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. 2023 Aug 28;31(10):2914–2928. doi: 10.1016/j.ymthe.2023.08.018

Figure 3.

Figure 3

Retargeting of Ad5 to murine fibroblasts using a human/mouse cross-reactive FAP DARPin

(A) Analysis of hFAP DARPins for binding to mFAP via ELISA. Purified DARPins, previously selected to be hFAP specific, were analyzed via ELISA for cross-reactivity to mFAP. The unselected, non-binding DARPin E3_5 was applied as a negative binding control. The maltose-binding protein (MBP)-specific DARPin off7 and recombinant MBP were applied as a technical positive binding control. The dashed line indicates a cut-off signal set on the negative binding control to select mFAP-binding DARPins. Bars represent mean transduction level of two biological replicates ± SD. Statistics: unpaired t test; ∗p < 0.05, ∗∗p < 0.005; p values are indicated for each sample with respect to the E3_5 control DARPin. (B) Flow cytometry analysis of mFAP expression of the NIH3T3 and NIH3T3mFAP cell line with mFAP antibody staining. (C) Cell-based DARPin binding assay on target and non-target cells. Purified DARPins selected as mFAP binders by ELISA were analyzed for binding on mFAP+ NIH3T3mFAP and mFAP− NIH3T3 cells. Binding was detected by flow cytometry upon FLAG tag antibody staining of the FLAG-tagged DARPin. The unselected, non-binding DARPin E3_5 was applied as a negative binding control. Bars represent specific binding signal of single point measurements. Representative data at 1 μM DARPin concentration of a titration experiment are shown. MFI, mean fluorescent intensity. (D) Transduction of target and non-target cells by mFAP adapter-retargeted Ad5. Recombinant Ad5 encoding iRFP670 was pre-incubated with the mFAP adapter no. 6 or the E3_5 blocking adapter and tested for transduction of mFAP+ NIH3T3mFAP and mFAP– NIH3T3 cells in comparison with the untargeted Ad5 at an MOI of 2 (PFU/cell). Transduction levels were determined via cellular expression of iRFP670 detected by flow cytometry. Bars represent mean transduction level of two biological replicates ± SD. Representative data of three independent experiments are shown. (E) Flow cytometry analysis for CAR expression of the NIH3T3 and NIH3T3mFAP cell line in comparison with the positive control A549 cell line upon CAR antibody staining.