Generation of autologous MAGE-B2-TCR-T cells in a fully closed
bioprocess
On day0, the fresh leukopak apheresis is washed and concentrated to
150e6 cells/mL in complete culture media using Sepax C-Pro cell
processing system (Cytiva) using the program CultureWash-Pro with the
CT600 kit. Immediately after sampling and determining the viable cell
density (VCD), cells are diluted to 1.2e9 nucleated cells and incubated
with ImmunoCult CD3/28/2 activator (StemCell Technologies) at a density
of 75e6 cells/mL using the Dilution program on the Sepax C-Pro. After a
1-hour incubation with the activator in a transfer bag at room
temperature, the cells are inoculated into a semi-static Xuri W25
bioreactor (Cytiva) pre-equilibrated at 37°C, 5% CO2, 0.1 L/min gas
flow rate, and 2 rpm at a 2° angle rocking agitation at seeding density
of 4e6 cells/mL with 300 mL complete culture media (CTS OpTmizer T Cell
Expansion SFM (Gibco) with 2.5% CTS Immune Cell SR (Gibco) and 300
IU/mL IL-2 (STEMCELL Technologies)). On day1, lentivirus for
MAGE-B2-speficic TCR corresponding to MOI = 1 functional titer is
transferred into the bioreactor through the feed port via gravity flow
after sampling and determining VCD. Until the volume within the Xuri
exceeds 300 mL, 150 mL medium is exchanged per day using semi-continuous
perfusion to maintain glucose and lactate levels above 2g/L and below
2g/L, respectively. Bioreactor volume is increased to 500 mL or 1 L to
maintain cell density at 4e6 cells/mL and above with corresponding
rocking agitations and semi-continuous perfusion rates until harvest(Supplemental Fig. 1G) . Once cells have expanded to reach the
desired yield (10-20e9), cells are harvested and washed with 0.9%
saline (Baxter) supplemented with 0.1% v/v human serum albumin
(InVitroCare) (HSA) using the Sefia S-2000 Cell Processing System
(Cytiva) under FlexCell program and CT-800.1 Cell Processing kit. Using
the same system, TCR-T cells are concentrated to cell density of
100-200e6 cells/mL into two cryobags (Origen Biomedical) and formulated
with 50% HyClone cryopreservation media (Cytiva) in 0.9% saline with a
final HSA concentration of 0.5% v/v. The cryobags are then frozen in a
rate-controlled freezer (VIA Freeze Quad freezer (Cytiva)) at a cooling
rate of -1°C/min until the temperature reaches -80°C. Cryobags are then
transferred to liquid nitrogen (-140°C) for long term storage.