CAR T-Cell Therapy Targeting the Membrane-Proximal C2-Set Domain of CD33 for Treatment of Acute Myeloid Leukemia and Other CD33-Expressing Hematopoietic Neoplasms

摘要 抗原特异性免疫治疗长期以来一直致力于改善急性髓细胞白血病的预后 （AML）。到目前为止，用于此目的的最多的是靶向CD 33的抗体，CD 33是细胞上展示的糖蛋白 在几乎所有的情况下，白血病原始细胞的表面和可能的白血病干细胞在一些情况下。提高生存率 用CD 33抗体-药物缀合物吉妥珠单抗（GO）治疗的一些患者证实了这一点 然而，许多CD 33 + AML患者并没有从GO中获益。这促使人们对发展 改进的CD 33导向疗法，最近有几种研究药物进入临床试验。 与GO类似，所有这些分子识别膜远端V组内的免疫显性表位 CD 33的结构域。然而，在我们对CD 33 V-set/CD 3双特异性抗体的初步研究中，我们观察到， 增强的免疫效应细胞功能和更高的细胞溶解效力，与CD 33的近膜结合。 因此，我们假设针对膜近端C2-set结构域的抗体将提供上级的免疫抑制。 与现有的治疗方法相比，CD 33靶向免疫治疗的方法。此外，由于V-集而不是C2-集 结构域在一些CD 33剪接变异体中缺失，C2-set结构域定向抗体可以识别所有天然- 在一些实施方案中，抗体是CD 33的存在变体（即“CD 33 PAN抗体”）。作为实现我们长期发展目标的第一步， 这种新形式的CD 33免疫疗法，我们最近已经产生了鼠和-使用人源化的 （“Trianni”）小鼠-针对人CD 33的C2-set结构域的完全人抗体。概念性验证研究 用鼠CD 33 PAN/CD 3双特异性分子证明了有效的抗白血病活性和有效的T细胞免疫应答。 订婚我们现在建议开发一种新的疗法，是基于表达嵌合抗原的T细胞 在一些实施方案中，本发明涉及具有人CD 33 PAN抗体结合序列的CAR受体（汽车），用于患者使用。我们计划严格测试 这些CAR-T细胞在体外和体内的抗白血病特性，同时研究CD 33 基因编辑的正常造血干细胞和祖细胞（HSPCs）可以减轻我们的过继细胞的毒性 治疗正常血细胞。为了实现这些目标，我们组建了一个多学科团队， 在CD 33导向免疫疗法、CAR T细胞技术和 基因修饰的HSPC移植。预期结果将指导CD 33 PAN CAR的进一步开发 T细胞单独或与工程造血细胞一起扩大其治疗窗口，这些细胞可以 为AML和其他CD 33+肿瘤提供了一种新的治疗选择， 不满意。

ABSTRACT Antigen-specific immunotherapies have long been pursued to improve outcomes in acute myeloid leukemia (AML). So far most exploited for this purpose are antibodies targeting CD33, a glycoprotein displayed on the cell surface of leukemic blasts in almost all cases and possibly leukemia stem cells in some. Improved survival of some patients treated with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this approach, but many patients with CD33+ AML do not benefit from GO. This has prompted interest in developing improved CD33-directed therapeutics, with several investigational drugs recently advancing to clinical testing. Similar to GO, all these molecules recognize immune-dominant epitope(s) within the membrane-distal V-set domain of CD33. In our preliminary studies with CD33V-set/CD3 bispecific antibodies, however, we have observed enhanced immune-effector cell function and higher cytolytic efficacy with membrane-proximal binding of CD33. We therefore hypothesize antibodies against the membrane-proximal C2-set domain will provide a superior approach to CD33-targeted immunotherapy than existing therapeutics. Moreover, since the V-set but not C2-set domain is missing in some CD33 splice variants, C2-set domain-directed antibodies can recognize all naturally- occurring variants of CD33 (i.e. are “CD33PAN antibodies”). As a first step toward our long-term goal of developing this new form of CD33 immunotherapy, we have recently generated murine and – with use of humanized (“Trianni”) mice – fully human antibodies against the C2-set domain of human CD33. Proof of concept studies with a murine CD33PAN/CD3 bispecific molecule demonstrated potent anti-leukemia activity and effective T-cell engagement. We now propose to develop a new therapy that is based on T-cells expressing chimeric antigen receptors (CARs) with human CD33PAN antibody binding sequences for patient use. We plan to rigorously test the anti-leukemia properties of these CAR T-cells in vitro and in vivo, while at the same time studying how CD33 gene-edited normal hematopoietic stem and progenitor cells (HSPCs) can mitigate toxicity of our adoptive cell therapy to normal blood cells. To accomplish these goals, we have assembled a multidisciplinary team of investigators with complementary expertise in CD33-directed immunotherapies, CAR T-cell technology, and transplantation of gene-modified HSPCs. Expected results will guide the further development of CD33PAN CAR T-cells. Alone or together with engineered hematopoietic cells to widen their therapeutic window, these cells may offer a new treatment option for AML and other CD33+ neoplasms for which outcomes continue to be unsatisfactory.