Fig 1: Expression and binding properties of CD98-specific TMs. (a) Antitumor activity of UniCAR T cells can be repeatedly switched ‘ON’ and ‘OFF’ in dependence of E5B9-tagged target modules (TMs). (b) The novel murine (mu) and humanized (hu) CD98 TM were generated by fusing the variable light (VL) and variable heavy (VH) domains of the aCD98 IgG1 mAb MEM-108 via flexible peptide linkers to the UniCAR epitope E5B9. The N-terminal murine Ig kappa leader sequence (L) mediates secretion, while the C-terminal hexahistidine (His6)-tag facilitates purification and detection of the recombinant proteins. (c, d) Ni-NTA purified TMs were separated by SDS-PAGE. (c) After staining with Coomassie Brilliant Blue G250, TM concentration was estimated based on a BSA standard. (d) Cell culture supernatant (S), wash fraction (W)1, W2 and eluate (E) were transferred to a nitrocellulose membrane. Recombinantly expressed TMs were subsequently detected via their C-terminal His6-Tag. (e, f) TM binding was analyzed by flow cytometry. (e) After incubation of tumor cells with 5 ng/µl of TM, TM binding was detected via the UniCAR epitope. As positive control, tumor cells were stained with an aCD98-APC-Vio770 Ab. Histograms show stained cells (blue) and respective negative controls (black). Numbers represent the percentage of CD98+ cells. Results of one out of three experiments are shown. (f) Tumor cells were incubated with increasing concentrations of muCD98 TM (upper panel) or huCD98 TM (lower panel) and subsequently stained with aHis-PE Ab. In order to determine TM affinity toward CD98, TM concentrations were plotted against the relative median fluorescence intensity (rel. MFI). Mean ± SEM of three independent experiments are shown.