Evaluation of in vivo activity of 20D9-ADC in xenograft mouse models. NSG mice were injected IV with 1 × 10⁵ luciferase expressing MOLM-13 cells (A-C) or 2 × 10⁶ luciferase expressing AML-573 PDX cells (E-F). Leukemic burden was monitored once or twice a week by BLI, and total flux was quantified. Mean ± SD is depicted. Treatment is indicated with triangles in dark blue (20D9-ADC, 3 mg/kg), light blue (20D9-ADC, 1 mg/kg), gray (PBS), or black (all groups as indicated). Dashed black line indicates imaging threshold. (A) One week after transplantation, mice were treated with 20D9-ADC (1 mg/kg or 3 mg/kg, IV) or PBS as control (n = 4/group) once a week for 6 weeks (1 mg/kg) or for 4 weeks (3 mg/kg). (B) BLI pictures of 1 representative mouse per group are shown. (C) One week after transplantation, mice were treated with either native or glycosylated 20D9-ADC or with either native or glycosylated IgG1-ADC (3 mg/kg; n = 3/group) once a week for 2 weeks. PBS control mice of experiment shown in (A) are included as control. (D) FLT3 RNA expression of AML PDX samples (n = 21) and primary patient samples³³ (n = 261) analyzed by single cell RNA barcoding sequencing.³⁴ Data presented as normalized log2 counts per million. Samples selected for ex vivo and in vivo analysis are marked in blue. (E) 20 days after transplantation, mice were treated at intermediate tumor burden with 20D9-ADC (3 mg/kg) or PBS as control (n = 3/group) once a week for up to 4 weeks (2 mice) or 5 weeks (1 mouse). (F) PBS-treated control mice from (E) (n = 3) were treated at day 41 after transplantation at advanced tumor burden with 20D9-ADC once a week for 4 weeks (2 doses of 3 mg/kg, followed by 2 doses of 1 mg/kg).