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Optimization of Human T Cell Activation and Expansion ProtocolsImproves Ef�ciency of Genetic Modi�cation and Overall Cell YieldJessie Yu1, Priscilla Chen1, Ashley Watson1, Danielle Truong1, Jennifer Antonchuk1, Andy I. Kokaji1, Steven M. Woodside1, Allen C. Eaves1,2, and Terry E. Thomas1
1STEMCELL Technologies Inc., Vancouver, BC, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver BC, Canada
Cancer immunotherapy using chimeric antigen receptor (CAR) T cells is a rapidly progressing field, and manufacturing these cells is a complex process that requires multiple optimization steps. We have developed reagents for the isolation, activation, and expansion of human T cells that will be available for clinical cell therapy manufacturing. Soluble ImmunoCult™ Human T Cell Activators induce T cell activation via cross-linking CD3 and co-stimulatory molecules on the surface of cells. Activated T cells then can be genetically modified and subsequently expanded in serum- and xeno-free ImmunoCult™-XF T Cell Expansion Medium. Here, we present several optimization strategies with ImmunoCult™ products in order to obtain high transfection efficiency and maximum cell yield. By evaluating activation dynamics of T cells and determining the optimal transfection time points, the transfection efficiency can be substantially improved in both CRISPR/Cas9- and lentiviral-mediated gene modification methods. Our study also suggests that diluting T cells to a lower cell density after the third day following activation greatly improves cell viability and cumulative cell growth, resulting in a more than 1000-fold expansion of total human T cells with > 85% viability over 10 - 12 days of culture. Expanded T cells co-express CD45RO+CD62L+. As an example, we applied the workflow described here to generate T cell receptor alpha beta (TCRαβ) knockout (KO) T cells from healthy donors with up to 90% knockout efficiency. The purity of TCRαβ KO cells can be further increased with the use of an EasySep™ Human TCRαβ depletion kit. Taken together, the processes outlined in this study can be easily and rapidlyimplemented to improve T cell manufacturing efficacy.
METHODS
Cells were stimulated with an ImmunoCult™ T cell activator in
ImmunoCult-XF™ for 1, 2 or 3 days
Cells were transduced with lentiviral vector encoding GFP
at MOI 25
Cells were cultured for 4 days post-
transduction
GFP expression was analyzed by flow
cytometry
RNP complex was delivered into T cells by
electroporation (1400 V, 30 ms, 1 pulse)
T cells were isolated using EasySep™ Human
T Cell Isolation Kit (17951)
Cells were cultured for at least 48 hrs to allow
editing to occur
Knockout efficiency was analyzed by T7 assay and
flow cytometry
Gene-edited cells were expanded and purified
(EasySep™ depletion of any non-edited cells)
Genome Editing withLentiviral Vectors
Genome Editing with CRISPR/Cas9
+
PAM site
DNA
5’
NGG
Cas9
gRNA+
RNP
T7 Assay
FACS
TCR
αβ
CD45
CD
4
FACS
GFP
B
A
FIGURE 3. EasySep™ Human TCRαβ Depletion Protocol. Gene-edited TCRαβ KO T cells were expanded for 7 - 10 days, harvested, and resuspended in EasySep™ Buffer at 5 x 107 cells/mL. Following a 13-minute EasySep™ TCRαβ depletion protocol, flow cytometry was performed to assess the depletion of TCRαβ+ cells.
100 3Day
Harvest
5
Data Analysis•
•
•
Static culture flasks
EasySep™-isolated T cells were seeded at 1 x 106 cells/mL in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) supplemented with recombinant human IL-2 (Catalog #78036)
24-well plate
T cells were stimulated with ImmunoCultTM human T cell activators
Diluted cells with ImmunoCultTM-XF + rhIL-2
Diluted cells with ImmunoCultTM-XF + rhIL-2
Diluted cells with ImmunoCultTM-XF + rhIL-2 every 2 - 3 days
Cells were inoculated into a 2 L Xuri CellbagTM Bioreactor and operated in fed-batch mode to maintain cell density at 0.5 x 106 cells/mL until total volume reached 1 L. Initiated perfusion at 500 mL/day when cell density exceeded 2 x 106 cells/mL.
Cell count and viability assessment
Activation assessment by CD25 and PD-1 expression level
Phenotype assessment by CD45RO and CD62L expression level
Xuri® W25 Cell Expansion System
Add EasySepTM Top up with EasySepTM buffer and place tube in magnet for 5 minutes
Add EasySepTM RapidSpheresTMHuman TCRαβ
Depletion Cocktail
Incubate 5 minutes
Incubate 3 minutes
FIGURE 7. Residual TCRαβ+ Cells can be Removed From Expanded TRAC KO Cell Populations Using EasySep™ Human TCRαβ Depletion Kit. Representative data plots are shown for the frequency of TCRαβ cells in the expanded TRAC KO cells before and after EasySep™ depletion.
FIGURE 6. ImmunoCult™ Activation Kinetics for Optimal Lentiviral Transduction and CRISPR/Cas9-Mediated TRAC Knockout of Human T Cells. (A) Activation with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator for 2 days resulted in the highest percentage of GFP-positive cells. (B) Activation with ImmunoCult™ Human CD3/CD28 T Cell Activator for 3 days resulted in the highest TRAC knockout efficiency.
