DEVELOPMENT & EVALUATION OF PRACTICABLE APPROACHES FOR GENERATION OF CYTOTOXIC &

白血病复发仍然是异基因造血干细胞移植成功的一个重要障碍，特别是对患者来说 在疾病的晚期移植急性白血病和MDS。供体来源的过继转移 抗原特异性T细胞已成为治疗和预防危及生命的疾病的一种很有前途的方法 移植后的病毒感染，也可以用来提供可扩展的杀瘤效应T 细胞转移到肿瘤宿主。在这个项目中，我们建议探索和开发切实可行的广泛适用的 产生供者来源的T细胞选择性反应差异表达决定因素的策略 对白血病细胞进行过继免疫治疗，以治疗或最终防止白血病复发 移植佩里奥德。在目标1中，我们建议开发和评估快速产生WT1的新策略 来自正常移植供者的至少表达一系列常见HLAI类中的一种的多肽特异性T细胞 或II等位基因，用可选择的可立即获取和补充的人工材料进行体外敏化 抗原提呈细胞被设计为表达关键的共刺激分子和单一的I类或II类等位基因 由已经加载了特定WT1表位或合成重叠池的供体共享 跨越WT1序列或转导表达WT1蛋白的十五肽。这些T细胞将 然后与负载WT1多肽的自体树突状细胞致敏的T细胞进行比较， 表型、多肽特异性反应性和杀白血病活性。在目标2中，我们将在体外开发和评估 产生和选择EBV或CMV病毒特异性T细胞，转导后也表达T细胞受体 针对由流行的第I类等位基因呈递的免疫原性WT1肽，或CD19特异性单链抗体- 基于嵌合抗原受体，并评估其抗WT1+和/或CD19+白血病的活性 淋巴瘤及其病毒抗原靶标。我们假设，白血病反应性WT1特异性基因的引入 TCR或CE19特异性CAR将消除转换同种异体反应性T细胞的风险，并可能增强 表达潜伏病毒抗原的细胞在体内持续刺激双受体T细胞的持久性。 在目标3中，我们建议比较评估在AIMS 1和CD19中产生的WT1特异性和CD19特异性T细胞 2对白血病异种移植瘤的迁移、蓄积、存留和诱导消退的能力 NOD/SCID小鼠，并评价WT1多肽对致敏T细胞和T细胞表达的影响 转导受体对正常造血细胞和白血病细胞植入和体内扩增的影响 在允许的NOD/SCIDyc“‘”小鼠模型中。相关性：这些研究可能会产生快速、可行和 广泛适用的方法和可补充试剂为收养产生白血病反应性T细胞 治疗，并应提供对这些T细胞的抗白血病效果的比较估计，这是计划所必需的 并确定临床试验的优先顺序

Leukemia Relapse remains a significant obstacle to the success of allogeneic HSCT, particularly for patients with acute leukemias and MDS transplanted in advanced stages of disease. Adoptive transfer of donor-derived antigen-specific T cells has emerged as a promising approach for the tretment and prevention of life-threatening viral infections post transplant, and may also be used to provide expandable populations of tumoricidal effector T cells to tumor-beariing hosts. In this project, we propose to explore and develop practicable broadly applicable strategies for generating donor-derived T cells that selectively react againstdeterminants differentially expressed on leukemia cells for adoptive immunotherapy to treat or, ultimately, prevent leukemia relapse in the post transplant peeriod. In Aim 1, we propose to develop and evaluate new strategies for rapid generation of WT1 peptide specific T cells from normal transplant donors expressing at least one of a series of common HLA class I or II alleles by in vitro sensitization with a selectable panel of immediately accessible and replenishable artificial antigen presented cells engineered to express critical costimulatory molecules and single class I or class II alleles shared by the donor which have been either loaded with specific WT1 epitopes or a pool of synthetic overlapping pentadecapeptides spanning the WT1 sequence or transduced to express the WT1 protein. These T cells will then be compared with T cells sensitized with autologous, WT1 peptide loaded dendritic cells as to yield, phenotype, peptide-speciflc reactivity and leukemocidal activity. In Aim 2, we will develop and evaluate in vitro generated and selected EBV or CMV virus-specific T cells transduced to also express either a T cell receptor specific for an immunogenic WT1 peptide presented by a prevalent class I HLA allele, or CD19-specific ScFv- based chimeric antigen receptor and evaluate them for their activity against WT1+ and/or CD19+ leukemias and lymphomas and their viral antigen targets. We hypothesize that introduction of a leukemia reactive WT1-specific TCR or CE19-specific CAR will abrogate the risk of transducing alloreactive T cells and may also enhance persistence of dual receptor T cells through ongoing stimulation in vivo by cells expressing latent viral antigens. In Aim 3, we propose to comparatively evaluate WT1 specific and CD19 specific T vcells generated in aims 1 and 2 for their capacity to migrate to, accumulate and persist in and induce regressions of leukemia xenografts in NOD/SCID mice, and to also assess the effects of the WT1 peptide sensitized T cells and T cells expressing transduced receptors on the engraftment and in vivo expansion of normal hematopoietic cells and leukemia blasts in the permissive NOD/SCIDyc"'" mouse model. Relevance: These studies may yield rapid, practicable and broadly applicable approaches and replenishable reagents for generating leukemia-reactive T cells for adoptive therapy and should provide comparative estimates of the anti-leukemia effects of such T cells essential to plan and prioritize clinical trials