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.

Oncolytic Vaccinia virus safely and effectively treats skin tumors in mouse models of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder of DNA repair. When exposed to genotoxic stress, XP patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. Unfortunately, given the nature of their underlying genetic defect, tumor-bearing XP patients cannot be treated with conventional DNA damaging therapies. Engineered strains of the poxvirus Vaccinia have been shown to cure cancer in numerous preclinical models, and based on promising Phase I/II clinical trials have recently been approved for late phase evaluation in humans. As poxviruses are nongenotoxic, we investigated whether clinical-candidate strains of Vaccinia can safely and effectively treat cancers arising from XP. In vitro, Vaccinia virus was highly cytotoxic against tumor-derived cells from XP patients, on average 10- to 100-fold more so than on nontumor derived control cells from similar patients. In vivo, local or systemic administration of Vaccinia virus led to durable tumor resolution in both xenograft and genetic models of XP. Importantly, Vaccinia virus was well tolerated in the genetic models, which are each null for a critical component of the DNA repair process. Taken together, our data suggest that oncolytic Vaccinia virus may be a safe and effective therapy for cancers arising from XP, and raise the possibility of similar therapeutic potential against tumors that arise in patients with other DNA repair disorders.

Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

Inhibitor of apoptosis protein cIAP2 is essential for lipopolysaccharide-induced macrophage survival.

The cellular inhibitor of apoptosis 2 (cIAP2/HIAP1) is a potent inhibitor of apoptotic death. In contrast to the other members of the IAP family, cIAP2 is transcriptionally inducible by nuclear factor-kappaB in response to multiple triggers. We demonstrate here that cIAP2-/- mice exhibit profound resistance to lipopolysaccharide (LPS)-induced sepsis, specifically because of an attenuated inflammatory response. We show that LPS potently upregulates cIAP2 in macrophages and that cIAP2-/- macrophages are highly susceptible to apoptosis in a LPS-induced proinflammatory environment. Hence, cIAP2 is critical in the maintenance of a normal innate immune inflammatory response.

Type I IFN blockade uncouples immunotherapy-induced antitumor immunity and autoimmune toxicity.

Despite its success in treating melanoma and hematological malignancies, adoptive cell therapy (ACT) has had only limited effects in solid tumors. This is due in part to a lack of specific antigen targets, poor trafficking and infiltration, and immunosuppression in the tumor microenvironment. In this study, we combined ACT with oncolytic virus vaccines (OVVs) to drive expansion and tumor infiltration of transferred antigen-specific T cells and demonstrated that the combination is highly potent for the eradication of established solid tumors. Consistent with other successful immunotherapies, this approach elicited severe autoimmune consequences when the antigen targeted was a self-protein. However, modulation of IFN-α/-ß signaling, either by functional blockade or rational selection of an OVV backbone, ameliorated autoimmune side effects without compromising antitumor efficacy. Our study uncovers a pathogenic role for IFN-α/-ß in facilitating autoimmune toxicity during cancer immunotherapy and presents a safe and powerful combinatorial regimen with immediate translational applications.

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.

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.

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.

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.

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.

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.

Loss of XIAP protein expression by RNAi and antisense approaches sensitizes cancer cells to functionally diverse chemotherapeutics.

Stable expression of short-hairpin RNAs (shRNAs) directed against the X-linked inhibitor of apoptosis (XIAP) resulted in the generation of three MDA-MB-231 cell lines (XIAP shRNA cells) with reductions in XIAP mRNA and protein levels > 85% relative to MDA-MB-231 cells stably transfected with the U6 RNA polymerase III promoter alone (U6 cells). This RNA interference (RNAi) approach dramatically sensitized these cells to killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, loss of XIAP also sensitized the cells to killing by taxanes but had no additional effects on killing by carboplatin and doxorubicin. The increased sensitivity of the XIAP shRNA cells to killing by TRAIL and taxanes correlated with enhanced caspase cleavage and activation, including caspase-8, and robust processing of poly(ADP-ribose) polymerase and BID compared to U6 cells. Additionally, increasing XIAP levels by adenovirus-mediated expression protected both XIAP shRNA and U6 cells from TRAIL killing in a dose-dependent manner. The effects observed by stable RNAi with respect to TRAIL sensitization were also achieved following downregulation of XIAP in Panc-1 cells treated with a second-generation, mixed-backbone antisense oligonucleotide, AEG 35156/GEM640. These data indicate that reducing XIAP protein expression by either RNAi or antisense approaches increases cancer cell susceptibility to functionally diverse chemotherapeutic agents and supports the notion that downregulation of XIAP in vivo may synergize with disease-relevant chemotherapeutic regimes, including TRAIL and taxanes, to increase the effectiveness of antineoplastic agents.

Cloning and characterization of the rat homologues of the Inhibitor of Apoptosis protein 1, 2, and 3 genes.

BACKGROUND: Inhibitor of Apoptosis (IAP) proteins are key intrinsic regulators of apoptosis induced by a variety of triggers. We isolated the rat Inhibitor of Apoptosis genes 1, 2 and 3 and characterized their tissue distribution and expression. RESULTS: Rat iap-1 encodes a protein of 67.1 kDa with 73 % and 89.2 % homology to human and mouse iap-1 respectively. Rat iap-2 encodes a protein of 66.7 kDa with 81.6 % and 89.3 % homology to human and mouse iap-2 respectively. Rat iap-3 encodes a protein of 56.1 kDa with 89.5 % and 93.1 % homology to human and mouse iap-3 respectively. We have generated rabbit polyclonal antibodies against all three rat IAP genes. Northern and Western blot analysis detected rat IAP transcripts and proteins in majority of the tissues examined. In addition, a shorter, alternatively spliced transcript corresponding to iap-2 was found in testes. CONCLUSIONS: We have identified three rat homologues of the IAP genes. The elevated expression of rat iap-1 and iap2 in testes suggests that these two genes play an important antiapoptotic role in spermatogenesis.

Virus-tumor interactome screen reveals ER stress response can reprogram resistant cancers for oncolytic virus-triggered caspase-2 cell death.

To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

Spurious splicing within the XIAP 5' UTR occurs in the Rluc/Fluc but not the betagal/CAT bicistronic reporter system.

X-chromosome-linked inhibitor of apoptosis, XIAP, has been shown to contain a strong internal ribosome entry site (IRES) within its 5' untranslated region (UTR) that promotes translation of XIAP mRNA under conditions of cellular stress. This claim came under scrutiny in a recent report demonstrating that the XIAP 5' UTR undergoes splicing when inserted between the two reporter cistrons of the dual luciferase plasmid Rluc/Fluc. In this paper, we demonstrate that the splicing within the XIAP 5' UTR specifically occurs only in the context of mRNA produced from the Rluc/Fluc but not the pbetagal/CAT bicistronic reporter plasmid.