Natural Flt3Lg-Based Chimeric Antigen Receptor (Flt3-CAR) T Cells Successfully Target Flt3 on AML Cell Lines

Abstract

Relapsed/refractory acute myeloid leukemia (AML) cannot be cured with chemotherapy alone, as the blasts survive the treatment. Chimeric antigen receptor (CAR) approaches for AML are being actively developed. CARs promote immune reactions through recognition of the target molecular epitopes at the surface of cancer cells. The recognition involves the extracellular portion of the CAR protein, which corresponds to either the antibody or the physiological binding partner of the targeted antigen. Here, we design a chimeric receptor with a full-length natural Flt3-ligand recognition module that targets Flt3 tyrosine kinase, known as an adverse marker in AML. We demonstrate specific killing of Flt3-positive THP-1 cells by Flt3-CAR T cells and the lack of cytotoxicity towards Flt3-negative U937 cells. We prove that the inherent cytolytic capacity of T cells is essential for the killing. Finally, we confirm the authenticity of targeting by its competitive dose-dependent inhibition with a soluble Flt3-ligand. The developed system can be viewed as a non-immunogenic functional equivalent of scFv-mediated targeting. The robust in vitro antitumor effects of Flt3-CAR T cells, combined with their low off-target cytotoxicity, hold promise for AML treatment.

Challenges and Advances in Chimeric Antigen Receptor Therapy for Acute Myeloid Leukemia

“…Multiple inhibitors of FLT3 are FDA approved for use in FLT3 + AML including gilteritinib, midosaurin, and quizartinib, so it is not surprising that FLT3 CARs are being investigated. Currently all published work is preclinical, but has showed promising activity against leukemic cell lines [114][115][116][117][118]. Many groups are using safety switches to allow for bone marrow recovery afterwards, given that FLT3 also depleted HSCs [38].…”

Recent Advances in the Development of Anti-FLT3 CAR T-Cell Therapies for Treatment of AML

“…The CARs were encoded on a bicistronic lentiviral vector using the same promoter and separated by a self-cleaving 2A peptide to ensure equal expression on the cell surface [ 35 ]. ( E ) A third generation anti-FLT3 CAR with two co-stimulatory domains, 4-1BB and inducible T-cell co-stimulator (ICOS) [ 36 ].…”

“…While most studies used an scFv derived from known antibodies, Sommer et al developed and screened their own scFvs from a phage-display library [ 33 ]. In contrast to using scFvs, Wang et al and Maiorova et al targeted FLT3 using its natural ligand, FLT3 ligand (FLT3L) [ 34 , 36 ]. This has the advantage of potentially eliminating problems with immunogenicity, and the natural ligand might display higher specificity than scFvs.…”

“…All second generation anti-FLT3 CAR T-cells with either CD28 or 4-1BB showed cytotoxicity against different FLT3-positive cell lines; Kasumi, OCI-AML3, MOLM-13, THP-1, EOL-1 or MV4-11. Maiorova et al chose to use ICOS and 4-1BB to generate a third generation CAR [ 36 ]. The authors used mKate2-expressing (red fluorophore) THP-1 cells and measured decreased red object count as a representation of THP-1 killing.…”

Natural Receptor- and Ligand-Based Chimeric Antigen Receptors: Strategies Using Natural Ligands and Receptors for Targeted Cell Killing

“…Importantly, treatment with CAR T cells did not inhibit hematopoietic cell colony formation when cultured with CD34+ healthy human umbilical cord blood stem cells despite FLT3 expression in hematopoietic stem cells (HSCs) [ 122 ], indicating little off-target toxicity in the hematopoietic compartment, at least in vitro. Maiorova et al [ 93 ] also developed a ligand-based CAR targeting FLT3 utilizing the full-length FLT3L. The authors found the enhanced killing of FLT3+ THP-1 cell lines by CAR T cells in vitro, though in vivo studies are still needed to determine efficaciousness.…”