Systematic Characterization of AML Cell Lines to Support Preclinical In Vitro CAR-T Research

The development of chimeric antigen receptor (CAR) T cell therapies for acute myeloid leukemia (AML) remains challenging, underscoring the need for reliable preclinical models. Although AML cell lines are widely used in CAR-T studies, their ability to reflect clinically relevant antigen expression patterns and functional characteristics of primary AML cells has not been systematically evaluated. We characterized nine AML cell lines (KG-1, HL-60, Kasumi-1, OCI-AML2, OCI-AML5, THP-1, U-937, MV4-11, and MOLM-13) as targets for in vitro CAR-T studies. We assessed the expression of established CAR-T target antigens (e.g., CLL1, CD33), cell lines similarity to minimal residual disease (MRD) cells and matched healthy monocytes from 161 AML patients, the expression of emerging AML-associated antigens (Siglec-6, CSF1R, and CD70), growth behavior, and susceptibility to non-specific killing by non-transduced T cells. AML cell lines demonstrated substantial heterogeneity in antigen expression with several cell lines showing antigen densities exceeding those observed in AML blasts. Several widely used lines, including U-937, were susceptible to non-transduced T-cell cytotoxicity, potentially confounding CAR-T functional assays and overestimating CAR-T activity in vitro. The examined parameters were integrated into a scoring framework to rank AML cell lines according to their suitability for in vitro CAR-T studies. Based on this framework and additional assay-related considerations, OCI-AML2, OCI-AML5, MV4-11, and MOLM-13 were identified as most suitable for in vitro evaluation of CAR-T products, particularly those targeting CD33 and CLL1. These findings may facilitate more informed selection of AML cell lines for preclinical development of CAR-T therapies for AML.