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).