Rae Myers

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 x 10(6) 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 10(8) and 4 x 10(8) 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 x 10(4) TCID50/kg (10(6) TCID50 per patient).

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 Ifnar(KO)xCD46 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.

Gliomas have a dismal prognosis, with the median survival of patients with the most common histology, glioblastoma multiforme, being only 12-15 months. Development of novel therapeutic agents is urgently needed. We have previously demonstrated that oncolytic measles virus strains derived from the Edmonston vaccine lineage have significant antitumor activity against gliomas [Phuong, L.K., Allen, C., Peng, K.W., Giannini, C., Greiner, S., Teneyck, C.J., Mishra, P.K., Macura, S.I., Russell, S.J., Galanis, E.C. (2003). Cancer. Res. 63, 2462-2469]. MV-CEA is an Edmonston vaccine lineage measles virus strain engineered to express the marker peptide carcinoembryonic antigen (CEA): CEA levels can serve as a correlate of viral gene expression. In support of a phase I clinical trial of intratumoral and resection cavity administration of MV-CEA to patients with recurrent gliomas, we assessed the neurotoxicity of MV-CEA in adult immune male rhesus macaques (Macaca mulatta). The animals ' immune status and administration schedule mimicked the trial population and proposed administration schema. Macaca mulatta represents the prototype animal species for assessment of measles neurotoxicity. The animals were stereotactically administered either vehicle (n = 1) or MV-CEA at 2 x 10(5)or 2 x 10(6) TCID(50) (each, n = 2) in the right frontal lobe in two injections on days 1 and 5. Macaques were closely monitored clinically for neurotoxicity. Body weight, temperature, complete blood count, CEA, clinical chemistries, coagulation, complement levels, immunoglobulin, measles antibody titers, viremia, and shedding (buccal swabs) were tested at multiple time points. Furthermore, cisterna magna spinal taps were performed on day 9 and 1 year after the first viral dose administration, and samples were analyzed for protein, glucose, cell differential, and presence of MV-CEA. Magnetic resonance imaging (MRI) was performed between 4 and 5 months after article administration to assess for subclinical neurotoxicity. To date, 36+ months from study initiation there has been no clinical or biochemical evidence of toxicity, including lack of neurological symptoms, fever, or other systemic symptoms and lack of immunosuppression. Quantitative RT-PCR analysis of blood, buccal swabs, and cerebrospinal fluid (CSF) was negative for MV-CEA at all time points, with the exception of viral genome deletion in the blood of one asymptomatic animal at the 2 x 10(6) TCID(50) dose level on day 85. Vero cell overlays of CSF cells and supernatant were negative for viral recovery. There was no detection of CEA in serum or CSF at any time point. MRI scans were negative for imaging abnormalities and showed no evidence of encephalitis. Our results support the safety of CNS administration of MV-CEA in glioma patients. A clinical trial of intratumoral and resection cavity administration of MV-CEA in patients with recurrent glioblastoma multiforme is currently ongoing.

The present study reports on the safety and toxicity of an oncolytic vesicular stomatitis virus (VSV)-based gene therapy in animal models in preparation for clinical trials for the treatment of hepatocellular carcinoma (HCC). To restrict spread of the oncolytic virus beyond the tumor, the VSV vector carries human interferon-β for its potent antiviral effect. The study design used rat and nonhuman primate models, and included an evaluation of safety. 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-β (VSV-hIFNβ) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 × 109 TCID50 (50% tissue culture infective dose) of VSV-hIFNβ 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-hIFNβ 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-hIFNβ in Buffalo rats was 107 TCID50. Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNβ, toxicity of VSV-rIFNβ (recombinant VSV expressing rat IFN-β) was greatly diminished in Buffalo rats (NOAEL, >1010 TCID50). Two groups of two adult male rhesus macaques received 109 or 1010 TCID50 of VSV-hIFNβ 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-α, and hIFN-β 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.