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治疗来改善急性髓系白血病(AML)的预后。所以 到目前为止，利用最多的是针对CD33的单抗，CD33是一种显示在细胞表面的糖蛋白 几乎所有病例中都有白血病母细胞，部分病例中可能有白血病干细胞。一些患者的生存时间更长 用CD33抗体-药物结合物getuzumab ozogamicin(GO)治疗可以验证这种方法，但GO 通常是无效的，促使人们努力开发改进的、更有效的CD33导向疗法。因为 AML细胞对辐射的敏感性呈剂量依赖性，放射性核素是理想的抗辐射药物。 CD33单抗。事实上，早期临床试验显示抗CD33单抗具有显著的抗急性髓系白血病疗效。 Lintuzumab(HuM195，SGN-33)，当与a发射器Actdium-225(225Ac)结合时。A-发射器提供了非常 仅在几个细胞直径上的高辐射量，从而能够精确和有效地杀死靶细胞， 使它们对具有放射免疫结合物(“RIT”)的AML的特异性靶向特别感兴趣。 然而，即使使用225Ac-lintuzumab，一个重要的缺点是CD33在正常髓系细胞上的表达， 这会导致“靶点上，肿瘤细胞外”的毒性，表现为严重的和长期的骨髓抑制 危及生命的后遗症(如感染)。因此，CD33导向的无即刻干细胞RIT的临床应用 救援目前仅限于次优药物剂量。我们最近在老鼠和非人类身上展示了 灵长类动物基于CRISPR/Cas9核酸酶的CD33编辑导致功能正常的造血 表达降低的CD33水平，并受到GO和CD33指导的T细胞参与治疗的保护。 我们假设CD33编辑的正常造血干细胞和祖细胞(HSPC)将抵抗CD33定向的 RIT具有a粒子发射的放射性核素，并使其能够在最大有效药物剂量下安全使用。然而， 基于CRISPR/Cas9的CD33基因编辑策略存在显著的脱靶活性，DNA倍增 链断裂(DSB)可导致更大的缺失和复杂的染色体重排，并导致 依赖TP53的DNA损伤反应和细胞周期停滞。为了解决这一限制，我们将优化和 描述一种新的基因编辑策略，以保护正常造血免受高效CD33的影响 RIT通过利用最近描述的基本编辑(BE)技术。BES诱导精确的核苷酸修饰 在不有意引入DSB的情况下，使其成为一种有吸引力的策略来生成CD33Null“正常” 造血细胞。我们已经组建了一支由多学科调查人员组成的团队，他们的专业知识互补 在CD33指导的治疗中，RIT的临床前优化，以及放射药剂学的良好控制 临床前使能研究开发基于BE的正常人HSPC的CD33工程用于临床 与a-发射体CD33导向的RIT一起用于AML和其他CD33表达障碍的患者。

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.