Download Hardcastle, A., et. al. Pharmacodynamic markers of response to

Pharmacodynamic markers of
response to novel anticancer
agents using a protein profiling
sandwich immunoassay format
Anthea Hardcastle,
Matthew Cordwell, Peter Fong, Lindsay Stimson,
Paul Workman and Wynne Aherne
Cancer Research UK Centre for Cancer Therapeutics
Institute of Cancer Research
Sutton, Surrey, UK
Molecular mechanism-based drug discovery
Medicinal chemistry, combinatorial chemistry
structure-based design
Target
Identification
Target
validation
Biochemical,
phenotypic,
virtual screens
Compound collections
Mechanistic &
cell-based
assays
In vivo
evaluation
Clinical
evaluation
‰ Mechanistic endpoint or pharmacodynamic
(PD) marker assays
‰ Pharmacokinetics and metabolism
‰ Assays required for all matrices
‰ Sample often limited so high sensitivity required
Techniques for PD marker measurement
‰ Western blotting (Gold Standard’)
‰ Immunohistochemistry
‰ Flow cytometry
‰ Microplate immunoassays (ELISA)
‰ MSD Protein Profiling sandwich immunoassays
¾ Multiplex ‘catalogue’ assays
¾ Single assay development ‘in-house’
¾ Option to multiplex ‘in-house’ and ‘catalogue’
assays
The ease of validation for GCLP is an important consideration for PD assays
as compliance with regulatory requirements is essential
Meso Scale Discovery (MSD) technology
Measured signal is light
LIGHT
z
Can these assays be applied to different matrices?
‰ In vitro cell lysates
‰ Human tumour xenografts
‰ Clinical samples eg. PBMCs, tumours and plasma
Aim : to carry out a feasibility study with inhibitors
of HSP90 and HDAC
PD markers for inhibitors of HSP90
‰ The molecular chaperone HSP90 maintains the
conformation, stability and function of oncogenic
client proteins (eg. ERBB2, AKT and CDK4)
‰ HSP90 inhibitors cause degradation of client
proteins, disruption of signalling pathways and
antitumour activity
‰ Several agents currently in Phase I and II trials
e.g. 17-AAG and 17-DMAG
‰ The molecular signature of HSP90 inhibition
includes a fall in ERBB2, AKT and pERK and
induction of HSP70
‘Off the shelf’ MSD duplexed assays
for PD markers of HSP90 inhibition
1
Phospho
protein
2
SULFO-TAGTM labelled,
detection antibody
BSA
A
B
BSA
1
2
3
4
Protein
Capture
Antibody
Total Protein
Working Electrode
pERK
Total ERK
35
Total ERBB2
25
20
15
10
1000
Ru/µg protein
Ru/µg protein
30
750
500
5
250
0
0
DMSO
Geld
DMSO
Geld
HCT116 cells,1µM geldanamycin,24h. 1.25-10µg protein/well
‘Off the shelf’ duplexed assays for PD
markers of HSP90 inhibition
Total AKT
pAKT
700
100
75
500
400
50
300
200
25
Phospho AKT
Total AKT
600
100
0
0
DMSO
Geld
LY
HCT116 cells,1µM Geldanamycin,20µM LY294002 (LY) 24h. 1.25-10µg protein/well
‘In-house’ MSD assay for HSP70
SULFO-TAGTM labelled,
Goat anti rabbit IgG
‰ DELFIA HSP70 assay validated to
Calibration curve
Ru counts
100000
50000
0
0
1000 2000 3000 4000 5000 6000 7000
fmoles HSP70/well
2
Rabbit anti
HSP70
A
HSP70
Capture HSP70
Monoclonal Antibody
B
Working
Electrode
fmole HSP70/µg lysate
GCLP
‰ In use for clinical trials of HSP90
inhibitors
‰ Assay transferred to MSD
‰ Potential for multiplexing HSP70
with client protein expression
1
1750
HCT116 ± 1 µM Geld 24h
1500
1250
1000
750
500
250
0
DMSO
Geld
PD markers for HDAC inhibitors
‰ HDACs catalyse the deacetylation of histones and are important
for the regulation of gene expression
‰ HDACs are involved in the development and progression of
malignancy
‰ HDACIs display antitumour activity and several compounds are
being evaluated clinically
‰ Their precise mechanism of action is not clear but the most
obvious result of HDAC inhibition is hyperacetylation of histones
‰ Hyperacetylation of histone H3 is used as a PD marker for HDAC
inhibition
‘In-house’ assay for acetyl histone H3 (AcH3)
SULFO-TAGTM labelled,
Goat anti rabbit IgG
Ac histone H3
Histones
Capture Pan Histone
monoclonal antibody
Ru counts
Rabbit anti AcH3
10000
7500
5000
Butyrate-treated
HeLa cell extract
2500
0
Working
Electrode
Calibration curve
12500
0
100
150
200
250
300
ng/well AcH3 standard
HCT116 5X GI50 SAHA 24h
ng equivalents / µg lysate
50
DMSO SAHA
1000
AcH3
100
GAPDH
10
1
DMSO
SAHA
Assay being successfully used to measure
the effect of novel HDACIs on AcH3 in
human tumour xenografts
Ex vivo treatment of PBMCs with HDACIs
‰ PBMCs and plasma separated from whole blood
‰ HDACIs SAHA and MS-275 or DMSO (control) added
‰ Incubated at 37°C for 4h
‰ PBMCs separated,washed and lysed
2
MS-275
AcH3
1
0
SAHA
ng equivalents AcH3
3
Ex vivo PBMNCs 4h 5XGI50
DMSO
‰ AcH3 in lysates measured by MSD assay and western blot
GAPDH
DMSO
SAHA
MS275
This assay is being validated to GCLP
for use in clinical trials of HDACIs
Summary
‰ Experience with the MSD format is encouraging
‰ robust
‰ sensitive
‰ minimal matrix interference
‰ quantitative, higher-throughput alternative to western blots
‰ both catalogue and in-house assays used
‰ Provided proof of principle for mechanism of action in 2 therapeutic
areas, HSP90 and HDAC, in different sample matrices
‰ Now used in a number of drug discovery projects in the Centre
‰ Assays are amenable to GCLP validation to comply with regulatory
requirements for clinical trials
‰ Multiplexing potential for ‘in-house’ assays to be investigated
Acknowledgements
Matthew Cordwell, Peter Fong, Lindsay Stimson,
Paul Workman and Wynne Aherne
Colleagues in the Cancer Research UK Centre for Cancer
Therapeutics
Members of the Analytical Technology and Screening Team
Tuc Ahmad from MSD