancer cells, OVs act through a multitude of events, including modification of the tumour micro/macro-environment as well as a complex modulation of the anti-tumour immune response by activation of danger signals and immunogenic cell death pathways. Most OVs show limited effects, depending on the viral platform and the interactions with the host. OVs used as monotherapy only in a minority of patients elicited a full response. Better outcomes were obtained using OVs in combination with other treatments, such as immune therapy or chemotherapy, suggesting that the full potential of OVs can be unleashed in combination with other treatment modalities. Here, we report the main described combination of OVs with conventional chemotherapeutic agents: platinum salts, mitotic inhibitors, anthracyclines and other antibiotics, anti-metabolites, alkylating agents and topoisomerase inhibitors. Additionally, our work provides an overview of OV combination with targeted therapies: histone deacetylase inhibitors, kinase inhibitors, monoclonal antibodies, inhibitors of DNA repair, inhibitors of the proteasome complex and statins that demonstrated enhanced OV anti-neoplastic activity. Although further studies are required to assess the best combinations to translate the results in the clinic, it is clear that combined therapies, acting with complementary mechanisms of action might be useful to target cancer lesions resistant to currently available treatments.

Combination therapy with oncolytic viruses and immune checkpoint inhibitors.

Introduction: Immune checkpoint inhibitors (ICI) have dramatically improved the outcome for cancer patients across multiple tumor types. However the response rates to ICI monotherapy remain relatively low, in part due to some tumors cultivating an inherently 'cold' immune microenvironment. Oncolytic viruses (OV) have the capability to promote a 'hotter' immune microenvironment which can improve the efficacy of ICI.Areas covered: In this article we conducted a literature search through Pubmed/Medline to identify relevant articles in both the pre-clinical and clinical settings for combining OVs with ICIs and discuss the impact of this approach on treatment as well as changes within the tumor microenvironment. We also explore the future directions of this novel combination strategy.Expert opinion: The imminent results of the Phase 3 study combining pembrolizumab with or without T-Vec injection are eagerly awaited. OV/ICI combinations remain one of the most promising avenues to explore in the success of cancer immunotherapy.

Combined therapy of oncolytic Newcastle disease virus and rhizomes extract of Rheum ribes enhances cancer virotherapy in vitro and in vivo.

Phytotherapy has been used to treat a different type of diseases including cancer for a long time, and it was a source for different active anti-tumor agents. Oncolytic Newcastle disease virus (AMHA1) are very promising anti-tumor therapy. Nevertheless, NDV-based monotherapeutics have not been very useful to some resistant tumors. Thus, the efficiency of oncolytic NDV must enhance by combining NDV with other novel therapies. The current study aimed to determine the possibility of improving the oncolytic effect induced by NDV through Rheum ribes rhizomes extract administration in vitro and in vivo. Methods, the in vitro study include exposure of the crude extract of Rheum ribes alone or NDV alone or combination of both agents for 72 h. The cancer cells tested were murine mammary adenocarcinoma AMN3, Human Rhabdomyosarcoma RD, and Human Glioblastoma AMGM5, and using rat embryo fibroblast REF as normal control cells. MTT cell viability assay was used and analyzed for possible synergism using the Chou-Talalay analysis method. In vivo experiment included study the combination and the monotherapeutic modalities in the transplanted murine mammary adenocarcinoma AM3 line and tumor sections analyzed by histopathology. Results, Combination therapy of NDV-R. ribes showed enhanced oncolytic activity on cancer cells. With no cytotoxicity on normal cells. In vivo study showed that monotherapeutic modalities had lower growth inhibitory effect on transplanted tumors in mice in compare to combination therapy. Histopathological examination revealed the broader area of necrosis in tumors treated by combination therapy. In conclusion, the novel combination recommended for clinical application for cancer therapy.

Considerations for Clinical Translation of MG1 Maraba Virus.

Oncolytic viral immunotherapy based on the MG1 Maraba platform has undergone extensive preclinical evaluation, resulting in the advancement of two programs into clinical trials. MG1 Maraba encoding tumor antigens (tumor associated antigens or viral antigens) are used to boost antitumor immunity, while MG1 Maraba infects tumors, causes oncolysis and transforms the tumor microenvironment. An overview of MG1 Maraba clinical development is outlined here, along with general considerations relating to the design of clinical trials for complex biologic products such as oncolytic viral immunotherapies. These include choice of patient population, optimized treatment regimen, and endpoints which provide early signals of activity and inform the late-stage development path of these agents with novel mechanisms of action.

