Measles (MV) is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths1,2. While it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to initially infect macrophages and dendritic cells of the airways using the signaling lymphocytic activation molecule (SLAM, CD150) as receptor3-6. These cells then cross the respiratory epithelium and ferry the infection to lymphatic organs where MV replicates vigorously7. How and where the virus crosses back into the airways has remained unknown. Based on functional analyses of surface proteins preferentially expressed on virus-permissive epithelial cell lines, we identified nectin-48 (poliovirus-receptor-like-4) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains MV entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is down-regulated in infected epithelial cells, including those of macaque tracheas. While other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that MV targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer9-11, which has implications for ongoing MV-based clinical trials of oncolysis12.

Combination chemotherapy regimen incorporating CD20 antibodies are commonly used in the treatment of CD20-positive non-Hodgkin's lymphoma (NHL). Fludarabine phosphate (F-araAMP), cyclophosphamide, and CD20 antibodies (Rituximab) constitute the FCR regimen for treating selected NHL, including aggressive mantle cell lymphoma (MCL). As an alternative to the CD20 antibody, we generated a CD20-targeted measles virus (MV)-based vector. This vector was also armed with the prodrug convertase purine nucleoside phosphorylase (PNP) that locally converts the active metabolite of F-araAMP to a highly diffusible substance capable of efficiently killing bystander cells. We showed in infected cells that early prodrug administration controls vector spread, whereas late administration enhances cell killing. Control of spread by early prodrug administration was also shown in an animal model: F-araAMP protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Enhanced oncolytic potency after extensive infection was shown in a Burkitt's lymphoma xenograft model (Raji cells): After systemic vector inoculation, prodrug administration enhanced the therapeutic effect synergistically. In a MCL xenograft model (Granta 519 cells), intratumoral (i.t.) vector administration alone had high oncolytic efficacy: All mice experienced complete but temporary tumor regression, and survival was two to four times longer than that of untreated mice. Cells from MCL patients were shown to be sensitive to infection. Thus, synergy of F-araAMP with a PNParmed and CD20-targeted MV was shown in one lymphoma therapy model after systemic vector inoculation. [Cancer Res 2007;67(22):10939-47]

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMPexpressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis. (Cancer Res 2006; 66(15): 7694-700)

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.