Characterization of CD123-6 and G4723A antibodies. (A) Binding of the CD123 antibodies to IL-3Rα, GM-CSFRα, and their swapped domain molecules as assessed by enzyme-linked immunosorbent assay. (B) The number of G4723A binding sites (CD123 ABC) on bone marrow cells from AML and healthy donors. Cell populations were defined by lineage markers as presented in supplemental Table 1. (C) Internalization of G4723A antibody by EOL-1 or HNT-34 cells. Cells were incubated with AF488-labeled G4723A or nonbinding huIgG1 (Ab, antibody); then the surface-bound fluorescence was quenched, and internalized AF488 signal was quantified by flow cytometry at indicated time points. (D) Processing of [³H]-propionate-G4723A antibody by HNT-34, MV4-11, and SHI-1 cells. Cells were exposed to G4723A antibody labeled with [³H]-propionate for 30 minutes followed by wash and 24 hours of incubation. The proteolytic catabolism of G4723A was quantified by comparing the protein and non-protein-associated radioactivity present in the cell lysate and culture medium. AML progen, AML progenitor; CLP, common lymphoid progenitors; CMP, common myeloid progenitors; GMP, granulocyte macrophage progenitors; HSC, hematopoietic stem cells; LSC, leukemic stem cells; MEP, megakaryocyte-erythroid progenitors; MPP, multipotent progenitors; NTD, N-terminal domain.