SOCS4 expressed by recombinant HSV protects against cytokine storm in a mouse model.

Oncolytic viruses are genetically engineered viruses designed for the treatment of solid tumors, and are often coupled with the antitumor immunity of the host. The challenge of using oncolytic herpes simplex virus (oHSV) as an efficacious oncolytic agent is the potential host tissue damage caused by the production of a range of cytokines following intratumoral oHSV injection. An HSV­suppressor of cytokine signaling 4 (SOCS4) recombinant virus was created to investigate whether it inhibits cytokine storm. Recombinant HSV­SOCS4 and HSV­1(F) were used to infect mice, and levels of several representative cytokines, including monocyte chemoattractant protein­1, interleukin (IL)­1ß, tumor necrosis factor­α, IL­6 and interferon γ, in serum and bronchoalveolar lavage fluid (BALF) of infected mice were determined, and immune cells in BALF and spleen were enumerated. Lung damage, virus titers in the lung, body weight and survival rates of infected mice were also determined and compared between the two groups. The cytokine concentration of HSV­SOCS4­infected mice was significantly decreased compared with that of HSV­1(F)­infected mice in BALF and serum, and a smaller number of cluster of differentiation (CD)11b+ cells of BALF, and CD8+CD62L+ T cells and CD4+CD62L+ T cells of the spleen were also identified in HSV­SOCS4­infected mice. HSV­SOCS4­infected mice exhibited slight lung damage, a decrease in body weight loss and a 100% survival rate. The results of the present study indicated that SOCS4 protein may be a useful regulator to inhibit cytokine overproduction, and that HSV­SOCS4 may provide a possible solution to control cytokine storm and its consequences following induction by oncolytic virus treatment.

The advent of oncolytic virotherapy in oncology: The Rigvir® story.

Oncolytic viruses are a fast-developing cancer treatment field. Numerous viruses have been tested in clinical trials and three are approved. The first, Rigvir, is an immunomodulator with anti-tumour effect for treatment of melanoma, local treatment of skin and subcutaneous metastases of melanoma, for prevention of relapse and metastasis after radical surgery registered in Latvia, Georgia, Armenia and Uzbekistan. The aim of the present review is to summarize the development of Rigvir. Approximately 60 viruses were screened preclinically. Clinical safety and efficacy trials were with 5 oncolytic enteroviruses. Safety of the selected and melanoma-adapted ECHO-7 virus Rigvir was tested in over 180 patients with no severe adverse events observed. Pre-registration efficacy studies involved over 700 cancer patients: over 540 melanoma patients, and patients with late stage stomach (ca. 90), colorectal cancer (ca. 60), and other cancers. Patients were treated with Rigvir for 3 years after surgery and compared to immunotherapy: 3- and 5-year overall survival appeared to be increased in Rigvir treated patients. In post-marketing retrospective studies, Rigvir-treated stage II melanoma patients showed a 6.67-fold decreased risk for disease progression in comparison to those that had been observed according to guidelines, and stage IB and stage II melanoma patients that had received Rigvir therapy had 4.39-6.57-fold lower mortality. The results are confirmed and extended by case reports. Several immunological markers have been measured. In conclusion, Rigvir is an oncotropic and oncolytic virus for treatment of melanoma; the results will be confirmed and updated by modern clinical studies.

Combining Tumor Vaccination and Oncolytic Viral Approaches with Checkpoint Inhibitors: Rationale, Pre-Clinical Experience, and Current Clinical Trials in Malignant Melanoma.

The field of tumor immunology has faced many complex challenges over the last century, but the approval of immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte-associated protein 4 [CTLA4] and anti-programmed cell death-1 [PD-1]/PD-ligand 1 [PD-L1]) and talimogene laherparepvec (T-VEC) for the treatment of metastatic melanoma have awakened a new wave of interest in cancer immunotherapy. Additionally, combinations of vaccines and oncolytic viral therapies with immune checkpoint inhibitors and other systemic agents seem to be promising synergistic strategies to further boost the immune response against cancer. These combinations are undergoing clinical investigation, and if successful, will hopefully soon become available to patients. Here, we review key basic concepts of tumor-induced immune suppression in malignant melanoma, the historical perspective around vaccine development in melanoma, and advances in oncolytic viral therapies. We also discuss the emerging role for combination approaches with different immunomodulatory agents as well as new developments in personalized immunization approaches.

