Fig. 3: AFM28 induces potent lysis of CD123⁺ LSPCs in AML and HR-MDS patient samples and spares healthy hematopoiesis.

BMMC patient-derived AML and MDS samples were treated with or without 100 pM of AFM28 for 24 h in the presence of IL-2-preincubated allogeneic NK cells, all derived from different donors, at an E:T ratio of 1:1. Analysis was performed using flow cytometry. LSPCs were defined as viable/CD45^low/CD34⁺/CD38⁻/CD117⁺ cells. The gating strategy is shown in Supplementary Fig. 2A. Cell counts of 0 pM treatment for LSPCs and CD123⁺ LSPCs were set to baseline. A Representative dot plot of LSPC lysis following treatment with 100 pM AFM28 or without AFM28 (indicated as 0 pM) of one AML patient sample. B, C Lysis of LSPCs from AML patients (B, n = 5) and from MDS patients (C, n = 5) in the presence of 100 pM AFM28 or without AFM28. Data are represented as mean ± SD and were analyzed using one-way and two-way ANOVA and Šídák’s multiple comparisons test. D, Schematic workflow of the CFU assay. Created in BioRender (https://BioRender.com/8l2brjy). E–G CFU assay results of AML (E, n = 5), MDS (F, n = 5) and healthy (G, n = 5) CD34⁺ cell samples treated with 0/10/100/1000 pM of AFM28 for 24 h in the presence of allogeneic NK cells at an E:T ratio of 1:1. “CD34⁺ only” describes culturing untreated CD34⁺ cells without allogeneic NK cells. Colonies were counted manually. Colony count of “CD34⁺ only” condition was normalized to 100%. Data are represented as mean ± SD and were analyzed using one-way ANOVA and Tukey’s multiple comparisons test. SD standard deviation.