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The tumor stroma acts as a barrier that limits the efficacy of systemically administered oncolytic viruses (OV). We previously demonstrated that stromal-selective, retargeted oncolytic measles viruses (MVs) delay in vivo tumor progression. To further characterize the contribution of stromal targeting to MV's overall in vivo efficacy in an experimental cancer model, a dual targeted oncolytic measles virus (MV-CD46-muPA) able to simultaneously infect murine stromal (via murine uPAR) and human cancer (via CD46) cells was developed. MV-CD46-muPA infected, replicated, and induced cytotoxicity in both murine and human cancer cells. Viral infection was successfully transferred from stromal to tumor cells in vitro, leading to tumor cell oncolysis. Systemic administration of MV-CD46-muPA led to improved antitumor effects in colon (HT-29) cancer xenografts compared to vehicle or CD46 only targeted MVs. These effects were associated with improved tumor viral deposition, increased apoptosis, and decreases in murine stromal endothelial cells and fibroblasts. MV-CD46-muPA modulated cell cycle, survival, proliferation, and metabolic pathways, as determined by functional proteomic analysis of treated tumors. The above findings further validate the concept that dual stromal and tumor cell viral targeting enhances the therapeutic effects of systemically administered OVs and support further preclinical and clinical development of stromal directed virotherapies.

The tumor microenvironment (TME) is a relevant target for novel biological therapies. MV-m-uPA and MV-h-uPA are fully retargeted, species-specific, oncolytic measles viruses (MV) directed against murine or human urokinase receptor (PLAUR/uPAR), expressed in tumor and stromal cells. The effects of stromal-selective targeting by uPAR-retargeted MVs were investigated. In vitro infection, virus-induced GFP expression, and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer-associated fibroblasts in a species-specific manner. In a murine fibroblast/human breast cancer 3D coculture model, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. Systemic administration of murine-specific MV-m-uPA in mice bearing human MDA-MB-231 xenografts was associated with a significant delay in tumor progression and improved survival compared with controls. Experiments comparing tumor (MV-h-uPA) versus stromal (MV-m-uPA) versus combined virus targeting showed that tumor and stromal targeting was associated with improved tumor control over the other groups. Correlative studies confirmed in vivo viral targeting of tumor stroma by MV-m-uPA, increased apoptosis, and virus-induced differential regulation of murine stromal genes associated with inflammatory, angiogenesis, and survival pathways, as well as indirect regulation of human cancer pathways, indicating viral-induced modulation of tumor-stroma interactions. These data demonstrate the feasibility of stromal-selective targeting by an oncolytic MV, virus-induced modulation of tumor-stroma pathways, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as antistromal agents. Implications: The current report demonstrates for the first time the biological, in vitro , and in vivo antitumor and molecular effects of stromal selective targeting by an oncolytic virus. Mol Cancer Res; 15(10); 1410-20. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.

Oncolytic measles virus (MV) induces cell fusion and cytotoxicity in a CD46-dependent manner. Development of fully retargeted oncolytic MVs would improve tumor selectivity. The urokinase-type plasminogen activator receptor (uPAR) is a tumor and stromal target overexpressed in multiple malignancies. MV-H glycoproteins fully retargeted to either human or murine uPAR were engineered and their fusogenic activity was determined. Recombinant human (MV-h-uPA) and murine (MV-m-uPA) uPAR-retargeted MVs expressing enhanced green fluorescent protein (eGFP) were rescued and characterized. Viral expression of chimeric MV-H was shown by reverse transcription-PCR and Western blot. In vitro viral replication was comparable to MV-GFP control. The receptor and species specificity of MV-uPAs was shown in human and murine cells with different levels of uPAR expression. Removal of the NH(2)-terminal fragment ligand from MV-uPA by factor X(a) treatment ablated the MV-uPA functional activity. Cytotoxicity was shown in uPAR-expressing human and murine cells. MV-h-uPA efficiently infected human endothelial cells and capillary tubes in vitro. I.v. administration of MV-h-uPA delayed tumor growth and prolonged survival in the MDA-MB-231 breast cancer xenograft model. Viral tumor targeting was confirmed by immunohistochemistry. MV-m-uPA transduced murine mammary tumors (4T1) in vivo after intratumor administration. MV-m-uPA targeted murine tumor vasculature after systemic administration, as shown by dual (CD31 and MV-N) staining of tumor capillaries in the MDA-MB-231 model. In conclusion, MV-uPA is a novel oncolytic MV associated with potent and specific antitumor effects and tumor vascular targeting. This is the first retargeted oncolytic MV able to replicate in murine cells and target tumor vasculature in a uPAR-dependent manner.

