Abstract
BackgroundChimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. However, treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation.MethodsThe FLT3L CAR-T using FLT3 ligand as recognizing domain was constructed. The specific cytotoxicity against FLT3+ leukemia cell lines, primary AML cells, and normal hematopoietic progenitor stem cells (HPSCs) in vitro were evaluated. In addition, FLT3+ AML mouse model was used to assess the effect of FLT3L CAR-T therapy in vivo.ResultsFLT3L CAR-T cells could specifically kill FLT3+ leukemia cell lines and AML patients’ bone marrow mononuclear cells in vitro (with or without FLT3 mutation) and have more potent cytotoxicity to FLT3-ITD cells. In a human FLT3+ AML xenograft mouse model, FLT3L CAR-T cells could significantly prolong the survival of mice. Furthermore, it was found that FLT3L CAR-T cells could activate the FLT3/ERK signaling pathway of FLT3+ leukemia cells with wild-type FLT3; meanwhile, it had no inhibitory effects on the colony formation of CD34+ stem cells derived from normal human umbilical cord blood.ConclusionsThe ligand-based FLT3L CAR-T cells could be a promising strategy for FLT3+ AML treatment, especially those carried FLT3 mutation.
Highlights
Chimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia
According to the expression of CD45RA and CCR7 surface markers, which were used to determine the distribution of T cell subpopulations (Tcm, Effector memory T cell (Tem), Terminally differentiated effector memory T cell (Temra), and native T cells), it was demonstrated that CAR-T cells had a higher percentage of CCR7+CD45RA− (Tcm); no significant difference was noted between CAR-T or VEC-T cells in Tem and Temra (Fig. 1d)
FLT3L CAR-T cells exhibited antigen-specific cytotoxicity against Fms-like tyrosine kinase 3 (FLT3)+ leukemia cells To evaluate the specific cytotoxicity of FLT3L CAR-T cells, FLT3 expression on several acute myeloid leukemia (AML) cell lines was analyzed by specific fluorescence indices (SFI) using flow cytometry and FLT3-positive (FLT3+) leukemia cell lines MV4-11 (SFI:1.45), MOML13 (SFI:1.77), REH (SFI:2.48), and THP-1 (SFI:1.88) were used as target cells, FLT3negative (FLT3−) cell line U937 (SFI:1.07) was used as control target cells, and VEC-T cells were used as control effector cells (Fig. 2a)
Summary
Chimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. Treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation. Acute myeloid leukemia (AML) remains a disease with a poor clinical prognosis. The development of FLT3 inhibitors might improve the clinical outcome, the only effective treatment for FLT3 mutant AML patients remained allogeneic HSCT currently [7]. It is necessary to investigate a new treatment strategy for AML patients with FLT3 mutation
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