Tumor-specific T cells Current Strategies to Reduce · Cost of Manufacture Juan Vera. 2 Infusion...
Transcript of Tumor-specific T cells Current Strategies to Reduce · Cost of Manufacture Juan Vera. 2 Infusion...
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Tumor-specific T cells
Current Strategies to Reduce
Cost of ManufactureJuan Vera
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Infusion
Tumor-specific T cells
Antigen
SpecificityP
atient Adoptive T cell
transfer
Blood draw
PBMCs
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T cell therapy for cancer
T cell
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Challenge: Tumor heterogeneity
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Immune escape
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• Simultaneously target multiple TAAs
• Target multiple epitopes (CD4 and CD8) within each antigen
• T cells with native T cell receptor specificity (non-engineered)
Our approach
7MultiTAA T cells
MultiTAA T cell therapy
MAGEA4
PRAME
Survivin
NYESO1
SSX2
8
0
20
40
60
80
100%
Positiv
e c
ells
MultiTAA-T Cell Phenotype
n=39CD4 CD8CD3 DC TCM TEMNK
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MultiTAA-T Cell Phenotype
0.1
1
10
100
1000
0 1 2 3 4 5 6
SF
C/2
x1
05
ce
lls
SSX2 MAGEA4PRAME SurvivinNYESO1
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MultiTAA-T Cell Phenotype
0%
10%
20%
0 0.5 1 1.5 2E:T of 20:1
% S
pecific
Lysis
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0
20
40
60
80
100
120
140
160
180
200
PRE MTH3 MTH9
SurvivinNYESO1MAGEA4SSX2PRAME
SF
C/2
x1
05
Pre Mth3 Mth9
Targeted antigens Non-targeted antigen
0
5
10
15
20
25
30
35
40
45
Pre Mth3 Mth9
MAGEC1
SF
C/2
x1
05
Mth 3Mth 9Pre-Infusion
Clinical response – DLBCL
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Activation
DC
Overlapping pepmixes
PBMCs MultiTAA T cells
Expansion
Marker current PROCESS Current process
7 days 10 days 7 days
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How can we expand
a T cell product in a
practical matter?
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Conventional cultureware limitations
• Limited media volume to allow gas exchange
O2
CO2
T75 flask
75cm2
O2
CO2
1cm2
24 well plate
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Limitations of conventional suspension cell culture methods
•Prolonged culture period
•Extensive manipulation
•Risk of contamination
• Labor intensive
• Highly trained personnel
• Excessive reagent use
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• Gas permeable membrane allows exchange of CO2 and O2
• Supports cell growth with large media volumes
• Reduces frequency of feeding/manipulation
• No rocking or stirring
100 cm2
100
0 m
L
1000U/IL2
1 cm2 = 5x105 cells
Vera JF et al. JIT. 2010
®
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G-Rex vs conventional
cultureware – cell expansion
0
3
6
9
12
15
G-RexPlate
Fold
exp
ansi
on
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Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
CFSE
24-well plate G-Rex
Superior cell numbers
not due to increased
cell division
Vera JF et al. JIT. 2010
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Anexin
PE
24-well plate
G-Rex
7AA-D Prcp
Day 1 Day 3 Day 5 Day 7
24-well plate G-Rex
Improved cell viability in G-Rex
SS
FS
Vera JF et al. JIT. 2010
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Low seeding density results in
greater fold expansion
-5
15
35
55
75
95
115
1.00E+06 5.00E+05 2.50E+05 1.25E+05 6.50E+04
Cells/cm2 (x 106)
Fold
Exp
ansi
on
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Low cell density = Greater cell expansion
Time
Cel
l nu
mb
ers
Max cell density 10E+07
cells/cm2
Op
timal tim
e f
or
cu
lture
harv
est
Optimal seeding density
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10ml of media/cm2 resulted in
maximum cell expansion
Volume of media/cm2
Cel
ls/c
m2
(x 1
06)
0
3
6
9
12
15
0.5 2 5 10 15 20
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10ml of media/cm2 resulted in
maximum cell expansion
Volume of media/cm2
Cel
ls/c
m2
(x 1
06)
0
3
6
9
12
15
0.5 2 5 10 15 2010
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Upfront media addition resulted in shortest culture period
0
2
4
6
8
10
12
14
16
0 3 6 9 13 16 20 24
10mL/cm2 (2.5mL*4)
10mL/cm2 (5mL*2)
10mL/cm2
Cel
ls/c
m2
(x 1
06)
Time in culture
25
0
2
4
6
8
10
12
14
0
50
100
150
200
250
300
Day 0 Day 3 Day 6 Day 9
GlucoseCells
Glu
cose
[mg/
dl] C
ells/cm2
Time in culture
Inverse correlation between cell number and glucose concentration
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Cel
l nu
mb
er (x
10
6)
Days in culture
0
20
40
60
80
100
120
140
0 4 8 12
Hemocytometer
Flow
Formula
Glucose assessment can be used to predict cell output
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Are these observations
reproducible?
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•25E+06 cells•1 L of media
Day 0
G-Rex 100M
•1.4E+09 cells
Day 12
G-Rex 100M
Combination of optimal culture conditions using the G-Rex
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1
10
100
1000
10000
Day 0 Day 12
CelGeneBaylorCity of Hope
Cel
l nu
mb
er (x
10
6)
Days in cultureBajgain et al. Mol Ther Methods Clin Dev. 2014
Multicenter study validates optimal G-Rex culture conditions
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SA: 5 cm2
Vol: 50 ml
G-Rex 5
G-Rex 500
SA: 500 cm2
Vol: 5000 mlSA: 100 cm2
Vol: 1000 ml
G-Rex 100
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G-Rex Culture conditions are
linearly scalable
Cel
l nu
mb
er (x
10
6)
0.1
1
10
100
1000
10000
Day 0 Day 10
G-Rex 5
G-Rex 100
G-Rex 500
Bajgain et al. Mol Ther Methods Clin Dev. 2014
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G-Rex 100
G-Rex 500
Available As A Closed System
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Rapid Cell Harvest
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Cell collection using GatheRex
Video
Cell harvest with the GatheRex
Bajgain et al. Mol Ther Methods Clin Dev. 2014
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Automated closed
manufacture
36BUSINESS CONFIDENTIAL AND PROPRIETARY
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SummaryMultiple AntigensTarget expression of multiple tumor antigens thereby
targeting more tumor cells
Clinical SafetyNo related SAEs or CRS in 60+ patients
No Genetic ModificationNative peptides presented in culture,
natural T cells expand with no mutagenesis risk
Lower CostEfficient process - requires no genetic modification or
extensive product manipulation
Clinical ImpactPotential of increased duration – prevents immune
escape with antigen spread
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Tumor-specific T cells
Current Strategies to Reduce
Cost of ManufactureJuan Vera