Oncolytic Viral Therapy and the Immune System: A Double-Edged Sword Against Cancer.

Oncolytic viral therapy is a new promising strategy against cancer. Oncolytic viruses (OVs) can replicate in cancer cells but not in normal cells, leading to lysis of the tumor mass. Beside this primary effect, OVs can also stimulate the immune system. Tumors are an immuno-suppressive environment in which the immune system is silenced in order to avoid the immune response against cancer cells. The delivery of OVs into the tumor wakes up the immune system so that it can facilitate a strong and durable response against the tumor itself. Both innate and adaptive immune responses contribute to this process, producing an immune response against tumor antigens and facilitating immunological memory. However, viruses are recognized by the immune system as pathogens and the consequent anti-viral response could represent a big hurdle for OVs. Finding a balance between anti-tumor and anti-viral immunity is, under this new light, a priority for researchers. In this review, we provide an overview of the various ways in which different components of the immune system can be allied with OVs. We have analyzed the different immune responses in order to highlight the new and promising perspectives leading to increased anti-tumor response and decreased immune reaction to the OVs.

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer.

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.

Report on the NCI Microbial-Based Cancer Therapy Conference.

The National Cancer Institute Inaugural Microbial-Based Cancer Therapy Conference was held in Bethesda, Maryland, on July 11-12, 2017. This interdisciplinary forum included industry leaders, academic investigators, and regulatory officers involved in the development of microbial-based therapies for the treatment of cancer. The aim of the meeting was to discuss the potential of virus- and bacteria-based therapies to halt tumorigenesis and induce immune responses in cancers where conventional therapy is inadequate. This summary highlights topics and viewpoints raised by the presenters and discussants and should not be viewed as the conclusions or recommendations of the workshop as a whole. Cancer Immunol Res; 6(2); 122-6. ©2017 AACR.

Temozolomide renders murine cancer cells susceptible to oncolytic adenovirus replication and oncolysis.

The preclinical evaluation of oncolytic adenoviruses (OAds) has been limited to cancer xenograft mouse models because OAds replicate poorly in murine cancer cells. The alkylating agent temozolomide (TMZ) has been shown to enhance oncolytic virotherapy in human cancer cells; therefore, we investigated whether TMZ could increase OAd replication and oncolysis in murine cancer cells. To test our hypothesis, three murine cancer cells were infected with OAd (E1b-deleted) alone or in combination with TMZ. TMZ increased OAd-mediated oncolysis in all three murine cancer cells tested. This increased oncolysis was, at least in part, due to productive virus replication, apoptosis, and autophagy induction. Most importantly, murine lung non-cancerous cells were not affected by OAd+TMZ. Moreover, TMZ increased Ad transduction efficiency. However, TMZ did not increase coxsackievirus and adenovirus receptor; therefore, other mechanism could be implicated on the transduction efficiency. These results showed, for the first time, that TMZ could render murine tumor cells more susceptible to oncolytic virotherapy. The proposed combination of OAds with TMZ presents an attractive approach towards the evaluation of OAd potency and safety in syngeneic mouse models using these murine cancer cell-lines in vivo.

Virotherapy Research in Germany: From Engineering to Translation.

Virotherapy is a unique modality for the treatment of cancer with oncolytic viruses (OVs) that selectively infect and lyse tumor cells, spread within tumors, and activate anti-tumor immunity. Various viruses are being developed as OVs preclinically and clinically, several of them engineered to encode therapeutic proteins for tumor-targeted gene therapy. Scientists and clinicians in German academia have made significant contributions to OV research and development, which are highlighted in this review paper. Innovative strategies for "shielding," entry or postentry targeting, and "arming" of OVs have been established, focusing on adenovirus, measles virus, parvovirus, and vaccinia virus platforms. Thereby, new-generation virotherapeutics have been derived. Moreover, immunotherapeutic properties of OVs and combination therapies with pharmacotherapy, radiotherapy, and especially immunotherapy have been investigated and optimized. German investigators are increasingly assessing their OV innovations in investigator-initiated and sponsored clinical trials. As a prototype, parvovirus has been tested as an OV from preclinical proof-of-concept up to first-in-human clinical studies. The approval of the first OV in the Western world, T-VEC (Imlygic), has further spurred the involvement of investigators in Germany in international multicenter studies. With the encouraging developments in funding, commercialization, and regulatory procedures, more German engineering will be translated into OV clinical trials in the near future.