As the mechanisms underlying tumorigenesis become better understood, the dynamic roles of cellular components of the tumor microenvironment, and their cross-talk with tumor cells, have come to light as key drivers of disease progression and have emerged as important targets of new cancer therapies. In the field of oncolytic virus (OV) therapy, stromal cells have been considered as potential barriers to viral spread, thus limiting virus replication and therapeutic outcome. However, new evidence indicates that intratumoral fibroblasts could support virus replication. We have demonstrated in a rat model of stromal-rich intrahepatic cholangiocarcinoma (CCA) that vesicular stomatitis virus (VSV) can be localized within intratumoral hepatic stellate cells (HSCs), in addition to tumor cells, when the virus was applied via hepatic arterial infusion. Furthermore, VSV was shown to efficiently kill CCA cells and activated HSCs, and co-culture of CCA and HSCs increased viral titers. Interestingly, this effect is also observed when each cell type is cultured alone in a conditioned medium of the other cell type, indicating that secreted cell factors are at least partially responsible for this phenomenon. Partial reduction in sensitivity to type I interferons was observed in co-culture systems, providing a possible mechanism for the increased viral titers. Together, the results indicate that targeting activated HSCs with VSV could provide an additional mechanism of OV therapy, which, until now has not been considered. Furthermore, these findings suggest that VSV is a potentially powerful therapeutic agent for stromal-rich tumors, such as CCA and pancreatic cancer, both of which are very difficult to treat with conventional therapy and have a very poor prognosis.

Supplementary Fig. S1. Schematic of study design.

Supplementary tables S1-3 and figure legends. Supplementary Table S1. Antibodies and antibody dilution used for immunohistochemical analyses. Supplementary Table S2. Characterization of human HCC Supplementary Table S3. Number of tumors per analyzed animal of DEN and McA model.

Supplementary Fig. S2. Macrophage and T-lymphocyte infiltration in DEN and McA animals.

Supplementary Fig. S4. Tumor vascularization and necrosis in human HCC.

Supplementary Fig. S3. aCGH analyses.

Vesicular stomatitis virus (VSV) represents an attractive oncolytic virotherapy platform because of its potent tumor cell-killing and immune-stimulating properties; yet the clinical translation of VSV faces numerous challenges, such as inefficient systemic delivery and severe side effects such as neurotoxicity. We hypothesized that we could overcome these limitations and simultaneously enhance the therapy, by combining VSV with adoptively transferred T cell receptor (TCR) transgenic T cells as carrier cells. We show that CD8(+) T central memory cells (CD8(+) T cm) can be efficiently loaded with VSV, they support intracellular virus production, and they can efficiently transfer VSV to tumor cells without compromising their own viability or antitumor reactivity. Loading VSV onto CD8(+) T cm not only improves the safety compared with systemic administration of naked virus, but this approach also allows for an effective delivery of virus to its tumor target, resulting in an effective combination therapy in NSG mice bearing subcutaneous human acute myeloid leukemia (AML) tumors. We conclude that the combination of potent tumor debulking provided by the oncolytic VSV with the added effector functions afforded by the cytotoxic immune carrier cells results in a potent and safer immunotherapeutic, which can be further developed for clinical translation.

The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

34 35 Oncolytic viruses represent an exciting new aspect of the evolving field of cancer 36 immunotherapy. We have engineered a novel hybrid vector comprising vesicular 37 stomatitis virus (VSV) and Newcastle disease virus (NDV), named rVSV-NDV, 38 wherein the VSV backbone is conserved, but its glycoprotein has been replaced by 39 the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) 40 envelope proteins of recombinant NDV. As opposed to wild-type VSV, which kills 41 cells through a classical cytopathic effect, the recombinant virus is able to induce 42 tumor-specific syncytia formation, allowing for efficient cell-to-cell spread of the virus 43 and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein 44 exchange substantially abrogates the off-target effects in brain and liver tissue 45 associated with wildtype VSV, resulting in a substantially enhanced safety profile, 46 even in immune-deficient NOD-SCID mice which are highly susceptible to wild-type 47 VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the 48 incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in 49 embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in 50 orthotopic HCC-bearing immune-competent mice resulted in significant survival 51 prolongation. This strategy, therefore, combines the beneficial properties of the 52 rapidly replicating VSV platform with the highly efficient spread and immunogenic cell 53 death of a fusogenic virus, without risking the safety and environmental threats 54 associated with either parental vector. Taken together, rVSV-NDV represents an 55 attractive vector platform for clinical translation as a safe and effective oncolytic virus. 56

