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\begin{document}

\title{A closed, autologous bioprocess optimized for TCR-T cell therapies}



\author[1]{Jennitte Stevens}%
\author[1]{Yijun Liu}%
\author[1]{Eugenia Zah}%
\author[1]{Carlos Arbelaez}%
\author[1]{Haejin Kim}%
\author[1]{Edwige Gros}%
\author[1]{Ranelle Buck}%
\author[1]{Kayley Cox}%
\author[2]{Sungeun Kim}%
\author[1]{Martina Kopp}%
\author[1]{Kathryn Henckels}%
\affil[1]{Amgen Inc}%
\affil[2]{Amgen Inc San Francisco}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Autologous cell therapy has proven to be an effective treatment for
hematological malignancies. Cell therapies for solid tumors are on the
horizon, however the high cost and complexity of manufacturing these
therapies remain a challenge. Routinely used open steps to transfer
cells and reagents through unit operations further burden the workflow
reducing efficiency and increasing the chance for human error. Here we
describe a fully closed, autologous bioprocess generating MAGE-B2 TCR-T
cells. This bioprocess yielded 5 -- 12e9 MAGE-B2-specific TCR-expressing
T cells, transduced at low MOIs, within 7 to 10 days, and cells
exhibited an enriched memory T cell phenotype and enhanced metabolic
fitness. It was demonstrated that activating, transducing, and expanding
leuko-apheresed cells in a single bioreactor without a T cell enrichment
step promoted lentivirus transduction efficiency while resulting in
comparable level of T cell purity (\textasciitilde{}97\%) as that of
leukopak cells that went through CD8+ and CD4+ positive selection. The
critical process parameters of the bioreactor, including culturing at a
high cell density (7e6 cells/mL), adjusting rocking agitations during
phases of scale up, lowering glycolysis through addition of
2-Deoxy-D-glucose (2-DG), and modulating IL-2 levels, were shown to
positively regulate TCR expression and cell doubling time, and promote
resistance to effector-associated apoptosis of TCR-T cells. The
bioprocess described herein supports scale-out feasibility by enabling
processing of multiple patients' batches in parallel within a Grade C
cleanroom.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Autologous T cell therapy has proven to be an effective treatment for
hematological malignancies, and there are currently six FDA approved
CAR-T therapies for the treatment of leukemia, lymphoma, and multiple
myloma (Albinger, Hartmann, \& Ullrich, 2021; Costa et al., 2021; Ma et
al., 2019; Munshi et al., 2020). Developing effective therapeutics
against solid tumors represent an unmet need, and there are several
ongoing clinical trials using CAR-T or TCR-T cell therapies for solid
malignancies (Morotti et al., 2021). As promising as these personalized
T cell therapies are, manufacturing them is challenging due to the high
cost of goods manufactured (COGM), inability to scale, complex supply
chain logistics, long end-to-end time of bioprocessing plus release
testing, and limited patient access. Treating solid tumors presents
further challenges; not only must the dose be higher but the engineered
T cells must be equipped to penetrate and function within the tumor
microenvironment. Doses for solid tumors reach into the 100e8/CAR+ or
TCR+/kg cells(Mirzaei, Rodriguez, Shepphird, Brown, \& Badie, 2017),
therefore a higher yield is required from the manufacturing process.
Additionally, producing cells with characteristics that will enable them
to home to the tumor site and attack cells expressing the tumor antigen
is necessary for an effective treatment.

Typical manufacturing processes for cell therapy rely on isolating white
blood cells from the patient through leukophoresis, followed by
enriching T cells from a leukopak using magnetic separation, activating
the cells with CD3 and CD28 activators, transducing with a viral vector
carrying the gene of interest in either static flasks or gas permeable
bags and expanding the T cells in a static vessel or rocking
bioreactor(Eyles et al., 2019). Many of these steps are open, requiring
the technician to handle the patient's cells inside a biosafety cabinet
and use pipettes or syringes to transfer cells into vessels. Repeated
physical manipulation of the cells causes sheer stress and can
negatively impact cell viability, phenotype and overall fitness(Fowell
\& Kim, 2021; Mastrogiovanni, Juzans, Alcover, \& Di Bartolo, 2020). Due
to the high risk of contamination and inability to sterilize the final
drug product, these processes must be performed in an ISO 7 clean room.
It is also recommended that each patient have a dedicated incubator
which increases the manufacturing footprint(Sekiya et al., 2012).
Furthermore, each manufacturing step requires costly single-use
consumables, increasing COGM and impacting the environmental
sustainability as patient numbers increase.

