Comparative studies of recombinant oncolytic viruses for the treatment of CD133-positive tumors
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Cancer stem cells (CSCs) have been found to mark the tumorigenic cell population within various malignancies. Among many putative markers, prominin-1 (also known as CD133) is frequently used to identify and isolate CSCs of the above mentioned malignancies. To date, there is no effective approach to reduce the rate of tumor recurrence ascribed to CSCs. Thus, targeting of CSCs is critical to achieve long-term success in cancer treatment. One strategy to selectively destroy CSCs is the precise targeting of this cell subset via targeted oncolytic viruses (OVs). One of the lead oncolytic agents in ongoing clinical trials is oncolytic measles virus (MV) based on the safe vaccine strain Edmonston B (Edm) that induced complete remission after one intravenous (i.v.) administration of 1E+11 infectious virus in one patient suffering from disseminated multiple myeloma (Russell et al., 2014). MV-Edm uses the ubiquitous membrane cofactor protein (MCP, also known as CD46) for cell entry, which is overexpressed on tumor cells. To restrict virus replication to tumor cells, the glycoprotein (gp) complex of MV can be engineered to redirect receptor usage to cell surface proteins of choice. Based on this approach, a CD133-retargeted MV (MV-CD133) was generated prior to this work through fusion of a CD133-specific single-chain antibody fragment (scFv) as targeting domain to the receptor attachment protein hemagglutinin (H) ablated for its natural receptors CD46, signal lymphocyte-activation molecule (SLAM) and nectin-4 (also known as Poliovirus receptor-related 4, PVRL4). MV-CD133 exhibited a stronger oncolytic activity on CD133-positive tumors compared to untargeted parental MVs using CD46 for cell entry (Bach and Abel et al., 2013).As tumor regression was incomplete, this thesis pursued strategies to enhance the oncolytic activity of MV-CD133 without having to compromise on safety. For that purpose, arming with the prodrug 5-fluorocytosine (5-FC) converting suicide gene supercytosinedeaminase (SCD), or with P/V/C genes from wild-type MVs, or receptor extension through omission of blinding mutations were pursued. Additionally, a chimeric vesicular stomatitis / measles virus (VSV-MV), in which the gp VSV-G was replaced by the MV-H and MV-F gp fused to a CD133-scFv was examined. The viruses were analyzed for their oncolytic activity in vitro and by in vivo models of the hepatocellular carcinoma (HCC) cell line HuH7 and of the primary glioma sphere cells NCH644 using NOD/Scid (Nonobese diabetic/severe combined immunodefiency) mice known to be devoid of human CD46 expression. All viruses were found to be highly selective for CD133-positive cells as demonstrated by infection of receptor transgenic chinese hamster ovary (CHO) cells. As CD133 is also expressed on early hematopoietic progenitor cells, the proliferative and differentiation capacity of infected human hematopoietic stem cells (HSCs) was evaluated using clonal outgrowth assays. None of the CD133-retargeted viruses impaired the hematopoietic capabilities of HSCs. On HuH7 cells, VSV-MV chimeric viruses yielded substantially higher titers than their parental viruses. VSV-CD133 and MV-SCD-CD133 (+ 5-FC) achieved the most rapid and efficient cytotoxicity on HuH7 cells. In a subcutaneous HuH7 model, VSV-CD133 most efficiently slowed down tumor growth resulting in a prolonged survival of mice treated intratumorally (i.t.) and i.v.. VSV-CD133 infected a more than 1E+04-fold larger area of the tumor than MV-CD133 did in the same period. In killing assays on NCH644 cells, all viruses killed cells dose-dependently. While MV-CD46/CD133 and MV-SCD-CD133 (+ 5-FC) caused a continuous decline in cell numbers over time even at low multiplicity of infection (MOI), VSV-MV chimeric viruses were only able to reduce cell viability within in the first 48 h after infection. In an orthotopic NCH644 model, MV-CD46/CD133 and MV-SCD-CD133 (+ 5-FC) were most effective in prolonging survival compared to mock-treated mice. Ex vivo sphere formation assays revealed that cells of those tumors explanted at termination showed impaired stemness properties compared to those of the other viruses. By contrast, VSV-CD133 led to severe neurotoxic symptoms within 10 days after infection in tumor bearing mice. The neurotoxicity was also apparent in tumor-naïve mice that were intracerebrally (i.c.) injected with VSV-CD133 as well as upon injection with nontargeted VSV-MV suggesting that deceased mice must have succumbed to another genesis than that of i.t. virus amplification or the scFv themselves. The role of the yet un-known neuronal MV receptor or a membrane fusion process that occurred independently from a contact with H need to be tested as potential causatives.Verknüpfung zu Publikationen oder weiteren Datensätzen
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