Figure 2
Determination of FMC63-scFv binding affinity to CD19 by steady-state analysis. (A) NALM6-GFP cells endogenously expressing WT-CD19 were incubated with various concentrations of His-tagged FMC63-scFv at 4 °C for either 0.5 or 4 h, as indicated, followed by staining with α-HIS-AF647 for subsequent flow cytometric analysis. (B) Titration of soluble FMC63-scFv on CD19 negative Jurkat cells transiently expressing Flag-tagged WT-CD19 or Flag-tagged SF-CD19. Incubations were performed with His-tagged FMC63 scFv at 4 °C for either 0.5 or 4 h. Secondary staining was performed with α-FLAG-PE and α-HIS-AF647. gMFI of FMC63-scFv binding of Flag-positive Jurkat cells was analyzed. Moreover, conditions were chosen to either promote or avoid ligand depletion, as indicated. (C) Representation of SF-CD19 in complex with the FMC63-scFv (PDB-ID 7URV) indicating in blue the three point mutations of the stability engineered SF-CD19 and in green the binding epitope of the FMC63-scFv on CD19. (D) Titration of SF-CD19 fusion protein (N-terminal His8-SUMO-tag) on FMC63:41BBz CAR expressing Jurkat cells. Secondary staining was performed with α-HIS-AF647. (A, B and D) Shown are averages ± standard deviations of background subtracted and normalized geometric mean fluorescence intensities (gMFIs) of three independent experiments. All data were fitted using a 1:1 binding model.
