Leveraging the TCR Repertoire to identify target neoantigens in FLT3-ITD positive Acute Myeloid Leukemia

摘要 异基因造血干细胞移植(HSCT)是治疗高血压性白血病的唯一有效方法。 有患急性髓系白血病(AML)的风险。然而，HSCT受到移植物抗宿主病(GvHD)和移植物抗宿主病(GVHD)的影响。 抗白血病(GVL)效应，两者都是由供体T淋巴细胞介导的，并显著影响治疗 成功，因此总的结果。AML患者通常携带Flt3/内部串联复制(Flt3-ITD)， 受体酪氨酸激酶Flt3的突变与预后不良有关。Flt3靶向治疗 已被证明具有临床益处，特别是在联合治疗中使用时。米多妥林(一种激酶抑制剂) 最近被批准用于移植前患有Flt3-ITD的患者，并结合标准治疗。此外 由于其直接的白血病抑制作用，Flt3抑制剂激活了白血病抗原特异性的T细胞反应。 T细胞受体(TCR)是一种表达在T细胞表面的蛋白质，可识别由 MHC分子。我们最近描述了接受匹配供者或联合移植的患者的TCR谱系。 单脐带造血干细胞移植。我们证明了GvHD和复发(相互排斥)与较低的 某些T细胞克隆的TCR谱系多样性和扩增。我们的数据表明， 免疫谱系显著影响AML患者的临床结果，并强调了 对大型TCR曲目进行全面的定量、功能和机理分析 急性髓系白血病患者队列。在这里，我们假设：1)TCR曲目(多样性、克隆扩增和V-片段 利用)影响临床结果(GvHD或复发)，因此可以用来识别GVL-和GvHD- 相关克隆；2)白血病细胞的体细胞突变(例如，Flt3-ITD)影响TCR谱系和 随后特定T细胞克隆的扩增；以及3)Flt3抑制剂(例如米多妥林)调节TCR HSCT患者的移植指征和功能及增强GVL效应。我们将进行一次前瞻性的 ~250白血病细胞TCR谱系和突变格局的纵向队列研究 FLT3-ITD患者60~80例。GVHD或复发将使用比例风险模型进行预测 基于TCR曲目特征的竞争风险。TCR序列和体细胞突变将是 使用我们开发的用于预测候选白血病新抗原的基于结构的预测算法进行分析 以及相关联的TCR克隆。新抗原将使用体外和小鼠模型进行验证。最后，功能 分析将检查米多妥林对TCR曲目和功能的影响。我们的发现将确立 TCR谱系作为预测HSCT临床结果和确定负责任的TCR克隆的有用工具。 鉴定与GVL抗Flt3-ITD细胞相关的TCR克隆将有助于 表达GVL相关TCR克隆的工程化T细胞的研制修改TCR曲目 通过针对特定体细胞突变的治疗来合成将促进优化的 联合治疗方法，例如在移植后方案中增加靶向治疗。

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with high- risk acute myeloid leukemia (AML). However, HSCT is affected by graft-versus-host disease (GvHD) and graft- versus-leukemia (GvL) effects, both are mediated by donor T lymphocytes and significantly impact treatment success and thus overall outcome. AML patients commonly harbor FLT3/internal tandem duplication (FLT3-ITD), a mutation in the receptor tyrosine kinase FLT3 that is associated with poor prognosis. FLT3 targeted therapies have proven clinical benefit particularly when used in combinational approaches. Midostaurin (a kinase inhibitor) was recently approved for pre-transplant patients with FLT3-ITD in combination with standard therapy. In addition to their direct leukemia suppressive effects, FLT3 inhibitors activate leukemia antigen-specific T-cell responses. T-cell receptors (TCRs) are proteins expressed on the surface of T cells that recognize antigens presented by MHC molecules. We recently characterized the TCR repertoire in patients who underwent matched donor or haplo-cord HSCT. We demonstrated that GvHD and relapse (exclusive of each other) are associated with lower TCR repertoire diversity and expansion of certain T-cell clones. Our data suggest that individual variations in the immune repertoire significantly impact the clinical outcome in AML patients and underscore the need for comprehensive quantitative, functional, and mechanistic analyses of the TCR repertoire in a large cohort of AML patients. Here, we hypothesize: 1) TCR repertoire (diversity, clonal expansion, and V-segment utilization) affects clinical outcome (GvHD or relapse) and can therefore be used to identify GvL- and GvHD- associated clones; 2) Somatic mutations in leukemic cells (e.g., FLT3-ITD) affect the TCR repertoire and subsequent expansion of specific T-cell clones; and 3) FLT3 inhibitors (e.g., midostaurin) modulate the TCR repertoire and function and enhance GvL effects in patients undergoing HSCT. We will conduct a prospective longitudinal cohort study characterizing the TCR repertoire and mutational landscape of leukemia cells in ~250 patients (~ 60–80 with FLT3-ITD). GvHD or relapse will be predicted using a proportional hazards model for competing risks based on TCR repertoire characteristics. TCR sequences and somatic mutations will be analyzed using a structure based prediction algorithms we developed to predict candidate leukemia neoantigens and associated TCR clones. Neoantigens will be validated using in vitro and murine models. Finally, functional analyses will examine the effect of midostaurin on TCR repertoire and function. Our findings will establish the TCR repertoire as a useful tool for predicting clinical outcomes of HSCT and identify responsible TCR clones. The identification of TCR clones associated with the GvL effect against FLT3-ITD+ cells will facilitate the development of engineered T cells expressing GvL-associated TCR clones. Modifying the TCR repertoire composition via therapies targeting specific somatic mutations will facilitate development of optimized combinational therapeutic approaches, such as the addition of targeted therapy to post-transplant regimens.