Scaling up the manufacture of Tumor Associated Antigen-specific T cells (TAA-T)

Abstract

Background & Aim Adoptive immunotherapy with ex vivo expanded TAA-T has shown promise for patients with various cancers. However, current manufacturing processes limit the cell yield (median of 1.85 × 108 for 56 products manufactured at CNMC's GMP facility), and potential clinical applications to this investigational therapy. The goal of this study was to demonstrate the potential to “scale up” the manufacture of TAA-T to increase cell yield whilst preserving product phenotype and maintaining/enhancing potency. Methods, Results & Conclusion Mononuclear cells (MNCs) from apheresis products were isolated by Ficoll and cryopreserved. MNCs were plated in plates or flasks and adherent cells were then cultured with GM-CSF and IL-4 and matured with a cytokine cocktail plus LPS. Dendritic cells (DC) were harvested, pulsed with overlapping TAA peptides of the proteins WT1, PRAME, and Survivin, irradiated, and co-cultured with non-adherent cells or MNCs. T cells were expanded and re-stimulated with peptide pulsed DCs and PHA blasts. The standard and scaled up processes are described in Table 1. DC yields were higher for the scaled-up process compared to the standard process (mean 11.3% vs 6.6%) while expression of DC markers (CD80/83/86) was similar. Fold expansion of T cells after 2 stimulations was lower when using the standard manufacturing method in plates (mean 4: range 2.2-4.9) when compared to the G-Rex100 (mean 12, range 3.1-19.3) but comparable for the 3rd stimulation in G-Rex10 (mean of 11.5, range 5.8-18.8) and G-Rex100 (mean of 12.3, range 6-18.6). Theoretical yields of TAA-T final products were increased with the scaled-up process (mean 1.4 × 1011 cells, range 21-260 × 109 cells) compared to the standard process (mean 3.9 × 109 cells, 0.8-111 × 108 cells). TAA-T were similar when comparing cell phenotype and average cell populations (standard versus scaled-up) were: 8% versus 16% CD4+, 48% versus 40% CD8+, 25% versus 27% NK cells, 0% B cells and DCs for both groups. T cells manufactured by the scaled-up process had a mean of 108 spots/105 cells (SFC) for WT1 (range 14-228), 245 SFC for PRAME (range 63-552) and 0 for Survivin after 2 stimulations by Elispot IFNγ, whereas one paired product manufactured with the standard process showed no specificity. In conclusion, our scaled-up process to manufacture TAA-T showed similar or improved product characteristics compared to the standard process, allowing for the expansion to >20 × 109 TAA-T, therefore enabling patients to be treated with higher and/or multiple doses.