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

具有双基因编辑（CD33 和 CLL-1）的人类供体干细胞/祖细胞能够实现急性髓系白血病的“两次打击”

• 批准号: 10311553
• 负责人: Abdullah M Ali
• 金额: $ 22.27万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-03 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311553
• 关键词: Ablation; Activities of Daily Living; Acute Myelocytic Leukemia; Alleles; Allogenic; Antibody-drug conjugates; Antigen Targeting; Antigens; Biological Assay; Blast Cell; Bone Marrow; CD34 gene; CRISPR/Cas technology; Cell Line; Cells; Clinical; Clinical Research; Clinical Trials; Clonal Expansion; Colony-Forming Units Assay; Disease; Disease model; Disease remission; Dose; Drug Efflux; Dysmyelopoietic Syndromes; Engineering; Engraftment; Flow Cytometry; Funding; Gemtuzumab Ozogamicin; Genes; Genetic; Genomics; Goals; Grant; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; IL3RA gene; IL6 gene; IL8 gene; Immunotherapeutic agent; Immunotherapy; In Vitro; Injections; Institutional Review Boards; Legal patent; Letters; Leukemic Cell; Lymphoid; Malignant Neoplasms; Measures; Mediating; Methods; Modeling; Monitor; Morbidity - disease rate; Mus; Mutation; Myelogenous; Myeloid Cells; Myeloproliferative disease; Myelosuppression; PTPRC gene; Patients; Phagocytosis; Phase; Postremission Therapy; Pre-Clinical Model; Preparation; Production; Publishing; Refractory; Relapse; Remission Induction; Residual Neoplasm; Resistance; Resistance development; Site; Stem cell transplant; TNF gene; Testing; Therapeutic; Toxic effect; Transplantation; Tumor Antigens; Veno-Occlusive Disease; Work; acute myeloid leukemia cell; bi-specific T cell engager; chimeric antigen receptor T cells; chronic T-cell leukemia; curative treatments; cytokine; cytokine release syndrome; deep sequencing; experimental study; high risk; improved outcome; in vitro Assay; in vivo; innovation; leukemia; macrophage; monocyte; mouse model; novel; novel strategies; novel therapeutic intervention; overexpression; patient derived xenograft model; peripheral blood; phase 1 study; pre-clinical; preservation; progenitor; relapse patients; relapse risk; resistance mechanism; stem; stem cells; therapeutic gene; translational study

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.

项目摘要/摘要： 本研究的目标是开发一种针对 AML（一种骨髓系恶性肿瘤）的新型治疗策略， 代表了一种尚未满足巨大治疗需求的疾病。 复发/难治性 AML 患者的唯一治疗选择，但高达 40% 的患者会进一步复发 在 HSCT 后，使用针对谱系特异性抗原（例如 CD33）的药物来靶向 AML 细胞。 抗体药物缀合物 Gemtuzimab Ozagamycin（GO；Mylotarg）改善了结果，但使用 Mylotarg 与严重的骨髓抑制有关，这是由于靶向白血病细胞和骨髓抑制所致。 也表达 CD33 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） （正常骨髓细胞同时被 Mylotarg 杀死，Mylotarg 还具有其他毒性，包括静脉毒性 高剂量下的闭塞性疾病）我们推断是通过使用基因组工程消除 CD33 表达。 通过在供体干细胞/祖细胞中的方法，我们可以产生具有抗性的用于移植的干/祖细胞 Mylotarg 治疗，同时使 AML 细胞对抗 CD33 治疗（例如低剂量）特别敏感 事实上，在最近发表的一篇文章中，Mylotarg、抗 CD33 CAR-T 或 CD33 双特异性 T 细胞接合剂 (BiTE) 的剂量。 “概念验证研究”，我们证明了该策略（单独的 Mylotarg、CART-33 或两者）能够实现 完全杀死植入的人类 CD33+ AML 细胞系，同时允许功能齐全的造血功能 CD33-HSPC 进行再增殖。 然而，在对原代人 AML 细胞进行的初步实验中，我们发现尽管 AML 被清除 治疗 16 周后，一小部分 AML 细胞 Mylotarg 仍存留在骨髓中。 是 CD33+，但也表达第二种谱系抗原 CLL-1+ (Clec12a)。 抗原将减少复发的机会。在这个提议中，我们计划检验长期的假设。 当连续或同时靶向 AML 细胞上的两种抗原时，即可实现白血病缓解。 为了验证这一假设，我们将测量 HSPC 的植入、分化和功能潜力， 经过基因编辑，可单独或与 CD33 联合消除 CLL-1 抗原的表达（目标 1）。 这种二次打击治疗方法使用原发性人类 AML 的患者衍生异种移植 (PDX) 模型，并且还 测试单细胞 (CLL-1) 和双细胞（CLL-1 和 CD33）的植入、分化和功能潜力 在此背景下删除 HSPC（目标 2）。本研究的长期目标是开发新的基于细胞的基因。 治疗策略与免疫治疗方法相结合，用于治疗 AML 及相关疾病 癌症，例如骨髓增生异常综合征。