Designing a high throughput multi-parameter assay for process development of antigen specific T cell therapy products

Abstract

Background & Aim Infusion of viral T cells(VST) can be an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches have become more rapid but are based on a few select growth conditions that have not been fully optimized. We designed a comprehensive high throughput flow cytometry-based assay for process development(PD) of VSTs that can evaluate all basic T cell characterization within one 96 well plate, measuring viability, function, growth, and differentiation in up to 40 different cytokine combinations. Methods, Results & Conclusion Peripheral blood mononuclear cells (PBMC) from consenting donors (n= 6) were seeded in individual wells with 105 cells, viral peptides (IE-1 and pp65 from CMV), and combinations of cytokines, including IL15, IL6, IL21, IFNα, IL12, IL18, IL4, and IL7. Cells were grown over a period of 10 days and subsequently stained with a 13 color flow cytometry panel to evaluate cell count, viability, expression of standard lymphocyte phenotype and memory markers, IFNγ and TNFα (function), and CD107a (killing). Combinations of IL15/IL6 and IL4/IL7 were the optimal combinations for expansion of viral specific CD3+ T cells, with CD3+ cells cultured in IL15/IL6 growing by 18-fold vs. 14-fold for cells cultured in IL4/IL7 (the standard approach for manufacture) which were both compared with media alone controls. CD8+ T cells expanded by 24-fold in IL15/IL6 containing media over the media alone control which was significantly more than when cells were cultured in the presence of IL4/IL7 (9-fold) (p