1. Phospho-EGFR kit using a generic format
Sales training
FABIENNE CHARRIER-SAVOURNIN LAURENCE JACQUEMART June 2012 – V0
April 2013 – V1 (specific formats)

2. Contents Phospho- EGFR Slides 3/4 EGFR background
Slide 5 Pathway and therapeutic areas
Slide 6 target customer
Slide 7 kit contents
Slides 8/9/10 protocol
Slides 11/12 validation
on human A431 cells (various agonist stimulation)
on human A431, SKOV3 (EGF stimulation)
Slide 13/14 inhibition of stimulated A431 (EGF)
by small molecules (erlotinib, lapatinib)
by antibody (cetuximab)
Slide 15/16/17 additional data (influence of a starvation step, stimulation kinetics, cell density)
Slide 18 troubleshooting
Slide 19/20 benefits
Slide 21 associated HTRF products
Slide 19 background
Slide 22 to 27 specific formats – update April 2013
Phospho-EGFR (V1)

3. EGFR - background
EGFR = Epidermal Growth Factor Receptor / Other names: HER1, ErbB-1, ERBB
Family: ErbB = tyrosine kinase receptor (RTK)
contains an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmic domain with tyrosine-kinase function.
Activation
compounds
Dimerization P
Autophosphorylation
Detection
In normal cells, ligand binding (7 known ligands: EGF, TGF-a….) results in receptor dimerization with formation of homodimers (EGFR-EGFR) or heterodimers (EGFR with an other ErbB receptor). This step leads to the activation of the kinase domain and the autophosphorylation of distinct tyrosine residues located in the C-terminal tail of the receptor These phospho- residues create docking sites for adaptor proteins such as GRB2 and SHC which initiate multiple signal transduction cascades, MAPK and PI3K/AKT pathways mainly .
Key fonctions
These pathways lead to the activation of various transcription factors that then impact cellular responses such as proliferation, migration, differentiation and survival.
Phospho-EGFR (V1)

4. Detection with Phospho-EGFR
EGFR - background (2/2)
Activation
compounds
Dimerization P
Autophosphorylation
Detection with Phospho-EGFR
Activation of dowstream signaling (MAPK, Akt, JNK)
Internalization
degradation
Phospho-EGFR (V1)

5. EGFR signaling pathway
Associated therapeutic areas
Associated therapeutic areas :
Mainly in oncology
e.g. colorectal cancer, head-and-neck cancer , lung cancer & breast cancer
Anti-EGFR molecules :
Monoclonal antibodies, e.g. Cetuximab
Small molecule Tyrosine Kinase Inhibitors (TKIs), e.g. Erlotinib
Phospho-EGFR (V1)

6. Target customer Users: Therapeutic area of interest: oncology
Phospho-EGFR
Users:
Therapeutic area of interest: oncology
Phospho-EGFR: a target
Scientists working on small molecules or therapeutic antibodies
Identify/ characterize customers: Key questions to ask:
Do you measure protein phosphorylation in cells (versus biochemical)?
What technologies do you use (ELISA, WB, Surefire, Meso Scale?,…..)
Are you more into profiling or screening?
Can you tell me more about your kinases of interest? …..
Phospho-EGFR (V1)

7. Store the kit at -60°C or below upon reception
Kit contents
Phospho-EGFR
Included in the kit:
- 1 vial of anti-EGFR Tb3+ cryptate
- 1 vial of anti phospho-Tyr d2
- 1 vial of control lysate
- 2 vials of EGFR stimulation buffer
- 1 vial of blocking reagent
- 4 vials of EGFR lysis buffer
- 2 vials of EGFR detection buffer
- a package insert
500 tests (20µL final) + 10,000 & 50,000 tests
Ref 64HR1PEG /H/I
Store the kit at -60°C or below upon reception
Phospho-EGFR (V1)

8. Phospho-EGFR kit
This assay is designed for the quantitative determination of phospho-EGFR.
Phospho-EGFR is measured by a sandwich assay format using 2 different antibodies, one anti-EGFR Ab labelled with Tb-Cryptate (donor), and a second anti-phospho-Tyr Ab labeled with d2 (acceptor).
The intensity of the signal obtained is proportional to the number of antigen-antibody complexes formed and therefore the fluorescence is proportional to the concentration of the phopho-EGFR
Phospho-EGFR (V1)

9. Two plate assay protocol
1) Cells are plated in a culture plate for 24h
2) Cells are treated with compounds (10 to 30 min)
3) Supernatant is discarded and cells are lysed
4) Cell lysates are transferred in a 384 SV plate
5) Phosphorylated EGFR is detected with anti-EGFR-Tb and anti-phospho-Tyr-d2 after incubating ON at RT
Phospho-EGFR (V1)

