Preclinical Development of FLT3-Redirected Chimeric Antigen Receptor T Cell Immunotherapy for Acute Myeloid Leukemia
Authors: Chien Christopher Daniel , Sauter Christopher Tor , Ishii Kazusa , Nguyen Sang Minh , Shen Feng , Tasian Sarah K , Chen Weizao , Dimitrov Dimiter S , Fry Terry J 
Published: November 2019

Abstract
Background: Outcomes for adults and children with acute myeloid leukemia (AML) are dismal with 20-40% and 60% 5-year event-free survival, respectively. Alternative therapeutic strategies for AML are thus needed to improve outcomes. Chimeric antigen receptor (CAR) T cell immunotherapy has induced remarkable clinical responses in multiple phase 1 clinical trials for patients with relapsed or chemorefractory B cell leukemias, encouraging great interest in developing similar approaches for AML. Prior studies have demonstrated efficacy of CD33 or CD123-redirected CAR T cells in AML models, although the genetic heterogeneity of AML will likely require identification of additional therapeutic targets. In the current studies, we report preliminary in vitro and in vivo efficacy of new CAR T cells targeting the FMS-like tyrosine kinase 3 (FLT3) in human AML. FLT3 mutations via internal tandem duplication or kinase domain point mutations occur in approximately 25% of AML and result in FLT3 surface protein overexpression, suggesting potential efficacy of FLT3-targeting therapies. Both types of FLT3 alterations induce ligand-independent activation of FLT3 signaling, further demonstrating a critical role of FLT3 in AML pathogenesis.
Hypothesis: FLT3 is a promising target for CAR T cell immunotherapy based treatment of AML.
Results: Quantitative flow cytometric analysis of human AML cell lines demonstrated FLT3 surface expression ranging from 1338 (MOLM-13), 2594 (MOLM-14), and 2710 (MV4;11) receptors/cell versus 623 receptors/cell on negative control U937 cells. We first generated FLT3-redirected CAR construct consisting of a single chain variable fragment (scFv) derived from a well-characterized anti-human FLT3 antibody coupled to T cell 4-1BB (CD137) costimulatory and CD3-zeta activation domains. CD33 CAR T cells based on Gemtuzumab created by identical methodologies were also used as AML CAR T cell controls. In vitro studies verified that human T cells transduced with the FLT3 CAR construct induced interferon-gamma and interleukin-2 production after co-culture with AML cell lines MOLM-13, MOLM-14, and MV4;11. One dose of FLT3 CAR T cells inhibited leukemia proliferation in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with FLT3-mutant MOLM-13 or MOLM14 cell lines. These first data demonstrate potent preclinical activity of FLT3 CAR T cells and warrant further study in additional AML models.
However, on target/off tumor toxicities can occur with AML antigen-targeted immunotherapies, as previously reported in studies of CD33 and CD123 CAR T cells. Normal expression of FLT3 has been mainly described on CD34+ hematopoietic progenitor stem cell populations, and FLT3-targeted therapies have potential to induce aplastic anemia. To address this question of hematologic toxicity of FLT3 CAR T cells, we created normal human hematopoiesis xenograft models in NOD scid gamma Il3-GM-SF (NSGS) mice engrafted with CD34+ cord blood cells for treatment with anti-AML CAR T cells. No difference in human granulocyte numbers was observed in marrows of engrafted mice treated with FLT3 CAR T cells, CD33 CAR T cells, or non-transduced T cells. A significant reduction in monocytes was observed in FLT3 CAR T cell-treated animals, however (p<0.05 by t test). To determine potential for increased hematologic toxicity in the presence of greater target antigen levels, we injected MOLM-14 into CD34+ cell-engrafted mice, then treated animals with control or anti-AML CAR T cells. We surprisingly found no decrement in defined hematopoietic stem cell (HSC) or granulocyte macrophage progenitor (GMP) populations, but did observe increased multipotent and common myeloid progenitor (MPP, CMP) cell numbers and an increase in total human cell engraftment 5 days after FLT3 CAR treatment in comparison to non-transduced T cell-treated animals. Relative to CD33 CAR T cells, FLT3 CAR T cells induced less toxicity to HSCs and MPPs and equivalent toxicity to CMPs and GMPs, indicating lower hematologic toxicity with FLT3 targeting.
Conclusions: Taken together, these initial data demonstrate potent in vitro and in vivo anti-AML activity with limited hematopoietic toxicity of FLT3 CAR T cell immunotherapy. Future studies are focused on testing the effectiveness on other AML cell lines with varying expression of FLT3.

Disclosures
No relevant conflicts of interest to declare.