Transient mRNA-based CD117 CAR T cells effectively target acute myeloid leukemia in vitro for potential use as a preconditioning strategy

Background Acute myeloid leukemia (AML) is a high-risk malignancy, particularly in patients with primary induction failure or relapsed/refractory disease. CD117 (c-Kit), expressed on both leukemic blasts and normal hematopoietic stem and progenitor cells (HSPCs), represents a potential therapeutic target but poses challenges due to the risk of severe myelotoxicity.

Materials and methods Retrospective flow cytometry analyses of samples from 27 AML patients and AML cell lines were carried out to assess CD117 expression. Second-generation CD117-specific chimeric antigen receptor (CAR) T cells were generated by either retroviral transduction or in vitro-transcribed (IVT) messenger RNA (mRNA) electroporation. The mRNA-based CD117 CAR T cells were evaluated for viability, immunophenotype, cytotoxic activity, and toxicity toward primary HSPCs using clonogenic assays, and compared with retroviral-based counterparts.

Results CD117 was expressed in AML patient samples and cell lines at varying levels. CD117 CAR T cells demonstrated potent and specific cytotoxicity against AML cells. The mRNA-based CAR T cells exhibited high transfection efficiency, good viability, and an immunophenotype similar to non-transduced T cells, and were functionally competent as early as 2 h post-electroporation. In long-term co-culture with a high tumor burden, repeated dosing of mRNA CAR T cells effectively eliminated CD117+ cells, comparable to viral vector-based CAR T cells. Notably, residual mRNA CAR T cells following AML clearance showed no detectable CAR expression and preserved HSPC colony-forming capacity.

Conclusions Our in vitro studies suggest the potential use of mRNA CD117 CAR T cells as a non-genotoxic preconditioning strategy for patients with high-risk or refractory AML.