Rapid Generation of Antigen-Specific T Cells for Pre-Clinical and Clinical Applications Using a Novel Mini Cell Bioreactor

Abstract

Although flasks, bags, or rocking bioreactors can readily expand T lymphocytes after non-specific stimulation, the requirements for antigen-driven expansion of cytotoxic T lymphocytes (CTLs) are more rigorous. Antigen-specific T cells proliferate optimally only in the 2 mL wells of 24-well plates and cannot reproducibly be adapted to growth in flasks or bags. Hence, preparation of antigen-specific T cells for adoptive immunotherapy of malignancies is extremely time-consuming, requiring between 4wks and 3mths to produce sufficient cells for therapeutic purposes, and expensive (media + plastics + cytokines + man hours). The extensive manipulation required during the culturing process increases the risk of contamination. In combination, these problems obstruct the broader clinical application of antigen-specific T cells. Antigen-specific T cell growth is limited by gas exchange, nutrients and waste buildup. Bioreactors developed to provide these requirements tend to be complex, involving mechanical rocking or stirring and continuous perfusion, which increases the expense of the procedure and limits the number of products to the number of mechanical devices that can be housed and maintained. We have now explored the use of a new static mini Cell Bioreactor for antigen-specific T cell expansion. This device is essentially a flask with a gas permeable membrane supported by a plastic lattice as its base. The O2/CO2 exchange from the base allows large volumes of media to be added thereby reducing nutrient limitations and waste build-up, and consequently the manipulation required to sustain cell expansion. We tested two different sizes of Cell Bioreactor, 10 cm2 and 100 cm2 that hold a maximum of 40mL and 2000mL of media, respectively. We were able to generate and expand Epstein-Barr virus antigen-specific cytotoxic T lymphocytes (EBVCTLs) from normal donors by coculturing antigen presenting cells (APC) (1.4E+05 × cm2) with established EBV-CTL (4.3E+03 × cm2) at an optimized cell density and stimulator: responder ratio (32:1). These culture conditions induced accelerated CTL expansion (42.5 fold ±14.8 vs 3.4 fold ±1.2 within 7 days) without media change. Manipulation was restricted to cytokine addition every 3–4 days and to LCL stimulation on a weekly basis. A single 100cm2 bioreactor could produce up to 800E+06 antigen-specific T cells, which would have required approximately 320 wells in 24 well plates (>13 plates) under standard culture conditions. The CD4:CD8 T cell ratio and phenotype of the Cell Bioreactor-expanded CTLs was similar to those expanded using the conventional method (CD27 48% vs 52.4%, CD28 65.2% vs 62.2%, CD62L 53.15% vs 54.5%, CD45RO 58.1% vs 55.7%, and CD45RA 51.1% vs 54.9%). Antigen specificity, as evaluated by tetramer analysis and IFN-g ELIspot assay demonstrated no significant differences between CTL expanded by each process. Finally, cytolytic function was confirmed using a standard chromium release assay where both sources of CTL had high specific killing of the autologous EBV-transformed LCL targets (85%±12% vs 77%±19%) and minimal killing of allogeneic targets (22%±9% vs 15%±12).In summary, we have successfully utilized the new mini Cell Bioreactor technology to induce optimal in vitro antigen-specific T cell expansion with minimal handling. Future work will evaluate the impact of the accelerated expansion on differentiation and memory markers. This new system is suited to the clinical grade expansion of other cell types including suspension cell lines, and mitogen-activated T cells, as well as T cell blasts engrafted with chimeric antigen receptors.