@phdthesis{Jetani2021,
  author    = {Jetani, Hardikkumar},
  title     = {Chimeric antigen receptor (CAR)-modified T cells targeting FLT3 in acute myeloid leukemia (AML)},
  doi       = {10.25972/OPUS-17909},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179096},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells targeting CD19 has shown remarkable therapeutic efficacy against B cell leukemia and lymphoma, and provided proof of concept for therapeutic potential in other hematologic malignancies. Acute myeloid leukemia (AML) is an entity with an unmet medical need for effective and curative treatments. Therefore, there is a strong desire for development of potentially curative CAR-T cell immunotherapy for AML treatment. FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein expressed uniformly by AML blasts. FLT3 plays a vital role in the survival of AML blasts and is a key driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3ITD) and tyrosine kinase domain (TKD) mutations. These attributes suggest that FLT3 could be an excellent target for CAR-T cell immunotherapy. Here, we engineered human CD4+ and CD8+ T cells to express FLT3-specific CARs and demonstrate that they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3-ITD. Further, we show that FLT3 CAR-T cells exert potent antileukemia activity in xenograft models of AML and induce complete remissions. We also demonstrate that FLT3-expression on FLT3-ITD+ AML cells can be augmented by FLT3 inhibitors, which lead to increased recognition by CARs and improved efficacy of FLT3 CAR-T cells. We confirmed this principle with three different FLT3 inhibitors which are at distinct stages of clinical development i.e. Phase II/III clinical trial (crenolanib, quizartinib) and clinically approved (midostaurin). Further, we observed the strongest anti-leukemia activity of FLT3 CAR-T cells in combination with crenolanib in vivo. FLT3 is known to be expressed by normal hematopoietic stem and progenitor cells. We evaluated FLT3-expression on normal hematopoietic stem cells (HSCs) using flow cytometry and confirmed lower level of FLT3-expression on HSCs and progenitors compared to AML cells. As anticipated, we found that FLT3 CAR-T cells recognize normal HSCs in vitro and in vivo, and compromise normal hematopoiesis, suggesting that adoptive therapy with FLT3 CAR-T cells will require successive CAR-T cell depletion and allogeneic HSC transplantation (HSCT) to reconstitute the hematopoietic system. Moreover, an FLT3 inhibitor treatment does not increase FLT3-expression on HSCs. Accordingly, we demonstrate that the depletion of FLT3 CAR-T cells is possible with inducible Caspase 9 (iCasp9) safety switch. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Our data demonstrate that FLT3 CAR-T cells act synergistically with FLT3 inhibitors in FLT3-ITD+ AML. i.e. FLT3 inhibitors-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSCs, the clinical use of FLT3 CART cells is likely restricted to a defined therapeutic window and must be followed by CART cell depletion and allogeneic HSCT for hematopoietic reconstitution. The data provide rational to use FLT3 CAR-T cells in combination with FLT3 inhibitors to augment the anti-leukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk of relapse with FLT3-negative AML variants, which could otherwise develop under therapeutic pressure. The data provide proof of concept for synergistic use of CAR-T cell immunotherapy and small molecule targeted therapy and encourage the clinical evaluation of this combination treatment in high-risk patients with FLT3-ITD+ AML.},
  subject      = {Chimeric antigen receptor (CAR)},
  language  = {en}
}