Analysis of hematotoxicity of 20D9-ADC. (A) Expression of FLT3 and CD64 in CD34⁺ healthy bone marrow (BM) cells measured in flow cytometry. Mean ± SD of n = 3 donors. (B-C) CD34⁺ cells were treated with 0.04 μg/mL, 0.2 μg/mL, or 1 μg/mL 20D9-ADC; 1 μg/mL IgG1-ADC; or PBS and analyzed in flow cytometry after 4 days. Kruskal-Wallis test; mean ± SD of n = 5. (B) Percentage of living cells measured with Annexin V/PI staining and normalized to PBS. (C) Differentiation assessment after staining to differentiation markers. CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; MEP, megakaryocyte/erythroid progenitors; MLP, multilymphoid progenitors; MPP, multipotent progenitors; HSC, hematopoietic stem cells. (D) Assessment of clonogenic capacity of healthy CD34⁺ BM cells. Cells were treated with 0.04 μg/mL, 0.2 μg/mL, or 1 μg/mL 20D9-ADC; 1 μg/mL IgG1-ADC; or PBS and plated afterwards for colony-forming unit (CFU) assay without further treatment. After 14 days, colonies were counted. GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte; GM, granulocyte, macrophage; M, macrophage; G, granulocyte; E, erythrocyte; BFU-E, burst-forming unit erythrocyte. 2-way analysis of variance; mean ± SD, n = 5. ∗P < .05. ns, not significant.