Rae Myers

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.)

Oncolytic measles virus strains in the treatment of gliomas.

Background: Recurrent gliomas have a dismal outcome despite use of multimodality treatment including surgery, radiation therapy and chemotherapy. Objective: In this article the authors discuss potential applications of oncolytic measles virus strains as novel antitumor agents in the treatment of gliomas. Methods: Important aspects of measles virus development as an anticancer therapeutic agent including engineering, retargeting and combination studies with other therapeutic modalities are discussed. The translational process that led to the first clinical trial of an engineered measles virus derivative in patients with recurrent glioblastoma multiforme is also described. Results/conclusions: Oncolytic measles virus strains hold promise as novel antitumor agents in the treatment of gliomas.

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.

Dynamics of multiple myeloma tumor therapy with a recombinant measles virus

Replication-competent viruses are being tested as tumor therapy agents. The fundamental premise of this therapy is the selective infection of the tumor cell population with the amplification of the virus. Spread of the virus in the tumor ultimately should lead to eradication of the cancer. Tumor virotherapy is unlike any other form of cancer therapy as the outcome depends on the dynamics that emerge from the interaction between the virus and tumor cell populations both of which change in time. We explore these interactions using a model that captures the salient biological features of this system in combination with in vivo data. Our results show that various therapeutic outcomes are possible ranging from tumor eradication to oscillatory behavior. Data from in vivo studies support these conclusions and validate our modeling approach. Such realistic models can be used to understand experimental observations, explore alternative therapeutic scenarios and develop techniques to optimize therapy.

Expression of immunomodulatory neutrophil-activating protein of Helicobacter pylori enhances the antitumor activity of oncolytic measles virus

Helicobacter pylori neutrophil-activating protein (NAP) is a major virulence factor and powerful inducer of inflammatory reaction and Th1-polarized immune response. Here, we evaluated the therapeutic efficacy of measles virus (MV) strains engineered to express secretory NAP forms against metastatic breast cancer. Recombinant viruses encoding secretory NAP forms (MV-lambda-NAP and MV-s-NAP) efficiently infect and destroy breast cancer cells by cell-to-cell viral spread and large syncytia formation independently of hormone receptor status. Intrapleural administration of MV-s-NAP doubled the median survival in a pleural effusion xenograft model: 65 days as compared to 29 days in the control group (P < 0.0001). This therapeutic effect correlated with a brisk Th1 type cytokine response in vivo. Secretory NAP was expressed at high levels by infected tumor cells and increased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12/23 cytokine concentrations were detected in the pleural effusion. In an aggressive model of lung metastatic breast cancer, MV-lambda-NAP and MV-s-NAP also significantly improved survival of the treated animals (P < 0.05) as compared to the control MV strain. These data suggest that potent immunomodulators of bacterial origin, such as H. pylori NAP, can enhance the antitumor effect of oncolytic viruses and support the feasibility and potential of a combined viroimmunotherapy approach.

Preclinical efficacy of the oncolytic measles virus expressing the sodium iodide symporter in iodine non-avid anaplastic thyroid cancer: a novel therapeutic agent allowing noninvasive imaging and radioiodine therapy.

Anaplastic thyroid cancer is an extremely aggressive disease resistant to radioiodine treatment because of loss of sodium iodide symporter (NIS) expression. To enhance prognosis of this fatal cancer, we validated the preclinical efficacy of measles virus (MV)-NIS, the vaccine strain of the oncolytic MV (MV-Edm), modified to include the NIS gene. Western blotting analysis confirmed that a panel of eight anaplastic thyroid cancer (ATC)-derived cell lines do not express NIS protein, but do express CD46, the MV receptor. In vitro cell death assays and in vivo xenograft studies demonstrate the oncolytic efficacy of MV-NIS in BHT-101 and KTC-3, ATC-derived cell lines. Radioactive iodine uptake along with single-photon emission computed tomography (SPECT)-computed tomography imaging of KTC-3 xenografts after 99Tcm administration confirmed NIS expression in vitro and in vivo, respectively, after virus treatment. Adjuvant administration of RAI, to MV-NIS-treated KTC-3 tumors showed a trend for increased tumor cell killing. As current treatment for ATC is only palliative, and MV-NIS is currently Food and Drug Administration approved for human clinical trials in myeloma, our data indicate that targeting ATC with MV-NIS could prove to be a novel therapeutic strategy for effective treatment of iodine-resistant ATC and will expedite its testing in clinical trials for this aggressive disease.