10. One plate assay protocol
1) Suspension cells are plated in a 384 SV plate
2) Cells are treated with compounds (10 to 30 min)
3) Cells are lysed and phosphorylated EGFR is detected with anti-EGFR-Tb and anti-phospho-Tyr-d2 after incubating ON at RT
Phospho-EGFR (V1)

11. Validation on human A431 cells activated with various agonists
Phospho-EGFR
2 plate-assay protocol / 25Kcells/96-well plate / activation 10’ / lysis 30’ / ON incubation at RT
Phospho-EGFR (V1)

12. Validation on various human cell lines activated with EGF
Phospho-EGFR
1 plate-assay protocol / 5Kcells (A431) – 20Kcells (SKOVO3) – 10Kcells ST-EGFR / 384-well plate sv / activation 10’ / lysis 30’ /
ON incubation at RT
Phospho-EGFR (V1)

13. Pharmacological effect of inhibitors: small molecules inhibitors
A431 cells, 25Kc/W, 2 plate assay, +/- erlotinib, lapatinib/ EGF (PQ401 = negative control)
Good inhibition of the phosphorylation of EGFR triggered by inhibitors
Protocol:
- 50µl C/w/ / C/W / Incubation ON at 37°C 5% CO2 / 25µl [Antago] 3X ; Incubation 30 min at 37°C / 25µl 100nM EGF 4X / Incubation 10' at 37°C / supernatant removed by flicking / 50µl lysis buffer, incubation 30' at RT under shaking / transfer in 384w-plate
Phospho-EGFR (V1)

14. Pharmacological effect of inhibitor: antibody
A431 cells, 25Kc/W, 2 plate assay, +/- cetuximab/ EGF
Good inhibition of the phosphorylation of EGFR triggered by cetuximab
Protocol:
- 50µl C/w/ / C/W / Incubation ON at 37°C 5% CO2 / 25µl [Antago] 3X ; Incubation 30 min at 37°C / 25µl 100nM EGF 4X / Incubation 10' at 37°C / supernatant removed by flicking / 50µl lysis buffer, incubation 30' at RT under shaking / transfer in 384w-plate
Phospho-EGFR (V1)

15. Effect of a starvation step on SKOV3 cells
Phospho-EGFR
Starvation does not decrease basal level on SKOV3 cells
2 plate-assay protocol / SKOV3 50Kcells / 96-well plate / with & without starvation step / activation 10’ / lysis 30’ /
ON incubation at RT
Phospho-EGFR (V1)

16. A431: stimulation kinetics trigered by EGF
Phospho-EGFR
On A431 cells activated by EGF, signal remains stable from 2’ to 15’ of stimulation
2 plate-assay protocol / A431 50Kcells / 96-well plate / activation 2’, 5’,10’ , 15’ / lysis 30’ / ON incubation at RT
Phospho-EGFR (V1)

17. Optimal cell density: 5000 cells in 384 sv-well plate
A431 Cell density
Phospho-EGFR
Optimal cell density: 5000 cells in 384 sv-well plate
1 plate-assay protocol / A431 5, 10 & 20 Kcells / 384-well plate sv/ activation 10’ / lysis 30’ / ON incubation at RT
Phospho-EGFR (V1)

18. Trouble shooting
Phospho-EGFR
Lysates used for the assay must be generated by using the EGFR kit specific buffers
Stimulation, lysis and detection buffers have been set for optimal assay performances
Do not use the lysis and conjugate buffers from the other cellular kinase assays!
Anti-EGFR labeled with Tb3+-cryptate
Conjugate buffer with no KF
Be careful for readers: use the Tb specific set-up
Blocking reagent: it is the same « universal » anti-phosphatase cocktail used in all HTRF cellular kinase assays. It prevents dephosphorylation of phosphorylated proteins from active Ser/Thr and Tyr phosphatases present in cell lysates
Phospho-EGFR (V1)

19. HTRF offer's concrete benefits
Phospho-EGFR
Cost effective
Easy to run and faster
High robustness
HTRF kit
SureFire (PE)
MSD
ELISA
So What?
Residues
all the Y susceptible to be phosphorylated
Y1068
Y1068 on request
Y1173
CST Y845/Y1068/Y1173
Millipore Y1068 / Y1173
Sigma Y1173
Thermo Y1173
R&D Y1068 & generic
Process
1 incubation step
2 incubation steps
Several washing steps
Easy to run and faster
Stability
YES
NO (ligh,T° sensitive, signal ↘ after readout)
NO (50% signal lost after 1min)
NO (color will fade after 30 min)
Robust
Price -2013/well (20µL final)
3 / 0.8 / 0.5* ($/€)
5.5 / 4 ($)
4 – 5 ($/€)
Cost effective
Depending of the packaging: from 500 tests to 50,000 tests
Confidential
Phospho-EGFR (V1)

