Impaired ex vivo T-cell expansion before leukapheresis is associated with out-of-specification tisagenlecleucel manufacturing in pediatric CD19+ALL

Introduction Chimeric antigen receptor (CAR)-T cell therapy requires individual production for each patient, starting with a heterogeneous apheresis product of variable cellular composition and function. This inter-patient variability presents a major challenge for standardizing manufacturing processes and may lead to delays or even production failure. The primary aim of this study was to identify pre-leukapheresis characteristics associated with subsequent manufacturing deviations. Methods Multiple T cell-related parameters including T cell receptor (TCR) Vβ repertoire, differentiation status, proliferation capacity (e.g. fraction diluted – DIL), and exhaustion were examined in n = 27 patients before leukapheresis for Kymriah ® immunotherapy. These parameters were analyzed for associations with manufacturing specification compliance and out-of-specification (OOS) events. Results Impaired T-cell proliferation was the parameter most strongly associated with manufacturing specification non-conformance. Six of 21 evaluable patients showed poor proliferation (DIL <80%), including all four patients with OOS manufacturing outcomes. In contrast, all remaining patients had DIL values ≥84.9%. Exploratory associations between selected TCR Vβ subsets and manufacturing specifications were also identified. Conclusion DIL was the parameter most strongly associated with manufacturing specification compliance and may represent a simple functional measure of pre-apheresis T-cell fitness.