Optimization of Suspension Cell Culture - PACT GROUP · PACT Meeting, Houston Vera 1 Optimization...
Transcript of Optimization of Suspension Cell Culture - PACT GROUP · PACT Meeting, Houston Vera 1 Optimization...
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PACT Meeting, Houston Vera
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Optimization of Suspension Cell Culture
Roopa Mucharla, Ann Leen, Natasha Lapteva, Helen Heslop, Adrian Gee, Malcolm Brenner, Cliona Rooney, and Juan Vera
• CAR/PSCA ‐Humanized
CAR targeting PSCA
‐Codon optimized
CAR‐PSCA
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CAR‐PSCA T cells kill prostate cancer cell line DU145
NT T cells50%sp=0.003
CAR‐PSCANT T cells
20%
30%
40%
ge o
f tum
or ly
si
0%
10%
20%
293T DU145
Per
cent
ag
T cells
*Antigen2
Obstacles associated with the expansion of CAR‐T cells
1
Blood draw
*Antigen Specificity*
3
Infusion
T‐cell product generation
3
4
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T cells
*Antigen2
Obstacles associated with the expansion of CAR‐T cells
1
Blood draw
*Antigen Specificity*
3
Infusion
T‐cell product generation
3
4
Extensive/complicated culture expansion
Our purpose:p p(i) Improve CAR expression and
expansion of T cells(ii) Simplify manufacture(ii) Simplify manufacture
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Our purpose:p p(i) Improve CAR expression and
expansion of T cells(ii) Simplified the manufacture(ii) Simplified the manufacture
T cell expansion and CAR expressionwhen using aAPCs
Artificial APC - K562t c a C 56
PSCA
CAR-PSCA T cells
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Artificial APC - K562
T cell expansion and CAR expressionwhen using aAPCs
t c a C 56
PSCA
CD86
CAR-PSCA T cells
CD8041BB
Increase in CAR expression afterco‐culture with aAPCs
Fold increase
/PSCA expressio
nF
CAR
/
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Increased expansion of CAR‐PSCA T cells after co‐culture with aAPCs
12
4681012
expa
nsio
n
Media K562 41BBmIL15
K562/PSCA
41BBCD80
41BB 41BBCD80CD86
ø024
T ce
ll
Our purpose:p p(i) Improve the CAR expression
and expansion of T cells(ii) Simplify manufacture(ii) Simplify manufacture
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Problems with Current protocols
‐Prolonged culture period ‐Extensive manipulation →risk of contamination‐Labor intensive‐Requires highly trainedRequires highly trained personnel‐Excessive use of reagents
Limited volume of media and gas exchange
Conventional Cultureware:
Limited volume of media and gas exchange
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Wilson Wolf ManufacturingUnique Gas Permeable Devices
• Gas permeable membrane allows optimal exchange of CO2 and O2
• Supports cell growth with large volumes of media• Reduces feeding frequency and manipulation• No rocking or stirring Vera JF et al. JIT. 2010
Wilson Wolf ManufacturingUnique Gas Permeable Devices
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SA: 100 cm2Vol: 2000 ml
G Rex 100
G-Rex 100L
SA: 10 cm2 Vol:
SA: 100 cm2 Vol: 500 ml
G-Rex 10
G-Rex 100
SA: 10 cm2 Vol: 40 ml
EBV‐CTL For Clinical Use ‐ 3x108 CTLday 0 – CTL initiation 2.4 x 107
day 9 ‐ restim 2 x 107
day 12 – IL2 feed
day 20 – split + IL2 feed
day 16‐harvest and reseed 6 x 107
Day 30 ‐ harvest/freeze
day 27 – split + IL2 feed
day 23 – harvest and reseed 1.2 x 108
3.6 x 108
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G-Rex
EBV‐CTL For Clinical Use – 3 x108 CTL
day 0 – CTL initiation 2.4 x 107
day 9 – restim (add LCL)
day 12 – IL2 feed
1 x 108
4 x 108
Production time halved
Day 16 –harvest/freeze
Superior expansion of EBV-CTLs in G-Rex vs 24w plate
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S d d l d iS d d l d i
CTLs Expanded Using Optimized Protocol Conserve Their Phenotype and Function
7080
s 7080
s
36.2% 10.7%44.4% 6.3%
QAK
Tet
ram
er P
E
RAK
Tet
ram
er P
E
QAK
Tet
ram
er P
E
RA
K P
E
QA
K P
E
RA
K P
E
QA
K P
E
GPCStandard culture device
CD 8 FITC CD 8 FITC
36.2% 10.7%44.4% 6.3%
QAK
Tet
ram
er P
E
RAK
Tet
ram
er P
E
QAK
Tet
ram
er P
E
RA
K P
E
QA
K P
E
RA
K P
E
QA
K P
E
GPCStandard culture device
CD 8 FITC CD 8 FITC
100
n
100
n
Days in culture 9 16 23
Days in culture Days in culture 9 16 23
Days in culture
Effector/target Ratio
40-1 20-1 10-1 5-10
102030405060 AUTO Plate
AUTO GPALO PlateALO GP
% o
f spe
cific
lysi
s
Effector/target Ratio
40-1 20-1 10-1 5-10
102030405060 AUTO Plate
AUTO GPALO PlateALO GP
% o
f spe
cific
lysi
s
0
20
40
60
80
CD
45R
o
CD
45R
o
CD
62L
CD
27
CD
56
CD
16
CD
25
CD
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CD
8
CD
3
% o
f exp
ress
ion
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20
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80
CD
45R
o
CD
45R
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CD
62L
CD
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CD
56
CD
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CD
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CD
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CD
8
CD
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% o
f exp
ress
ion
Cell Harvest
Conventional G-RexCultureware
Harvest time – 20mins/plate
4hr to harvest 12 plates
Harvest time?
