Mary Harvey

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.

Antibody-targeted cell fusion

Membrane fusion has many potential applications in biotechnology. Here we show that antibody-targeted cell fusion can be achieved by engineering a fusogenic viral membrane glycoprotein complex. Three different single-chain antibodies were displayed at the extracellular C terminus of the measles hemagglutinin (H) protein, and combinations of point mutations were introduced to ablate its ability to trigger fusion through the native viral receptors CD46 and SLAM. When coexpressed with the measles fusion (F) protein, using plasmid cotransfection or bicistronic adenoviral vectors, the retargeted H proteins could mediate antibody-targeted cell fusion of receptor-negative or receptor-positive index cells with receptor-positive target cells. Adenoviral expression vectors mediating human epidermal growth factor receptor (EGFR)-targeted cell fusion were potently cytotoxic against EGFR-positive tumor cell lines and showed superior antitumor potency against EGFR-positive tumor xenografts as compared with control adenoviruses expressing native (untargeted) or CD38-targeted H proteins.

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).

The use of a tropism-modified measles virus in folate receptor-targeted virotherapy of ovarian cancer.

Purpose: Attenuated measles viruses are promising experimental anticancer agents currently being evaluated in a phase I dose escalation trial for ovarian cancer patients. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors (CD46 and SLAM). To minimize potential toxicity due to measles virus–associated immunosuppression and infection of nontarget tissues, we sought to develop an ovarian cancer exclusive fully retargeted measles virus. Experimental Design and Results: Interactions of measles virus with its natural receptors were ablated, and a single-chain antibody (scFv) specific for α-folate receptor (FRα), a target overexpressed on 90% of nonmucinous ovarian cancer, was genetically engineered on the viral attachment protein (MV-αFR). Specificity of virus tropism was tested on tumor and normal cells. Biodistribution of measles virus infection was evaluated in measles-susceptible CD46 transgenic mice, whereas antitumor activity was monitored noninvasively by bioluminescence imaging in xenograft models. Tropism and fusogenic activity of MV-αFR was redirected exclusively to FRα without compromise to virus infectivity. In contrast to the parental virus, MV-αFR has no background infectivity on normal human cells. The antitumor activity of MV-αFR, as assessed by tumor volume reduction and overall survival increase, was equal to the parental virus in two models of human ovarian cancer (s.c. and i.p.). Conclusions: A FR-exclusive ovarian cancer targeted oncolytic virus was generated and shown to be therapeutically effective, thus introducing a new modality for FR targeting and a candidate measles virus for clinical testing.

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.

Immunoglobulin G Antibody-Mediated Enhancement of Measles Virus Infection Can Bypass the Protective Antiviral Immune Response

ABSTRACT Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.

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.

73. Fully Retargeted Oncolytic Measles Viruses for Cancer Therapy

Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer and glioma. The virus is selectively oncolytic, causing extensive cell to cell fusion via CD46, which is more highly expressed on tumor cells than normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM. CD46 is found on all human nucleated cells. SLAM (signaling lymphocyte activation molecule) is only expressed on activated T and B cells, dendritic cells and macrophages. Therefore, the native tropism has possibility to cause immunosupression, unwanted damage to normal tissues, and toxicity of oncolytic measles virus.

281. Modulating the Immune Response to Oncolytic Measles Viruses

Attenuated measles viruses are promising oncolytic agents that will soon be tested in phase I clinical trials. However, we predict that their therapeutic potential will be limited by the antiviral immune response of the host. Initial delivery of the virus via the bloodstream will be impeded by antiviral antibodies and subsequent intratumoral spread of the viruses will be limited by cellular immunity. Successive doses of the virus are likely to be more severely impeded than the initial dose due to progressive amplification of host antiviral immunity after each exposure. We are therefore interested to develop immunosuppressive protocols that will suppress both humoral and cellular immune responses to oncolytic measles viruses, thereby enhancing their therapeutic potential. MV-CEA is an oncolytic measles virus expressing a soluble marker peptide (the extracellular domain of CEA) which facilitates noninvasive monitoring of viral gene expression. When MV-CEA was administered intraperitoneally to naive, measles-susceptible (CD46 transgenic, IFN receptor knockout) mice, plasma CEA expression peaked after 3 days and disappeared by day 8. However, no plasma CEA was detected when mice previously challenged with MV-Edmonston were challenged at a later date with the same dose of MV-CEA. We therefore compared the profiles of CEA expression, anti-measles antibody responses and susceptibility to second virus challenge in mice that received immunosuppressive therapy prior to an intraperitoneal injection of 10e7 infectious units of MV-CEA. The following protocols were tested: gamma radiation 250 rads or 500 rads 24 hours before MV-CEA; Dexamethasone 1.5 mg/kg ip 4 hours before MV-CEA; Cyclophosphamide 250 mg/kg ip 4 hours before MV-CEA; Saline control ip 4 hours before MV-CEA. Cyclophosphamide treatment prolonged the CEA expression profile from 8 days to 29 days, reduced the anti-measles antibody titers (measured on day 36) and resulted in significant levels of CEA gene expression 3 days post MV-CEA rechallenge (without additional cyclophosphamide day 53). Gamma radiation led to a dose dependent suppression of the antiviral response, more pronounced at 500 than at 250 rads, but less marked than with cyclophosphamide. Dexamethasone did not significantly impact the kinetics of the antiviral response. QRT-PCR analysis was conducted for detection of measles RNA in the spleens of mice day 59 after a single ip dose of MV-CEA. Interestingly, despite the greatly prolonged duration of viral gene expression that was observed in the irradiated and cyclophosphamide treated mice, no viral RNA was detected in their spleens. In contrast, viral RNA was readily detected in the spleens of control or dexamethasone treated mice.