Peng, Kah-Whye

ABSTRACTA novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. StandardMus musculuslaboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whetherMus paharicould serve as a model for XMRV, primaryMus paharifibroblasts and mice were infected with cell-free XMRV. Infection of cellsin vitroresulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV intoMus paharimice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP+CD4+T cells and CD19+B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3in vivo. These data demonstrate infection ofMus pahariby XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infectionin vivo. These data support the use ofMus paharias a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.

Abstract Edmonston vaccine strains of measles virus (MV) have shown significant antitumor activity in preclinical models of ovarian cancer. We engineered MV to express the marker peptide carcinoembryonic antigen (MV-CEA virus) to also permit real-time monitoring of viral gene expression in tumors in the clinical setting. Patients with Taxol and platinum-refractory recurrent ovarian cancer and normal CEA levels were eligible for this phase I trial. Twenty-one patients were treated with MV-CEA i.p. every 4 weeks for up to 6 cycles at seven different dose levels (103–109 TCID50). We observed no dose-limiting toxicity, treatment-induced immunosuppression, development of anti-CEA antibodies, increase in anti-MV antibody titers, or virus shedding in urine or saliva. Dose-dependent CEA elevation in peritoneal fluid and serum was observed. Immunohistochemical analysis of patient tumor specimens revealed overexpression of measles receptor CD46 in 13 of 15 patients. Best objective response was dose-dependent disease stabilization in 14 of 21 patients with a median duration of 92.5 days (range, 54–277 days). Five patients had significant decreases in CA-125 levels. Median survival of patients on study was 12.15 months (range, 1.3–38.4 months), comparing favorably to an expected median survival of 6 months in this patient population. Our findings indicate that i.p. administration of MV-CEA is well tolerated and results in dose-dependent biological activity in a cohort of heavily pretreated recurrent ovarian cancer patients. Cancer Res; 70(3); 875–82

Abstract 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)

AbstractUp to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50% of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover, the naive and memory phenotypes of transduced resting B cells were maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-cell chronic lymphocytic leukemia cells, blocked in G0/G1 early phase of the cell cycle. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B cell–based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells.

ABSTRACT Gene transfer vectors containing adenovirus (Ad) serotype 35 (Ad35) fibers have shown promise for cancer and stem cell gene therapy. In this study, we attempted to improve the in vitro and in vivo infection properties of these vectors by increasing their affinity to the Ad35 fiber receptor CD46. We constructed Ad vectors containing either the wild-type Ad35 fiber knob (Ad5/35) or Ad35 knob mutants with 4-fold- and 60-fold-higher affinity to CD46 (Ad5/35+ and Ad5/35++, respectively). In in vitro studies with cell lines, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not translate into correspondingly higher transduction efficiencies, regardless of the CD46 receptor density present on cells. However, in vivo, in a mouse model with preestablished CD46 high liver metastases, intravenous injection of Ad5/35++ resulted in more-efficient tumor cell transduction. We conclude that Ad5/35 vectors with increased affinity to CD46 have an advantage in competing with non-CD46-mediated sequestration of vector particles after intravenous injection.

ABSTRACT Enveloped viruses trigger membrane fusion to gain entry into cells. The receptor affinities of their attachment proteins vary greatly, from 10 −4 M to 10 −9 M, but the significance of this is unknown. Using six retargeted measles viruses that bind to Her-2/neu with a 5-log range in affinity, we show that receptor affinity has little impact on viral attachment but is nevertheless a key determinant of infectivity and intercellular fusion. For a given cell surface receptor density, there is an affinity threshold above which cell-cell fusion proceeds efficiently. Suprathreshold affinities do not further enhance the efficiency of membrane fusion.