Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) significantly reduces the rate of relapse in Acute Myeloid Leukemia (AML) but comes at the cost of significant Treatment Related Mortality (TRM). Despite the reduction in relapse overall, it remains common, especially in high risk groups. There is increasing evidence that many cases of relapse are driven by residual AML that can be detected with minimal residual disease techniques. A large proportion of the morbidity that prevents most patients accessing allo-HSCT is due to nonspecific toxic conditioning agents which are required to remove recipient Hematopoietic Stem and Progenitor Cells (HSPC) allowing for successful engraftment. We propose that a targeted conditioning agent against AML and HSPC can facilitate a reduction in relapse and TRM in allo-HSCT.

CD300f is expressed evenly across HSPC subtypes. CD300f has equivalent transcription and protein expression as CD33 on AML. We have developed an anti-CD300f antibody which efficiently internalises into target cells. Harnessing this ability to internalise, we have generated a highly potent anti-CD300f Antibody Drug Conjugate (ADC) with a pyrrolobenzodiazepine (PBD) warhead which selectively depletes AML cell lines in vitro. Colony Forming Units were inhibited when cultured in the presence of the ADC. The anti-CD300f ADC has a rapid onset of action with >99% cytotoxicity of the AML cell line HL-60 occurring within 24 hours. The ADC synergises with fludarabine, making it a natural combination to use in a minimal toxicity conditioning regimen.

The growth of subcutaneous tumours in mice engrafted with the AML cell line U937 was significantly reduced by a single dose of our ADC (at 150 μg/kg or 300 μg/kg). In a bone marrow engraftment model with HL-60, our ADC prolongs the survival at a single dose of 300 μg/kg (Fig 1A). In a humanised mouse model, 85% of all CD34+ cells and 90% of the primitive CD34+ CD38- CD90+ population were depleted with a single injection of our ADC at 300 μg/kg (Fig1 B). There was significant reduction in primary AML cells with a single injection of our ADC at 300 μg/kg (Fig 1C).

This proof of principle work establishes an anti-CD300f ADC as an attractive potential therapeutic to develop which will reduce the toxicity of allo-HSCT and the relapse rate afterwards. We have demonstrated that CD300f is expressed across HSPC and AML. Our antibody to CD300f is efficiently internalised and capable of delivering a PBD warhead. Our ADC synergised with fludarabine and has a rapid onset of action. We have demonstrated that our ADC prolongs survival in mouse cell line models as well as depletes both HSPC and primary AML in vivo. A targeted conditioning agent has the potential to reduce relapse risk and toxicity of allo-HSCT in AML.

同种异体造血干细胞移植（allo-HSCT）显着降低了急性髓细胞性白血病（AML）的复发率，但以治疗相关死亡率（TRM）为代价。尽管总体上减少了复发，但它仍然很普遍，尤其是在高危人群中。越来越多的证据表明，许多复发病例是由残留AML驱动的，而残留AML可以用最少的残留疾病技术检测出来。阻止大多数患者使用异基因造血干细胞移植的大部分疾病是由于非特异性有毒调理剂所致，这些非必需的有毒调理剂需要去除受体造血干细胞和祖细胞（HSPC）才能成功植入。我们建议针对AML和HSPC的靶向调理剂可以促进异源HSCT的复发率和TRM降低。

CD300f在HSPC亚型中均匀表达。CD300f在AML上具有与CD33相同的转录和蛋白质表达。我们已经开发了可有效内化到靶细胞中的抗CD300f抗体。利用这种内在化能力，我们产生了具有高效吡咯并苯并二氮杂（PBD）战斗部的抗CD300f抗体药物偶联物（ADC），该战斗部选择性地消耗了AML细胞株。在ADC存在下培养时，菌落形成单位被抑制。抗CD300f ADC起效迅速，在24小时内对AML细胞HL-60的细胞毒性> 99％。ADC与氟达拉滨具有协同作用，使其自然结合，可用于最低限度的毒性调节方案。

通过单剂量的ADC（150μg/ kg或300μg/ kg），移植了AML细胞系U937的小鼠皮下肿瘤的生长明显降低。在HL-60的骨髓移植模型中，我们的ADC以300μg/ kg的单剂量延长了生存期（图1A）。在人源化的小鼠模型中，单次注射300μg/ kg的ADC即可消耗掉所有CD34 +细胞中的85％和原始CD34 + CD38- CD90 +中的90％（图1B）。通过单次注射300μg/ kg的ADC，原代AML细胞明显减少（图1C）。

这项原理性工作证明，抗CD300f ADC具有诱人的潜在治疗作用，可降低同种HSCT的毒性并降低其复发率。我们已经证明CD300f在HSPC和AML中表达。我们的CD300f抗体可以有效地内化并能够提供PBD战斗部。我们的ADC与氟达拉滨协同作用，起效迅速。我们已经证明，我们的ADC可以延长小鼠细胞系车型的生存以及耗尽都HSPC和初级AML体内。有针对性的调理剂具有降低AML中allo-HSCT复发风险和毒性的潜力。