Suzanne Greiner

Rescue and propagation of fully retargeted oncolytic measles viruses.

Live attenuated measles viruses of the Edmonston lineage (MV-Edm) have potent anti-tumor activity but are not entirely tumor-specific owing to widespread distribution of their native receptors, CD46 and SLAM. We have therefore developed a pseudoreceptor system that allows rescue and propagation of fully retargeted viruses displaying single-chain antibody fragments. Viruses retargeted to tumor-selective CD38, epidermal growth factor receptor (EGFR) or EGFR mutant vIII (EGFRvIII) efficiently entered cells through their respective targeted receptors in vitro and in vivo, but not through CD46 and SLAM. When administered intratumorally or intravenously to mice bearing human CD38 or EGFR-positive human tumor xenografts, the targeted viruses demonstrated specific receptor-mediated anti-tumor activity. These data provide an in vivo demonstration of antibody-directed tumor destruction by retargeted oncolytic viruses.

Engineering microRNA responsiveness to decrease virus pathogenicity.

The cellular tropisms of eukaryotic viruses are shaped by their need for entry receptors and intracellular transcription factors. Here we show that viral tropisms can also be regulated by tissue-specific microRNAs (miRNAs). Target sequences complementary to muscle-specific miRNAs were inserted into the 3′ untranslated region (UTR) of an oncolytic picornavirus that causes lethal myositis in tumor-bearing mice. The recombinant virus still propagated in subcutaneous tumors, causing total regression and sustained viremia, but could not replicate in cells expressing complementary miRNAs and therefore did not cause myositis. This altered tropism was not due to insertional attenuation, as a control virus containing a 3′ UTR insert with a disrupted miRNA target sequence fully retained its lethal myotropism. Tissue-specific destabilization of viral genomes by miRNA target insertion provides a potentially versatile new mechanism for controlling the tropism of replicating viruses for therapy and may serve as a new modality for attenuating viruses for vaccine purposes.

Preclinical Pharmacology and Toxicology of Intravenous MV-NIS, an Oncolytic Measles Virus Administered With or Without Cyclophosphamide

MV‐NIS is an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS). Here, we report the results of preclinical pharmacology and toxicology studies conducted in support of our clinical protocol “Phase I Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or Refractory Multiple Myeloma.” Dose–response studies in the KAS‐6/1 myeloma xenograft model demonstrated a minimum effective dose of 4 × 106 TCID50 (tissue culture infectious dose 50)/kg. Toxicity studies in measles‐naive squirrel monkeys and measles‐susceptible transgenic mice were negative at intravenous doses up to 108 and 4 × 108 TCID50/kg, respectively. Abundant viral mRNA, maximal on day 8, was detected in cheek swabs of squirrel monkeys, more so after pretreatment with cyclophosphamide. On the basis of these data, the safe starting dose of MV‐NIS for our clinical protocol was set at 1−2 × 104 TCID50/kg (106 TCID50 per patient).

Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma

The oncolytic measles virus Edmonston strain (MV‐Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV‐Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus‐infected cells. We evaluated recombinant MV‐Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV‐Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV‐Edm in vitro and in vivo was assessed by CEA concentration, 125I‐uptake, and 123I‐imaging studies. Toxicology studies were performed in IfnarKO×CD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV‐Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV‐Edm resulted in high‐level expression of transgenes in vitro and in vivo. MV‐Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV‐Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV‐Edm may be a potent and novel cancer gene therapy system for HCC. MV‐Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner. (HEPATOLOGY 2006;44:1465–1477.)

Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m–2 was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m–2 should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

Oncolytic activities of approved mumps and measles vaccines for therapy of ovarian cancer.

Oncolytic viruses are promising cytoreductive agents for cancer treatment but extensive human testing will be required before they are made commercially available. Here, we investigated the oncolytic potential of two commercially available live attenuated vaccines, Moraten measles and Jeryl-Lynn mumps, in a murine model of intraperitoneal human ovarian cancer and compared their efficacies against a recombinant oncolytic measles virus (MV-CEA) that is being tested in a phase I clinical trial. The common feature of these viruses is that they express hemagglutinin and fusion therapeutic proteins that can induce extensive fusion of the infected cell with its neighbors, resulting in death of the cell monolayer. In vitro, the three viruses caused intercellular fusion in human ovarian cancer cells but with marked differences in fusion kinetics. MV-CEA was the fastest followed by Jeryl-Lynn mumps virus while Moraten measles virus was the slowest, although all viruses eventually caused comparable cell death 6 days postinfection. Tumor-bearing mice treated with 106 or 107 pfu (one thousand times the vaccine dose) of each of the three viruses responded favorably to therapy with significant prolongations in survival. All three viruses demonstrated equivalent antitumor potency. Commercially available Moraten measles and Jeryl-Lynn mumps vaccines warrant further investigation as potential anticancer agents.