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EBV-CTL harvested from G-Rex
Collect 1000E+06 Cell Approx. 4 minutes processMinimal manipulation
G-Rex decrease production cost
24 well plate G-Rex24 well plate G RexStarting cell # 2x107 PBMCs 2x107 PBMCsFinal cell # 1x1010 CTLs 1x1010 CTLsTech time 129 hours $ 3,096 2.5 hours $ 60GMP charge $ 2.280 $ 300Cultureware 310 plates $ 2325 2 (G-Rex40)
8 to 16 (G-$ 2200
8 to 16 (GRex500)
Media/cytos 11160 ml $ 1674 3280 ml $ 492Plastics 618 $ 309 20 $ 10Release Tests $1843 $ 1843Final cost $11,527 (1.15x1E+06 cells) $4,905 (0.49x1E+06 cells)
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G-Rex decrease production cost
24 well plate G-Rex# of interventions 222,720 34Risk of contamination ++++ +Biohazard ++++ +Time for generation 59 days 23 days g y y
(i) Establish the optimal culture conditions for the expansion of suspension cells
•Optimal seeding density
•Establish the minimum volume of media needed to achieve maximal cell expansion
•Optimal seeding density
•Characterize an accurate readout of the culture status
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(i) Establish the optimal culture conditions for the expansion of suspension cells
•Optimal seeding density
•Establish the minimum volume of media needed to achieve maximal cell expansion
•Optimal seeding density
•Characterize an accurate readout of the culture status
Expected results: Lower cell density = Higher expansion
M ll d it 10E 07
Optimal seeding density
numbe
rs
Max cell density 10E+07 cells/cm2
mal
tim
e fo
r re
har
vest
Time
Cell
Opt
imcu
ltu
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Lower cell density = Higher expansion Optimal seeding density 1.2E+05 cells/cm2
ld expansio
nFo
1E+06 cells x cm2
(i) Establish the optimal culture conditions for the expansion of suspension cells
•Optimal seeding density
•Establish the minimum volume of media needed to achieve maximal cell expansion
Optimal seeding density From 1.2 to 2.5+05 cells/cm2
•Characterize an accurate readout of the culture status
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Establish optimal culture conditions using a test device
•Small scale representation of the G‐Rex•Test multiple conditions simultaneously•Test multiple conditions simultaneously •Facilitates the identification of optimal conditions
‐cost effective
Maximum cell number directly correlates with the increment of media
Cell Num
ber x 10
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•What is the best way of providing this volume to the culture?
• What is the best way of providing this volume?
1200
1400
E+0
6 ce
lls
400
600
800
1000
1200 10ml/cm2
1
0
200
400
0 3 6 9 13 16 20 24
Days in culture
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• What is the best way of providing this volume?
1200
14005ml/cm2
1E+0
6 ce
lls
400
600
800
1000
1200
10ml/cm2
1
Days in culture
0
200
0 3 6 9 13 16 20 24
• What is the best way of providing this volume?
