Fig. 2: Oncolytic measles virus encoding BiKEs.
A Schematic of recombinant MV-BiKE. (i) The BiKE transgene is encoded downstream of the P open reading frame within the MV genome. Hemagglutinin (HA) and hexa histidine (His6) tags enable purification and detection. Kozak and Igκ leader sequences mediate efficient translation and secretion. (G4S)3, glycine-serine peptide linker; HMA, human muscle aldolase linker; scFv, single chain variable fragment with variable heavy (VH) and variable light (VL) chain; TAA, tumor-associated antigen. (ii) Transgene-encoding virus can be rescued via a reverse genetics system and triggers (iii) BiKE secretion upon infection. BiKEs included in this study target either human carcinoembryonic antigen (CEA) as model tumor antigen or human high molecular weight melanoma-associated antigen (HMWMAA) as a non-relevant control. B Replication kinetics of MV-BiKE. Vero producer cells were inoculated with MeVac P-BiKE or unmodified MeVac at MOI 0.03. Viral progeny at designated time points were determined via serial dilution titration assay to obtain a multistep growth curve. C Direct cytotoxic effect of MV-BiKE. Cells were infected with respective viruses at MOI 0.03 for Vero (i) or at MOI 1 for colorectal cancer cell lines KM12 (ii) and HT-29 (iii). Cell viability was determined via XTT assay and normalized to mock-infected control. D, E BiKE expression kinetics on infected colorectal cancer cells. KM12 and HT-29 cells were inoculated with MeVac P-BiKE or MeVac at MOI 1. Supernatants and cell lysates were collected at indicated time points. BiKE concentrations were determined via ELISA using anti-His6 and anti-HA antibodies and purified vBiKEs as standard. D Kinetics of BiKE concentrations in culture supernatants after infection with MV-BiKE. E Concentrations of cell-associated BiKE in tumor cell lysates and BiKE release into culture supernatants. In C–E mean values of technical triplicates are shown. Error bars indicate SD.
