Schematic drawing and binding characteristics of CiTE, sctb, and control molecules. (A) Modular composition of CiTE, sctb, and BiTE-like molecule. (B) Binding analysis of CiTE and sctb to MOLM-13:PD-L1 cells and HD T cells at 15 ng/µL concentration by flow cytometry. (C) Binding analysis of PD-1_ex (in PD-1_ex.αCD3) and αPD-L1 scFv (in αPD-L1.αCD3) to HEK:PD-L1, MOLM-13:PD-L1, and HEK:PD-L1_ind. cells at 1.5 ng/µL concentration. Gray line indicates unspecific staining by the secondary antibody. Histograms show 1 of 3 experiments with similar results. (D) Binding of αPD-L1 mAB (clone MIH1) to MOLM-13:PD-L1 cells in the presence or absence of CiTE, sctb, or controls at 150 nM concentration as measured by flow cytometry. Mean values show n = 3 independent experiments with standard error of the mean (SEM) as error bars. For statistical analysis, unpaired Student t test with Welch correction was applied. *P < .05, **P < .01, ***P < .001. MFI, mean fluorescence intensity.