Selective replication of TILT-123 was studied in A549 lung adenocarcinoma cells (positive control, purchased from ATCC, Manassas, VA, USA), human primary hepatocytes (Lonza Verviers SPRL, Bruxelles, Belgium), human MRC-5 fibroblasts (ATCC), and human vascular endothelial HUVEC cells (Millipore). The cells were plated on 24-well plates and infected with TILT-123, wild type Adenovirus 5 (VR-1516, ATCC), Ad5/3 replicative control virus (a similar selection device as TILT-123 but no cytokine payload), or Ad5/3-Luc1 non-replicative control virus [10 (link),19 (link)]. Infected cells and cell culture supernatants were harvested 72 h post-infection by centrifugation.
The virus was released from the cells by repeating freeze–thaw cycles four times in total. DNA was extracted from the samples with the phenol/chloroform/isoamyl alcohol (25:24:1) method and the virus copy numbers determined by detecting the adenovirus E4 copy number in samples by qPCR [20 (link)]. Infectious virus particles were determined with TCID50 assay, where a sample dilution series was plated on A549 cells and cytopathic effect formation was followed for ten days. TILT-123 transgene production was measured from the cell culture supernatants with ELISA, as described above.
The virus was released from the cells by repeating freeze–thaw cycles four times in total. DNA was extracted from the samples with the phenol/chloroform/isoamyl alcohol (25:24:1) method and the virus copy numbers determined by detecting the adenovirus E4 copy number in samples by qPCR [20 (link)]. Infectious virus particles were determined with TCID50 assay, where a sample dilution series was plated on A549 cells and cytopathic effect formation was followed for ten days. TILT-123 transgene production was measured from the cell culture supernatants with ELISA, as described above.
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