Development of 211Astatine-Conjugated Anti-CD45 Antibody-Based Conditioning for Hematopoietic Stem Cell Gene Therapy and Editing

摘要 血红蛋白（Hb）遗传性疾病是世界范围内最常见的单基因疾病，即使在 在发达国家，这与高发病率和预期寿命缩短有关。同种异体 造血细胞移植（HCT）在临床上被用作治疗这些疾病的根本原因的手段。 疾病-患者造血干细胞和祖细胞（HSPC）的遗传缺陷。但这 这种方法受到大多数患者中HLA匹配供体的可用性的限制， 免疫并发症使用用功能性b-血红蛋白基因转导的自体HSPC 或用最近开发的基因组编辑技术进行修改，将克服目前的限制， 同种异体血细胞比容。特别是，自然发生的遗传性胎儿血红蛋白的重演 原则上，使用基因编辑的HSPC中的HPFH突变可以逆转这些HSPC的临床表型。 紊乱然而，就像同种异体HCT一样，仍然需要预处理以促进移植物的植入。 这些细胞。迄今为止，这是用g-束全身照射（TBI）或烷化剂如 白消安具有显著毒性的风险，包括不育、生长迟缓， 报告-继发性恶性肿瘤。因此，下一代移植的一个关键因素是 方法和基因治疗/编辑将是非遗传毒性调节方案的发展， 最小的毒性并允许同种异体或修饰的自体HSPC的稳健植入。一个有希望 放射免疫治疗的策略是使用放射免疫治疗（RIT），放射性核素与靶向抗体结合 CD 45是一种在除血小板和红细胞以外的几乎所有造血细胞上表达的抗原， 他们的祖先。与b型发射体相比，a型发射体在几个电池上提供更高的能量 直径，用于有效、精确和高效的靶向细胞杀伤，并将对非靶向环境的毒性降至最低 细胞由于半衰期为7.2小时，211 At是理想的患者应用。基于我们之前 在狗中进行的研究表明，211 At-抗CD 45 RIT可以替代g-束TBI作为同种异体移植前的预处理， HCT，我们目前正在活动性恶性血液病患者中使用211 At-抗CD 45 RIT。我们现在计划 开发211 At-抗CD 45 RIT作为基因修饰的HSPCs自体移植前的预处理， 血红蛋白病患者，利用抗体的Fc工程，以进一步减少非特异性 与RIT相关的毒性。我们假设，优化的211 At-抗CD 45 RIT将使植入 在g-珠蛋白基因座处用CRISPR/Cas9编辑的自体HSPC，以再现HPFH突变，并且具有 与高剂量G-束标准预处理相比，脱靶毒性显著降低，耐受性更好 创伤性脑损伤由于我们对快速临床转化我们的发现感兴趣，并且已经收集了大量数据， 为了证明可行性，我们将在我们建立的非人灵长类动物自体移植模型中测试这一假设。 血红蛋白病的HCT。

ABSTRACT The inherited disorders of hemoglobin (Hb) are the most common monogenic diseases worldwide and, even in developed countries, associated with substantial morbidity and shortened life expectancy. Allogeneic hematopoietic cell transplantation (HCT) is clinically pursued as a means to treat the underlying cause of these disorders – the genetic defect in the patients’ hematopoietic stem and progenitor cells (HSPCs). However, this approach is limited by the availability of HLA-matched donors in the majority of patients and associated immunological complications. Use of autologous HSPCs either transduced with a functional b-hemoglobin gene or modified with recently-developed genome-editing technologies would overcome the current limitations of allogeneic HCT. In particular, the recapitulation of naturally-occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in HSPCs using gene editing can, in principle, reverse the clinical phenotype of these disorders. However, just like with allogeneic HCT, there is still need for conditioning to facilitate engraftment of these cells. To date, this is accomplished with g-beam total body irradiation (TBI) or alkylating agents such as busulfan which carry the risk of significant toxicities including infertility, growth retardation, and – as has already been reported – secondary malignancies. Thus, a critical remaining factor for next-generation transplant approaches and gene therapy/editing will be the development of nongenotoxic conditioning regimens that have minimal toxicity and allow robust engraftment of allogeneic or modified autologous HSPCs. One promising strategy is the use of radioimmunotherapy (RIT) with a-emitting radionuclides conjugated to antibodies targeting CD45, an antigen expressed on almost all hematopoietic cells except platelets and erythrocytes and some of their progenitors. Compared to b-emitters, a-emitters deliver a higher amount of energy over just a few cell diameters for potent, precise, and efficiently targeted cell kill and minimized toxicity to non-targeted surrounding cells. With a half-life of 7.2 hours, astatine-211 (211At) is ideal for patient application. Based on our previous studies in dogs demonstrating that 211At-anti-CD45 RIT can replace g-beam TBI as conditioning before allogeneic HCT, we are currently using 211At-anti-CD45 RIT in patients with active hematologic malignancies. We now plan to develop 211At-anti-CD45 RIT as conditioning before autologous transplantation of gene-modified HSPCs for people with hemoglobinopathies, exploiting Fc engineering of antibodies to further minimize non-specific toxicities associated with RIT. We hypothesize that optimized 211At-anti-CD45 RIT will enable engraftment of autologous HSPCs edited with CRISPR/Cas9 at the g-globin gene locus to reproduce HPFH mutations and have significantly less off-target toxicities and better tolerability than the standard conditioning with high-dose g-beam TBI. As we are interested in rapid clinical translation of our findings and have already collected substantial data demonstrating feasibility, we will test this hypothesis in our established nonhuman primate model of autologous HCT for hemoglobinopathies.