Download Michael G Tovey

Establishment of a Cell-Based Assay Specific for a Growth Factor
that Shares a Common Receptor Chain and Overlapping
Biological Activities with Other Cytokines and Growth Factors
Michael G Tovey,
INSERM Director of Research, Laboratory of Biotechnology &
Applied Pharmacology,
ENS CACHAN
[email protected]
Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF)
¾ GM-CSF is a hematopoietic growth factor that plays a central
role in in the generation of neutrophils, macrophages, and
DCs
¾ GM-CSF acts together with IL-3 and IL-5 to regulate the
survival proliferation, differentiation, and functional activation
of hematopoietic cells
¾ GM-CSF also regulates its own activity by via the induction of
CIS, a SOCS family member SH2-domain protein that inhibits
Jak2/STAT5 phosphorylation & signaling.
¾ CIS and SOCS3 also regulate EPO induced Jak2/STAT5
signaling
Quantification of GM-CSF Activity
¾ Current methods for quantifying human GM-CSF activity, are
bioassays based on the ability of GM-CSF to support the
proliferation of cell lines such as TF-1, or UT-7, that require
GM-CSF for their growth
¾ Due to overlapping biological activities, IL-3 or EPO, can also
support the proliferation of these cells and act synergistically
together with GM-CSF.
¾ M-CSF & IL-1 can also enhance GM-CSF dependent cell
proliferation
¾ TGF-β and IFNα, or IFN β, can antagonize this activity
¾ IL-3 & IL-5 share a common receptor βc chain with GM-CSF
¾ Thus, proliferation based assays for GM-CSF are subject to
non specific interference.
Development of a Cell-based Assay
Specific for GM-CSF: Strategy
¾ Use a cell line that possesses functional GM-CSF receptors,
but does not require GM-CSF or other related growth factors
for proliferation
¾ U937 cells possess functional GM-CSF receptors but not
express functional EPO receptors
¾ U937 cells do not require GM-CSF, EPO or other related
growth factors for proliferation
¾ Transfect U937 cells with a GM-CSF responsive reportergene construct
Reporter-Gene Assay: Construction
SV40
CM-CSF-Specific
Regulatory Element Min. prom.
Luciferase
SV40
Poly A
Intron Human β-globine
However,
GM-CSF, IL-3, & IL-5 share a common
receptor βc chain
GM-CSF Receptor
¾ GM-CSF binds to a heterodimeric receptor comprised of a
GM-CSF specific α subunit and a common receptor βc chain
that is shared with IL-3 & IL-5
¾ GM-CSF receptor does not possess intrinsic tyrosine kinase
activity; associates with Jak2 required for βc transphosphorylation, initiation of signaling, & biological activity
¾ Receptors for GM-CSF, IL-3, & IL-5 are expressed at very low
levels (100-1,000 receptors/cell)
¾ GM-CSF, IL-3, & IL-5 each bind with low affinity to their
specific Rα chain (Kd = 0.2 – 100 nM)
¾ In the presence of the βc receptor chain each cytokine binds
with high affinity (Kd = 100 pM) resulting in dimerization of
both sub-units and receptor activation
Development of a Cell-based Assay
Specific for GM-CSF: Strategy
¾ Overexpress the GMRα, GM-CSF ligand-specific binding subunit, of the human GM-CSF receptor
¾ Hypothesis, GMRα receptor chain will compete with IL-3 or IL5 specific binding sub-units for the pool of the βc signaling
receptor sub-unit common to the GM-CSF, IL-3, and IL-5
heterodimeric receptors.
