Background Despite advances in the development of novel strategies against acute myeloid leukemia (AML), treatment options are limited and most patients relapse. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate the outgrowth of the disease, highlighting the need of targeting LSCs to improve patient survival. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen. CD47 is a ubiquitously expressed immune checkpoint upregulated on LSCs where it acts as an immune escape mechanism. CD47 transmits a ‘don’t eat me’ signal upon its interaction with the signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123+ AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity.
Materials and Methods The bifunctional SIRPα-CD123 antibody was generated by fusing an extracellular domain of the SIRPα receptor, which functions as the CD47 blocking domain, to the CD123 antibody. The biological activity of the SIRPα-CD123 antibody was examined using AML-derived MOLM-13 cells, primary AML patient material and patient-derived xenografted (PDX) AML cells with NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice.
Results The SIRPα fusion improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody. The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity via NK cells. Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells. Furthermore, an improved cytotoxicity towards leukemia initiating AML PDX cells was observed with the SIRPα-CD123 antibody using luciferase bioluminescence in vivo imaging. With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in both allogenic and autologous setting. We were also able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity.
Conclusions The bifunctional SIRPα-CD123 fusion antibodies target the CD123+CD47+ cells and stimulate their phagocytosis by blocking the inhibitory CD47 signal. The dual mode of action of the SIRPα-CD123 has the potential to deplete the AML LSCs through NK cell cytotoxicity and macrophage-mediated phagocytosis while restricting the CD47 related on-target off-leukemia toxicity.
Support H2020-EU grant agreement no 641549
Disclosure Information S. Tahk: None. S.M. Schmitt: None. B. Vick: None. C. Augsberger: None. L. Pascual Ponce: None. I. Jeremias: None. G. Wittmann: None. M. Subklewe: None. N.C. Fenn: None. K. Hopfner: None.
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