Treatment combination of 20D9-ADC and TKIs. (A-B) Upregulation of FLT3 cell surface expression in MOLM-13 cells after treatment with kinase inhibitors compared with untreated control. Cells were analyzed in flow cytometry after treatment. Dotted line represents MFI ratio of untreated cells. Mean ± SD of n = 2 is depicted. (A) Cells were treated with 5, 25, or 50 nM midostaurin, quizartinib, and sorafenib for 6, 24, 48, or 72 hours. (B) Cells were treated with 5 nM midostaurin, 1 nM quizartinib, 5 nM sorafenib, or 1 μM dasatinib for 72 hours. (C-F) Treatment combination of 20D9-ADC and midostaurin (C-D) or quizartinib (E-F) in MOLM-13 cells compared with treatment with 20D9-ADC, midostaurin, or quizartinib as single agent. Viability was determined after 96 hours by resazurin fluorescence and normalized to dimethyl sulfoxide−treated control. (C,E) Each dot represents 1 biological replicate; the horizontal line indicates the mean. Combination indices (CIs) with standard deviation were determined using CompuSyn software; CI < 1 indicates synergy and is underlined; CI = 1 additivity; CI > 1 antagonism. (D-F) The synergy score was calculated by Synergy Finder software using zero interaction potency (ZIP) modeling. Gray triangles indicate increasing drug concentrations. A positive Synergy score value δ and the red coloring indicate synergism. (G) Treatment combination of 20D9-ADC and midostaurin in vivo. NSG mice were injected IV with 1e5 luciferase-expressing MOLM-13 cells. Leukemic burden was monitored once or twice a week by BLI, and total flux was quantified (left). Mean ± SD is depicted. Dashed black line indicates imaging threshold. One week after transplantation, mice were treated for 3 weeks with 20D9-ADC (1 mg/kg IV, once per week), midostaurin (50 mg/kg by mouth, 5 days per week), a combination of both, or PBS as control (n = 4/group). Bioluminescence imaging of 1 representative mouse of each group at days 6 and 16 (right). Two-way analysis of variance: ∗∗∗P < .001; ∗∗∗∗P < .0001.