Optimization of Suspension Cell Culture - PACT GROUP · PACT Meeting, Houston Vera 1 Optimization...

PACT Meeting, Houston Vera

1

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


2

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


3

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


4

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


7

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

4

CD

8

CD

3

% o

f exp

ress

ion

0

20

40

60

80

CD

45R

o

CD

45R

o

CD

62L

CD

27

CD

56

CD

16

CD

25

CD

4

CD

8

CD

3

% 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


•Characterize an accurate readout of the culture status


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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




Optimal seeding density From 1.2 to 2.5+05 cells/cm2



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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


18


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


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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•Optimal seeding density / 2


From 1.2 to 2.5+05 cells/cm2

10 mls/cm2 (best when provided upfront)


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


•Optimal seeding density / 2


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?


Artificial APC K562

PSCA

G-Rex

CAR‐PSCA T cells

CD8041BB


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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


23



+6)

1000

1500


r of T

cel

ls (1

E+

0

500

0 10 18 26 34 42 58Time in days

Num

ber



+6) 87%

1000

1500


r of T

cel

ls (1

E+

35%

39%

0

500

0 10 18 26 34 42 58Time in days

Num

ber


24



+6)

1000

1500


r of T

cel

ls (1

E+

0

500

0 10 18 26 34 42 58Time in days

Num

ber


100

20406080 Conventional method

Optimized manufacture

% e

xpre

ssio

n

0

CD45RA

CD45R0

CD62L

CCR7

CD27

CD28

CD4

CD16

CD56

CD3


25


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

Optimization of Suspension Cell Culture - PACT GROUP · PACT Meeting, Houston Vera 1 Optimization...

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