Effects of CLL1-ADC and CD33-ADC on in vitro and in vivo differentiation of normal CD34⁺ stem cells. (A-C) Enriched CD34⁺ cells from a pool of 3 donors were treated with PBS or the indicated doses of IgG-ADC (nonbinding control), CLL1-ADC, or CD33-ADC. Treated cells were seeded in methylcellulose-based hematopoietic colony-forming media (250 cells per plate) in triplicate and scored for erythroid (E), granulocyte (G), monocyte/macrophage (M), or granulocyte macrophage (GM) colony formation (average and standard deviation of n = 3). (A) The percentage of cells plated that gave rise to each colony type is shown. The percentage of cells that gave rise to (B) erythroid or (C) combined G, M, or GM myeloid colonies is shown. *P < .05; **P < .01; ***P < .001 for CD33-ADC compared with CLL1-ADC or CLL1-ADC compared with IgG-ADC applied at the same concentration using the 2-tailed equal variance Student t test. (D-E) Sublethally irradiated NSG mice (6-7 animals per group) were dosed with 2 × 10⁶ CD34⁺ cells (pooled from 3 healthy G-CSF–mobilized peripheral blood donors) and treated with 0.5 mg/kg IgG-ADC, CLL1-ADC, or CD33-ADC 24 hours after cell administration. (D) Fourteen days following cell dosing, overall bone marrow engraftment is shown as the percentage of human CD45⁺ cells and (E) myeloid engraftment is shown as the percentage of human CD45⁺CD33⁺ cells. (F) Kaplan-Meier survival analysis for 28 days following cell infusion is shown for a similar in vivo study for sublethally irradiated NSG mice (8 animals per group) dosed with 2 × 10⁶ CD34⁺ cells from a pool of three different mobilized peripheral blood donors. One day following irradiation, mice were dosed with vehicle (PBS) only or CD34⁺ cells. Twenty-four hours after cell administration, mice were treated with PBS or 0.5 mg/kg IgG-ADC, CLL1-ADC, or CD33-ADC. *P = 0.0226 for CLL1-ADC vs CD33-ADC survival using the log-rank (Mantel-Cox) test.