Optimizing the graft versus leukemia effect for pediatric ALL

优化儿童 ALL 的移植物抗白血病效果

• 批准号: 8157749
• 负责人: Terry Fry
• 金额: $ 33万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157749
• 关键词: Childhood Acute Lymphocytic Leukemia; leukemia

Under the first aim of this project we have utilized a precursor B cell leukemia derived from mice that express an E2aPBX1 transgene. E2aPBX1 leukemia cells from a single mouse were initially passaged in vivo and subsequently conditioned to grow in vitro. Injection of these cells intravenously reproducibly results in leukemia development from as few as 10,000 cells with distribution to bone marrow, blood, lymph nodes, spleen, liver and central nervous system. A subline has been transfected with luciferase and leukemia development can be tracked in situ using imaging. The cell line was confirmed to be immunogeneic as vaccination with irradiated leukemia cells protects against subsequent challenge with E2aPBX1 but not other tumors. Using monoclonal antibodies to deplete cell subsets, we demonstrated that protection in immunized mice requires both CD4 and CD8 T cells and is impaired following NK cell depletion. Under aim 2 we next performed syngeneic bone marrow transplantation experiments in which mice were injected with E2aPBX1 one week following transplant. This was followed by adoptive transfer of T cells from donors primed with an irradiated E2aPBX1 vaccine. While leukemia development was delayed compared to BMT recipients receiving purified T cells from unprimed mice, all recipients eventually succumbed to leukemia. Together, this data indicates that whole-cell vaccination induces a tumor-specific, T cell mediated immune response that is unable to prevent the develop of leukemia following syngeneic BMT. Importantly, these experiments also demonstrate that ALL can be targeted by T cells but requires prior priming to leukemia antigens The use of allogeneic bone marrow rather than syngeneic bone marrow introduces minor histocomaptibility antigens potential targets on leukemia cells. Indeed, allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells resulted in cure of leukemia in all mice but weight loss and histologic changes consistent with moderate GVHD. Interestingly, priming T cell donors with recipient (and leukemia) strain non-malignant B cells did not cure the mice indicating that both minor antigens and leukemia-associated antigens are responsible for cure in this model. We next sought to separate the anti-leukemic GVL effect from GVHD by selecting for T cells subsets. Neither CD4 or CD8 T cells from primed donors alone were sufficient to cure all of the mice. However, selection for CD62L (l-selectin) positive T cells (consistent with a central memory subtype in primed donors) was capable of mediating a selective, curative GVL effect without GVHD. Aim 3 is ongoing and involves identification of leukemia-associated targets. we have established that E2aPBX1 overexpresses the Wilm's Tumor 1 gene, also overexpressed on approximately 70-80% of human leukemias and validated as potential target in patients. In order to obtain large numbers of WT-1 specific T cells, we are in the process of generating mice that express a T cell receptor specific for the dominant epitope derived from WT-1. The T cell receptor has been cloned and multiple founders have been generated that express large numbers of WT-1 specific T cells. Once the murine lines have been expanded, T cells from these mice will be tested in the E2aPBX1 model. If, successful, subsequent experiments following allogeneic transplantion will help validate and characterize the use of leukemia directed T cells as a modality to treat or prevent relapse.

在这个项目的第一个目标下，我们利用了来自表达E2aPBX1转基因的小鼠的前体B细胞白血病。来自单个小鼠的E2aPBX1白血病细胞最初在体内传代，随后在体外培养。静脉注射这些细胞可再生地导致白血病的发展，从少至10,000个细胞分布到骨髓、血液、淋巴结、脾脏、肝脏和中枢神经系统。一个亚系被荧光素酶转染，白血病的发展可以通过成像原位追踪。该细胞系被证实具有免疫基因性，因为接种辐照白血病细胞可保护其免受随后的E2aPBX1侵袭，但对其他肿瘤无效。使用单克隆抗体来消耗细胞亚群，我们证明免疫小鼠的保护需要CD4和CD8 T细胞，并且在NK细胞消耗后受损。在目的2下，我们接下来进行了同基因骨髓移植实验，在小鼠移植后一周注射E2aPBX1。随后，将供体的T细胞过继转移至辐照过的E2aPBX1疫苗。虽然与接受未启动小鼠的纯化T细胞的BMT受体相比，白血病的发展延迟，但所有受体最终都死于白血病。综上所述，这些数据表明，全细胞疫苗接种诱导肿瘤特异性T细胞介导的免疫反应，但无法预防同基因BMT后白血病的发生。重要的是，这些实验还表明，ALL可以被T细胞靶向，但需要事先启动白血病抗原。使用同种异体骨髓而不是同基因骨髓会引入次要的组织相容性抗原，这些抗原可能是白血病细胞的靶点。事实上，同种异体移植后，E2aPBX1白血病攻击，随后转移启动的同种异体T细胞，在所有小鼠中导致白血病治愈，但体重减轻和组织学改变与中度GVHD一致。有趣的是，用受体（和白血病）非恶性B细胞注入T细胞供体并不能治愈小鼠，这表明在该模型中，次要抗原和白血病相关抗原都能治愈小鼠。接下来，我们试图通过选择T细胞亚群来分离抗白血病GVL和GVHD的作用。单独来自供体的CD4或CD8 T细胞都不足以治愈所有小鼠。然而，选择CD62L （l-选择素）阳性的T细胞（与启动供体中的中枢记忆亚型一致）能够介导选择性的、治疗性的GVL效应，而不会产生GVHD。Aim 3正在进行中，涉及白血病相关靶点的鉴定。我们已经确定E2aPBX1过表达Wilm's Tumor 1基因，该基因在大约70-80%的人类白血病中也过表达，并被证实为患者的潜在靶点。为了获得大量的WT-1特异性T细胞，我们正在培养表达WT-1显性表位特异性T细胞受体的小鼠。T细胞受体已经被克隆，并且产生了多个表达大量WT-1特异性T细胞的创始细胞。一旦小鼠细胞系被扩增，来自这些小鼠的T细胞将在E2aPBX1模型中进行测试。如果成功，同种异体移植后的后续实验将有助于验证和表征白血病定向T细胞作为治疗或预防复发的一种方式。