We have developed a fully closed bioprocess for autologous T cell
therapies that eliminates the need for syringes, gas permeabilized bags,
and static incubators. Through integrating unit operations including
activation, transduction of leuko-apheresed cells, and cell expansion in
a rocking bioreactor, we achieved high transgene expression and desired
T cell yield sufficient for dose escalation studies for solid tumor
indications. This bioprocess provides a new approach for TCR-T cell
manufacturing resulting in a significant reduction in COGM compared to
the state-of-the-art T cell bioprocesses.

\textbf{Results}

\textbf{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
activation\textsuperscript{5, 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 \textbf{(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 \textbf{(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\selectlanguage{ngerman}°
is effective in enabling T cell activation \textbf{(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\textbf{(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 (\textbf{Fig. 2} ).

This fully closed bioprocess yields an average of 10e9 T cells from
\textasciitilde{}1e9 leukopak cells as starting material within 7-8
days, and a peak of 20-30e9 cells can be reached in 10 days\textbf{(Fig.
1C)} . Viability dips midway through the process due to non-T cell
death, but rebounds to \textgreater{}97\% by day6 of expansion
\textbf{(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\%
\textbf{(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 \textbf{(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\%\textbf{(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 \textbf{(Fig. 1G)} . Staining with a Vbeta
antibody V\selectlanguage{greek}β\selectlanguage{english}13.2 targeting the TCR \selectlanguage{greek}β \selectlanguage{english}chain confirmed that CD4+ cells are
transduced, but CD4+ TCR expression is undetectable by dextramer
staining \textbf{(Supplemental Fig. 1C)} . 97\% purity of CD3+ T cells
was achieved at EOP regardless of CD3+ percentage of leukopak starting
material (20-80\%) \textbf{(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 \emph{in vitro} functional
assays \textbf{(Fig. 1I)} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Optimizing Xuri W25 bioreactor parameters}

To further shorten the end-to-end process time, we evaluated whether
several bioreactor parameters, including seeding density, dissolved
oxygen (DO) level and rocking agitation, could accelerate cell growth.
The same donor apheresis was split into five tested bioreactor
conditions listed in \textbf{Table 1} .

Xuri\#1, the standard condition where cells were seeded at lower density
(3.5e6 cells/ml) with lower agitation (3 rpm x 3\selectlanguage{ngerman}°), yielded \textless{}
4e9 engineered T cells at day9 while Xuri\#2, Xuri\#3, and Xuri\#5 all
significantly promoted cell growth to around 10e9 cells through
increasing rocking agitation or gassing oxygen directly (DO feedback
loop control) to promote a greater level of oxygen transfer\textbf{(Fig.
3A)} . Moreover, Xuri\#2, Xuri\#3, and Xuri\#5 with increased rocking
speed and angle demonstrated that enhancing agitation also improved
in-process cell viability from day6 to day9 compared to Xuri\#1
\textbf{(Fig. 3B)} . In addition, shortening the bioprocess from 11 days
to nine days did not adversely affect T cell purity, as comparable \%
CD3 T cells were found for all conditions upon harvest\textbf{(Fig. 3C)}
.

While enhanced oxygen mixing through increasing agitation or direct
oxygen input promoted cell growth, those two approaches contrasted in
their impact on transduction efficiency. Having direct and steady oxygen
delivery through DO feedback loop control from the start (Xuri\#3)
compromised transduction efficiency and resulted in the lowest TCR
surface expression \textbf{(Fig. 3D)} . In contrast, DO feedback loop
control at a later stage when the bioreactor was fully scaled up
(Xuri\#5) or enhancing oxygen mixing through rocking agitation at 500 mL
volume (Xuri\#2) led to TCR levels similar to standard conditions
(Xuri\#1) \textbf{(Fig. 3D)} . In addition, a higher inoculation density
at 7e6 cells/ml (Xuri\#4) led to a \textasciitilde{}15\% increase in
transduction efficiency compared with conditions at 3.4e6 cells/ml
(Xuri\#2) \textbf{(Fig. 3D)} . Correlating with higher TCR expression in
cells from Xuri\#4 and \% Tcm for cells from Xuri\#2, INF-\selectlanguage{english}g release was
also modestly enhanced compared with Xuri\#1 when co-cultured with
MAGE-B2 peptide loaded T2 cells for 24 hours at E:T ratio at 1
\textbf{(Fig. 3E)} . No distinct differences were found for \% T2 cells
lysis at 48 hours due to saturation of T2 cell lysis above 99\%
\textbf{(Fig. 3F)} .

