TY - JOUR
T1 - Controlled activation modulates T-cell expansion and phenotype in stirred-tank bioreactors
AU - Costa, Margarida S.
AU - Costa, Constança M.
AU - Matos, Leonor N.
AU - Sebastião, Maria João
AU - Duarte, Nádia
AU - Costa, Marta H.G.
AU - Serra, Margarida
N1 - Funding Information:
This work was supported by Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia (FCT) through the CARTool project ( https://doi.org/10.54499/2022.07312.PTDC ). This work was also funded by FCT/Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health ( UIDB/04462/2020 and UIDP/04462/2020 ) and the Associate Laboratory LS4FUTURE ( LA/P/0087/2020 ). The project was also funded by the European Union's European Innovation Council Transition program under grant agreement no. 101113067 . MSC was supported by FCT PhD fellowship 2022.12494.BD .
Publisher Copyright:
© 2025 International Society for Cell & Gene Therapy
PY - 2025/6
Y1 - 2025/6
N2 - Background aims: 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. Methods: 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. Results: 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. Conclusions: 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.
AB - Background aims: 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. Methods: 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. Results: 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. Conclusions: 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.
KW - controlled activation
KW - immunotherapy
KW - scalable manufacturing
KW - stirred-tank bioreactor
KW - T-cell phenotype
UR - http://www.scopus.com/inward/record.url?scp=85219023906&partnerID=8YFLogxK
U2 - 10.1016/j.jcyt.2025.02.003
DO - 10.1016/j.jcyt.2025.02.003
M3 - Article
C2 - 40019461
AN - SCOPUS:85219023906
SN - 1465-3249
VL - 27
SP - 774
EP - 781
JO - Cytotherapy
JF - Cytotherapy
IS - 6
ER -