GM-CSF Receptor Structure and Signal
Transduction
STAT5
STAT5
P
STAT5
P
STAT5
IL5
IL3
IL5Rα
IL3Rα
GMRα
IL5
IL3
IL5R
α
IL3R
α
GM-CSF Gene Reporter Assay (FireFly Luciferase)
GM-CSF
Stat5 Stat5
+1
(T/A)TTCnnnGAA(T/A)
FireFly Luciferase
SV40
Poly A
TATA
Intron
STAT5 Consensus Sequence:
(T/A)TTCnnnGAA(T/A)
STAT5 con. Ax4
TTCCCCGAAATGATGAGCTAGGAGCCTGATTTCCCCGAAATGATGAGCTAGGAGCCTGATTTCCCCGAAATGATGCTAGGAGCCTGATTTCC
CCGAAATGATctGtTAG
STAT5 con. Bx6
GAGGCTCTGATTTCCGGGAAACTGATTCCCGGAATACGTTTTCCGGGAATACGTTTCCGGGAAACGTATTCCGGGAAAACTGATT
CCGGGAAATGATCTGTTAG
STAT5 con. Cx6
GATTTCTAGGAATTCttctcagaaGAATTCttctgagaaGAATTCttctcagaaGAATTCttctgagaaGAATTCttctcagaaGGAATTCAAATCG
STAT5 con. Dx4
GATTTCTAGGAATTCAAATCGGATCTAGATTTCTAGGAATTCAAATCGGATCTAGATTTCTAGGAATTCAAATCGGATCT
AGATTTCTAGGAATTCAAATCG
STAT 5 Reporter Gene Constructs – Firefly luciferase A, B, C or D
Transcient transfection in U937 cells
12
Fold induction luciferase
10
8
6
Ctl
500ng/ml rhGM-CSF
4
2
0
A
B
STAT5-luc
C
D
STAT5-luc
STAT5 Reporter Gene – Firefly luciferase (C or D) relative to Renilla luciferase
expression
Transcient transfection in K562 and U937 cells
1800
Ratio (Firefly / Renilla luciferase)
1600
1400
1200
1000
Ctl
800
500ng/ml rhGM-CSF
600
400
200
0
STAT5 - luc
C
D
K562
STAT5 is expressed
constitutively in K562 cells
C
D
U937
Effect of rhEPO and rhIFNγ on STAT5 – luciferase (C or D) Reporter Gene
Transcient transfection in U937 cells
12
10
Fold induction
8
Ctl
500ng/ml rhGM-CSF
6
500ng/ml rhEPO
10000 IU/ml IFNg
4
2
0
STAT5 C
STAT5 D
Analysis of Clones Transfected with the US5‐Luc Construct
16,0
14,0
FireFly Fo ld Induc o n
12,0
10,0
8,0
6,0
4,0
2,0
0,0
1
2
3
4
5
6
7
8
9
10
11
12
13
Clone N°
14
15
16
17
18
19
20
21
22
23
24
Selection of stable clones transfected by STAT5-luc C in U937 cell line
13
11
Fold induction
9
Clone 2
7
Clone 3
Clone 6
5
3
1
0.01
0.1
1
Log rh GM-CSF (ng/ml)
10
Firefly Luciferase induction in U937/STAT5-luc stable cell line
50
45
Firefly Luciferase Fold Induction
40
35
30
rh GM-CSF 4h
25
rh GM-CSF 18h
rh EPO 4h
20
rh EPO 18h
15
10
5
0
0.0005
0.005
0.05
0.5
5
ng/ml
50
500
5000
Cell number optimisation for the GM-CSF assay
Stimulation with 200ng/ml rhGM-CSF (Gibco®)
4.E+06
4.E+06
Firefly luciferase RLU
3.E+06
3.E+06
2.E+06
2.E+06
1.E+06
5.E+05
0.E+00
0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
cell number
2.5E+05
3.0E+05
3.5E+05
4.0E+05
GM-CSF Standard Curves
14
12
Fold Induction
10
Standard
NIBSC
8
6
4
2
0
0.000001 0.00001
0.0001
0.001
0.01
0.1
GM-CSF ng/ml
1
10
100
1000
GM-CSF Gene Reporter Assay (FireFly /
Renilla)
GM-CSF
Stat5 Stat5
+1
(T/A)TTCnnnGAA(T/A)
FireFly Luciferase
SV40
Poly A
TATA
Intron
+1
CMV
+1
GM-CSF-Rα
Renilla Luciferase
SV40
Poly A
IRES
Intron