Targeting tumor-associated vascular endothelium by replication-competent viral vectors is a promising strategy for cancer gene therapy. Here we describe the development of a viral vector based on the Edmonston vaccine strain of measles virus targeted to integrin alpha(v)beta3, which is expressed abundantly on activated but not quiescent vascular endothelium. We displayed a disintegrin, M28L echistatin that binds with a high affinity to integrin alpha(v)beta3 on the COOH terminus of the viral attachment (H) protein and rescued the replication-competent recombinant virus by reverse genetics. The new targeted virus was named measles virus echistatin vector (MV-ERV). Its native binding to CD46 was purposefully retained to allow virus infection of tumor cells expressing this receptor. MV-ERV correctly displayed echistatin on the outer surface of its envelope and produced interesting ring formation phenomena due to cell detachment upon infection of susceptible Vero cells in vitro. MV-ERV grew to 10(6) plaque-forming units/mL, slightly lower than the parental Edmonston strain of measles virus (MV-Edm), but it selectively infected Chinese hamster ovary cells expressing integrin alpha(v)beta3. It also selectively infected both bovine and human endothelial cells on matrigels and unlike MV-Edm, MV-ERV infected newly formed blood vessels in chorioallantoic membrane assays. In animal models, MV-ERV but not the control MV-Edm caused the regression of s.c. xenografts of resistant multiple myeloma tumors (MM1) in severe combined immunodeficient mice. The tumors were either completely eradicated or their growth was significantly retarded. The specificity, potency, and feasibility of MV-ERV infection clearly show the potential use of MV-ERV in gene therapy for targeting tumor-associated vasculature for the treatment of solid tumors.

Objective. The Edmonston vaccine strain of measles virus (MV- Edm) is a novel oncolytic agent with potent in vitro and in vivo antitumor activity. In order to improve viral tumor specificity, suitable targets need to be identified. The “ideal” target for an oncolytic virus should be biologically relevant, overexpressed in tumor compared to normal cells, and preferably overexpressed by tumor stroma, to amplify the virus' antitumor effects. To test the retargeted virus' effects in appropriate syngeneic models, it should be relevant for murine tumors. The urokinase receptor (uPAR) is a biologically validated tumor and stromal target that has been associated with an aggressive tumor phenotype in several cancer types. Our goals are to determine if uPAR is a suitable target for MV oncolytic virotherapy in human and mouse cancer models.Methods and Results. The receptor binding, aminoterminal fragment (ATF) of human (h) and murine (m) uPA displayed into the C-terminus of a CD46 “blind” mutant MV-H glycoprotein (Y481A, R533A, S548L, F549S-Nakafumi et al., 2005-). Co-transfection of human and murine cells with uPAR retargeted H and F genes demonstrated potent in vitro fusion in uPAR overexpressing tumor cells, but not in uPAR non-overexpressing tumor or normal cells. CD46-ablated, eGFP expressing uPAR retargeted MV-Edm [MV-ATF (h) and (m)] were successfully rescued and expanded. Expression of chimeric H-ATF was successfully demonstrated by RT-PCR of MV-ATF-infected VERO-His cells, and by Western Blot of viral particles. Viruses retargeted to (h) and (m) uPAR entered cells selectively through their respective targeted receptors and induced cell fusion, syncitia formation and cytotoxicity. Since uPA/ uPAR binding is species specific, evidence of uPAR target specificity was further demonstrated by the absence of cell fusion of human uPAR(+) cells after infection with MV-ATF (murine) and vice versa. Successful uPAR dependent viral replication of MV-ATF (m) was demonstrated in uPAR (+) murine tumorigenic cells. Murine mammary cancer cells (4T1) were implanted in immunocompetent Balb/C and SCID mice. Mice received intratumor (IT) injections of MV-GFP retargeted against (m) uPAR and sacrificed 48-96 hours after the last dose. IT administration of MV-ATF (m) resulted in successful infection and syncytia formation in tumor cells as well as tumor vasculature in vivo in immunocompetent and SCID tumor bearing mice, as demonstrated by confocal laser microscopy.Conclusions: uPAR mediated cell fusion is feasible in human and murine cancer cells. uPAR retargeted MV-Edm specifically and efficiently induces fusion and cytotoxicity in human and murine cancer cells in a uPAR dependent manner. Our results demonstrate for the first time successful replication of a fully retargeted measles virus in murine tumor cells in vitro and in vivo, and suggest targeting of tumor vasculature. MV-ATF is a novel, promising viral oncolytic agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the antitumor effects of this novel virus in syngeneic and xenograft models are underway.Molecular Therapy (2006) 13, S166–S167; doi: 10.1016/j.ymthe.2006.08.498 [ABSTRACT FROM AUTHOR]