T cell activation and expansion usually comes at the cost of
differentiation into effector phenotype (CD45RO+, CCR7-, CD95+) from
na\selectlanguage{ngerman}ïve (CD45RA+, CCR7-, CD95-), stem (CD45RA+, CCR7+, CD95+) and central
memory (CD45RO+, CCR7+, CD95+) pool, a phenomenon associated with a
shortened life span, T cell exhaustion and compromised therapeutic
persistence in the patient(Henning, Roychoudhuri, \& Restifo, 2018;
Wherry, 2011). Cells from Xuri\#2, Xuri\#3, and Xuri\#5, which had
greater oxygen/mass mixing compared to Xuri\#1, contained significantly
higher \% Tcm cells than cells from Xuri\#1 and Xuri\#4 when compared at
yield of 10e9 cells \textbf{(Fig. 3G)} . It is also notable that while
cells from \#Xuri4 had the highest TCR surface expression \textbf{(Fig.
3D)} among all the conditions tested, cell growth and \%Tcm was lower
than its counterpart conditions tested. On the other hand, while cells
from Xuri\#3 showed higher \% Tcm and enhanced cell growth, TCR surface
expression of those cells was 3-fold less than cells from
Xuri\#2\textbf{(Fig. 3D)} . This suggests that while oxygen promotes T
cell growth, it may negatively impact transduction when supplied in
excess during T cell activation. Overall, higher inoculation density and
greater rocking agitation adopted at 0.5 L culture volume enables a
shorter end-to-end bioprocess time with optimal T cell phenotype and
potency.

\textbf{Inhibiting glycolysis skews T cells towards a more memory
phenotype}

Upon activation, T cells undergo a metabolic switch from oxidation of
fatty acids to glycolysis to enable effector function(Chang \& Pearce,
2016; Edwards-Hicks, Mitterer, Pearce, \& Buescher, 2020). The switch
into effector function is critical for immune surveillance however is
undesirable during \emph{ex vivo} manufacturing due to the loss of
memory function of T cell and its association with reduced therapeutic
efficacy and persistence(Delgoffe et al., 2021; Eyles et al., 2019). We
hypothesized that limiting glucose intake could sustain the memory
phenotype of T cells and potentially lead to increased efficacy and
persistence. Cells were activated and expanded in the presence of a
glycolysis inhibitor, 2-DG, at 2mM on day0 until harvest on day9 or
gradually removed at day6(Pelicano, Martin, Xu, \& Huang, 2006) through
semi-continuous perfusion (1L/day) until harvest. No inhibition of cell
growth was observed in the presence of 2-DG independent of when 2-DG was
removed \textbf{(Fig. 4A)} . While cells still continuously
differentiated from Tn to Tscm and eventually enriched to a mixed pool
of Tcm and Tem, glycolysis inhibition by 2-DG noticeably increased the
\%Tscm and Tcm at different timepoints during expansion with data shown
for two representative donors. \textbf{(Fig. 4B)} . \% Tscm was higher
in conditions with 2-DG on day4 which led to 10\% to 20\% higher of \%
Tcm + Tscm on day 7 or day9 compared to the condition lacking
2-DG\textbf{(Fig. 4B)} . In addition to enriching total memory T cells,
glycolysis inhibition by 2-DG also increased TCR expression by 10\% at
time of harvest. \textbf{(Fig. 4C)} .