1200
1400 2.5ml/cm2
l/
E+0
6 ce
lls
400
600
800
1000
1200 5ml/cm2
10ml/cm2
1
Days in culture
0
200
0 3 6 9 13 16 20 24
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(i) Establish the optimal culture conditions for the expansion of suspension cells
•Optimal seeding density / 2
•Establish the minimum volume of media needed to achieve maximal cell expansion
From 1.2 to 2.5+05 cells/cm2
10 mls/cm2 (best when provided upfront)
•Characterize an accurate readout of the culture status
Inverse correlation of cell number and glucose concentration
on
300
350 10 E+07
se co
ncentratio Cell num
ber 100150
200
250
3008E+07
6E+07
4E+07
2E+07
Glucos
0
50
day 0 day 3 day 6 day 9 day10
1E+07
Number of days in culture
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Glucose can be used to predict cell numbers in culture
140H t tH t t
406080
100120 Hemocytometer
FlowFormula
06 c
ells
x c
m2
GlucoseFlow Hemocytometer
02040
day0 day4 day7 day11
1E+0
(i) Establish the optimal culture conditions for the expansion of suspension cells
•Optimal seeding density / 2
•Establish the minimum volume of media needed to achieve maximal cell expansion
From 1.2 to 2.5+05 cells/cm2
10 mls/cm2
•Characterize an accurate readout of the culture status Glucose measurement
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What does this mean for expansion of CAR‐T cells?
What does this mean for expansion of CAR‐T cells?
Artificial APC K562
PSCA
G-Rex
CAR‐PSCA T cells
CD8041BB
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What does this mean for expansion of CAR‐T cells?
Preliminary data from G-Rex100
•Media volume - 1L
Day 0
•25E+06 CAR T cells 54%
K 62 PSCA BB 80
Day 10
•2.3E+09 CAR PSCA T cells 82%
•K562-PSCA-BB-80
•100U of IL2/ml
•IL-2 administration x3 per week
2500Optimized manufacture
Improved and simplified manufacture of CAR T cells
+6)
1000
1500
2000Optimized manufactureConventional method
r of T
cel
ls (1
E+
0
500
0 10 18 26 34 42 58Time in days
Num
ber
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2500Optimized manufacture
Improved and simplified manufacture of CAR T cells
+6)
1000
1500
2000Optimized manufactureConventional method
r of T
cel
ls (1
E+
0
500
0 10 18 26 34 42 58Time in days
Num
ber
2500Optimized manufacture
Improved and simplified manufacture of CAR T cells
+6) 87%
1000
1500
2000Optimized manufactureConventional method
r of T
cel
ls (1
E+
35%
39%
0
500
0 10 18 26 34 42 58Time in days
Num
ber
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2500Optimized manufacture
Improved and simplified manufacture of CAR T cells
+6)
1000
1500
2000Optimized manufactureConventional method
r of T
cel
ls (1
E+
0
500
0 10 18 26 34 42 58Time in days
Num
ber
Improved and simplified manufacture of CAR T cells
100
20406080 Conventional method
Optimized manufacture
% e
xpre
ssio
n
0
CD45RA
CD45R0
CD62L
CCR7
CD27
CD28
CD4
CD16
CD56
CD3
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Improved and simplified manufacture of CAR T cells
60%
70%
20%
30%
40%
50%
60%
% tu
mor
lysi
s
0%
10%
20%
1 2
%
Optimized manufacture
Conventional manufacture
Similar cell expansion when G‐Rex was scaled‐up
ells
10000
umbe
r of c
e
100
1000G‐Rex 10G‐Rex 100G‐Rex 600
Tota
l nu
1
10
0 10
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Robust manufacture process
2000
2500E+
6) 1G.CAR-Muc1
1000
1500
2000
Optimized manufactureConventional method
er o
f T c
ells
(1E
0
500
0 10 14 19 23
Num
be
Time in days
Summary (i) •Combination of CD80/41BB optimal for enrichment and expansion of CAR T cells
O GOptimal G-Rex conditions;• Seeding density 1.2‐2.5E+05 cells/cm2
•Media volume 10ml of media/cm2
•Readout GlucoseReadout Glucose•Robust manufacture platform•200 fold expansion of CAR T cells in 10 days•No additional feeding/manipulation required
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Summary (ii)
•Trivirus CTLs (EBV, Adv, CMV)•Primary T cells y•Genetically modified T cells and CTLs•TILs•NK T Cells•EBV LCL•EBV‐LCL•K562•Virtually any suspension cell type
Cell culture G‐Rex Optimization
Roopa MucharlaNatasha LaptevaAnn LeenHelen HeslopAdrian GeeCliona RooneyMalcolm Brenner