402. Bioengineering of Peripheral Blood Derived Gamma Delta T Cells in a Serum-Free Expansion Medium
Authors: Kathryn Sutton, Anindya Dasgupta, McCarty David, Christopher Doering, H. Trent Spencer
Journal: Molecular Therapy (2016)

To date the main source of effector cells in preclinical and clinical immunotherapy trials has been αβ T cells. Although cellular products utilizing these cells have shown some promise, there is a need for better control of antitumor immunogenicity, especially in the context of chimeric antigen receptor technologies. Therefore, as an alternative, we are investigating the use of a T cell subset, γΔ T cells. These are a potentially attractive cellular therapeutic as they can be expanded to clinically useful levels and have potent MHC independent intrinsic anti-tumorigenicity driven by their recognition of stress antigens on tumor cells. Our efforts have focused on development of a clinically reliable expansion and manufacturing process of genetically engineered γΔ T cells. In the current study we describe the optimization of culture conditions in serum free media (SFM) that can support robust expansion followed by genetic modification of the expanded cells. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2 mM L-glutamine, zoledronic acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 lU/ml added twice weekly). Of the SFM cultures, robust γΔ T cell expansion was only observed in OpTmizer supplemented with high-dose IL-2, which resulted in expansion and γΔ T cell percentages to levels sufficient for clinical use. Specifically, in OpTmizer, the percentage of γΔ T cells increased from a mean of 2.4% (0.8%-3.6%) in the starting product to 66.4% (35%-94.5%, 6 of 9 cultures were >70% γΔ T cell), resulting in 80-fold expansion at 14 days. The SFM-expanded γΔ T cells were evaluated for their anti-tumor cytotoxicity and shown to be equally effective at killing both hematologic and solid cancer cell lines as γΔ T cells expanded in serum containing media. Transduction efficiency was tested using GFP encoding self-inactivating lentiviral vectors (LV). These studies demonstrated that SIV and HIV transduce expanded γΔ T cells equally, and that compared to EF1α, cassettes employing an MSCV promoter provide greater transgene expression. The timing of LV addition was optimized with respect to i) γΔ T cell expansion, ii) LDL Receptor (LDL-R) expression, and iii) LV dose (3-4 consecutive transductions over 48 hours). The greatest transduction efficiencies were achieved in cultures with the greatest expansion. As expected a range of transduction efficiencies was observed for various donors with a mean of approximately 20%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and rates of expansion. Therefore, an optimized strategy to expand and transduce γΔ T cells with demonstrated anti-cancer properties was developed. The utilization of SFM to expand these cells to clinically useful doses and redirection toward tumor cells via bioengineering can result in a clinically safe, widely applicable, and potentially more efficacious cellular immunotherapy.