The Edmonston vaccine strain of measles virus (MV-Edm) propagates efficiently in a broad range of human tumor cells, killing them selectively. However, the oncolytic potency of MV-Edm in different human tumor xenograft therapy models is highly variable and there is no convenient way to map the distribution of virus-infected tissues in vivo. To enhance the oncolytic potency of MV-Edm against radiosensitive malignancies and to facilitate noninvasive imaging of infected tissues, we generated a recombinant MV-Edm encoding the human thyroidal iodide symporter (NIS). MV-NIS replicated almost as efficiently as unmodified MV-Edm, and human tumor cells efficiently concentrated radioiodine when infected with MV-NIS. Intratumoral spread of MV-NIS was noninvasively demonstrated by serial gammacamera imaging of iodine-123 ( 123 I) uptake both in MV-sensitive KAS-6/1 myeloma xenografts, which regressed completely after a single intravenous dose of MV-NIS, and in MM1 myeloma xenografts, which were unresponsive to MV-NIS therapy. However, MV-resistant MM1 tumors regressed completely when 131 I was administered 9 days after a single intravenous injection of MV-NIS (radiovirotherapy). 131 I alone had no effect on MM1 tumor growth. While the potential hematopoietic toxicity of this new therapy requires further evaluation, image-guided radiovirotherapy is a promising new approach to the treatment of multiple myeloma, an incurable but highly radiosensitive plasma cell malignancy. Testing in other radiosensitive cancers is warranted. IntroductionMultiple myeloma is a disseminated malignancy of antibodysecreting plasma cells that reside in active bone marrow. Clinical features of the disease include bone pain, lytic lesions, pathologic fractures, hypercalcemia, anemia, suppression of humoral immunity, and renal dysfunction caused by the tumor-derived monoclonal immunoglobulin. 1 In most patients, the fraction of proliferating cells is less than 1% until late in the disease. 2 Standard therapy is with alkylating agents (melphalan, cyclophosphamide) plus prednisone or combination chemotherapy (vincristine, doxorubicin, and dexamethasone) followed by high-dose melphalan with stem cell rescue. 1,3 At relapse, patients can be offered thalidomide or investigational drugs such as PS-341. [3][4][5] The disease, however, remains incurable and new therapeutic approaches are required.Myeloma cells are highly radiosensitive, and local radiotherapy provides effective palliation for painful bone lesions. 6 However, the disseminated nature of myeloma precludes curative external beam radiation therapy due to unacceptable end organ toxicity. 7 Bone-seeking radioisotopes that bind to bone mineral are being tested in multiple myeloma, 8 but their appeal and efficacy are limited by their inability to penetrate into the centers of myelomatous bone marrow deposits.Replicating viruses have considerable potential as cytoreductive agents for cancer. 9,10 Of the oncolytic viruses currently under investigation, measles virus (MV) is naturally lymphotrophic 11 ...

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.

ABSTRACT

MV-NIS is an engineered measles virus that is selectively destructive to myeloma plasma cells and can be monitored by noninvasive radioiodine imaging of NIS gene expression. Two measles-seronegative patients with relapsing drug-refractory myeloma and multiple glucose-avid plasmacytomas were treated by intravenous infusion of 1011 TCID50 (50% tissue culture infectious dose) infectious units of MV-NIS. Both patients responded to therapy with M protein reduction and resolution of bone marrow plasmacytosis. Further, one patient experienced durable complete remission at all disease sites. Tumor targeting was clearly documented by NIS-mediated radioiodine uptake in virus-infected plasmacytomas. Toxicities resolved within the first week after therapy. Oncolytic viruses offer a promising new modality for the targeted infection and destruction of disseminated cancer.

IntroductionAttenuated viruses with tumor specificity have attracted considerable interest as novel anticancer agents, and clinical testing of several such agents is under way. 1 Tumor selectivity of these viruses has been attributed to various intracellular restrictions to their life cycles that are strongly inhibitory to virus propagation in nontransformed cells but that are overridden by cellular factors present in neoplastic cells. [2][3][4][5][6] Measles is an acute viral disease caused by a negative-strand RNA virus of the family Paramyxoviridae, which remains responsible for approximately 1 million deaths each year. 7 In the early stages of measles virus (MV) infection, the lymphoid organs and tissues are predominant sites for viral replication, leading to the formation of giant reticuloendothelial (Warthin-Finkeldey) cells in the tissues. 7 Measles is prevented by the use of a live attenuated virus vaccine now routinely administered during childhood. The Edmonston-B vaccine strain of measles virus (MV-Edm) was attenuated by serial tissue culture passage of a clinical isolate. 7 Despite its profound attenuation as a human pathogen, MV-Edm replicates more efficiently than nonattenuated measles virus in many primate cell lines, inducing cell cell fusion and the formation of characteristic multinucleated syncytia. 8 Here, we compared the ability of MV-Edm to replicate in neoplastic myeloma cells and normal cells, and we investigated its potential as an antitumor agent in human myeloma xenografts in vivo. We report that the virus replicated selectively in a panel of 6 myeloma cell lines and in CD138-sorted myeloma cells from 6 patients and that it caused potent cytopathic effects. When administered intratumorally or intravenously into mice bearing established myeloma xenografts, MV-Edm caused growth inhibition or total regression of 2 different myeloma xenograft models. Materials and methods Cell cultureThe multiple myeloma ARH-77 cell line (ATCC CRL-1621) was obtained from American Type Culture Collection (Rockville, MD). The rest of the multiple myeloma cell lines were kind gifts of Dr John Lust (RPMI 8226), Dr Diane Jelinek (KAS-6/1), and Dr Rafael Fonseca (KMS-11, MM1, JJN-3) from the Mayo Clinic (Rochester, MN). All the human myeloma cell lines were maintained in RPMI-1640 (Gibco BRL, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) except for KAS-6/1, which was grown in media supplemented with 1 ng/mL interleukin (IL)-6 (Sigma, St Louis, MO). Bone marrow aspirates were obtained after institutional review board approval and informed patient consent. Bone marrow samples were drawn into a tube containing heparin and centrifuged on a Ficoll-Hypaque gradient (Amersham Pharmacia, Piscataway, NJ) to enrich for mononuclear cells. Mononuclear cells were then incubated with MACS CD138 microbeads (Miltenyi Biotech, Auburn, CA) for 15 minutes in a 8°C water bath. CD138 ϩ primary myeloma cells were then isolated, washed, and maintained in 10% FBS-RPMI supplemented with 1 ng/mL IL-6. Peripheral blood lym...