Abstract With the clinical approval of Talimogen laherparepvec (T-VEC), an oncolytic Herpes-simplex-Virus 1, in 2015, oncolytic viruses have gained much attention as a versatile and promising platform for cancer therapy. Vesicular stomatitis virus (VSV) represents an attractive oncolytic agent due to its inherent ability to preferentially infect and kill tumor cells, boost an antitumor immune response, and its flexibility to efficiently express transgenes. In order to overcome potential hurdles for the systemic administration of VSV, which include neutralization and nonspecific uptake, we aim to develop a combination therapy which employs T-cell receptor (TCR) transgenic T cells directed against an epitope of Myeloperoxidase (Klar et al. 2014, Leukemia). As genetically engineered T cells are currently undergoing clinical trials, they have the potential to provide an ideal vehicle for a combination therapy with VSV. In our work, we have shown that human CD8+ central memory T cells (CD8+ Tcm) not only transport and deliver infectious virus to their tumor target while eliciting their own potent cytotoxic effector functions, but they also support viral amplification in the absence of significantly decreased cell viability in culture after 24h of viral infection. Moreover, we demonstrate in coculture assays with ML2 leukemia cells, that TCR transgenic CD8+ Tcm profit from the additional virus-mediated tumor cell killing as compared to a monotherapy with TCR transgenic CD8+ Tcm alone. Although viral titers in coculture assays with TCR transgenic cells were slightly decreased compared to those achieved in cocultures with T cells not expressing the TCR, we consider this to be an additional benefit of the combination therapeutic approach to reduce VSV-mediated off-target side effects, as VSV is known for its neurotoxic and hepatotoxic effects in a dose dependent manner. This is supported by our findings that highly immune-deficient NSG mice tolerate similar amounts of virus better if applied together with CD8+ Tcm. Furthermore, we demonstrate that the combination therapy for tumor-bearing NSG mice leads to enhanced transduction efficiency of VSV compared to that achieved by delivery of naked virus, as well as faster tumor cell killing compared to monotherapies with TCR transgenic CD8+ T cm or VSV alone when applied at the maximum tolerated dose. We therefore conclude that the combination of these monotherapies is a crucial step toward a broader and safer option for cancer treatment, as each offers a complimentary approach to tumor cell killing while simultaneously reducing the weaknesses of the other treatment option. We speculate that immune evasion can be efficiently overcome by application of the combination therapy while providing an efficient targeting and delivery vehicle of systemically applied oncolytic VSV with a concomitant reduction of toxicity. As both, rVSV and transgenic T cells are already undergoing clinical testing, our proposed combination therapy has the potential for a seamless translation to clinical application. Citation Format: Michael Karl Melzer, Lisa Zeitlinger, Sabine Mall, Katja Steiger, Angela Krackhardt, Oliver Ebert, Jennifer Altomonte. TCR transgenic T cells improve the anticancer potential of oncolytic vesicular stomatitis virus as cell carriers and as synergistic therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B14.