Mitochondrial membrane potential (MMP) and mitochondrial mass were also
quantified based on the mean fluorescent intensity (MFI) of
DilC\textsubscript{1}(5) and MitoTracker Red acquired by flow cytometry
to investigate whether glycolysis inhibition improved metabolic fitness
of TCR-T cells. After encountering target cells, TCR-T cells from all
conditions exhibited elevated mitochondrial metabolism as MMP became
more polarized and mitochondrial mass increased in order to sustain
energetic metabolism for effector function \textbf{(Fig. 4D \& 4E)} .
Interestingly, MMP and mitochondrial mass were less elevated for TCR-T
cells activated and expanded in the presence of 2-DG than those cultured
without 2-DG. TCR-T cells generated in the presence of 2-DG had similar
lysis capacity (E:T=1) \textbf{(Fig. 4F)} with lower MMP and
mitochondrial mass than cells cultured in the absence of 2-DG.
Thissuggests that 2-DG cells have a greater mitochondrial capacity and
fitness to meet the increased energy demand for effector function
(Sukumar et al., 2016; van der Windt et al., 2012).

\textbf{Removing IL-2 in-process promotes metabolic fitness and reduced
background killing}

Over-activation of T cells prior to transduction and expansion can lead
to pre-exhaustion of EOP TCR-T cells with compromised persistence and
potentially contribute to cytokine release syndrome (CRS) due to high
background reactivity(Alizadeh et al., 2019; Gett, Sallusto,
Lanzavecchia, \& Geginat, 2003; Schluns \& Lefrançois, 2003). We
hypothesized that in-process IL-2 withdrawal from complete culture media
together with semi-continuous perfusion would attenuate activation
signaling and generate T cells with lower background activity and
enhanced fitness. When evaluated with two donor samples, we found that
less than 5\% T cells were positive for CD25 upon harvest when IL-2 was
withdrawn on day5 or day6 while cells cultured with IL-2 until harvest
had 50-80\% CD25+ T cells \textbf{(Fig. 5A)} . The delay in CD25
downregulation post IL-2 withdrawal is expected with a semi-continuous
perfusion system where IL-2 containing media was slowly perfused out of
the bioreactor resulting a gradual decline in IL-2 concentration.
Importantly, cells with lower CD25 expression exhibited reduced
nonspecific killing while maintaining specific target cell lysis when
E:T ratio was increased \textbf{(Fig. 5B)} . In the two donors tested,
withdrawing IL-2 on day6 or day5 differentially affected TCR expression
compared to the control condition (with IL-2), although the differences
were not significant. \textbf{(Fig. 5C)} .

MMP, ROS, and apoptosis at both resting state and effector state against
target cells were measured based on fluorescent intensity (MFI) of
DilC\textsubscript{1}(5), CellROS Orange, and Annexin of viable T cells
using flow cytometry. When encountering target cells, MMP became more
polarized, and cells generated more ROS \textbf{(Fig. 5D \& 5E)} .
Spontaneous apoptosis associated with effector function measured by
annexin level was elevated upon target cell activation regardless of
IL-2 conditions \textbf{(Fig. 5F)} . However, T cells from the IL-2
withdrawal condition had lower levels of MMP and ROS at both resting
state and effector state compared to T cells that were supplemented with
IL-2 throughout the bioprocess \textbf{(Fig. 5D \& 5E)} .

\textbf{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
in\textbf{Supplemental 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 (\textbf{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 \textbf{(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.

\textbf{Methods}

\textbf{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\textbf{(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.

\textbf{Lentivirus vector construction and production}

DNA sequences encoding MAGE-B2-TCR alpha and beta chains separated by a
self-cleaving T2A peptide were cloned into the pALD-Lenti expression
vector (Aldevron) by isothermal assembly, with an EF1a promoter driving
TCR gene expression. A total of five MAGE-B2 TCRs were tested. DNA
sequences encoding CARs or GFP driven by a MSCV promoter were also
cloned into a lentiviral expression vector for evaluation. Lentivirus
was generated by transient transfection of HEK293 cells with third
generation LVV packaging plasmids (p-ALD-Lenti, Aldevron) and the
expression construct. Lentivirus was harvested from the supernatant 3
days post-transfection,concentrated using high-speed centrifugation,
formulated in a sucrose solution and stored at -80 degC(Gandara,
Affleck, \& Stoll, 2018). Infectious titers were determined by using a
flow cytometry cell-based assay on HEK293 and Jurkat cells(Kutner,
Zhang, \& Reiser, 2009).

