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

摘要 遗传性血红蛋白(Hb)疾病是世界上最常见的单基因疾病，即使在 在发达国家，发病率很高，预期寿命较短。同种异体 造血细胞移植(Hct)是临床上治疗这些疾病的一种手段。 疾病-患者的造血干细胞和祖细胞(HSPC)的遗传缺陷。不过，这个 方法受到大多数患者和相关患者中可获得的人类白细胞抗原相合供者的限制 免疫并发症。转导功能性b-血红蛋白基因的自体HSPC的应用 或用最新开发的基因组编辑技术进行修改，将克服目前 异基因血细胞移植。特别是对自然发生的胎儿血红蛋白遗传性持久性的概述 使用基因编辑的HSPC中的(HPFH)突变原则上可以逆转这些患者的临床表型 精神错乱。然而，就像同种异体HCT一样，仍然需要条件化来促进植入 这些细胞。到目前为止，这是通过g-射线全身照射(TBI)或烷化剂实现的，如 它具有严重的毒性风险，包括不孕不育、生长迟缓和--就像已经做过的那样 已报告-继发性恶性肿瘤。因此，下一代移植的关键剩余因素 方法和基因治疗/编辑将是开发具有以下特点的非遗传毒性调节方案 毒性最小，并允许同种异体或修饰的自体HSPC牢固植入。一件有希望的事 策略是使用放射免疫疗法(Rit)，将a发射的放射性核素结合到抗体靶标上。 CD45是一种抗原，表达在除血小板和红细胞以外的几乎所有造血细胞上，以及一些 他们的祖先。与b-发射器相比，a-发射器仅通过几个电池就能提供更多的能量。 有效、精确和高效的靶向细胞杀伤直径，并将对非靶向环境的毒性降至最低 细胞。阿司他丁-211(211 At)的半衰期为7.2小时，是患者应用的理想药物。基于我们之前的 犬211At-抗CD45RIT可替代g-BEAM作为同种异体移植前预适应的研究 目前，我们正在活动性血液系统恶性肿瘤患者中应用~(211)At-抗CD45 RIT。我们现在计划 211At-抗CD45RIT作为基因修饰HSPC自体移植前预适应的实验研究 患有血红蛋白疾病的人，利用Fc工程抗体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CD45 RIT将使植入 用CRISPR/Cas9编辑的自体HSPC在g-珠蛋白基因座复制HPFH突变，并具有 与使用高剂量g-BEAM的标准预适应相比，脱靶毒性显著减少，耐受性更好 TBI。由于我们对我们的研究结果的快速临床翻译很感兴趣，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自体非人类灵长类动物模型中测试这一假设。 红细胞压积用于治疗血红蛋白疾病。

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.