Figure Legends
Fig. 1 . Process and final T cell product characterization of a closed autologous bioprocess. Transduction efficiency of lentivirus coding for CAR, TCR, and GFP measured at day8 (A). T cell purity of leukapheresis at start and following activation and expansion compared to T cell purity following CD4 and CD8 antibody positive enrichment (B). In-process measurements of cell growth (C), viability (D), glucose (GLC & E), lactate (LAC & F). End-of-process TCR expression of CD8 and CD3 T cells detected by PE-dextramer (G). Donor leukopak and EOP CD3 T cell purity (H) and cytotoxicity against MAGE-B2 target cells (I).
Fig. 2 . Fully closed end-to-end bioprocessing system generating TCR-T cell for autologous T cell therapy. Illustration of autologous bioprocess, including washing and activating leukopak apheresis, lentiviral vector transduction and cell expansion in a bioreactor, harvesting and formulating final TCR-T cell product into on desired cell concentration, and finally cryopreservation.
Fig. 3 . Optimization of bioreactor parameters to improve lentiviral vector transduction efficiency and end-to-end process time. In-process cell growth (A), viability (B), CD3 cells percentage (C), TCR expression (D), secretion of IFN-gamma after coculturing with MAGE-B2 peptide loaded T2 cells (E), cytotoxicity against T2 cells pulsed with MAGE-B2 peptide (H), and the expression of CD45RA and CCR7 of CD8+ cells to indicate Tscm, Tcm, and Tem (G).
Fig. 4 . Enrichment of memory T cell subset in final TCR-T cell product via glycolysis inhibition. Glycolysis inhibitor 2-DG at 2mM was added in culture media starting from inoculation until day6 or day9. In-process cell growth (A) was monitored. CD8 T cell differentiation was compared for their percentage of Tscm, Tcm, and Tem subsets in-process at day4 and at harvest for two presentative donors (B). EOP TCR-T cells of CD8+ cells were shown with two representative donors (C). MMP (D), and mitochondrial mass (E) at basal and activated state, and cytotoxicity against MAGE-B2 peptide (+) and (-) T2 cells (F).
Fig. 5. IL2 withdraw from culture medium at day5 or day6 reduced background activation of EOP TCR-T cells with improved metabolic fitness. Impact of IL-2 level on CD25 expression (A), cytotoxicity of TCR-T cells against MAGE-B2 peptide loaded and no peptide loaded T2 Luc cells at E:T ratio 0.2, 1, and 5 (B), EOP TCR expression detected by PE-dextramer (C), levels of mitochondrial membrane potential (D), annexin (F), and ROS (E) of TCR-T cells.
Supplemental Fig. 1. Expression of CD25 of CD8+ T cells following ImmunoCult CD3/28/2 activation in Xuri W25 bioreactor (A). TCR expression at MOI from 0.25 to 10 (B). Vbeta vs Dextramer expression specific to MAGEB2 TCR of CD8 and CD4 cells at harvest (C). Representative percentage of B cells, monocytes, and NK cells of leukocytes following activation in Xuri W25 bioreactor (D). CD8 T cell differentiation as a function of cell growth (E). TCR expression of CD8+ T cells activated by ImmunoCult CD3/28/2 activator and TransAct at MOI of 1 (F). Rocking agitation parameters optimized at different volumes (G).
Supplemental Fig. 2 . A leukopak was split and processed through either the integrated bioprocess developed in current study or a representative autologous bioprocess. In the integrated and fully closed bioprocess, leukopak cells were washed, and 1.2e9 cells were directly activated with ImmunoCult CD3/28/2 and transduced in a 2L Xuri W25 bioreactor, as described in Fig. 2 and A . In the presentative and semi-closed bioprocess, the remaining leukopak from the same donor was enriched for T cells using CD4 and CD8 antibody positive selection on the CliniMACS Plus (Miltenyi Biotec) and 0.5e9 T cells were activated in PL240-2G PermaLife Cell Culture Bag (OriGen Biomedical) with plate-bound CD3 and soluble CD28 antibodies (Miltenyi Biotec) at day0. Cells were transduced at MOI of 1 in both conditions on day1. On day2, T cells activated and transduced in PL240-2G PermaLife Cell Culture Bag were transferred into a Xuri W25 bioreactor, scaled up from 500ml to 1L on day3, followed by semi-continuous perfusion at 500ml -1L/day until harvest (Fig.3A) . In-process cell growth (B), CD25 expression (C), viability (D), percentage of CD3 positivity (E), TCR expression were monitored (F). FACS analysis of CD8+ T cell differentiation when cell yield was at 8e9(G). Cytotoxicity against MAGE-B2 peptide pulsed T2 Luc cells at E:T ratio of 1:1 and 1:5 (H). IFN-gamma secretion of TCR-T cells cocultured with MAGE-B2 peptide pulsed T2 cells at 10nM, 1uM, 0uM concentrations (I).