Background/Objectives: Angiogenesis plays a central role in tumor growth and metastasis and tyrosine kinases are crucial in the modulation of growth factor signaling. Several side effects of tyrosine kinase inhibitors have been reported, including diarrhea due to pancreatic insufficiency. The suspected mechanism is the anti-angiogenetic effect of the inhibited vascular endothelial growth factor (VEGF) causing a disturbance of the microvasculation. The aim of the present study was to determine the volume of the pancreas before and after a therapy both with the multi-tyrosine kinase inhibitor Sorafenib and Bevacizumab, which is a humanized monoclonal immunoglobulin GI antibody against VEGF.Methods: Retrospective monocentric study including 42 patients who received either Sorafenib, Bevacizumab combined with Flourouracil and/or Irinotecan, or singly Flourouracil and Irinotecan for different non-pancreatic malignancies. The volume of the pancreas was measured before and after therapy by CT-scan based volumetry.Results: The pancreatic volume was statistically significantly lower after treatment with Sorafenib (75.4 mL vs. 71.0 mL; p = 0.006) or Bevacizumab and Fluorouracil Irinotecan (71.8 mL vs. 62.6 mL; p = 0.020). The pancreatic volume did not change statistically significantly after treatment with Fluorouracil +/- Irinotecan only (51.1 mL vs. 49.9 mL; p = 0.142).Conclusions: Pancreatic volume decreases statistically significantly under treatment with both the multi tyrosine kinase inhibitor Sorafenib and the angiogenesis inhibitor Bevacizumab. This volume reduction is most likely due to a reduced microvasculation by inhibition of VEGF. (C) 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Background & Aims: Codrituzumab, a humanized monoclonal antibody against Glypican-3 (GPC3) that is expressed in hepatocellular carcinoma (HCC), interacts with CD16/FccRIIIa and triggers antibody-dependent cytotoxicity. Codrituzumab was studied vs. placebo in a randomized phase II trial in advanced HCC patients who had failed prior systemic therapy.Methods: Patients with advanced HCC who had failed prior systemic therapy, >= 18 years, Eastern cooperative oncology group (ECOG) 0-1, Child-Pugh A were randomized 2: 1 to biweekly codrituzumab 1600 mg vs. placebo. Patients were stratified based on GPC3 immunohistochemical expression: 2+/3+, 1+, and 0. Primary endpoint was progression free survival. Secondary endpoints include overall survival (OS), tolerability, pharmacokinetics, and an exploratory endpoint in biomarkers analysis.Results: 185 patients were enrolled: 125 received codrituzumab and 60 placebo: Median age 64/63, 85/75% male, 46/42% Asian, ECOG 0 65/63%, 74/77% having vascular invasion and/or extrahepatic metastasis. 84%/70% had prior sorafenib. Drug exposure was 98.4% of planned dose, with an identical adverse events profile between the 2 groups. The median progression free survival and overall survival in the codrituzumab vs. placebo groups in months were: 2.6 vs. 1.5 (hazard ratios 0.97, p = 0.87), and 8.7 vs. 10 (hazard ratios 0.96, p = 0.82). Projected Ctrough at cycle 3 day 1 based exposure, high CD16/FccRIIIa on peripheral immune cells, and GPC3 expression in the tumor, were all associated with prolonged progression free survival and overall survival.Conclusions: Codrituzumab did not show clinical benefit in this previously treated HCC population. Whether higher codrituzumab drug exposure or the use of CD16 and GPC3 as potential biomarkers would improve outcome remain unanswered questions.Lay summary: Codrituzumab is a manufactured antibody against a liver cancer protein called glypican-3. In this clinical trial, codrituzumab was not found be effective against liver cancer. It was suggested though that a higher dose of codrituzumab or selecting patients with high level of glypican-3 or its mediator CD16 might improve outcome. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Hepatocellular carcinoma (HCC) is a refractory malignancy with a high mortality and increasing worldwide incidence rates, including the United States and central Europe. In this study, we demonstrate that a specific inhibitor of signal transducer and activator of transcription 3 (STAT3), NSC74859, efficiently reduces HCC cell proliferation and can be successfully combined with oncolytic virotherapy using vesicular stomatitis virus (VSV). The potential benefits of this combination treatment are strengthened by the ability of NSC74859 to protect primary hepatocytes and nervous system cells against virus-induced cytotoxicity, with an elevation of the VSV maximum tolerated dose in mice. Hereby we propose a strategy for improving the current regimen for HCC treatment and seek to further explore the molecular mechanisms underlying selective oncolytic specificity of VSV.

