A retargeted measles virus strain MV-GFP-H(AA)-scEGFR was generated by engineering the MV-NSe Edmonston vaccine strain to incorporate both CD46 (Y481A) and signaling lymphocyte activation molecule (SLAM) (R533A) ablating mutations in the hemagglutinin protein in combination with the display of a single-chain antibody against epidermal growth factor receptor (EGFR) at the C terminus of hemagglutinin. The unmodified MV-GFP virus was used as a positive control. Specificity of the EGFR retargeted virus was demonstrated in non-permissive Chinese hamster ovary (CHO) cells stably transfected to express either the natural receptors CD46 or SLAM or the target receptors EGFR and EGFRvIII. In vitro, the retargeted virus had potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multi-forme (GBM) cell lines that was comparable to the activity of the unmodified MV-GFP virus. Intratumoral administration of MV-GFP-H(AA)-scEGFRvIII in orthotopic GBM12 xenografts resulted in tumor regression, as demonstrated by bioluminescence imaging and significant prolongation of survival, that was comparable to the effect of the unmodified strain. In contrast to MV-GFP, central nervous system administration of the targeted MV-GFP-H(AA)-scEGFR virus in measles replication-permissive Ifnar(ko) CD46 transgenic mice resulted in no neurotoxicity. In conclusion, EGFR-retargeted measles virus strains have comparable therapeutic efficacy to the unmodified virus in glioma cells overexpressing EGFR or EGFRvIII in vivo and in vitro, and improved therapeutic index, a finding with potential translational implications in glioma virotherapy.
The majority of glioblastoma multiforme (GBM) tumors (80%) overexpress interleukin-13 receptor α2 (IL-13Rα2), but there is no expression of IL-13Rα2 in normal brain. Vaccine strains of measles virus have significant antitumor activity against gliomas. We tested the hypothesis that measles virus entry could be retargeted via the IL-13Rα2. MV-GFP-HAA-IL-13 was generated from the Edmonston-NSe vaccine strain, by displaying human IL-13 at the C-terminus of the H protein, and introducing CD46 and signaling lymphocyte activation molecule (SLAM)-ablating mutations in H. The IL-13 retargeted virus showed significant cytopathic effect (CPE) against IL-13Rα2 overexpressing glioma lines, and lack of CPE/viral replication in normal human astrocytes and normal human fibroblasts not expressing IL-13Rα2. In vivo treatment of orthotopically implanted GBM12 xenografts demonstrated significant prolongation of survival in mice treated with the retargeted strain (P < 0.0001), and comparable activity between the IL-13R retargeted strain and MV-GFP (P = 0.6377). In contrast to MV-GFP-treated mice, administration of the retargeted strain in the central nervous system of measles replication-permissive Ifnarko CD46 Ge mice resulted in lack of neurotoxicity. Strains of measles virus retargeted against the glioma-specific IL-13Rα2 receptor have comparable therapeutic efficacy, and improved specificity as compared with the unmodified measles virus strain MV-GFP in vitro and in vivo.
Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months.We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RTwith MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase^mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality^or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RTcombination^treated cells, as compared with single agent MV-CEA^treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore,TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination^treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/ RT^induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1blocked MV/RT^induced PARP cleavage whereas the Fas agonistic antibody CH11increased PARP cleavage in MV/RTcombination^treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway.These results could have translational implications in glioma therapy.
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.
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