Professor Georgia Institute of Technology Atlanta, Georgia, United States
Introduction:: T-cell-based therapies, such as CAR-Ts, can be used as life-saving medicines to treat or cure complex diseases. However, the manufacturing of these cell therapies remains a bottleneck for them to be accessible and affordable to the public. Recently, our group reported a degradable microcarrier scaffold (DMS) T cell activation method for high stem-like memory T cell generation, which was shown to be correlated with high therapeutic efficacy in previous clinical trials. This method was proven effective in static T-flask culture but had not been scaled up to a suspension bioreactor environment to demonstrate scalability. In this study, we used vertical wheel bioreactors to create a suspension environment to culture T cells. T cells and DMS were seeded in the bioreactor at the same time to enable a one-step unit operation process. Our overall objective is to study how static vs suspension environment affects DMS-activated T cell growth and identify T cell memory subsets upon the end of the manufacturing process.
Materials and Methods:: In this study, we used human CD3+ T cells purchased from Charles Rivers Laboratory, PBS Mini bioreactors from PBS Biotech, TexMACS media from Miltenyi Biotec. DMS was fabricated by using cultispher microcarriers from Sigma Aldrich conjugated with anti-CD3 and anti-CD28 via biotinylation. Fresh CD3+ T cells were thawed, activated by DMS, and seeded into bioreactor. IL2-supplemented media were fed in the bioreactor until it reaches a full vessel volume. Mixing was set to 10 RPM or 50 RPM depending on the experimental condition. Small-scale T flasks were used as static control. After expansion, cells were stained with panel including live/dead, CD3, CD4, CD8, CD45RA, CD45RO, CCR7, and CD95 to quantify percentages of each T cell subset via flow cytometry analysis.
Results, Conclusions, and Discussions:: 3 expansions were performed for each experimental condition. For yield, bioreactors under 50RPM showed 4.32 ± 0.38 e8 cells, bioreactors under 10 RPM showed 1.09 ± 0.62 e8 cells, while T-flasks showed 8.21 ± 0.26 e6 cells at the end of the expansion process, demonstrating that full suspension environment was conducive for DMS-activated T cell growth and bioreactor vessels can achieve a clinically relevant dose at the end of the manufacturing process. Surface marker data showed various distributions of memory subsets with each experimental condition. With CD8+ T stem cell memory population, which is one of the most potent memory phenotypes, on average, T-flask showed 29.5% expression, 10RPM showed 38.4% expression, and 50RPM showed 46.5% expression. DMS activation for T cells was successfully scaled up in the vertical wheel bioreactor, and T cells showed superior growth under a full suspension environment, this could be due to better nutrient transfer or better contact of DMS with T cells. Moreover, DMS also provides a more physiologically relevant environment for T cell activation by mimicking the lymph nodes where T cells are activated in clusters. High stem cell memory subsets were observed with DMS activation, especially in a full suspension environment. In conclusion, DMS has the potential to be a novel activator to be used in a T cell manufacturing process to produce a large number of highly potent T cell therapies.
Further experiments can include time course cell and media monitoring in the bioreactor to understand whether there are metabolic differences in the cells being expanded in different conditions and if there are any cell or media signatures that we can identify early on to enable predictions of T cell expansion outputs.
Acknowledgements (Optional): : This research is funded by the Marcus Foundation through the Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M), Cell Manufacturing Technologies (CMaT), and GTRI Independent Research and Development (IRAD).