Double transcient transfection STAT5-luc (C or D) and pCMV-GM-CSF-Rα in U937 cells
20
18
16
Fold induction
14
12
Ctl
10
500ng/ml rhGM-CSF
8
6
4
2
0
Control
GM-CSF-Rα
STAT5 C
Control
GM-CSF-Rα
STAT5 D
Firefly Luciferase induction in the U937 cell line transciently tranfected with STAT5-luc and GMCSF-Rα treated with GM-CSF, IL-3 or IL-5
STAT5-luc
STAT5-luc + GM-CSF-Ralpha
22
20
Firefly Luciferase fold induction
18
16
14
12
10
8
6
4
2
0
Ctl
100
500
rh GM-CSF
Gibco® (ng/ml)
1000
100
500
rh IL-3
Immunotools® (ng/ml)
1000
100
500
1000
rh IL-5
Immunotools® (ng/ml)
Analysis of US5-Luc Clones Expressing Renilla Luciferase
and the GM-CSFRα Receptor Chain
4500
4000
3500
RLU Renilla
3000
2500
2000
1500
1000
500
0
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
Clone N°
33
35
37
39
41
43
45
47
49
51
53
55
Analysis of RUS5-Luc clones Expressing Renilla
Luciferase and GM-CSFα Receptor Chain
18
16
FireFly Fold Induction
14
12
10
8
6
4
2
0
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
Clone N°
33
35
37
39
41
43
45
47
49
51
53
55
US5 Dose Response Curve (16h Incubation)
30
25
Fold
20
GM-CSF
15
IL-3
IL-5
10
5
0
0.001
0.01
0.1
1
ng/ml
10
100
US5 GM-CSF (4h incubation)
4.5
US5 GM-CSF (16h incubation)
12
4
10
3.5
Fold Induction
Fold
3
2.5
2
1.5
8
6
4
1
2
0.5
0
0.00001
0.001
0.1
GMCSF ng/ml
10
0
0.00001
0.001
0.1
GMCSF ng/ml
10
GM-CSF Gene Reporter Assay (FireFly /
Renilla)
GM-CSF
Stat5 Stat5
+1
(T/A)TTCnnnGAA(T/A)
FireFly Luciferase
SV40
Poly A
TATA
Intron
+1
CMV
+1
GM-CSF-Rα
Renilla Luciferase
SV40
Poly A
IRES
Intron
Transient Cotransfection of U937 Cells
60
STAT5Luc+RGM-CSF/hRL
50
Fold Induction
STAT5-Luc+EGFP
40
FireFly Activity
30
20
10
0
Control
GM-CSF 100ng/ml
60
STAT5Luc+RGM-CSF/hRL
Renilla Activity
Fold Induction
50
STAT5-Luc+EGFP
40
30
20
10
0
Control
GM-CSF 100ng/ml
Normalized Reporter Gene Assay for
GM-CSF
¾ Unexpectedly, constitutive expression of Renilla luciferase is
influenced (2x) by STAT5 activation in response to GM-CSF
treatment
¾ Consequence effective dynamic range of assay reduced
¾ Mechanism unclear, no Stat5 recognition sequences in
promoter construct
¾ Solution change promoter use SV40 constitutive promoter &
Herpes simplex TK promoter
¾ Use humanized Gaussia luciferase gene instead of Renilla
luciferase
GM-CSF Induced FL Expression Normalized Relative to
Gaussia Expression (4 H Induction)
30
US5 17.5.21 SV
US5 17.5.8 TK
Normalized Fold Induction
25
20
15
10
5
0
0.000001
0.00001
0.0001
0.001
0.01
GM-CSF ng/ml
0.1
1
10
100
GM-CSF Induced FL Expression Normalized Relative to
Gaussia Expression (16 H Induction)
50
US5 17.5.21 SV
45
US5 17.5.8 TK
Normalized Fold Induction
40
35
30
25
20
15
10
5
0
0.000001
0.00001
0.0001
0.001
0.01
