Figure 2.

Establishing the Ad-LV system. (A,B) Flow cytometric analysis of the surface expression of the H protein fused to the original scFv or a mutant variant (Opt) on HEK293T cells by either staining for the C-terminal HIS-tag (A) of the H protein or with fluorescently labeled biotin (B) or untreated cells (w/o). Data are represented as mean ± SD of 4 independent experiments. (C) LV productivity for concentrated LV supernatant was determined on biotinylated cells. Data are represented as mean ± SD of 3 independent experiments. (D) The optimal order of combination of the three components (LVs, cells, and adapter) required for transduction was evaluated via transduction of Raji cells with a GFP-encoding Ad-LV (0.05 TU/cell) using an LLE-CD20-mAB (clone: LT20, 1000 ng/mL). While cells with vector were preincubated for 30 min at 37 °C, cells with adapter or vector with adapter were preincubated for 30 min at 4 °C. Afterwards, the respective third component was added. Transduction without the addition of any adapter (w/o) was used to confirm specificity. MV-LV directly targeting CD20 (α-CD20-LV) was used as positive control. Transduction efficiency was analyzed 3 days post transduction via quantification of the GFP-positive cells using flow cytometry. Data are represented as mean ± SD of 3 technical replicates. Statistic ordinary one-way ANOVA using Tukey’s multiple comparisons test; ns, non-significant; * p < 0.05, *** p < 0.001, **** p < 0.0001.