Base-Edited Hematopoietic Stem and Progenitor Cells To Enable Safe Use Of Highly Potent CD33-Targeted Radioimmunotherapy

摘要 长期以来，人们一直在寻求抗原特异性疗法来改善急性髓性白血病（AML）的预后。所以 最常用的是靶向CD 33的单克隆抗体（mAb），CD 33是一种展示在细胞表面的糖蛋白 几乎所有的白血病原始细胞，有些可能是白血病干细胞。一些患者的生存时间更长 用CD 33抗体-药物缀合物吉妥珠单抗（gemtuzumab ozogamicin，GO）治疗证实了这种方法，但GO 通常是无效的，这促使人们努力开发改进的、更有效的CD 33导向治疗剂。因为 AML细胞以剂量依赖的方式对辐射非常敏感，放射性核素是理想的武装抗AML细胞的方法。 CD 33单克隆抗体。事实上，早期临床试验证明了抗CD 33 mAb的显著抗AML功效 当与α-发射体锕-225（225 Ac）偶联时，林妥珠单抗（HuM 195，SGN-33）。a型发射器提供了一个非常 在几个细胞直径上的大量辐射，从而能够精确和有效地杀死靶细胞， 使得它们对于用放射性免疫缀合物（“RIT”）特异性靶向AML特别有意义。 然而，即使使用225 Ac-林妥珠单抗，一个重要的缺点是正常骨髓细胞上的CD 33表达， 这导致“靶向、非肿瘤细胞”毒性，表现为严重和长期的骨髓抑制， 危及生命的后遗症（如感染）。因此，临床使用CD 33导向的RIT而不直接干细胞， 抢救目前仅限于次优药物剂量。我们最近在小鼠和非人类身上证明了 基于CRISPR/Cas9核酸酶的CD 33编辑导致功能正常的造血， 表达降低水平的CD 33，并且被保护免受GO和CD 33定向的T细胞接合治疗剂的影响。 我们假设CD 33编辑的正常造血干细胞和祖细胞（HSPC）将抵抗CD 33定向的造血干细胞。 RIT与α粒子发射放射性核素，并使其安全使用最大有效的药物剂量。然而，在这方面， 基于CRISPR/Cas9的CD 33基因编辑策略具有显著的脱靶活性， 链断裂（DSB）可产生较大的缺失和复杂的染色体重排， TP 53依赖的DNA损伤反应和细胞周期阻滞。为了解决这个问题，我们将优化和 描述一种新型基因编辑策略，以保护正常造血免受高效CD 33介导的影响 RIT通过利用最近描述的基础编辑器（BE）技术。BE诱导精确的核苷酸修饰 没有故意引入DSB，使其成为产生CD 33 null“正常”的有吸引力的策略 造血细胞我们已经组建了一个多学科的调查小组， 在CD 33导向疗法、RIT的临床前优化和放射性药剂学中， 临床前IND使能研究，以开发用于临床的正常人HSPC的基于BE的CD 33工程 与α-发射体CD 33导向的RIT一起用于AML和其他表达CD 33的疾病患者。

ABSTRACT Antigen-specific therapies have long been pursued to improve outcomes in acute myeloid leukemia (AML). So far most exploited are monoclonal antibodies (mAbs) targeting CD33, a glycoprotein displayed on the cell surface of leukemic blasts in almost all cases and possibly leukemia stem cells in some. Longer survival of some patients treated with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this approach, but GO is often ineffective, prompting efforts to develop improved, more potent CD33-directed therapeutics. Because AML cells are exquisitely sensitive to radiation in a dose-dependent fashion, radionuclides are ideal to arm anti- CD33 mAbs. Indeed, early phase clinical trials demonstrated substantial anti-AML efficacy of the anti-CD33 mAb lintuzumab (HuM195, SGN-33) when coupled with the a-emitter actinium-225 (225Ac). a-emitters deliver a very high amount of radiation over just a few cell diameters, thereby enabling precise and efficient target cell kill, rendering them particularly interesting for specific targeting of AML with radioimmunoconjugates (“RIT”). However, even with 225Ac-lintuzumab, an important shortcoming is CD33 expression on normal myeloid cells, which leads to “on-target, off-tumor cell” toxicities that manifest as severe and prolonged myelosuppression with life-threatening sequelae (e.g. infection). Thus, clinical use of CD33-directed RIT without immediate stem cell rescue is currently limited to suboptimal drug doses. We have recently demonstrated in mice and nonhuman primates that CRISPR/Cas9 nuclease-based editing of CD33 results in functionally normal hematopoiesis that expresses reduced levels of CD33 and is protected from GO and CD33-directed T cell-engaging therapeutics. We hypothesize CD33-edited normal hematopoietic stem and progenitor cells (HSPCs) will resist CD33-directed RIT with a-particle-emitting radionuclides and enable their safe use at maximally effective drug doses. However, the CRISPR/Cas9-based CD33 gene editing strategy suffers from significant off-target activity, and DNA double strand breaks (DSBs) can generate larger deletions and complex chromosomal rearrangements and cause TP53-dependent DNA damage response and cell cycle arrest. To address this limitation, we will optimize and characterize a novel gene-editing strategy to protect normal hematopoiesis from highly potent CD33-directed RIT by utilizing the recently described base editor (BE) technology. BEs induce precise nucleotide modifications without intentional introduction of DSBs, making them an attractive strategy to generate CD33null “normal” hematopoietic cells. We have assembled a multidisciplinary team of investigators with complementary expertise in CD33-directed therapies, preclinical optimization of RIT, and radiopharmaceutics to conduct well-controlled preclinical IND-enabling studies to develop BE-based CD33 engineering of normal human HSPCs for clinical use with a-emitter CD33-directed RIT for patients with AML and other CD33-expressing disorders.