CAR-T cell expansion platforms yield distinct T cell differentiation states

Area of Science:

• Cellular immunotherapy
• Biotechnology
• Bioprocess engineering

Background:

• Engineered T cell therapies like CAR-T are expanding to new targets and patient groups.
• Scaling CAR-T cell manufacturing is essential, particularly for solid tumors requiring higher doses.
• The influence of manufacturing platforms on CAR-T cell product phenotype and function remains largely unknown.

Purpose of the Study:

• To compare the impact of four common T cell manufacturing platforms on CAR-T cell expansion and phenotype.
• To investigate the role of dissolved oxygen levels in influencing T cell differentiation during manufacturing.
• To identify optimal manufacturing strategies for preserving naïve T cell phenotypes for enhanced therapeutic efficacy.

Main Methods:

• Four manufacturing platforms (CliniMACS Prodigy, Xuri, G-Rex, static bag) were used with identical culture conditions.
• CD4+CD8+ T cells were transduced with a CAR targeting FGFR4.
• Cell expansion, phenotype (e.g., CCR7+CD45RA+ naïve/stem central memory cells), gene expression, and metabolism were analyzed.
• Hypoxic conditions (1% O2) were tested in bag and G-Rex cultures to assess their effect on T cell phenotype.

Main Results:

• Significant differences in cell expansion were observed, with static bag cultures yielding the highest expansion and Prodigy the lowest.
• The Prodigy platform enriched for naïve/stem central memory-like T cells (46%) compared to bag (16%) and G-Rex (13%).
• Prodigy CAR-T cells exhibited a more naïve, less cytotoxic, and less exhausted phenotype, with distinct metabolic profiles.
• Culturing in 1% O2 significantly increased the percentage of naïve/stem central memory-like cells (>60%), suggesting oxygenation influences T cell differentiation.

Conclusions:

• Bioreactor system selection profoundly impacts CAR-T cell expansion capacity and differentiation state.
• Dissolved oxygen levels are a key factor influencing T cell phenotype during manufacturing.
• Hypoxic culture conditions represent a potential strategy to prevent T cell differentiation and preserve a more potent naïve phenotype for CAR-T cell therapy.