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

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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.