Noninvasive methods are needed to study the kinetic properties of viruses in living organisms. Oncolytic viruses are used increasingly for cancer therapy but there is currently no satisfactory way to measure efficiency of tumor transduction, changing levels of viral gene expression or the timing of virus elimination. We therefore generated trackable oncolytic measles viruses expressing inert (nonimmunogenic, nonfunctional and accurately measurable) soluble marker peptides. The marker peptides did not compromise virus replication. Ex vivo and in vivo kinetics of the trackable viruses could be easily followed by measuring the concentrations of virally encoded marker peptides in culture supernatant or in serum. When mice bearing human tumor xenografts were challenged with the trackable viruses, distinct kinetic profiles of marker-gene expression could be correlated with distinct therapeutic outcomes. Oncolytic viruses expressing inert soluble marker polypeptides should greatly facilitate the rational development of effective, individually tailored cancer virotherapy.

Purpose: Preexisting antiviral antibodies in cancer patients can quickly neutralize oncolytic measles virus (MV) and decrease its antitumor potency. In contrast to "naked" viruses, cell-associated viruses are protected from antibody neutralization. Hence, we hypothesized that measles virotherapy of ovarian cancer in measles-immune mice might be superior if MV-infected mesenchymal stem cell (MSC) carriers are used. Experimental Design: Antimeasles antibodies titers in ovarian cancer patients were determined. The protection of MV by MSC from antimeasles antibodies, the in vivo biodistribution profiles, and tumor infiltration capability of MSC were determined. Measles-naïve or immune tumor-bearing mice were treated with naked virus or MSC-associated virus and mice survivals were compared. Results: MSC transferred MV infection to target cells via cell-to-cell heterofusion and induced syncytia formation in the presence of high titers of antimeasles antibody, at levels that completely inactivated naked virus. Athymic mice bearing i.p. human SKOV3ip.1 ovarian tumor xenografts passively immunized with measles-immune human serum were treated with saline, naked MV, or MV-infected MSC. Bioluminescent and fluorescent imaging data indicated that i.p. administered MSC localized to peritoneal tumors, infiltrated into the tumor parenchyma, and transferred virus infection to tumors in measles naïve and passively immunized mice. Survival of the measles-immune mice was significantly enhanced by treatment with MV-infected MSC. In contrast, survivals of passively immunized mice were not prolonged by treatment with naked virus or uninfected MSC. Conclusions: MSC should be used as carriers of MV for intraperitoneal virotherapy in measles-immune ovarian cancer patients. (Clin Cancer Res 2009;15(23):7246-55)

Measles virus offers an ideal platform from which to build a new generation of safe, effective oncolytic viruses. Occasional "spontaneous" tumor regressions have occurred during natural measles infections, but common tumors do not express SLAM, the wild-type MV receptor, and are therefore not susceptible to the virus. Serendipitously, attenuated vaccine strains of measles virus have adapted to use CD46, a regulator of complement activation that is expressed in higher abundance on human tumor cells than on their non transformed counterparts. For this reason, attenuated measles viruses are potent and selective oncolytic agents showing impressive antitumor activity in mouse xenograft models. The viruses can be engineered to enhance their tumor specificity, increase their antitumor potency and facilitate noninvasive in vivo monitoring of their spread. A major impediment to the successful deployment of oncolytic measles viruses as anticancer agents is the high prevalence of pre-existing anti measles immunity, which impedes bloodstream delivery and curtails intratumoral virus spread. It is hoped that these problems can be addressed by delivering the virus inside measles-infected cell carriers and/or by concomitant administration of immunosuppressive drugs. From a safety perspective, population immunity provides an excellent defense against measles spread from patient to carers and, in fifty years of human experience, reversion of attenuated measles to a wild type pathogenic phenotype has not been observed. Clinical trials testing oncolytic measles viruses as an experimental cancer therapy are currently underway.