CD3/CD28/CD2 CD3/CD28
30
20
10
0
% G
FP+ C
ells
40
2 Days3 Days
1 Day
FIGURE 5. Large-scale T Cell Expansion in the Xuri™ W25 Cell Expansion System Bene�ted from Early Cell Dilution for T Cell Expansion. T cells were stimulated on day 0 with ImmunoCult™ Human CD3/CD28 T Cell Activator in culture flasks. On day 3 of culture, fresh medium was added to increase the total culture volume by 2-, 4-, and 8-fold. (A) The highest fold expansion (263) was observed with the 8-fold volume increase on day 3 following 11 days of culture as outlined in Figure 1. (B) Viability was similar across the different cell dilutions.
321682 4
Fold ExpansionViability
05101520
Fold
Expan
sion
o
n D
ay 5
0
10
2060
80
100
% V
iab
ility
Fold Increase in Medium Volumeon Day 3
4 80
200
400
600
800
1000*
Fold Increase in Medium Volumeon Day 3
Tota
l Fo
ld E
xpan
sio
n
on
Day
10
FIGURE 4. Diluting T Cells To A Lower Cell Density Three Days Following Initial T Cell Activation Improved T Cell Growth and Viability. T cells were stimulated on day 0 with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator. (A) An 8-fold volume increase on day 3 resulted in better viability and a higher fold expansion at day 5. (B) Increasing the culture volume by 8-fold on day 3 followed by a further volume increase by 4-fold on days 5 and 7 until day 10 of culture resulted in an overall 405 ± 174 total fold expansion (mean ± SD, n = 14). (C) The recommended human T cell expansion protocol with ImmunoCult™ reagents in static culture. (D) Representative FACS plots of expanded T cells using ImmunoCult™ products after 10 days of culture.
Day0 3 5 7 10
Activate human T cells at 1 x 106 cells/mL in
ImmunoCultTM-XF + rhIL-2
Dilute cells by increasing volume 8-fold or
to a cell density of 1 - 2.5 x 105 cells/mL
Dilute cells by increasing volume at least 4-fold or
to a cell density of 1 - 2.5 x 105 cells/mL
Dilute cells by increasing volume at least 4-fold or
to a cell density of 1 - 6 x 105 cells/mL
Harvest and phenotype analysis
CD3/CD28/CD2CD3/CD28
2 Days3 Days
20
40
60
80
0
Kn
ock
ou
t Ef
�ci
ency
(% T
CRαß
- CD
3- Cel
ls)
100
After
-103
103
104
105
106
-104 -103 104 105 106
CD45CD45
TCR
TCR
-103
103
104
105
106
-104 -103 104 105 106
Before
αβ αβ
CD45RO
CD62
L
CD8+ Cells
-100
10-1
101
102
103
104
-100 101 104103102
CD45RO
CD62
L
CD4+ Cells
-100
10-1
101
102
103
104
102 103 107106105104
CD4
CD
8a
-100
10-3
100
101
102
-100100 105104103
103
Viable Cells
ABSTRACT
A B
A B
D
C
1
10
100
1000
0 3 5 7 10
4-fold of Initial Volume8-fold of Initial Volume
Medium Increase on Day 3
Tota
l Fo
ld E
xpan
sio
n
Culture Time (Days)
Culture Time (Days)
40
60
80
100
% V
iab
ility
4-fold8-fold
2-foldA B
1
10
100
1000
Culture Time (Days)0 3 5 6 7 8 110 3 5 6 7 8 11
4-fold8-fold
2-fold
Tota
l Fo
ld E
xpan
sio
n
Summary
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FIGURE 2. Experimental Work�ow of Human Primary T Cell Gene Editing. Cryopreserved T cells were stimulated with T cell activation reagents for 1, 2, or 3 days in ImmunoCult™-XF T Cell Expansion Medium supplemented with recombinant human (rh) IL-2. (A) Activated cells were transduced with lentiviral vectors encoding GFP or (B) The ArciTect™ CRISPR-Cas9 RNP complex was delivered into activated T cells to knock out the TCR alpha constant (TRAC) locus.
FIGURE 1. A General Work�ow of Human Primary T Cell Isolation, Activation, and Expansion. Purified T cells were seeded at 1 x 106 cells/mL and stimulated with ImmunoCult™ Human CD3/CD28/CD2 or CD3/CD28 T Cell Activator (Catalog #10970 or 10971, respectively). Fresh culture medium was added to the cultures every 2 - 3 days until day 10 of culture. Cell counts and viability assessments were performed during the course of the expansion. Fold expansion during culture was analyzed relative to the initial cell seeding number.
RESULTS
An optimized T cell expansion protocol using ImmunoCult™ reagents has been developed for robust growth of viable T cells
Expanded T cells using ImmunoCultTM reagents have a less-differentiated phenotype (CD45RO+CD62L+)
ImmunoCultTM-activated human T cells can be genetically modified with high editing efficiency
A combination of ImmunoCultTM, ArciTectTM CRISPR-Cas9 system, and an EasySepTM cell isolation/depletion strategy provides a complete workflow for the production of TCRαβ KO cells from leukapheresis samples
