Regulation and therapeutic implication of TIM-3 in pediatric B-precursor acute lymphoblastic leukemia

小儿急性淋巴细胞白血病（ALL）的治疗在过去的几十年中不断得到改进。免疫治疗方案，如双特异性T细胞融合剂（Blinatumomab）或用抗CD19嵌合抗原受体（抗CD19 CAR）工程化的T细胞，已在I/II期试验中进行了测试，最近获批用于复发性/难治性ALL。然而，新疗法无法为大多数患者提供长期益处。需要研究复发的新机制。我们的初步数据确定了骨髓CD4 + T细胞上的耗竭标志物TIM-3作为ALL复发的强预后标志物。T细胞上的TIM-3表达可以通过与白血病细胞的相互作用来诱导，从而降低T细胞活化和增殖。此外，我们发现抗CD19 CAR T细胞可以具有高水平的TIM-3。该项目由基础研究和转化研究两部分组成。基础研究部分旨在揭示白血病介导的TIM-3在原代T细胞中的诱导机制。将进行RNA测序和全基因组CRISPR筛选以及随后的单细胞RNA测序，以鉴定TIM-3上调的关键分子、受体和转录因子。基于对白血病诱导的TIM-3上调的更好理解，翻译部分将提供新的免疫学方法来逆转TIM-3介导的T细胞抑制。通过CRISPR介导的敲入，我们将抗CD19 CAR插入TIM-3基因座。靶向整合到TIM-3基因中允许转基因的同时TIM-3敲除和敲入，并迫使转基因处于TIM-3的内源启动子控制下。功能性测定将揭示该构建体是否能够改善抗白血病CAR功能并抵抗白血病细胞衍生的共抑制信号。该项目的目的是更好地了解原始T细胞相互作用，并改善目前治疗儿童晚期ALL的T细胞疗法。

The treatment of pediatric acute lymphoblastic leukemia (ALL) has been improved constantly during the last decades. Immunotherapeutic regimens such as bispecific T-cell engagers (Blinatumomab) or T cells engineered with a chimeric antigen receptor against CD19 (Anti-CD19 CAR) have been tested in phase I/II trials and were recently approved for relapsed/refractory ALL. Nevertheless, new therapies are not able to provide long-term benefit for the majority of patients. New mechanisms of relapse need to be investigated. Our preliminary data identified the exhaustion marker TIM-3 on bone marrow CD4+ T cells as strong prognostic marker for ALL relapse. TIM-3 expression on T cells can be induced by interaction with leukemic cells and thus decreases T-cell activation and proliferation. Moreover, we showed that Anti-CD19 CAR T cells can have high levels of TIM-3. The proposed project consists of a basic and a translational research part. The basic research part aims to uncover the mechanism of leukemia-mediated TIM-3 induction in primary T cells. RNA sequencing and a genome-wide CRISPR screen with subsequent single-cell RNA sequencing will be performed to identify key molecules, receptors and transcription factors for TIM-3 upregulation. Based on a better understanding of leukemia-induced TIM-3 upregulation, the translational part will provide new immunotherapeutic approaches to reverse TIM-3-mediated T-cell inhibition. By CRISPR-mediated knock-in, we will insert an Anti-CD19 CAR into the TIM-3 locus. Targeted integration into the TIM-3 gene allows simultaneous TIM-3 knockout and knock-in of the transgene and forces the transgene under endogenous promoter control of TIM-3. Functionality assays will reveal, if this construct is able to improve anti-leukemic CAR functionality and resist co-inhibitory signals derived by leukemic cells. Aim of this project is a better understanding of blast-T-cell interactions and improvement of current T-cell therapies for treatment of pediatric advanced ALL.