Abstract Purpose: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. Experimental Design: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. Results: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1–3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. Conclusions: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies. Clin Cancer Res; 21(19); 4440–50. ©2015 AACR. See related commentary by Weber et al., p. 4254

Hepatocellular carcinoma (HCC) is a disease with rising incidence, limited treatment options and poor prognosis, representing a major worldwide health concern. Due to the vast deficit of viable treatment options, oncolytic viruses have emerged as promising alternative therapies to specifically target and lyse tumor cells. We have previously demonstrated the safety and efficacy of oncolytic vesicular stomatitis virus (VSV) applied via hepatic arterial infusion to treat orthotopic HCC in immune-competent rats, and based on encouraging data such as these, a recombinant VSV vector has recently entered the clinic in phase I trials for HCC in patients. Although systemic administration of oncolytic viral therapies is ideal for targeting metastatic disease, the efficacy of such an approach is extremely compromised due to rapid clearance from circulation as a result of neutralizing blood components and non-specific uptake by liver and spleen. Furthermore, the complex microenvironment in the liver poses a unique set of challenges to the fate of oncolytic viral therapies directed at hepatic tumors. Although attempts to shield viruses from adverse interactions have led to increased circulation times in vivo, they are also associated with decreased infectivity of the target cells. We have therefore developed several strategies, including novel cell carrier and synthetic shielding approaches, to simultaneously protect VSV from nonspecific interactions and deliver it specifically to the tumor target, where it can exert its oncolytic effect. Because a variety of immune cells possess the inherent ability to take up oncolytic virus and subsequently home to tumor beds, they offer the unique dual benefit of delivering viruses in stealth, while contributing to antitumor immune responses. Alternatively, synthetic polymers can provide highly effective shielding through surface modification of oncolytic viruses and have the advantage of being adaptable to optimize the shielding effect. Through the incorporation of targeting ligands or selectively cleavable linkers, the ability of the virus to infect is then rescued locally and specifically at the tumor site. Furthermore, advancement of imaging technologies allows us the possibility to monitor the biodistribution of these shielded viruses to observe the circulation time and replication kinetics noninvasively and in real-time, which will further facilitate the clinical translation of oncolytic virus therapies. A summary of these novel approaches to enhance oncolytic VSV accumulation within orthotopic HCC tumors and improve the therapy outcome, as well as virus imaging strategies, will be presented.

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2(*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2(*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome.

4102 Background: RO5137382/GC33 (GC33) is a humanized monoclonal antibody against GPC3 that is frequently expressed in HCC. GC33 interacts with CD16/FcγR3 and triggers antibody-dependent cytotoxicity. GC33 was compared with placebo in a randomized phase II study in advanced HCC patients (pts) who had failed prior systemic therapy. Methods: Pts with advanced HCC who had failed prior systemic therapy, ≥18 years, ECOG 0-1, Child-Pugh A were randomized in a 2:1 ratio to GC33 1600 mg Q2W after two weekly doses versus placebo. Prior to randomization, pts were assigned into 3 cohorts based degree of GPC3 immunohistochemical expression: A (GPC3 2-3+), B (GPC3 1+) and C (GPC3 no expression). Primary endpoint was progression free survival (PFS). Secondary endpoints include overall survival (OS), time to progression (TTP), tolerability, and pharmacokinetics (PK). Tumor assessment was based on RECIST1.0 criteria and safety on CTCAE 4.0. Results: Between February 2012 to March 2013, 185 pts were enrolled: 121 received GC33 and 64 placebo: Median age 64/63, 85/75% male, 46/42% Asian, ECOG 0 65/63%, 74/77% having vascular invasion and/or extra-hepatic metastasis. 49/41% had prior single agent sorafenib. Drug exposure was 98.6% of planned dose, with an identical adverse events profile between the 2 groups. The median PFS, OS, and TTP in the GC33 vs placebo groups in months were: 2.6 vs 1.5 (HR 0.97, p=0.87), 6.8 vs 6.7 (HR 0.99, p=0.97), and 2.9 vs 1.7 (HR 0.96, p=0.85). A subsequent exposure-efficacy analysis showed that increased exposure based on projected Ctroughat cycle 3 day 1 was associated with prolonged PFS and OS. Median PFS for high GC33 exposure group (n=60) was 4 vs 1.5 months for placebo (n=60), and OS 9.7 vs 6.7 months, respectively. Combining higher exposure with FcγR3A-158V polymorphism or CD16 expression intensity may correlate with improved PFS and OS. Conclusions: GC33 did not show a clinical benefit in this advanced, previously treated HCC population, potentially due to suboptimal dosing. Further studies are needed to investigate whether higher GC33 drug exposure and a favored CD16/FcgR3 immune environment may help. Clinical trial information: NCT01507168.