Establishing a fully closed bioprocess to generate autologous
TCR-T cells
To streamline our TCR-T autologous bioprocess and lower COGM, we
identified the costliest materials and evaluated if they were necessary
for generating a high yield of transgene positive T cells. Published T
cell bioprocesses enrich T cells from the leukopak prior to
activation5, 9,10, however the CD4 and CD8 magnetic
beads and single-use kit used for selection make up a significant
portion of the raw material costs of manufacturing. We demonstrated that
eliminating the T cell enrichment step resulted in comparable T cell
purity following expansion to T cells enriched from a CliniMACS Plus at
day8 (Fig 1B & Supplemental Fig. 1D) . Unexpectedly, these
leuko-apheresed cells also had higher transduction efficiency compared
to T cells enriched from CD4+ and CD8+ positive selection independent of
whether the transgene was a CAR, GFP or TCR when tested with three
different donors in small-scale studies (Fig. 1A) .
Many established bioprocesses involve open steps where cells are moved
between culture vessels for T cell activation and transduction, mainly
due to the use of plate-bound activators and transduction enhancers.
Some processes begin expansion in static vessels or bags before
transferring to a rocking bioreactor(Eyles et al., 2019; Highfill &
Stroncek, 2019). We hypothesized that these separated unit operations
and associated open steps adversely affect process performance by
subjecting T cells to an unfavorable environment such as deprivation of
heterogeneous cell contact, cytokines, and insufficient/timely medium
exchange due to lack of perfusion(Chan & Shlomchik, 2000; Deola et al.,
2008). We envisioned integrating these steps into one single bioreactor
to simplify the workflow and aid in the efficiency of cell engineering
and expansion. To enable this, a soluble activator was used to eliminate
the need to pre-coat bags. Through optimization of process parameters,
we demonstrated that adding ImmunoCult CD3/28/2 to leuko-apheresed cells
in a semi-static bioreactor with rocking speed and angle at 2 rpm x 2°
is effective in enabling T cell activation (Supplemental Fig.
1A) . Cost of lentiviral vector limits the number of cells that can be
transduced, and transductions with a multiplicity of infection (MOI) of
5 or higher must be performed in a gas permeable bag due to the low
volume necessary for 1-2e8 cells at an optimal cell density. We found a
MOI of 1 was efficient for transduction of multiple TCRs(Supplemental Fig. 1B ). Due to the low amount of lentivirus
needed for efficient transduction, we concluded that it is feasible to
transduce roughly 7e8 cells in the bioreactor on day1. Together, these
findings enabled an integrated workflow for TCR-T cell production by
incorporating cell activation, transduction, and expansion in a single
bioreactor immediately after the leukopak wash step using the Sepax Pro
(Fig. 2 ).
This fully closed bioprocess yields an average of 10e9 T cells from
~1e9 leukopak cells as starting material within 7-8
days, and a peak of 20-30e9 cells can be reached in 10 days(Fig. 1C) . Viability dips midway through the process due to
non-T cell death, but rebounds to >97% by day6 of
expansion (Fig. 1D & Supplemental Fig. 1C) . Variation is
observed in growth curve and in-process viability due to donor-to-donor
variability, however, end-of-process viability is consistently above
90% (Fig. 1D) . Semi-continuous perfusion ensures timely
replenishment of nutrients, removal of undesired metabolites and cell
debris and maintains glucose levels above 2g/L and lactate levels below
2 g/L, respectively (Fig. 1E & 1F) . Cells harvested at the end
of process (EOP) showed TCR expression of CD8+ T cells detected by MHC
dextramer, ranging from 30% to 70% with mean expression at 55%(Fig. 1G) . Mean TCR expression of CD3+ T cells was 30%,
however, due to MHC class I tetramer being only able to detect TCR
expression on CD8+ cells (Fig. 1G) . Staining with a Vbeta
antibody Vβ13.2 targeting the TCR β chain confirmed that CD4+ cells are
transduced, but CD4+ TCR expression is undetectable by dextramer
staining (Supplemental Fig. 1C) . 97% purity of CD3+ T cells
was achieved at EOP regardless of CD3+ percentage of leukopak starting
material (20-80%) (Fig. 1H) . Harvested TCR-T cells showed
potent and specific cytotoxicity against MAGE-B2 peptide loaded T2-Luc
cells at E:T ratio of 1 after 24 hours during in vitro functional
assays (Fig. 1I) .