Binding competition of tafasitamab and CART19 to the B-cell surface marker CD19. (A) Detection of FMC63 using anti-His-PE (left) and tafasitamab using anti-Fcγ PE (right). First, Nalm6 was incubated with either FMC63-based His-tagged Fab (3.3 μg/mL = 66 nM), tafasitamab (10 μg/mL = 66.67 nM), or no antibody as a negative control. Second, either FMC63-based anti-CD19 Fab, tafasitamab, or no antibody was added and further incubation was performed. Third, a washing step, incubation with PE-labeled detection antibodies binding to FMC63-Fab (anti-His) or tafasitamab (anti-IgG, Fcγ-specific), another washing step, and flow cytometric analysis were performed (mean ± standard deviation [SD], ∗∗∗∗P < .0001, 1-way analysis of variance [ANOVA]; 2 independent experiments, 3 replicates). (B) Cytotoxicity assay of CART19 against CD19⁺ luciferase⁺ JeKo-1 in the presence of increasing doses of tafasitamab (10-400 μg/mL). Isotype IgG antibody was used as a control. At 24 hours, cytotoxicity was assessed by luminescence relative to controls (mean ± standard error of the mean [SEM], ∗P < .05, ∗∗P = .01, ∗∗∗P < .001, ∗∗∗∗P < .0001, 2-way ANOVA; n = 3, 2 replicates). (C) CART19 cell antigen-specific proliferation assay in the presence of increasing doses of tafasitamab (10-400 μg/mL). Isotype IgG antibody was used as a control. Lethally irradiated JeKo-1 were used for antigen stimulation. On day 5 of coculture, the absolute number of CD3⁺ T cells were quantified using volumetric flow cytometry (mean ± SEM, ∗∗P < .01, 1-way ANOVA; n = 3, 2 replicates). (D) CART19 cell antigen-specific CD107a degranulation assay in the presence of increasing doses of tafasitamab (10-400 μg/mL). Isotype IgG antibody was used as a control (mean ± SEM, ∗∗∗∗P < .0001, 2-way ANOVA; n = 3, 2 replicates). n.s., not significant; UTD, untransduced T cells.