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Abstract

Chimeric antigen receptor (CAR)-T cell therapy has proven efficacious in hematological malignancies, with success in CD19-targeted therapy for leukemia/lymphoma and B cell maturation antigen (BCMA)-targeted therapy in multiple myeloma. However, a significant portion of patients still relapse post-therapy. Factors such as T cell exhaustion and T cell memory phenotypes have been thought to contribute, given that they may impact persistence which has been correlated with poor outcomes after CAR-T cell therapy. In this project, we explore whether telomere maintenance plays a role in CAR-T cell persistence, given that shortened telomeres result in a loss of proliferative capacity. During CAR-T cell manufacturing, the T cells undergo a rapid expansion phase, which may lead to telomere shortening. Thus, methods were explored to measure telomerase activity, telomere length, cell cycle progression, senescence-associated 𝛽-galactosidase (SA 𝛽-gal) activity, and various cell surface markers. Through repetitive stimulations, telomerase activity decreased with each round of stimulations, and cells reached a plateau in growth. SA 𝛽-gal increased with each round of stimulation and an increased proportion of cells were observed in G₀/G₁. To address this, an 8-day and 12-day manufacturing process was assessed, to determine if this may better preserve cell phenotype. The 8-day process had an increased proportion of the cells in G₂/M compared to the 12-day manufacture, however had increased levels of SA 𝛽-gal. Lastly, epigenetic age has been explored as a potential predictor of patient outcomes and was assessed in healthy donor samples and patient samples from a CD19-directed CAR-T cell clinical trial. Responders were found to have a lower epigenetic age than non-responders, providing the rationale for further study.