Pharmacodynamic markers of response to novel anticancer .../media/files/customer...GCLP In use for clinical trials of HSP90 inhibitors Assay transferred to MSD Potential for multiplexing

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 TherapeuticsInstitute of Cancer Research

Sutton, Surrey, UK

Molecular mechanism-based drug discovery

Target Identification

Compound collections

Medicinal chemistry, combinatorial chemistrystructure-based design

In vivoevaluation

Mechanistic & cell-based

assays

Biochemical, phenotypic,

virtual screens

Target validation

Mechanistic endpoint or pharmacodynamic(PD) marker assays

Pharmacokinetics and metabolism

Clinical evaluation

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

Multiplex ‘catalogue’ assaysSingle assay development ‘in-house’Option to multiplex ‘in-house’ and ‘catalogue’

assaysThe ease of validation for GCLP is an important consideration for PD assaysas compliance with regulatory requirements is essential

Meso Scale Discovery (MSD) technologyMeasured signal is light

z

LIGHT

Can these assays be applied to different matrices?In vitro cell lysatesHuman tumour xenograftsClinical 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 oncogenicclient 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 2A

B

Total Protein

1 2

3 4

BSAPhosphoprotein

BSA

SULFO-TAGTM labelled,detection antibody

Working Electrode

Capture Antibody

Protein

HCT116 cells,1µM geldanamycin,24h. 1.25-10µg protein/well

DMSO Geld0

5

10

15

20

25

30

35

Total ERBB2

Ru/ µ

g pr

otein

DMSO Geld0

250

500

750

1000

pERKTotal ERK

Ru/ µ

g pr

otein

‘Off the shelf’ duplexed assays for PD markers of HSP90 inhibition

DMSO Geld LY0

100

200

300

400

500

600

700 pAKTTotal AKT

0

25

50

75

100To

tal A

KT

Phospho AK

T

HCT116 cells,1µM Geldanamycin,20µM LY294002 (LY) 24h. 1.25-10µg protein/well

‘In-house’ MSD assay for HSP70

Capture HSP70 Monoclonal Antibody

SULFO-TAGTM labelled,Goat anti rabbit IgG

HSP70

Rabbit anti HSP70

Working Electrode

1 2A

B

DELFIA HSP70 assay validated to GCLP

In use for clinical trials of HSP90 inhibitors

Assay transferred to MSDPotential for multiplexing HSP70

with client protein expression

Calibration curve

0 1000 2000 3000 4000 5000 6000 70000

50000

100000

fmoles HSP70/well

Ru c

ount

s

HCT116 ± 1 µM Geld 24h

DMSO Geld0

250

500

750

1000

1250

1500

1750

fmole

HSP7

0/µg

lysa

te

PD markers for HDAC inhibitorsHDACs 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

Rabbit anti AcH3

Ac histone H3

Working Electrode

HistonesCapture Pan Histonemonoclonal antibody

Calibration curve

0 50 100 150 200 250 3000

2500

5000

7500

10000

12500

Butyrate-treated HeLa cell extract

ng/well AcH3 standard

Ru c

ount

s

Assay being successfully used to measure the effect of novel HDACIs on AcH3 in

human tumour xenograftsDMSO SAHA

1

10

100

1000HCT116 5X GI50 SAHA 24h

ng e

quivalen

ts /

µg

lysa

te DMSO SAHA

AcH3

GAPDH

Ex vivo treatment of PBMCs with HDACIsPBMCs 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

AcH3 in lysates measured by MSD assay and western blot

Ex vivo PBMNCs 4h 5XGI50

DMSO SAHA MS2750

1

2

3

ng e

quivalen

ts A

cH3

AcH3

GAPDH

DM

SO

SAH

A

MS-

275

This assay is being validated to GCLP for use in clinical trials of HDACIs

SummaryExperience with the MSD format is encouraging

robust sensitiveminimal matrix interferencequantitative, higher-throughput alternative to western blotsboth 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