Autologous hematopoietic stem cell transplantation (Auto-HSCT) with gene-modification represents a potential cure for multiple genetic diseases, but its broad curative potential, is limited because of morbidity/mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed antibody drug conjugates (ADC) targeting CD117 (C-KIT) to specifically deplete the hematopoietic stem and progenitor cells (HSPC). To validate CD117 ADC-mediated depletion prior to HSCT, we developed an optimized non-human primate (NHP) tool anti-CD117 ADC and evaluated it in an auto-gene modified HSCT in a rhesus model.

The CD117-ADC is potent on primary human and NHP CD34+ cells in vitro (Figure 1A). Humanized NSG mice treated with a single dose had full depletion of human HSPCs in the bone marrow, while maintaining peripheral immune cells. In rhesus a single administration was fully myeloablative (>99% HSPC depletion) and comparable to HSPC depletion observed following busulfan conditioning (6 mg/kg/day x4). There was no effect of the ADC on the peripheral and bone marrow lymphocytes and the ADC was well tolerated compared to busulfan where the animals had diarrhea, loss of appetite and weight loss. To facilitate use in HSCT, the CD117-ADC was engineered to have a fast clearance and the half-life was < 10 h in NHP.

We next explored whether the tool CD117-ADC could enable auto gene modified HSCT in the rhesus model. Two rhesus NHP were mobilized with GCSF and plerixafor. The selected CD34+ cells were transduced with β-globin encoded lentivirus and cryopreserved. The tool CD117-ADC was dosed on day -6 and the CD34+ cells were thawed and infused on day 0. Bone marrow aspirates analyzed on the day of infusion (day 0) demonstrated >99% depletion of the HSPCs and maintenance of the bone marrow lymphocytes (Figure 1B). The primates engrafted neutrophils (day 8 and 10) and platelets (day 10 and 11), and the peripheral lymphocytes were maintained throughout the transplant (Figure 1C-D). The gene marking in the granulocytes is comparable to busulfan conditioned animals previously reported (Tisdale, Molecular Therapy 2019). Longer follow up and data from additional animals will be presented.

In summary, we have developed a tool CD117 ADC that shows potent activity on NHP CD34+ cells, is fully myeloablative, has a favorable safety profile, spares the immune system and is cleared rapidly as designed. In a rhesus model of auto-gene modified HSCT, a single dose of the ADC enables engraftment of gene modified HSC. These proof of concept studies validate the use of CD117-ADC for targeted HSPC depletion prior to transplant and support its use as a new conditioning agent for auto-gene modified HSCT. This targeted approach for safer conditioning could improve the risk benefit profile for patients undergoing HSCT and enable more patients to benefit from these potentially curative therapies.

具有基因修饰的自体造血干细胞移植（Auto-HSCT）代表了多种遗传疾病的潜在治疗方法，但其广泛的治疗潜力受到了限制，这是由于基于细胞毒性化学疗法的条件下的发病率/死亡率。为了克服这些局限性，我们开发了靶向CD117（C-KIT）的抗体药物偶联物（ADC），以专门消耗造血干细胞和祖细胞（HSPC）。为了在HSCT之前验证CD117 ADC介导的耗竭，我们开发了一种优化的非人类灵长类动物（NHP）工具抗CD117 ADC，并在恒河猴模型的自动基因修饰HSCT中对其进行了评估。

CD117-ADC在体外对原代人和NHP CD34 +细胞有效（图1A）。用单剂量治疗的人源化NSG小鼠骨髓中的人类HSPC完全耗尽，同时维持外周免疫细胞。在恒河猴中，单次给药完全是清髓性的（> 99％HSPC消耗），与在白消安条件下观察到的HSPC消耗相当（6 mg / kg /天x4）。ADC对外周和骨髓淋巴细胞没有作用，与环丁砜相比，ADC具有良好的耐受性，在白消安中，动物腹泻，食欲不振和体重减轻。为了便于在HSCT中使用，CD117-ADC经过精心设计，具有快速清除功能，在NHP中的半衰期小于10小时。

接下来，我们探讨了CD117-ADC工具是否可以在恒河猴模型中启用自动基因修饰的HSCT。用GCSF和plerixafor调动了两个恒河猴NHP。用β-球蛋白编码的慢病毒转导所选的CD34 +细胞并冷冻保存。在第-6天配药工具CD117-ADC，在第0天解冻并注入CD34 +细胞。在输注当天（第0天）分析的骨髓抽吸物显示，HSPC耗竭> 99％，并且骨髓得以维持淋巴细胞（图1B）。灵长类动物植入了中性粒细胞（第8和10天）和血小板（第10和11天），并且外周淋巴细胞在整个移植过程中都得到了维持（图1C-D）。粒细胞中的基因标记与先前报道的丁硫丹条件化动物相当（Tisdale，Molecular Therapy 2019）。

总而言之，我们开发了CD117 ADC工具，该工具对NHP CD34 +细胞显示出有效的活性，完全清髓，具有良好的安全性，可节省免疫系统，并按设计快速清除。在自体基因修饰的HSCT恒河猴模型中，单剂量ADC可植入基因修饰的HSC。这些概念验证研究验证了CD117-ADC在移植前用于靶向HSPC耗竭的情况，并支持将其用作自动基因修饰的HSCT的新调节剂。这种有针对性的安全调理方法可以改善接受HSCT的患者的风险收益状况，并使更多患者受益于这些潜在的治疗方法。