Download Reporter Gene Immunotherapy Bioassays

Reporter Gene Immunotherapy Bioassays
Expand the Tool Box for Drug Development in
Individual and Combination Immunotherapy
Jey Cheng, Ph.D.
Promega Corporation
Promega Corporation
©2015 Promega Corporation.
BEBPA Bioassay Conference
September 30, 2016
Presentation Outline
 Considerations in developing reporter gene
immunotherapy Bioassays
 Case studies of assay designs for MOA-based bioassays
 T Cell Activation (Anti-CD3 bispecific antibody, CAR)
 Immune checkpoint modulation
 Combination therapy
 Summary
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Considerations in developing Reporter Gene Immunotherapy Bioassays
1. Control cells as critical reagent
 Use engineered cell lines to replace primary cells
 “Thaw-and-Use” cells: no need of cell culture, convenient and timesaving, day to day consistency
 Cell line stability monitored and established, >P40
 Multi-tiers of cell banks and controlled cell manufacture (CellSTACK,
spinner, G-Rex, Triple-layer)
 QC tests: Cell ID, STR, mycoplasma, viability, function assay
Thaw-and-Use Format
Resuspend and Plate
Cells for Assay
Measure
Luminescence
B io lu m in e s c e n c e (R L U )
Thaw Cells
Monitor Cell Line Stability
4 .0  1 0
03
3 .0  1 0
03
T IG IT E ffe c to r c e ll lin e p a s s a g e #
p16
p28
p40
2 .0  1 0
03
1 .0  1 0
03
p54
0
Time to complete: <24 hours
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-3
-2
-1
0
1
2
L o g [ a n ti- T IG IT ],  g /m l
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Considerations in developing Reporter Gene Immunotherapy Bioassays
2. Optimized and robust protocol
 Simple and streamlined assay procedure, no washing and spinning
steps: Add-Mix-Read format
 Short assay time: one day; hands on time: a couple of hours
 Standard assay reagents and instrument:
• Medium, serum from multiple vendors
plate Effector Cells
Add
Test
• Lab equipped luminescence readers
Biologic
 Identify sources of assay variations
(single factor and DOE): cell number,
E:T ratio, incubation time, assay buffer
 Standard 96-well format, compatible
for 384-format by automation
Induce
(6-24 h)
Plate Antigenexpressing
Tumor Cells
Add Bio-Glo™
Reagent
Measure
Luminescence
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Considerations in developing Reporter Gene Immunotherapy Bioassays
3. Pre-qualified according to ICH guideline
M e a s u r e d R e la t iv e P o t e n c y , %






specificity
precision
accuracy
linearity
Range
Robustness
Dilutional Linearity/Range
250
Y=1.0883X-0.0838
R2=0.997
200
150
100
50
0
0
50
100
150
200
250
E x p e c t e d R e la t iv e P o t e n c y , %
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Assay qualification for PD-1/PD-L1 Blockade Bioassay
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Considerations in developing Reporter Gene Immunotherapy Bioassays
4. Test suitability for drug development life cycle




Early research and candidate screening
Potency determination
Stability study
Neutralizing antibody monitoring
Target
Discovery
Screen and
Optimization
384-well format by automation
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Pre- & Clinical Studies
Antibody Manufacture
Stability-Indicating
Post-Launch Monitoring
Human Serum Tolerance
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Case 1: T Cell Activation Bioassays
Platform assays for modulators of T cell activation
Inhibition:
• Orencia
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Redirected activation
• CD3 bispecific Ab
• CAR-T
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T Cell Activation Assay: Assay Design
Two TCR/CD3 Effector Cells Respond Similarly to TCR/CD3 signaling
TCR/CD3 (NFAT) Effector Cell:
• Jurkat cells engineered with an
NFAT-RE driving luciferase
expression.
• Responds to TCR/CD3 activation,
and responds minimally to CD28
co-stimulation .
IL-2 promoter
luciferase
2000000
B io lu m in e s c e n c e (R L U )
B io lu m in e s c e n c e (R L U )
NFAT-RE
EC50= 40ng/ml
Fold induction = 53
1500000
1000000
500000
0
-4
TCR/CD3 (IL-2) Effector Cell:
• Jurkat cells engineered with an
IL-2 promoter driving luciferase
expression.
• Responds to TCR/CD3 and CD28
stimulation.
