Establishment of a Cell-Based Assay Specific for a Growth … · [email protected]....
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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
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)
GM-CSF is a hematopoietic growth factor that plays a central role in in the generation of neutrophils, macrophages, and DCsGM-CSF acts together with IL-3 and IL-5 to regulate the survival proliferation, differentiation, and functional activation of hematopoietic cellsGM-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.CIS and SOCS3 also regulate EPO induced Jak2/STAT5 signaling
Quantification of GM-CSF Activity
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 growthDue to overlapping biological activities, IL-3 or EPO, can also support the proliferation of these cells and act synergistically together with GM-CSF. M-CSF & IL-1 can also enhance GM-CSF dependent cell proliferation TGF-β and IFNα, or IFN β, can antagonize this activityIL-3 & IL-5 share a common receptor βc chain with GM-CSFThus, proliferation based assays for GM-CSF are subject to non specific interference.
Development of a Cell-based Assay Specific for GM-CSF: Strategy
Use a cell line that possesses functional GM-CSF receptors, but does not require GM-CSF or other related growth factors for proliferationU937 cells possess functional GM-CSF receptors but not express functional EPO receptorsU937 cells do not require GM-CSF, EPO or other related growth factors for proliferationTransfect U937 cells with a GM-CSF responsive reporter-gene construct
CM-CSF-SpecificRegulatory Element
LuciferaseSV40
Min. prom.
Reporter-Gene Assay: Construction
SV40Poly A
Intron Human β-globine
GM-CSF Receptor
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-5GM-CSF receptor does not possess intrinsic tyrosine kinase activity; associates with Jak2 required for βc trans-phosphorylation, initiation of signaling, & biological activityReceptors for GM-CSF, IL-3, & IL-5 are expressed at very low levels (100-1,000 receptors/cell)GM-CSF, IL-3, & IL-5 each bind with low affinity to their specific Rα chain (Kd = 0.2 – 100 nM)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
Overexpress the GMRα, GM-CSF ligand-specific binding sub-unit, of the human GM-CSF receptorHypothesis, GMRα receptor chain will compete with IL-3 or IL-5 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.
TATA
GM-CSF Gene Reporter Assay (FireFly Luciferase)
SV40Poly A
Intron
+1
Stat5 Stat5 GM-CSF
(T/A)TTCnnnGAA(T/A) FireFly Luciferase
STAT5 Consensus Sequence: (T/A)TTCnnnGAA(T/A)
GATTTCTAGGAATTCAAATCGGATCTAGATTTCTAGGAATTCAAATCGGATCTAGATTTCTAGGAATTCAAATCGGATCTAGATTTCTAGGAATTCAAATCG
STAT5 con. Dx4
GATTTCTAGGAATTCttctcagaaGAATTCttctgagaaGAATTCttctcagaaGAATTCttctgagaaGAATTCttctcagaaGGAATTCAAATCG
TTCCCCGAAATGATGAGCTAGGAGCCTGATTTCCCCGAAATGATGAGCTAGGAGCCTGATTTCCCCGAAATGATGCTAGGAGCCTGATTTCCCCGAAATGATctGtTAG
GAGGCTCTGATTTCCGGGAAACTGATTCCCGGAATACGTTTTCCGGGAATACGTTTCCGGGAAACGTATTCCGGGAAAACTGATTCCGGGAAATGATCTGTTAG
STAT5 con. Cx6
STAT5 con. Ax4
STAT5 con. Bx6
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STAT 5 Reporter Gene Constructs – Firefly luciferase A, B, C or DTranscient transfection in U937 cells
STAT5-luc STAT5-luc
A B C D
STAT5 Reporter Gene – Firefly luciferase (C or D) relative to Renilla luciferase expression
Transcient transfection in K562 and U937 cells
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K562 U937STAT5 is expressed
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STAT5 - luc C D C D
Effect of rhEPO and rhIFNγ on STAT5 – luciferase (C or D) Reporter GeneTranscient transfection in U937 cells
STAT5 C STAT5 D0
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500ng/ml rhEPO
10000 IU/ml IFNg
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Clone N°
Analysis of Clones Transfected with the US5‐Luc Construct
Selection of stable clones transfected by STAT5-luc C in U937 cell line
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rh GM-CSF 18h
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Firefly Luciferase induction in U937/STAT5-luc stable cell line
