The study was designed to evaluate 3 different dosing schedules of ASP1235 administered as 30-minute IV infusions. In dosing schedule A, patients received 1 dose of ASP1235 administered once every 3 weeks (Q3W) of a 21-day cycle. In dosing schedule B, patients would have received 1 dose of ASP1235 every other week of a 28-day cycle. No patients were treated on schedule B. In dosing schedule C, patients were to receive 1 fractionated dose weekly for the first 3 weeks of a 28-day cycle. A fractionated dose was the selected schedule A dose divided into 3 weekly doses in schedule C. Dose escalation in dosing schedule A was initially performed using an accelerated titration design with 1 patient per dose cohort until a grade ≥ 2 adverse event (AE) occurred (Supplemental Fig. S1). There were 6 dose cohorts in the schedule A Q3W dosing schedule (cohort 1, 0.8 mg/kg; cohort 2, 1.6 mg/kg; cohort 3, 3.2 mg/kg; cohort 4, 4.8 mg/kg; cohort 5, 6.0 mg/kg; cohort 6, 7.5 mg/kg). Two additional cohorts were added based on specific circumstances: cohort -1 (de-escalation cohort if the starting dose from cohort 1 was not tolerated) and cohort 6b (an additional cohort was evaluated to assess the 7.5-mg/kg dose level with the implementation of the protocol-required prophylactic eye regimen). The initial dose in dosing schedule B or C would be calculated based on the pharmacokinetic and safety data in dosing schedule A. After completion of Q3W dosing in schedule A, the data review team recommended once-weekly dosing (dosing schedule C) which followed a 3 + 3 design; the starting dose was 7.5 mg/kg split (fractionated) into 3 doses of 2.5 mg/kg per week for the first 3 weeks of a 4-week cycle.

The study was designed to evaluate 3 different dosing schedules of ASP1235 administered as 30-minute IV infusions. In dosing schedule A, patients received 1 dose of ASP1235 administered once every 3 weeks (Q3W) of a 21-day cycle. In dosing schedule B, patients would have received 1 dose of ASP1235 every other week of a 28-day cycle. No patients were treated on schedule B. In dosing schedule C, patients were to receive 1 fractionated dose weekly for the first 3 weeks of a 28-day cycle. A fractionated dose was the selected schedule A dose divided into 3 weekly doses in schedule C. Dose escalation in dosing schedule A was initially performed using an accelerated titration design with 1 patient per dose cohort until a grade ≥ 2 adverse event (AE) occurred (Supplemental Fig. S1). There were 6 dose cohorts in the schedule A Q3W dosing schedule (cohort 1, 0.8 mg/kg; cohort 2, 1.6 mg/kg; cohort 3, 3.2 mg/kg; cohort 4, 4.8 mg/kg; cohort 5, 6.0 mg/kg; cohort 6, 7.5 mg/kg). Two additional cohorts were added based on specific circumstances: cohort -1 (de-escalation cohort if the starting dose from cohort 1 was not tolerated) and cohort 6b (an additional cohort was evaluated to assess the 7.5-mg/kg dose level with the implementation of the protocol-required prophylactic eye regimen). The initial dose in dosing schedule B or C would be calculated based on the pharmacokinetic and safety data in dosing schedule A. After completion of Q3W dosing in schedule A, the data review team recommended once-weekly dosing (dosing schedule C) which followed a 3 + 3 design; the starting dose was 7.5 mg/kg split (fractionated) into 3 doses of 2.5 mg/kg per week for the first 3 weeks of a 4-week cycle.

The in vivo antitumor activity of ASP1235 was evaluated in a FLT3 positive THP-1 xenograft model. THP-1 cells were subcutaneously inoculated in the right frank of mice at 5 x10⁶ cells/head. The vehicle of each drug is shown as follows: ASP1235 (20 mM Histidine/L-Histidine-HCl, 5.5% trehalose dihydrate, 0.01% Polysorbate 20, pH6.0), venetoclax (60% phosal 50PG, 30% PEG400, 10% ethanol), azacitidine (PBS). ASP1235 was intravenously administrated to each mouse once per week. One day before ASP1235 treatment, each mouse received intraperitoneal injection of 20 mg/kg of nonspecific human IgG1 antibody. Venetoclax was orally administered daily at 100 mg/kg. Azacitidine was intravenously injected at 3 mg/kg for five consecutive days in the first week of treatment.

The in vivo antitumor activity of ASP1235 was evaluated in a FLT3 positive THP-1 xenograft model. THP-1 cells were subcutaneously inoculated in the right frank of mice at 5 x10⁶ cells/head. The vehicle of each drug is shown as follows: ASP1235 (20 mM Histidine/L-Histidine-HCl, 5.5% trehalose dihydrate, 0.01% Polysorbate 20, pH6.0), venetoclax (60% phosal 50PG, 30% PEG400, 10% ethanol), azacitidine (PBS). ASP1235 was intravenously administrated to each mouse once per week. One day before ASP1235 treatment, each mouse received intraperitoneal injection of 20 mg/kg of nonspecific human IgG1 antibody. Venetoclax was orally administered daily at 100 mg/kg. Azacitidine was intravenously injected at 3 mg/kg for five consecutive days in the first week of treatment.

The in vivo antitumor activity of ASP1235 was evaluated in a FLT3 positive THP-1 xenograft model. THP-1 cells were subcutaneously inoculated in the right frank of mice at 5 x10⁶ cells/head. The vehicle of each drug is shown as follows: ASP1235 (20 mM Histidine/L-Histidine-HCl, 5.5% trehalose dihydrate, 0.01% Polysorbate 20, pH6.0), venetoclax (60% phosal 50PG, 30% PEG400, 10% ethanol), azacitidine (PBS). ASP1235 was intravenously administrated to each mouse once per week. One day before ASP1235 treatment, each mouse received intraperitoneal injection of 20 mg/kg of nonspecific human IgG1 antibody. Venetoclax was orally administered daily at 100 mg/kg. Azacitidine was intravenously injected at 3 mg/kg for five consecutive days in the first week of treatment.

The in vivo antitumor activity of ASP1235 was evaluated in a FLT3 positive THP-1 xenograft model. THP-1 cells were subcutaneously inoculated in the right frank of mice at 5 x10⁶ cells/head. The vehicle of each drug is shown as follows: ASP1235 (20 mM Histidine/L-Histidine-HCl, 5.5% trehalose dihydrate, 0.01% Polysorbate 20, pH6.0), venetoclax (60% phosal 50PG, 30% PEG400, 10% ethanol), azacitidine (PBS). ASP1235 was intravenously administrated to each mouse once per week. One day before ASP1235 treatment, each mouse received intraperitoneal injection of 20 mg/kg of nonspecific human IgG1 antibody. Venetoclax was orally administered daily at 100 mg/kg. Azacitidine was intravenously injected at 3 mg/kg for five consecutive days in the first week of treatment.