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 (SIRPα). 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 SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 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. SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. SIRPα-αCD123 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 SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.

中文翻译：

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SIRPα-αCD123 融合抗体靶向 CD123 结合 CD47 阻断增强 AML 起始细胞的清除

急性髓性白血病 (AML) 干细胞 (LSC) 会导致疾病复发。CD47“不要吃我的信号”在 LSC 上上调，并通过与骨髓特异性信号调节蛋白 α (SIRPα) 相互作用来抑制吞噬作用，从而有助于免疫逃避。通过阻断 CD47 激活巨噬细胞已取得成功，但 CD47 在健康细胞上的普遍表达对此类疗法造成了潜在的限制。相比之下，CD123 是众所周知的 LSC 特异性表面标记，用作治疗靶点。在这里，我们报告了 SIRPα-αCD123 融合抗体的发展，该抗体将 CD47/SIRPα 信号传导的破坏定位到 AML，同时专门增强 LSC 清除。SIRPα-αCD123 抗体是通过将 SIRPα 的细胞外结构域与 αCD123 抗体融合而产生的。通过流式细胞术和表面等离子体共振分析抗体的结合特性。融合抗体的功能特征是通过使用原代 AML 患者细胞的抗体依赖性细胞吞噬作用和抗体依赖性细胞毒性测定来确定的。最后，利用体内移植试验来评估 LSC 靶向。即使在 CD47+ 健康细胞存在的情况下，SIRPα-αCD123 融合抗体也表现出对 CD123+ CD47+ AML 细胞的增加的结合和优先靶向。此外，SIRPα-αCD123 融合抗体将 CD47-SIRPα 轴的破坏局限于 AML 细胞。体外实验表明，SIRPα-αCD123 抗体极大地增强了异基因和自体巨噬细胞介导的 AML 细胞吞噬作用。而且，SIRPα-αCD123 融合抗体有效靶向具有体内移植潜力的 LSC。SIRPα-αCD123 抗体将局部 CD47 阻断与特定 LSC 靶向结合在单个分子中，最大限度地降低靶向健康细胞的风险并有效消除 AML LSC。这些结果验证了 SIRPα-αCD123 抗体作为 AML 的有希望的治疗干预措施。