Development of effective therapies against acute myeloid leukemia (AML) will likely require elimination of the leukemic stem cell (LSC), which maintains the AML hierarchy. Based on high expression of the IL-3 receptor α chain (CD123) on AML-LSC, we tested a neutralizing antibody against CD123 (7G3) for its in vitro and in vivo effects on AML cells. By flow cytometric analysis, high expression of CD123 on CD34+/38− AML-LSC was confirmed. In vitro, 7G3 inhibited IL-3-induced proliferation in 31/34 primary AML specimens tested. Moreover, exposure of primary AML cells to 7G3 in vitro caused inhibition of IL-3-induced tyrosine phosphorylation and downstream signaling of the IL-3 receptor βc chain in a concentration-dependent fashion. Ex vivo treatment of primary AML cells with 7G3 (10 μg/ml, 2 h) profoundly reduced leukemic engraftment of human CD45+ (huCD45+) cells in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice in 9/10 evaluable samples (mean ± SEM, 89.7 ±1.9% inhibition relative to IgG2a control, P=0.013), establishing that the AML-LSC was targeted. The overall survival of engrafted mice was significantly improved (median survival of control IgG2a-treated group 11.5 weeks versus 24 weeks for the 7G3-treated group, n = 10 per group, P=0.019). Moreover, ex vivo 7G3 treatment reduced the short-term homing efficiency of AML cells to the bone marrow by 65% compared with IgG2a control-treated cells, measured at 24 h post inoculation. In contrast, ex vivo exposure to 7G3 had minor, if any, effects on the engraftment of normal bone marrow cells (23.5 ±8.9% inhibition relative to IgG2a control, n=5). Direct delivery of 7G3-treated AML cells into the bone marrow by intra-femoral injection did not completely restore engraftment, suggesting a specific biological role of 7G3 in inhibiting homing, lodgement and proliferation of AML-LSC. Despite some sample to sample heterogeneity, thrice-weekly administration of 7G3 to NOD/SCID mice with pre-established AML consistently reduced AML dissemination to peripheral blood, spleen and liver, and reduced infiltration of a single AML sample in the bone marrow. 7G3 treatment was also associated with down-regulation of CD123 expression on AML cells in vivo. AML cells harvested from 7G3-treated mice were unable to repopulate secondary recipient mice, with engraftment reduced to 5.7 ±4.1% (n=7) huCD45+ cells in the femoral bone marrow compared with 26.8 ±6.6% in IgG2a-treated controls (n=9, P=0.024), indicating that CD123 neutralization targeted AML-LSC self-renewal ability, specifically reducing the proportion of AML-LSC. These results suggest that blocking CD123 inhibits homing, lodgement and proliferation of AML-LSC in the bone marrow microenvironment and prevents subsequent organ infiltration, and support additional investigations into 7G3 and its potential as a novel therapy for AML.

Author notes

Disclosure:Employment: SB, DG and GV are employees of CSL Ltd. Consultancy: AL and RL are consultants of CSL Ltd. Research Funding: AL, AB, JD and RL receive research funding from CSL Ltd. Financial Information: AL receives royalty on the 7G3 patent.