-3
-2
-1
L o g [a n ti- C D 3 ],  g /m l
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0
luciferase
12000
EC50= 28ng/ml
Fold induction = 39
9000
6000
3000
0
-4
-3
-2
-1
0
L o g [a n ti- C D 3 ],  g /m l
8
TCR/CD3 (IL-2) or (NFAT) Effector Cells
Respond Differentially to CD28 Activity
Abatacept is a CTLA-4/IgG fusion protein used to prevent
overactive immune system.
80000
60000
40000
20000
TCR/CD3 (NFAT)
1500000
TCR/CD3 (IL-2)
L u m in e s c e n c e R L U
L u m in e s c e n c e R L U
100000
A reminder to
choose the right
assay system:
a Fit-for-Purpose
Bioassay
1000000
500000
0
0
-5
-4
-3
-2
-1
L o g [a b a ta c e p t] u M
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0
1
-5
-4
-3
-2
-1
0
1
L o g [a b a ta c e p t] u M
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T Cell Activation Bioassay for Anti-CD3
Bispecific Antibody
Assay Design
Antigen-expressing
Target Cells
antigen
Bispecific Ab
B io lu m in e s c e n c e ( R L U )
TCR/CD3 (NFAT)
Adherent SK-BR3 as target cells
4 1 0 06
3 1 0 06
2 1 0 06
1 1 0 06
0
-3
-2
-1
0
1
2
Catumaxomab
(Removab)
CD3xEpCAM
L o g [c a tu m a x o m a b ], n g /m l
T Cell
Receptor
RE
Luciferase
B io lu m in e s c e n c e (R L U )
1 .2  1 0
Suspension Raji as target cells
07
R a ji c e lls
n o R a ji c e lls
8 .0  1 0
06
4 .0  1 0
06
Blinatumomab (Blincyto)
CD3xCD19 Bispecific T
Cell Engager (BiTE)
0
-3
-2
-1
0
1
2
3
L o g [b lin a tu m o m a b ], p M
TCR/CD3 Effector Cells
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Similar results seen with TCR/CD3 (IL-2) Effector Cells
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T Cell Activation Bioassay for CAR-T Cell
Activity
Assay Design
Antigen-expressing
Target Cells
antigen
aCD20-CAR
T Cell
Receptor
CAR
aCD19-CAR
aCD19-CAR
RE
aCD20-CAR
Luciferase
TCR/CD3 Effector Cells
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Similar results seen with TCR/CD3 (IL-2) Effector Cells
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Case 2: Immune Checkpoint Bioassays
Co-inhibitory
 PD-1
 TIGIT
 CTLA-4
 LAG3
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Co-stimulatory
 CD40
 GITR
 4-1BB
 OX40
 HVEM
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MOA of Immune Checkpoint Modulation
Blocking Ab for immune
inhibitory receptors (PD1, CTLA4) release the
brakes.
Agonist Ab for immune
co-stimulatory receptors
(GITR, OX40, CD27,
CD137) push the gas.
T cells are activated and
kill the tumor cells.
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PD-1/PD-L1 Blockade Bioassay
Assay Design
Reflecting MOA
Anti-PD-1
PD-1
RE
Luciferase
B io lu m in e s c e n c e (R L U )
Assay specificity
T Cell
Receptor
2 .5  1 0
06
2 .0  1 0
06
1 .5  1 0
06
1 .0  1 0
06
5 .0  1 0
05
P D -1 A b
P D -L 1 A b
P D -L 2 A b
0
-9
PD-1 Effector Cells
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+ α-CTLA4
Anti-PD-L1
+ α-PD-L1
TCR
Activator
PD-L1 - cells
PD-L1
❸
PD-L1 + cells
❶ ❷
+ α-PD-1
PD-L1 aAPC Cells
-8
-7
-6
-5
-4
L o g [te s t a n tib o d y ], g /m l
14
TIGIT/CD155 Blockade Bioassay
Assay Design
CD155 aAPC Cells
TCR
Activator
Anti-TIGIT
T Cell
Receptor
TIGIT
CD226
B io lu m in e s c e n c e (R L U )
CD155
6 .0  1 0
A n ti- T IG IT
03
A n ti- P D -1
4 .0  1 0
03
2 .0  1 0
03
0
RE
Luciferase
TIGIT Effector Cells
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-2
-1
0
1
2
L o g [te s t a n tib o d y ],  g /m l
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CTLA-4 Blockade Bioassay
