Journal: bioRxiv
Article Title: FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment
doi: 10.1101/2022.12.19.520931
Figure Lengend Snippet: (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. (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 #6 or the E3_5 blocking adapter and tested for transduction of mFAP + NIH3T3mFAP and mFAP − NIH3T3 cells in comparison to 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 to the positive control A549 cell line upon CAR antibody staining.
Article Snippet: Mouse FAP (mFAP)-binding DARPins were screened via ELISA using recombinant mFAP (R&D Systems), and recombinant maltose-binding protein (MBP; kindly provided by Joana Marinho, University of Zurich) for an MBP binder as a positive control.
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Flow Cytometry, Expressing, Staining, FLAG-tag, Concentration Assay, Titration, Transduction, Incubation, Blocking Assay, Comparison, Positive Control
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