Culture expansion of CAR T cells evokes aberrant DNA methylation that is associated with adverse clinical outcome (Methylation)

Cellular immunotherapy using autologous, genetically modified T cells has delivered remarkable initial response rates in various hematological malignancies, including acute lymphoblastic leukemia (ALL) or lymphoma. Upon treatment with these chimeric antigen receptor (CAR) T cells, approximately 30–40% of patients show long-term remission. There is a growing perception that not only CAR constructs, but also in vitro manufacturing processes may determine therapeutic efficacy. However, little is known about how duration of culture expansion affects molecular and functional features of CAR T cells. A better understanding of molecular signatures, reflecting therapeutic activity of CAR T cells, may help to further optimize the manufacturing process. In this study, we therefore investigated DNAm changes during culture expansion of T cells and CAR T cells during a period of up to three weeks. The results of our current study indicate that culture expansion of CAR T cells leads to DNAm changes that are continuously acquired during culture expansion. Further analysis of culture-associated methylation profiles in CAR T cells from clinical trials support the notion that these chnages are disadvantageous with regards to therapeutic outcome. A shortened cultivation period to avoid dysfunctional methylation programs might be a promising manufacturing strategy to improve therapeutic efficacy of adoptive cell therapies. Our epigenetic signature for culture-associated DNAm may provide a biomarker for quality control of CAR T cell productions and to identify patients at risk for adverse events. Bisulphite converted DNA from the 32 CAR T and T cell samples from distinct culture conditions that were harvested during distinct time points of in vitro expansion were hybridised to the Illumina Infinium Methylation850 BeadChips.