Current approaches to conditioning prior to transplant and gene therapy employ non-specific genotoxic agents that are associated with severe acute and long-term toxicities. Consequently, many transplant eligible patients either refuse transplant or receive reduced intensity regimens, which still have adverse toxicities with increased risk of relapse and graft failure. Importantly, patients with non-malignant disease may choose to forgo a potentially curative transplant due to risks of conditioning-associated toxicities, potentially limiting access of these patients to transplant and emerging HSC-based gene therapy. Thus, safe and effective targeted conditioning agents would have broad application to transplant in both malignant and non-malignant settings as well as gene therapy.

We developed a CD117 antibody conjugated to amanitin (anti-CD117-AM) to specifically deplete hematopoietic stem and progenitor cells (HSPCs) as a novel approach to patient preparation for allogeneic and autologous transplant. ADCs have significant single agent anti-tumor activity in the clinic, however, achieving suitable tolerability at efficacious doses has presented a clinical challenge. We engineered the properties of the anti-CD117-AM to provide a broad therapeutic window across pre-clinical models.

Through modification of the linker moiety, the maximum tolerated dose (MTD) of the ADC in C57Bl6 mice was improved 17-fold compared to previous versions. The engineered anti-CD117-AM maintained potent cytotoxicity on primary human CD34+ cells and a CD117+ AML cell line (<10 pM IC50). In humanized NSG mice, a single dose (0.1 mg/kg) of the ADC selectively depleted >95% of human HSPCs (Fig. 1A). Single doses of the anti-CD117-AM demonstrated robust tumor control in xenograft models of AML, despite its purposefully shortened half-life as a targeted agent for transplant indications (Fig. 1B).

The tolerability of the engineered anti-CD117-AM was assessed in cynomolgus NHPs. The ADC was well-tolerated with an MTD at ≥ 2 mg/kg (Fig. 1C). A single dose of the ADC was able to drive marked depletion of HSPCs and downstream progeny. The pharmacokinetics of the ADC exhibited linear clearance supporting tolerability well above target saturation.

In summary, optimization of the chemical linker between antibody and payload yielded a well-tolerated ADC with wide safety margins that could eliminate HSPCs and confer survival benefit in AML xenograft models. ADC-mediated conditioning through selective depletion of CD117+ cells may provide an improved approach to patient preparation for transplant in malignant and non-malignant disease including gene therapy, broadening patient access to potentially curative treatments.

当前在移植和基因治疗之前进行调节的方法采用与严重急性和长期毒性相关的非特异性遗传毒性剂。因此，许多符合移植条件的患者要么拒绝移植，要么接受强度降低的治疗方案，这些方案仍具有不良毒性，增加了复发和移植失败的风险。重要的是，由于与条件相关的毒性的风险，患有非恶性疾病的患者可能选择放弃潜在的治愈性移植，从而潜在地限制了这些患者进行移植和基于HSC的基因治疗的机会。因此，安全有效的靶向调理剂将广泛应用于恶性和非恶性环境以及基因治疗的移植。

我们开发了与金刚霉素结合的CD117抗体（抗CD117-AM），以专门消耗造血干细胞和祖细胞（HSPC），作为异体和自体移植患者制备的新方法。ADC在临床上具有显着的单药抗肿瘤活性，但是，在有效剂量下实现合适的耐受性已成为临床挑战。我们设计了抗CD117-AM的特性，以为临床前模型提供广阔的治疗窗口。

通过修饰接头部分，与以前的版本相比，C57B16小鼠中ADC的最大耐受剂量（MTD）提高了17倍。经过工程改造的抗CD117-AM对原代人CD34 +细胞和CD117 + AML细胞系（<10 pM IC50）保持了有效的细胞毒性。在人源化NSG小鼠中，单剂量（0.1 mg / kg）的ADC选择性地消耗了> 95％的人类HSPC（图1A）。单剂量的抗CD117-AM在AML异种移植模型中显示出强大的肿瘤控制能力，尽管它有目的地缩短了作为移植适应症靶向药物的半衰期（图1B）。

在食蟹猴NHP中评估了工程改造的抗CD117-AM的耐受性。ADC的MTD≥2 mg / kg具有良好的耐受性（图1C）。单剂量的ADC能够驱动HSPC和下游后代的明显消耗。ADC的药代动力学表现出线性间隙，支持耐受性远高于目标饱和度。

总之，优化抗体和有效负载之间的化学接头可产生耐受性良好的ADC，具有宽泛的安全裕度，可以消除HSPC并在AML异种移植模型中赋予生存优势。通过选择性清除CD117 +细胞而进行的ADC介导的调节可为患者准备好用于恶性和非恶性疾病移植的改进方法，包括基因治疗，从而扩大了患者获得潜在治愈性治疗的机会。