Human Donor Stem/Progenitor Cells with Dual Gene Editing (CD33 and CLL-1) Enable "Two Hit" targeting of Acute Myeloid Leukemia"

项目总结/摘要： 本研究的目的是开发一种新的治疗AML的策略，AML是一种髓系恶性肿瘤， 代表了一种有巨大治疗需求的疾病。造血干细胞移植（HSCT）是 复发性/难治性AML患者的唯一治愈性治疗选择，但高达40%的患者进一步复发 HSCT后使用针对谱系特异性抗原如CD 33的试剂靶向AML细胞， 抗体药物偶联物吉妥珠单抗奥扎霉素（GO; Mylotarg）具有改善的结果，但是使用 Mylotarg与重度骨髓抑制相关。这是由于靶向白血病细胞和 也表达CD 33的正常骨髓细胞（包括供体移植物中的干细胞、祖细胞和骨髓细胞 (the正常骨髓细胞同时被Mylotarg杀死，Mylotarg也有其他毒性，包括静脉毒性， 在高剂量下的闭塞性疾病）。我们推断，通过使用基因工程消除CD 33表达， 在供体干/祖细胞中，我们可以产生用于移植的干/祖细胞， Mylotarg治疗，同时使AML细胞对抗CD 33治疗（如低- 剂量Mylotarg、抗CD 33 CAR-T或CD 33双特异性T细胞增殖剂（BiTE）。事实上，在最近出版的 在“概念验证研究”中，我们证明了这种策略（单独使用Mylotarg，CART-33或两者）能够 完全杀死移植的人CD 33 + AML细胞系，同时允许完全功能性造血 通过CD 33-HSPC再增殖。 然而，在用原代人AML细胞进行的初步实验中，我们发现，尽管用 治疗16周后，一小部分AML在骨髓中持续存在。这些AML细胞 是CD 33+，但也表达第二谱系抗原CLL-1+（Clec 12 a）。我们推断，针对多个 抗原将减少复发的机会。在这项提案中，我们计划测试长期的假设， 当连续或同时靶向AML细胞上的两种抗原时，实现白血病缓解。 为了验证这一假设，我们将测量HSPC的植入、分化和功能潜力， 被基因编辑以消除CLL-1抗原单独或与CD 33（Aim 1）组合的表达。我们将测试 这种使用原发性人AML的患者来源的异种移植物（PDX）模型的两次打击治疗方法， 测试单细胞（CLL-1）和双细胞（CLL-1和CD 33）的植入、分化和功能潜力 在这种情况下删除HSPC（目的2）。本研究的长期目标是开发新的基于细胞的基因 联合免疫疗法治疗AML及相关疾病的治疗策略 癌症如骨髓增生异常综合征。

PROJECT SUMMARY/ABSTRACT: The goal of this study is to develop a novel therapeutic strategy for AML, a malignancy of myeloid lineage, that represents a disease with enormous unmet therapeutic need. Hematopoietic stem cell transplant (HSCT) is the only curative treatment option for patients with relapsed/refractory AML but up to 40% patients further relapse after HSCT. Targeting of AML cells using agents directed against a lineage specific antigen such as CD33 using the antibody drug conjugate Gemtuzimab Ozagamycin (GO; Mylotarg) has improved outcomes, but the use of Mylotarg is associated with severe myelosuppression. This is due to targeting of both the leukemia cells and normal myeloid cells (including stem, progenitor and myeloid cells in the donor graft) that also express CD33 (the normal myeloid cells are concomitantly killed by Mylotarg, which also has other toxicities, including veno- occlusive disease at high doses). We reasoned that by ablating CD33 expression using genomic engineering methods in donor stem/progenitor cells, we could generate stem/progenitor cells for transplant that are resistant to Mylotarg treatment, while rendering the AML cells uniquely sensitive to anti-CD33 therapy such as low- dose Mylotarg, anti-CD33 CAR-T or CD33 bi-specific T cell engagers (BiTEs). Indeed, in a recently published “proof of concept study”, we demonstrated that this strategy (either Mylotarg alone, CART-33 or both) enabled the complete killing of an engrafted human CD33+ AML cell line, while allowing fully functional hematopoietic repopulation by CD33- HSPCs. In preliminary experiments with primary human AML cells, however, we found that despite AML clearance with Mylotarg, a small fraction of AML persisted in the bone marrow after 16 weeks of treatment. These AML cells are CD33+ but also express a second lineage antigen, CLL-1+ (Clec12a). We reasoned that targeting multiple antigens will result reduce the chances of relapse. In this proposal, we plan to test the hypothesis that long-term leukemia remission is achieved when two antigens on AML cells are targeted, either serially or simultaneously. To test this hypothesis, we will measure the engraftment, differentiation, and functional potential of HSPCs that are gene-edited to ablate expression of CLL-1 antigen alone or in combination with CD33 (Aim 1). We will test this two-hit treatment approach using patient derived xenograft (PDX) models of primary human AML and also test the engraftment, differentiation, and functional potential of single (CLL-1) and double (CLL-1 and CD33) deletion HSPC in this context (Aim 2). The long-term objective of this study is to develop novel cell-based gene therapeutic strategies in combination with immunotherapeutic approaches for the treatment of AML and related cancers such myelodysplastic syndromes.