Safety Studies on Intrahepatic or Intratumoral Injection of Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Rodents and Nonhuman Primates

Toxicology studies were performed in rats and rhesus macaques to establish a safe starting dose for intratumoral injection of an oncolytic vesicular stomatitis virus expressing human interferon-beta (VSV-hIFNbeta) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 x 10(9) TCID(50) (50% tissue culture infective dose) of VSV-hIFNbeta into the left lateral hepatic lobe of Harlan Sprague Dawley rats. Plasma alanine aminotransferase and alkaline phosphatase levels increased and platelet counts decreased in the virus-treated animals on days 1 and 2 but returned to pretreatment levels by day 4. VSV-hIFNbeta was also injected into normal livers or an intrahepatic McA-RH7777 HCC xenograft established in Buffalo rats. Buffalo rats were more sensitive to neurotoxic effects of VSV; the no observable adverse event level (NOAEL) of VSV-hIFNbeta in Buffalo rats was 10(7) TCID(50). Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNbeta, toxicity of VSV-rIFNbeta (recombinant VSV expressing rat IFN-beta) was greatly diminished in Buffalo rats (NOAEL, >10(10) TCID(50)). Two groups of two adult male rhesus macaques received 10(9) or 10(10) TCID(50) of VSV-hIFNbeta injected directly into the left hepatic lobe under computed tomographic guidance. No neurological signs were observed at any time point. No abnormalities (hematology, clinical chemistry, body weights, behavior) were seen and all macaques developed neutralizing anti-VSV antibodies. Plasma interleukin-6, tumor necrosis factor-alpha, and hIFN-beta remained below detection levels by ELISA. On the basis of these studies, we will be proposing a cautious approach to dose escalation in a phase I clinical trial among patients with HCC.

Pharmacokinetics of oncolytic measles virotherapy: eventual equilibrium between virus and tumor in an ovarian cancer xenograft model

Because of their ability to replicate, the dose–response relationships of oncolytic viruses cannot easily be predicted. To better understand the pharmacokinetics of virotherapy in relation to viral dose and schedule, we administered MV-CEA intraperitoneally in an orthotopic mouse model of ovarian cancer. MV-CEA is an attenuated oncolytic measles virus engineered to express soluble human carcinoembryonic antigen (CEA), and the virus is currently undergoing phase I clinical testing in patients with ovarian cancer. Plasma CEA levels correlate with numbers of virus-infected tumor cells at a given time, and were used as a surrogate to monitor the profiles of viral gene expression over time. The antineoplastic activity of single- or multiple-dose MV-CEA was apparent over a wide range of virus doses (103–108 TCID50), with little reduction in observed antitumor efficacy, even at the lowest tested dose. However, analysis of CEA profiles of treated mice was highly informative, illustrating the variability in virus kinetics at different dose levels. The highest doses of virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced survival of the mice.

Constitutive Interferon Pathway Activation in Tumors as an Efficacy Determinant Following Oncolytic Virotherapy

