Generation and utility of a biotinylated cetuximab. (A) Model is based on the crystal structure file 1YY9 (http://www.pdb.org/pdb/explore/explore.do?structureId = 1YY9), which provides the structure of the 4 extracellular domains (red, green, teal, and purple), and the bound cetuximab Fab (yellow and orange). The transmembrane helix (magenta) is modeled on known helix structure. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of nonreduced (left) and reduced (right) final biotinylated cetuximab product. (C) Titration of biotinylated cetuximab. A total of 10⁶ EGFR⁺ cells were stained with 0 μg (black), 1.45 μg (red), 0.145 μg (orange), 14.5 ng (yellow), 1.45 ng (green), 0.145 ng (blue), or 14.5 pg (purple) of biotinylated cetuximab followed by 0.5 μg PE-conjugated streptavidin and analyzed by flow cytometry. A total of 14.5 ng of biotinylated cetuximab was deemed sufficient for future staining of 10⁶ cells. (D) Biotinylated cetuximab can detect huEGFRt on transduced H9 cells. H9 cells were transduced with the CD19CAR-T2A-EGFRt containing lentivirus at a multiplicity of infection of 10 and analyzed 2 days later by flow cytometry using biotinylated cetuximab or biotinylated anti-Fc Ab (to detect the CD19CAR) followed by SA-PE (black histograms). Gray histograms represent nontransduced parental H9. Percentage positive staining is indicated in each histogram.