Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Deletion of immunomodulatory genes as a novel approach to oncolytic vaccinia virus development.

Vaccinia virus (VV) has emerged as a promising platform for oncolytic virotherapy. Many clinical VV candidates, such as the double-deleted VV, vvDD, are engineered with deletions that enhance viral tumor selectivity based on cellular proliferation rates. An alternative approach is to exploit the dampened interferon-based innate immune responses of tumor cells by deleting one of the many VV immunomodulatory genes expressed to dismantle the antiviral response. We hypothesized that such a VV mutant would be attenuated in non-tumor cells but retain the ability to effectively propagate in and kill tumor cells, yielding a tumor-selective oncolytic VV with significant anti-tumor potency. In this study, we demonstrated that VVs with a deletion in one of several VV immunomodulatory genes (N1L, K1L, K3L, A46R, or A52R) have similar or improved in vitro replication, spread, and cytotoxicity in colon and ovarian cancer cells compared to vvDD. These deletion mutants are tumor selective, and the best performing candidates (ΔK1L, ΔA46R, and ΔA52R VV) are associated with significant improvement in survival, as well as immunomodulation, within the tumor environment. Overall, we show that exploiting the diminished antiviral responses in tumors serves as an effective strategy for generating tumor-selective and potent oncolytic VVs, with important implications in future oncolytic virus (OV) design.

Characterizing Cellular Responses During Oncolytic Maraba Virus Infection.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials.

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Development and applications of oncolytic Maraba virus vaccines.

Oncolytic activity of the MG1 strain of the Maraba vesiculovirus has proven efficacy in numerous preclinical cancer models, and relied not only on a direct cytotoxicity but also on the induction of both innate and adaptive antitumor immunity. To further expand tumor-specific T-cell effector and long-lasting memory compartments, we introduced the MG1 virus in a prime-boost cancer vaccine strategy. To this aim, a replication-incompetent adenoviral [Ad] vector together with the oncolytic MG1 have each been armed with a transgene expressing a same tumor antigen. Immune priming with the Ad vaccine subsequently boosted with the MG1 vaccine mounted tumor-specific responses of remarkable magnitude, which significantly prolonged survival in various murine cancer models. Based on these promising results, we validated the safety profile of the Ad:MG1 oncolytic vaccination strategy in nonhuman primates and initiated clinical investigations in cancer patients. Two clinical trials are currently under way (NCT02285816; NCT02879760). The present review will recapitulate the discoveries that led to the development of MG1 oncolytic vaccines from bench to bedside.

Transforming the prostatic tumor microenvironment with oncolytic virotherapy.

Prostate cancer (PCa) was estimated to have the second highest global incidence rate for male non-skin tumors and is the fifth most deadly in men thus mandating the need for novel treatment options. MG1-Maraba is a potent and versatile oncolytic virus capable of lethally infecting a variety of prostatic tumor cell lines alongside primary PCa biopsies and exerts direct oncolytic effects against large TRAMP-C2 tumors in vivo. An oncolytic immunotherapeutic strategy utilizing a priming vaccine and intravenously administered MG1-Maraba both expressing the human six-transmembrane antigen of the prostate (STEAP) protein generated specific CD8+ T-cell responses against multiple STEAP epitopes and resulted in functional breach of tolerance. Treatment of mice with bulky TRAMP-C2 tumors using oncolytic STEAP immunotherapy induced an overt delay in tumor progression, marked intratumoral lymphocytic infiltration with an active transcriptional profile and up-regulation of MHC class I. The preclinical data generated here offers clear rationale for clinically evaluating this approach for men with advanced PCa.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Activating Peripheral Innate Immunity Enables Safe and Effective Oncolytic Virotherapy in the Brain.

The oncolytic mutant vesicular stomatitis virus VSVΔ51 achieves robust efficacy in multiple extracranial tumor models. Yet for malignancies of the brain, direct intratumoral infusion of VSVΔ51 causes lethal virus-induced neuropathology. Here, we have developed a novel therapeutic regime that uses peripheral immunization with a single sub-lethal dose of VSVΔ51 to establish an acute anti-viral state that enables the safe intracranial (IC) infusion of an otherwise lethal dose of VSVΔ51 within just 6 hr. Although type I interferons alone appeared insufficient to explain this protective phenotype, serum isolated at early time points from primed animals conferred protection against an IC dose of virus. Adaptive immune populations had minimal contributions. Finally, the therapeutic utility of this novel strategy was demonstrated by peripherally priming and intracranially treating mice bearing aggressive CT2A syngeneic astrocytomas with VSVΔ51. Approximately 25% of animals achieved complete regression of established tumors, with no signs of virus-induced neurological impairment. This approach may harness an early warning system in the brain that has evolved to protect the host against otherwise lethal neurotropic viral infections. We have exploited this protective mechanism to safely and efficaciously treat brain tumors with an otherwise neurotoxic virus, potentially widening the available treatment options for oncolytic virotherapy in the brain.

