Controlled activation modulates T-cell expansion and phenotype in stirred-tank bioreactors.

Abstract

Autologous cell therapies using chimeric antigen receptor (CAR) T cells have shown significant clinical success in hematologic cancers. However, current production platforms face challenges in scaling up to produce sufficient numbers of cells to meet the demands of multi-dose regimens. Additionally, tight control over critical process parameters during the distinct stages of cell production is required to maximize key phenotypic characteristics of CAR T-cell products that correlate with improved clinical responses. To address these issues, we propose an integrated manufacturing process in stirred-tank bioreactors (STBs) for controlled T-cell activation and expansion. By tailoring the stirring profile of STBs (Ambr® 15 bioreactors; Sartorius, Göttingen, Germany), microbeads functionalized with anti-CD3/CD28 antibodies allow control over the initiation/termination of T-cell activation without requiring additional washing steps to remove the activation signaling cues. This strategy resulted in up to a 10-fold increase in T-cell numbers compared with conventional static culture systems, resulting in a final cell concentration of 2.5 × 10⁷ cells/mL after 10 days of culture. Importantly, a higher proportion of CD8⁺ T cells and lower expression of exhaustion markers programmed cell death protein 1, lymphocyte activation gene 3 and T-cell immunoglobulin and mucin domain 3 (<8%) were obtained in STBs relative to static cultures. Additionally, the anti-CD3/CD28-functionalized microbeads were as efficient as the standard TransAct™ (Miltenyi Biotec, Bergisch Gladbach, Germany) stimuli in activating and expanding T cells in STBs. Overall, this approach presents a promising strategy for the scalable and tightly controlled manufacturing of T-cell therapies, particularly focusing on the T-cell activation step while minimizing manual operations, thus contributing towards more effective and cost-efficient immunotherapies.