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 急性白血病和骨髓增生异常综合征的晚期患者捐赠者衍生的 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有前途的方法。 移植后病毒感染，并且也可以用于提供可扩展的杀肿瘤效应T细胞群体。 细胞到携带肿瘤的宿主。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 产生选择性反应差异表达决定簇的供体来源T细胞的策略 对白血病细胞进行过继免疫治疗，以治疗或最终预防白血病复发。 移植周期。在目标1中，我们建议开发和评估快速生成WT 1的新策略 来自正常移植供体的肽特异性T细胞，其表达一系列常见HLA I类中的至少一种 或II等位基因，通过用可选择的一组可立即获得的和可降解的人工 经工程改造以表达关键共刺激分子和单个I类或II类等位基因的抗原呈递细胞 这些表位或者装载有特异性WT 1表位，或者装载有合成的重叠表位库 十五肽跨越WT 1序列或转导以表达WT 1蛋白。这些T细胞将 然后与用自体的、负载WT 1肽的树突状细胞致敏的T细胞进行比较， 表型、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 产生并选择转导的EBV或CMV病毒特异性T细胞，其也表达T细胞受体 特异于由流行的I类HLA等位基因呈递的免疫原性WT 1肽，或CD 19特异性ScFv- 基于嵌合抗原受体的细胞，并评估它们对WT 1+和/或CD 19+白血病的活性， 淋巴瘤及其病毒抗原靶点。我们假设，白血病反应性WT 1特异性 TCR或CE 19特异性CAR将消除转导同种异体反应性T细胞的风险，并且还可以增强免疫应答。 双受体T细胞通过表达潜伏病毒抗原的细胞在体内持续刺激而持续存在。 在目标3中，我们提出比较评估目标1中产生的WT 1特异性和CD 19特异性T细胞， 2，因为它们能够迁移到白血病异种移植物中，在白血病异种移植物中积累和持续存在并诱导白血病异种移植物消退。 NOD/SCID小鼠，并且还评估WT 1肽致敏的T细胞和表达WT 1肽的T细胞的作用。 转导的受体对正常造血细胞和白血病母细胞的植入和体内扩增的影响 在允许的NOD/SCIDyc小鼠模型中。相关性：这些研究可能产生快速、可行和 用于产生过继免疫的白血病反应性T细胞的广泛适用的方法和可扩增的试剂 治疗，并应提供抗白血病作用的比较估计这样的T细胞必不可少的计划， 优先考虑临床试验

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