Discussion
Autologous cell therapy would benefit from a cost-effective, streamlined
manufacturing process that yields cell numbers relevant for solid-tumor
indications with high transgene expression and the desired T cell
phenotype. The raw materials and the complexities of unit operations
employed by typical bioprocesses, particularly the costly antibodies for
magnetic cell selection and high number of touchpoints requiring skilled
labor, drive up COGM(ten Ham et al., 2020). The closed autologous T cell
bioprocess described here demonstrates that eliminating T cell
enrichment by starting with washed leuko-apheresis material not only
reduced COGM without compromising EOP CD3+ T cell purity, but also
increased transduction efficiency. Expansion conditions including high
seeding cell density (7e6 cells/mL), volume, rocking agitation, and low
glycolytic environment (inhibiting glycolysis using 2-DG at 5mM) in the
bioreactor also positively regulated TCR expression and T cell doubling
time.
T cell manufacturing processes involving open steps require cells to be
manually transferred between culture wares or fed in a biosafety cabinet
within a Grade B cleanroom(Dietz, Padley, & Gastineau, 2007),
increasing the risk of cross-contamination or microbial contamination
and negatively affecting the efficiency and robustness of T cell
manufacturing due to engineering control constraints. The only
commercially available all-in-one T cell processing unit, the CliniMACS
Prodigy (Miltenyi Biotec), is also limited by its chamber size,
scale-out ability and fixed operation programs. Presented here is a
fully closed T cell bioprocessing system utilizing single-use disposable
transfer bags for raw material transfer and a single bioreactor for
flexible and efficient T cell activation, transduction, and expansion.
This approach not only reduces the end-to-end processing time and
enhances key critical quality attributes of engineered T cells, it
increases manufacturing efficiency by enabling processing of multiple
lots in parallel within a Grade C cleanroom.
The critical quality attributes (CQA) of commercial autologous T cell
therapy include identity, viability, purity, % transgene positive,
potency, absence of impurities and sterility(Aijaz et al., 2018;
Lipsitz, Timmins, & Zandstra, 2016). To develop and control an
autologous bioprocessing system that meets these specifications while
accommodating the inherent donor variabilities requires a delicately
designed workflow guided by quality-by-design principles(Lipsitz et al.,
2016). The integrated operation developed here contrasted with
representative CAR-T cell bioprocess where T cells are enriched from
leukopak, activated, and transduced in gas permeable bags, and
transferred in a bioreactor for expansion, as depicted inSupplemental Fig. 2A . We have found that an integrated
operation resulted in superior critical quality attributes of engineered
T cells, including TCR-T cell yield, TCR expression, % memory subset,
and cytotoxicity (Supplemental Fig. 2B-I ), during a study using
a same donor leukopak. This enhancement of COA is possibly attributed to
collective factors from soluble activators and non-T cell population,
such as B cells and platelets, and bioreactor parameters(Bieback,
Fernandez-Munoz, Pati, & Schafer, 2019; Canestrari, Steidinger,
McSwain, Charlebois, & Dann, 2019; Chan & Shlomchik, 2000; Deola et
al., 2008). Indeed, we found that activation plays a vital role in
transduction efficiency and that ImmunoCult CD3/28/2 (StemCell
Technologies) dramatically increased TCR expression compared to TransAct
(Miltenyi Biotech) at the same MOI (Supplemental Fig. 1F) .
Stimulation drives differentiation of naïve T cell lymphocytes to
effector cells and subjects them to activation-mediated apoptosis(Gett
et al., 2003). The fitness of engineered T cells correlates with their
therapeutic efficacy and long-term persistence post-transplantation(Gett
et al., 2003). It also depends on the cellular characteristics that the
manufacturing bioprocess cultivates, mainly through the cellular signals
that activation antigen and cytokines trigger and the metabolic and
mechanobiological environment of the bioreactor(Franco, Jaccard, Romero,
Yu, & Ho, 2020; Rushdi et al., 2020; Scharping et al., 2021; Schluns &
Lefrançois, 2003). Consistent with findings that mTOR-driven anabolic
growth drives T cell differentiation from a naïve and memory-like state
to effector mode after activation(Huang, Long, Zhou, Chapman, & Chi,
2020), we found that limiting glucose, the major carbon source of
glycolysis, by using the glucose analog 2-DG effectively attenuated the
differentiation of naïve T cells into Tem and preserved Tcm during
expansion. 2-DG does not affect glycolysis as a single event but may
result in a holistic starvation signal that leads to an overall lowered
energetic profile(Sukumar et al., 2013). The slight increase in TCR
expression that accompanied the enhanced memory phenotype may be a
result of redirection of energetic metabolites from mitochondrial
oxidation to biosynthesis. After antigen activation, both endogenous and
exogenous IL-2 is engaged and further drive the antigen response while
regulating pro-survival molecules, such as B cell lymphoma-2 (Bcl-2),
until metabolic homeostasis is reached(Gett et al., 2003; Schluns &
Lefrançois, 2003). In turn, the strength of activation signals have a
profound impact on T cell fitness related to persistence(Gett et al.,
2003; Schluns & Lefrançois, 2003). Increased activation signals lead to
sustained activation post-production and could elicit life-threatening
CRS(Hay, 2018; Obstfeld et al., 2017). Here it is shown that IL-2
withdrawal from feed medium coupled with semi-continuous perfusion
resulted in a gradual decline of the activation state of T cells to less
than 5% without expanding the population of FoxP3+ Tregs (data not
shown). The low activation state of harvested T cells correlated with
reduced non-specific killing of T2 cells without affecting their
specific killing of target cells. More importantly, the lowered
activation signals via IL-2 withdrawal enhanced metabolic fitness of T
cells indicated by less polarized mitochondria and mitochondrial mass
when in effector mode(Sukumar et al., 2016). Taken together, these
results suggest that fine tuning activation signals during T cell
manufacturing can impact the phenotypic characteristics of TCR-T cells.
These optimizations enabled a robust manufacturing bioprocess that
generated a high yield of quality TCR-T cells with a memory phenotype
that has been shown to correlate with increased clinical efficacy while
also reducing COGM and eliminating many of the manual touchpoints
necessary in typical autologous manufacturing processes.