SIRP[alpha]-[alpha]CD123 fusion antibodies targeting CD123 in conjunction with CD47 blockade enhance the clearance of AML-initiating cells

Background Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRP[alpha]). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRP[alpha]-[alpha]CD123 fusion antibodies that localize the disruption of CD47/SIRP[alpha] signalling to AML while specifically enhancing LSC clearance. Methods SIRP[alpha]-[alpha]CD123 antibodies were generated by fusing the extracellular domain of SIRP[alpha] to an [alpha]CD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. Results SIRP[alpha]-[alpha]CD123 fusion antibodies exhibited increased binding and preferential targeting of CD123.sup.+ CD47.sup.+ AML cells even in the presence of CD47.sup.+ healthy cells. Furthermore, SIRP[alpha]-[alpha]CD123 fusion antibodies confined disruption of the CD47-SIRP[alpha] axis locally to AML cells. In vitro experiments demonstrated that SIRP[alpha]-[alpha]CD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRP[alpha]-[alpha]CD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. Conclusions SIRP[alpha]-[alpha]CD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRP[alpha]-[alpha]CD123 antibodies as promising therapeutic interventions for AML. Keywords: CD47, Acute myeloid leukaemia, CD123, Leukemic stem cells, Phagocytosis, Immunotherapy