Customized Viral Immunotherapy for HPV-Associated Cancer.

The viral-transforming proteins E6 and E7 make human papillomavirus-positive (HPV+) malignancies an attractive target for cancer immunotherapy. However, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. We designed a strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18 that is incorporated into MG1-Maraba virotherapy (MG1-E6E7). Mutations introduced to the transgene abrogate the ability of E6 and E7 to perturb p53 and retinoblastoma, respectively, while maintaining the ability to invoke tumor-specific, multifunctional CD8+ T-cell responses. Boosting with MG1-E6E7 significantly increased the magnitude of T-cell responses compared with mice treated with a priming vaccine alone (greater than 50 × 106 E7-specific CD8+ T cells per mouse was observed, representing a 39-fold mean increase in boosted animals). MG1-E6E7 vaccination in the HPV+ murine model TC1 clears large tumors in a CD8+-dependent manner and results in durable immunologic memory. MG1-Maraba can acutely alter the tumor microenvironment in vivo and exploit molecular hallmarks of HPV+ cancer, as demonstrated by marked infection of HPV+ patient tumor biopsies and is, therefore, ideally suited as an oncolytic treatment against clinical HPV+ cancer. This approach has the potential to be directly translatable to human clinical oncology to tackle a variety of HPV-associated neoplasms that cause significant morbidity and mortality globally. Cancer Immunol Res; 5(10); 847-59. ©2017 AACR.

NK-Cell Recruitment Is Necessary for Eradication of Peritoneal Carcinomatosis with an IL12-Expressing Maraba Virus Cellular Vaccine.

Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death from abdominal cancers. Immunotherapies have been effective for selected solid malignancies, but their potential in PC has been little explored. Here, we report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex vivo with an oncolytic Maraba MG1 virus expressing IL12, promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of IFNγ-induced protein-10 (IP-10) from dendritic cells. The recruitment of cytotoxic, IFNγ-secreting NK cells was associated with reduced tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors > 8 mm), a complete radiologic response was demonstrated within 8 to14 weeks, associated with 100% long-term survival. The impact of MG1-IL12-ICV upon NK-cell recruitment and function observed in the murine system was recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. These findings suggest that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year. Cancer Immunol Res; 5(3); 211-21. ©2017 AACR.

Oncolytic virus synergizes with Smac mimetic compounds to induce rhabdomyosarcoma cell death in a syngeneic murine model.

Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo. Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies.

The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.

High-throughput screening to enhance oncolytic virus immunotherapy.

High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms - including those based on herpes simplex virus, reovirus, and vaccinia virus - have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research.

Big Data Offers Novel Insights for Oncolytic Virus Immunotherapy.

Large-scale assays, such as microarrays, next-generation sequencing and various "omics" technologies, have explored multiple aspects of the immune response following virus infection, often from a public health perspective. Yet a lack of similar data exists for monitoring immune engagement during oncolytic virus immunotherapy (OVIT) in the cancer setting. Tracking immune signatures at the tumour site can create a snapshot or longitudinally analyse immune cell activation, infiltration and functionality within global populations or individual cells. Mapping immune changes over the course of oncolytic biotherapy-from initial infection to tumour stabilisation/regression through to long-term cure or escape/relapse-has the potential to generate important therapeutic insights around virus-host interactions. Further, correlating such immune signatures with specific tumour outcomes has significant value for guiding the development of novel oncolytic virus immunotherapy strategies. Here, we provide insights for OVIT from large-scale analyses of immune populations in the infection, vaccination and immunotherapy setting. We analyse several approaches to manipulating immune engagement during OVIT. We further explore immunocentric changes in the tumour tissue following immunotherapy, and compile several immune signatures of therapeutic success. Ultimately, we highlight clinically relevant large-scale approaches with the potential to strengthen future oncolytic strategies to optimally engage the immune system.

Reciprocal cellular cross-talk within the tumor microenvironment promotes oncolytic virus activity.

Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-ß (TGF-ß) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.

Going viral with cancer immunotherapy.

Recent clinical data have emphatically shown the capacity of our immune systems to eradicate even advanced cancers. Although oncolytic viruses (OVs) were originally designed to function as tumour-lysing therapeutics, they have now been clinically shown to initiate systemic antitumour immune responses. Cell signalling pathways that are activated and promote the growth of tumour cells also favour the growth and replication of viruses within the cancer. The ability to engineer OVs that express immune-stimulating 'cargo', the induction of immunogenic tumour cell death by OVs and the selective targeting of OVs to tumour beds suggests that they are the ideal reagents to enhance antitumour immune responses. Coupling of OV therapy with tumour antigen vaccination, immune checkpoint inhibitors and adoptive cell therapy seems to be ready to converge towards a new generation of multimodal therapeutics to improve outcomes for cancer patients.

Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease.

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Maraba virus as a potent oncolytic vaccine vector.

The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.

Triptolide-mediated inhibition of interferon signaling enhances vesicular stomatitis virus-based oncolysis.

Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.