Mutated Flt3Lg Provides Reduced Flt3 Recycling Compared to Wild-Type Flt3Lg and Retains the Specificity of Flt3Lg-Based CAR T-Cell Targeting in AML Models.

The cells of acute myeloid leukemia are defined by clonal growth and heterogenous immunophenotypes. Chimeric antigen receptors (CARs) commonly recognize molecular targets by single-chain antibody fragments (scFvs) specific to a tumor-associated antigen. However, ScFvs may form aggregates, thus stimulating tonic CAR T-cell activation and reducing CAR T-cell functioning in vivo. Harnessing natural ligands as recognition parts of CARs, specific targeting of membrane receptors can be achieved. Previously, we presented ligand-based Flt3-CAR T-cells targeting the Flt3 receptor. The extracellular part of Flt3-CAR consisted of full-size Flt3Lg. Meanwhile, upon recognition, Flt3-CAR may potentially activate Flt3, triggering proliferative signaling in blast cells. Moreover, the long-lasting presence of Flt3Lg may lead to Flt3 downregulation. In this paper, we present mutated Flt3Lg-based Flt3m-CAR ('m'-for 'mutant') T-cells targeting Flt3. The extracellular part of Flt3m-CAR consists of full-length Flt3Lg-L27P. We have determined that ED50 for recombinant Flt3Lg-L27P produced in CHO cells is at least 10-fold higher than for the wild-type Flt3Lg. We show that the mutation in the recognizing domain of Flt3m-CAR did not affect the specificity of Flt3m-CAR T-cells when compared to Flt3-CAR T-cells. Flt3m-CAR T-cells combine the specificity of ligand-receptor recognition with reduced Flt3Lg-L27P bioactivity, leading to potentially safer immunotherapy.

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

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.