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Cell number optimisation for the GM-CSF assayStimulation with 200ng/ml rhGM-CSF (Gibco®)
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GM-CSF Standard Curves
StandardNIBSC
TATA
GM-CSF Gene Reporter Assay (FireFly / Renilla)
SV40Poly A
Intron
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Stat5 Stat5 GM-CSF
(T/A)TTCnnnGAA(T/A) FireFly Luciferase
CMVSV40Poly A
Intron
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GM-CSF-Rα+1
Double transcient transfection STAT5-luc (C or D) and pCMV-GM-CSF-Rα in U937 cells
Control GM-CSF-Rα Control GM-CSF-Rα
STAT5 DSTAT5 C
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rh IL-3Immunotools® (ng/ml)
rh IL-5Immunotools® (ng/ml)
Firefly Luciferase induction in the U937 cell line transciently tranfected with STAT5-luc and GM-CSF-Rα treated with GM-CSF, IL-3 or IL-5
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Analysis of RUS5-Luc clones Expressing RenillaLuciferase and GM-CSFα Receptor Chain
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TATA
GM-CSF Gene Reporter Assay (FireFly / Renilla)
SV40Poly A
Intron
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Stat5 Stat5 GM-CSF
(T/A)TTCnnnGAA(T/A) FireFly Luciferase
CMVSV40Poly A
Intron
+1 Renilla LuciferaseIRES
GM-CSF-Rα+1
Transient Cotransfection of U937 Cells
FireFly Activity
Renilla Activity
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STAT5-Luc+EGFP
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STAT5-Luc+EGFP
Normalized Reporter Gene Assay for GM-CSF
Unexpectedly, constitutive expression of Renilla luciferase is influenced (2x) by STAT5 activation in response to GM-CSF treatmentConsequence effective dynamic range of assay reducedMechanism unclear, no Stat5 recognition sequences in promoter construct Solution change promoter use SV40 constitutive promoter & Herpes simplex TK promoterUse humanized Gaussia luciferase gene instead of Renilla luciferase
GM-CSF Induced FL Expression Normalized Relative to Gaussia Expression (4 H Induction)
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US5 17.5.21 SV
US5 17.5.8 TK
GM-CSF Induced FL Expression Normalized Relative to Gaussia Expression (16 H Induction)
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US5 17.5.21 SV
US5 17.5.8 TK
Relationship Between Drug Induced FL Expression and Cell Number
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Response of Reporter Cells to Serum Matrix Effects
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RA Sera 10%
RA sera 20%
Normal Sera 10%
Control
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RA sera 20%
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Control
Stability Studies: Sensitivity
Time (Hrs) Passage # EC50 (pg/ml) LLOQ (pg/ml)4 10 40 104 20 80 204 40 20 518 10 400 20018 20 400 20018 40 200 100
Stability Studies: Proliferation
Passage # Doubling Time (Hrs)
Max Cell Density (x106/ml)
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10 25 0.95
20 24 1.2
40 24 1.1
Conclusions - I
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 proliferationThe cell does, however, contain a functional receptor/signal transduction system for the growth factor of interestThe assay can be rendered specific by over-expression of a growth factor specific binding receptor sub-unit
Conclusions - II
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.
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Stat4‐kozak ‐ + ‐ ‐ + ‐ +
CX2RB1 ‐ ‐ + ‐ ‐ + +
CX2RB2 ‐ ‐ ‐ + + + +
STAT4‐Luc + + + + + + +
Reconstitution of a Functional Cytokine Signaling Pathway in Human U937 cells
Stat4‐kozak ‐ + ‐ ‐ + ‐ +
CX2RB1 ‐ ‐ + ‐ ‐ + +
CX2RB2 ‐ ‐ ‐ + + + +
STAT4‐luc + + + + + + +
Reconstitution of a Functional Cytokine Signaling Pathway in Human HEK cells
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Conclusions - III
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
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.
Scientific Organizing Committee
Dr. Michael Tovey, ChairDr. Shalini Gupta, AmgenDr. Susan Kirshner, FDADr. Daniel Kramer, Merck SeronoDr. Robin Thorpe, NIBSC
www.coralgablessymposia.org