\emph{In vitro} \textbf{T cell activation and lentivirus transduction
Grex vessels (WilsonWolf) and Permalife cell culture bags (Origen
Biomedical)}

T cells or leuko-apheresed cells were stimulated \emph{in vitro} with
ImmunoCult CD3/28/2 activator or Immunocult CD3/28 activator at 25 uL
per 1 mL of cells in complete culture media for 16-24 hours before
transduction. Prior to transduction, viable cell counts were determined
using NC-200. Amount of lentivirus at MOI=1 according to their
functional titer were used in all experiments except for the lentivirus
titration studies. 24 hours following transduction, culture media were
topped to 5 mL in small-scale Grex experiments.

To activate and transduce T cells in Permalife cell culture bags,
Permalife cell culture bags were coated with OKT CD3 antibody (Milteniyi
Biotech) overnight at 4°C prior to cell inoculation. Enriched T cells
were transferred to Permalife cell culture bags with soluble 15E8 CD28
antibody (Milteniyi Biotech) followed by transduction at MOI=1 24 hours
after activation. Cells were transferred into Xuri W25 bioreactor 1 day
after transduction for scale-up expansion.

\textbf{T cell enrichment}

Using Sepax C-pro, the fresh leukopak was washed and concentrated using
CliniMACS PBS/EDTA Buffer (Miltenyi Biotech) supplemented with 0.1\% HSA
and then incubated with antibodies specific to CD4 and CD8 (Miltenyi
Biotec) at room temperature for 30 minutes. Labeled leukopak cells were
then transferred on CliniMACS Plus (Miltenyi Biotech) and positive
fraction of flow through was collected according to manufacturers'
instructions.

\textbf{Flow cytometry immunophenotyping}

Human antibodies specific for CD3 (SK7, Biolegend), CD4 (SK3,
Biolegend), CD8 (RPA-T8, BD Biosciences), CD45RA (HI100, Biolegend),
CD45RO (UCHL1, Biolegend), CCR7 (REA108, Miltenyi Biotec), CD95 (DX2 ,
Biolegend) were used for low cytometry acquisition for the determination
of Tn, Tcm, Tem, Tte phenotype. 8 color immunophenotyping kit specific
for CD3, CD5/16, CD19, CD14, CD45 (Miltenyi Biotec) were used for flow
cytometry acquisition for the determination of T cell purity, B cell, NK
cells, NKT cells, and monocytes percentage. MAGE-B2 TCR dextramer
(Immudex) and anti-human TCR V\selectlanguage{greek}β\selectlanguage{english}13.2 (H132, Biolegend) were used to
detect MAGE-B2 TCR expression on CD8+ T cells and CD4 + T cells. FACS
Symphony or FACS Fortessa (BD Biosciences) was used for flow cytometry
acquisition. Samples were analyzed with FlowJo software.

\textbf{Reactive oxygen species (ROS), mitochondrial membrane potential
(MMP), mitochondrial mass, and Annexin}

For analysis of intracellular ROS, TCR-T cells were incubated with
CellROX Orange at 5 \selectlanguage{greek}µ\selectlanguage{english}M in complete culture media for 30 minutes at 37\selectlanguage{ngerman}°C.
For detection of effector TCR-T cells intracellular ROS level, TCR-T
cells were incubated with MAGE-B2 peptide-loaded T2 Luc Red cells at E:T
ratio=1 for 24 hours, cell mixtures were then incubated with CellROS
Orange at 5 uM in complete culture media for 30 minutes at 37°C. Cells
were then labeled with human antibody against CD3 and CD8 for 15 minutes
at 4°C, and ROS level was determined by flow cytometry analysis using
mean fluorescence intensity of CellROS Orange gated on CD8+ and CD3+
double positive cells.

Similarly, MMP and mitochondrial mass was measured by labeling cells
either by themselves or after co-cultured with T2 Luc Red cells at E:T
ratio=1 for 24 hours with DilC\textsubscript{1}(5) at 50 nM for 15
minutes at 37°C or MitoTracker Red CMXRos at 25 nM for 15 minutes at
37°C, respectively. Cells were then labeled with antibodies specific for
CD3+ and CD8+, followed by flow cytometry analysis.