Assay Design
aAPC/Raji Cells
a n ti-C T L A -4 ip ilim u m a b
CD80/86
T Cell
Receptor
Anti-CTLA-4
CD28
CTLA-4
B io lu m in e s c e n c e (R L U )
TCR
Activator
16000
a n ti-H E R 2 tra s tu z u m a b
a n ti-P D -L 1 A b
12000
8000
4000
0
RE
Luciferase
-3
-2
-1
0
1
2
3
L o g [te s t a n tib o d y ],  g /m l
CTLA-4 Effector Cells
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Case 3: Combination Therapy Bioassays




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PD-1 + CTLA-4
PD-1 + TIGIT
PD-1 + CD3 bispecific Ab
PD-1 + CAR
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PD-1+CTLA4 Combination Bioassay
Assay Design
PD-L1+ CD80 aAPC Cells
PD-L1
CD80
n iv o lu m a b
CD28
CTLA-4
PD-1
RE
Luciferase
B io lu m in e s c e n c e (R L U )
8 .0  1 0
03
Ip ilim u m a b
n iv o lu m a b + ip ilim u m a b
6 .0  1 0
03
4 .0  1 0
03
2 .0  1 0
03
0
-9
PD-1+ CTLA-4 Effector Cells
Promega Corporation
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-6
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-4
L o g [te s t a n tib o d y ], g /m l
18
PD-1+TIGIT Combination Bioassay
Assay Design
PD-L1+CD155 aAPC Cells
PD-L1
CD155
CD226
TIGIT
PD-1
B io lu m in e s c e n c e (R L U )
5 .0  1 0
Is o ty p e C o n tr o l
04
A n ti-P D -1 A b
4 .0  1 0
04
3 .0  1 0
04
2 .0  1 0
04
1 .0  1 0
04
A n ti-T IG IT A b
A n ti-P D - 1 + A n ti-T IG IT
0
RE
Luciferase
-3
-2
-1
0
1
2
L o g [ t e s t a n t ib o d y ] ,  g /m l
PD-1+TIGIT Effector Cells
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19
Combination Bioassay for PD-1 Blockade and
CAR
Assay Design
PD-L1+ antigen-expressing
Target Cells
PD-L1
antigen
Effector Cells + antigen+ target cells
J u rk a t/P D -1 + H E K 2 9 3 /C D 1 9
J u r k a t/P D - 1 + H E K 2 9 3 /C D 1 9 /P D - L 1
J u rk a t/P D -1 /a - C D 1 9 -C A R + H E K 2 9 3 /C D 1 9
CAR
PD-1
RE
Luciferase
PD-1 Effector Cells
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B io lu m in e s c e n c e (R L U )
T Cell
Receptor
2 .0  1 0
05
1 .5  1 0
05
1 .0  1 0
05
5 .0  1 0
04
J u rk a t/P D -1 /a - C D 1 9 -C A R + H E K 2 9 3 /C D 1 9 /P D -L 1
0
-9
-8
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-6
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L o g [n iv o lu m a b ], g /m l
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Combination Bioassay for PD-1 Blockade and
CD3 Bispecific Ab
Assay Design
PD-L1+ antigen-expressing
Target Cells
CD19
PD-L1
T a r g e t c e ll / C D 3 b is p e c if ic A b
C D 1 9 + t a r g e r c e lls / N o b is p e c if ic A b
C D 1 9 + ta r g e t c e ll / A d d B lin a tu m o m a b
C D 1 9 /P D -L 1 + ta r g e t c e ll / A d d B lin a tu m o m a b
T Cell
Receptor
PD-1
RE
Luciferase
PD-1 Effector Cells
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B io lu m in e s c e n c e (R L U )
BiTE
1 .0  1 0
06
5 .0  1 0
05
0
-9
-8
-7
-6
-5
-4
L o g [ n iv o lu m a b ] , g /m l
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Summary
 Reporter-based immunotherapy bioassays are able to quantitatively
measure the desired biological activities for targeted drug candidates.
 Combination bioassays can be used to monitor individual and synergetic
effects from multiple immunotherapy strategies.
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Acknowledgement
Pete Stecha
Jamison Grailer
Jun Wang
Michael Beck
Julia Gilden
Jim Hartnett
Mei Cong
Frank Fan
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