GM-CSF ng/ml
0.1
1
10
100
300000
0.6
250000
0.5
200000
0.4
RLU (FL/RL)
RLU (FL)
Relationship Between Drug Induced FL Expression and Cell Number
150000
0.2
50000
0.1
0.001
0.1
Drug (ng/ml)
10
40.000
20.000
100000
0
0.00001
80.000 cells
0.3
0
0.00001
10.000
0.001
0.1
Drug (ng/ml)
10
Response of Reporter Cells to Serum Matrix Effects
80
45
RA Sera 10%
RA Sera 10%
Fold Induction
60
50
RA sera 20%
35
Normal Sera 10%
30
Control
Fold Induction
70
40
40
30
RA sera 20%
Normal Sera 10%
Control
25
20
15
20
10
10
0
0.0001
5
0.01
1
Drug (ng/ml)
100
0
0.0001
0.01
1
Drug (ng/ml)
100
Stability Studies: Sensitivity
Time (Hrs)
Passage #
EC50 (pg/ml)
LLOQ (pg/ml)
4
10
40
10
4
20
80
20
4
40
20
5
18
10
400
200
18
20
400
200
18
40
200
100
Stability Studies: Proliferation
Passage #
Doubling Time
(Hrs)
Max Cell Density
(x106/ml)
4
27
1.0
10
25
0.95
20
24
1.2
40
24
1.1
Conclusions - I
¾ A cell-based assay specific for a growth factor can be
established by the use of a cell-line that does not require the
growth factor or other related grow factors for proliferation
¾ The cell does, however, contain a functional receptor/signal
transduction system for the growth factor of interest
¾ The assay can be rendered specific by over-expression of a
growth factor specific binding receptor sub-unit
Conclusions - II
¾ When a growth factor signals through multiple pathways
(MAPK, NFkB, STAT1-5 etc) it may be more useful to
reconstitute a complete receptor-signaling transduction
system in a cell that does not respond to the cytokine of
interest.
Reconstitution of a Functional Cytokine Signaling Pathway in Human U937 cells
16
14
Fold Induction
12
10
8
6
4
2
0
Stat4‐kozak
‐
+
‐
‐
+
‐
+
CX2RB1
‐
‐
+
‐
‐
+
+
CX2RB2
‐
‐
‐
+
+
+
+
STAT4‐Luc
+
+
+
+
+
+
+
Reconstitution of a Functional Cytokine Signaling Pathway in Human HEK
cells
16
14
Fold Induction
12
10
8
6
4
2
0
Stat4‐kozak
‐
+
‐
‐
+
‐
+
CX2RB1
‐
‐
+
‐
‐
+
+
CX2RB2
‐
‐
‐
+
+
+
+
STAT4‐luc
+
+
+
+
+
+
+
Conclusions - III
¾ Reconstitution of a cellular receptor-signal transduction
system in a cell that does not respond to the cytokine or
growth factor of interest is a powerful tool for the
establishment of Specific cell-based assays for the
quantification of the activity and neutralizing antibody
response to therapeutic proteins
www.coralgablessymposia.org
Gables Symposium 2012
provides a unique forum
for thought leaders to
address the principal
concerns regarding the
immunogenicity of
biopharmaceuticals; in their
development, regulation,
and clinical use.
www.coralgablessymposia.org
Scientific Organizing
Committee
Dr. Michael Tovey, Chair
Dr. Shalini Gupta, Amgen
Dr. Susan Kirshner, FDA
Dr. Daniel Kramer, Merck Serono
Dr. Robin Thorpe, NIBSC