20. Value proposition Easier, Faster, Cheaper than ELISA and WB
Phospho-EGFR
Easier, Faster, Cheaper than ELISA and WB
Time and labor saving
Cost effective (miniaturizable)
No washing steps
One single distribution of reagents
A given lysate can be studied in parallel on 2 targets
Robust
Mix and read
Very low rate of interferences
Long signal stability
Multiple reading, can be measured as frequently as needed
Flexibility
Simple detection, large choice of instruments ( > 20 certified readers)
Several experiments can be run with a single kit (500 tests = 5 ELISA kits)
Phospho-EGFR (V1)

21. Associated HTRF® products
Phospho Product name Cat# Total Product name Cat#
Phospho-Erk1/2 (Cellul’erk) 64ERKPEG/H/I Total ERK1/2 64NRKPEG/H/I
Phospho-AKT (Ser473) 64AKSPEG/H/I Total AKT 64NKTPEG/H/I
Phospho-AKT (Thr308) 64AKTPEG/H/I Total AKT 64NKTPEG/H/I
Phospho-S6RP (Ser235/236) 64RP6PEG/H/I
Phospho-JNK (Thr183/Tyr185) 64JNKPEG/H/I
Phospho-c Jun (Ser63) 64JUNPEG/H/I
Phospho-STAT3 (Tyr705) 62AT3PEG/H/I Total-STAT3 64NT3PEG/H/I
Phospho-p38 MAPK (Thr180/Tyr182) 64P38PEG/H/I
Phospho-IKKb (Ser177/181) 64KKBPEG/H/I
Phospho-mTOR (Ser2448) 64TORPEG/H/I
Phospho-CREB (Ser133) 64CREPEG/H/I
Phospho-EGFR 64HR1PEG/H/I
Phospho-MEK1 (Ser218/222) 64ME1PEG/H/I Total-MEK1 64NE1PEG/H/I
Phospho-MEK1/2 (Ser218/222) 64ME2PEG/H/I*
Phospho-EGFR (V1)

22. Specific formats EGFR phosphorylation sites
Looking for a specific phosphorylation site on EGFR (Y845, Y1086, Y 1173, T669 …)
Contact our
Cisbio Custom Services group

23. EGFR phosphorylation sites
various biological functions
Phosphorylation sites immature form *
Y869
Y998
Y1016
Y1069
Y1092
Y1110
Y1172
Y1197
S695
S991
S1026
S1070
S1071
S1190
T678
T693
Phosphorylation sites mature form *
Y845
Y974
Y992
Y1045
Y1068
Y1086
Y1148
Y1173
S671
S967
S1002
S1046
S1047
S1166
T654
T669
apoptosis
inhibited X
Cell adhesion
alterated
Cell cycle regulation
Cell growth
Cell mobility
Cytoskeletal reorganization x
endocytosis
transcription
activation
Enzymatic activity
induced
inhibition
Intracellular localization
Molecular association
regulation
phosphorylation
Protein conformation
Protein degradation
Receptor desensitization
Receptor internalization
Receptor recycling
ubiquitination
Amino Acid numbers on the 1rst line of the table referred to the immature form of EGFR. AA numbers on the 2nd line correspond to the mature form (cleavage of peptide signal composed of the 24 first amino acids e.g. Y839 corresponds to Y845)
Anti-Phospho Y845, Y1086, Y1173, T669 validated
Phospho-EGFR (V1)

24. Detection of EGFR auto phosphorylation in A431 cells
Generic format versus specific format (P-Y1173 detection)
Generic detection = specific P-Y1173 detection
Phospho-EGFR (V1)

25. Detection of EGFR auto phosphorylation in A431 cells
Assay protocol
 Two plate assay protocol
Day 1 : Plate 25Kc/W in a 96-well tissue culture treated plate (in 50 µL of complete medium) and incubate 24h, at 37°C in CO2 atmosphere.
Day 2 :
- Ligand dose-response : add 50 µL of ligand (2x) diluted in serum-free medium and incubate 10 min at RT.
- Inhibition assays : add 25 µL of compound (3x), incubate 30 min at RT, then add 25 µL of ligand (4x) and incubate 10 min at RT.
Remove carefully cell supernatant.
Add 50 µL of supplemented lysis buffer and incubate 30 min at RT with shaking.
Transfer 16 µL of cell lysate in a 384-well small volume black plate.
Add 2 µL of each HTRF conjugates (anti-phospho-EGFR-d2 / anti-total EGFR-Tb) and incubate 4h at RT.
Phospho-EGFR (V1)

26. HTRF detection on differents phosphorylation sites
Conjugates : total mAb –Tb (Ab15) (0.25 nMf) / antiphospho-d2 (12 nMf)
Phospho-EGFR (V1)

27. Validated anti-phospho EGFR from Milllipore
Millipore ref #
AM014
antipThr669 (clone 5F10)
04-281
AM015
AntipY845 (clone 12A3)
04-283
AM016
AntipY1086 (clone 8B8)
04-286
AM017
antipY1173 (clone 9H2)
05-483
Phospho-EGFR (V1)

28. Thank you