For analysis of Annexin V, TCR-T cells or cell mixtures of TCR-T cell
and MAGE-B2 peptide-loaded T2 Luc Red cells after 24-hour co-incubation
were labeled with antibodies specific for CD4+ and CD8+ then washed and
labeled with FITC Annexin V in Annexin V Binding Buffer at 1e6 cells/ml
for 15 minutes at room temperature in the dark. Cells were diluted with
Annexin V Binding Buffer to 0.2e6 cells/mL and analyzed by flow
cytometry.

\textbf{T cell killing assay}

T2 cells engineered with luciferase reporter (Luc) were maintained in
IMDM supplemented with 2mM L-glu, 20\% FBS, 2 ug/mL Blasticidin, and
0.25 ug/mL Puromycin. T2 Luc cells were pulsed with MAGE-B2 peptide at
10 \selectlanguage{greek}µ\selectlanguage{english}M for 4 hours. Previously frozen TCR-T cells were thawed and mixed
with MAGE-B2 peptide-pulsed T2 Luc cells at various E:T ratio in CTS
OpTmizer T Cell Expansion SFM (Gibco) with 2.5\% CTS Immune Cell SR
(Gibco). Cell mixtures were incubated at 5\% CO\textsubscript{2} and
37\selectlanguage{ngerman}°C for 48 hours. Steady-Glo® Luciferase (Promega) was added to cell
mixture according to manufacturer's protocol. Luminescence was read
using EnVision plate reader (PerkimElmer) 5 minutes after reaction.

\selectlanguage{english}\textbf{Human Interferon-gamma (IFN-g)}

TCR-T cells were incubated with MAGE-B2 peptide-loaded T2 Luc cells at
various E:T ratios for 24 hours in CTS OpTmizer T Cell Expansion SFM
(Gibco) with 2.5\% CTS Immune Cell SR (Gibco) at 1e6 cells/mL.
Supernatant was collected and analyzed for IFN-g \selectlanguage{english}using AlphaLISA Human
Interferon-gamma Kit (PerkinElmer) according to manufacturer's manual.
Sample and standard data was collected using EnVision plate reader
(PerkimElmer).

\textbf{Metabolites, cell counts, and viability measurements}

Cell culture supernatant was collected and analyzed for glucose (Glc),
Lactate (Lac), pyruvate (Pyr), and lactate dehydrogenase (LDH) using
Cedex Bio Analyzer (Roche) according to manufacturer's instructions.
Cell counts and viability were collected using NucleoCounter\selectlanguage{ngerman}® NC-200
(ChemoMetec) according to manufacturer's protocol.

\textbf{Statistics}

Data sets were compared using 1- or 2-tailed unpaired Student's t test.
For all analyses, a P value less than 0.05 was considered statistically
significant. * = P \textless{} 0.05; ** = P \textless{} 0.01;

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\textbf{Figure Legends}

\textbf{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).

\textbf{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.

\textbf{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).

\textbf{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).

\textbf{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.

\textbf{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).

\textbf{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 \textbf{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 \textbf{(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).

\textbf{Table 1. Bioreactor parameters tested in optimization study}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Bioreactor} & \textbf{Inoculation density (e6cells/ml)} &
\textbf{DO (\%)} & \textbf{Bioreactor end volume (L)} &
\textbf{Agitation}\tabularnewline
\midrule
\endhead
\textbf{Xuri \#1} & 3.5 & None & 1 & +\tabularnewline
\textbf{Xuri \#2} & 3.5 & None & 0.5 & ++\tabularnewline
\textbf{Xuri \#3} & 3.5 & 100\% since Day0 & 0.5 & ++\tabularnewline
\textbf{Xuri \#4} & 7 & None & 0.5 & +\tabularnewline
\textbf{Xuri \#5} & 7 & 100\% since Day5 & 0.5 & ++\tabularnewline
\bottomrule
\end{longtable}

Figure 1\selectlanguage{english}
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Supplemental Figure 1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Supplemental Figure 2\selectlanguage{english}
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