Background Attenuated measles virus (MV) strains are promising agents currently being tested against solid tumors or hematologic malignancies in ongoing phase I and II clinical trials; factors determining oncolytic virotherapy success remain poorly understood, however. Methods We performed RNA sequencing and gene set enrichment analysis to identify pathways differentially activated in MV-resistant (n = 3) and -permissive (n = 2) tumors derived from resected human glioblastoma (GBM) specimens and propagated as xenografts (PDX). Using a unique gene signature we identified, we generated a diagonal linear discriminant analysis (DLDA) classification algorithm to predict MV responders and nonresponders, which was validated in additional randomly selected GBM and ovarian cancer PDX and 10 GBM patients treated with MV in a phase I trial. GBM PDX lines were also treated with the US Food and Drug Administration-approved JAK inhibitor, ruxolitinib, for 48 hours prior to MV infection and virus production, STAT1/3 signaling and interferon stimulated gene expression was assessed. All statistical tests were two-sided. Results Constitutive interferon pathway activation, as reflected in the DLDA algorithm, was identified as the key determinant for MV replication, independent of virus receptor expression, in MV-permissive and -resistant GBM PDXs. Using these lines as the training data for the DLDA algorithm, we confirmed the accuracy of our algorithm in predicting MV response in randomly selected GBM PDX ovarian cancer PDXs. Using the DLDA prediction algorithm, we demonstrate that virus replication in patient tumors is inversely correlated with expression of this resistance gene signature (ρ = -0.717, P = .03). In vitro inhibition of the interferon response pathway with the JAK inhibitor ruxolitinib was able to overcome resistance and increase virus production (1000-fold, P = .03) in GBM PDX lines. Conclusions These findings document a key mechanism of tumor resistance to oncolytic MV therapy and describe for the first time the development of a prediction algorithm to preselect for oncolytic treatment or combinatorial strategies.

Abstract POSTER-THER-1419: Chk1 inhibitor (MK8776) enhances cytotoxicity of gemcitabine in select patient-derived ovarian tumorgraft models

Purpose: Treatment of recurrent ovarian cancer is limited by low response rates to salvage chemotherapy. Novel small molecule inhibitors to Chk1 (checkpoint kinase 1) have been proposed to potentiate the cytotoxic effect of one such salvage treatment, gemcitabine, by interrupting the DNA damage repair pathway. Patient derived tumorgrafts were characterized by responsiveness to gemcitabine +/- MK8776 (Chk1 inhibitor). Response was correlated with markers of Chk1 activity. Methods: Patient-derived tumorgraft models were created from patients undergoing surgery for epithelial ovarian cancer. Gemcitabine response was characterized in five high-grade serous models (PH044, PH048, PH070, PH080, and PH110). Two models (PH048 and PH080) were treated with gemcitabine monotherapy (240 mg/kg qweek x 4 doses), MK8776 monotherapy (32 mg/kg qweek x 4 doses), and with combination therapy (Gemcitabine 190 mg/kg and MK8776 32 mg/kg qweek x 4 doses). Animals were monitored for cytotoxicity, and tumors were measured weekly with ultrasound. Response to gemcitabine and to combination therapy was correlated to immunohistochemistry of γH2AX and immunoblotting for p345-Chk1 and p296-Chk1. Results: Gemcitabine monotherapy did not affect tumor growth in two models (PH044 and PH048). Significant in vivo response to gemcitabine was seen in PH070 and PH080. Significant growth attenuation but not tumor regression was seen in PH110. MK8776 did not have any effect on tumor growth as monotherapy or in combination with gemcitabine in the gemcitabine resistant model, PH048. In contrast, in a gemcitabine sensitive model, PH080, MK8776 monotherapy significantly attenuated tumor growth, and there was a trend for MK8776 to potentiate the cytotoxic effects of gemcitabine in PH080 (p=0.16). Final tumor weights in the PH080 model after 4 doses of chemotherapy were 1.7 g (90%CI: 1.5-1.9g), 0.3g (90%CI: 0.18-0.35g), and 0.14g (90%CI: 0.08-.19g) in the control, gemcitabine alone, and gemcitabine with MK8776 groups respectively. Decrease in the tumor auto-phosphorylation of Chk1 at Serine-296 was observed in some, but not all models in response to MK8766 treatment. γH2AX increased in response to the addition of MK8776 to gemcitabine. Conclusions: Chk1 inhibition may enhance the activity of gemcitabine in some, but not all ovarian tumors. Further work is needed to find markers that will predict response to Chk1 inhibitors to help select patients for future clinical trials. While pChk1-S296 previously demonstrated potential utility as a biomarker of Chk1 inhibition, in vivo assessments of its differential expression may demonstrate the unreliability of this marker. This work supports the use of patient derived tumorgrafts in drug development for ovarian cancer. Citation Format: A Kumar, S Weroha, M Becker, X Hou, S Greiner, L Karnitz, P Haluska. Chk1 inhibitor (MK8776) enhances cytotoxicity of gemcitabine in select patient-derived ovarian tumorgraft models [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1419.