5 minutes of fame

1. The added value of
cellular analysis in
Early (SAD/MAD)
Clinical Trials
Exciting possibilities…
Roger Greathead

2. Fast Track to Proof of Concept
Choices in Early Clinical Development: Maximal effect, Minimal costs
Discovery Toxicology Phase I/IIa Phase IIb Phase III
Attrition
Attrition Risk
Risk
Phase I/IIa
Phase I/IIa
Costs per Compound # Compounds
2 12/12/12

3. Cellular Analysis
• What is Flow cytometry (short)
• Results of Studies at PRA

4. What is flow cytometry (FACS)?
• Flow=fluid stream, cyto=cell, metry=measuring
• Fluorescence Activated Cell Sorting
– Uses fluorochrome-tagged (colored) antibodies
– Antibodies can be specific for:
• Membrane proteins (receptors, CD-markers)
• Intracellular proteins (phosphorylated proteins)
• Cytokines (intracellular staining)
• The fluorescent signals of each single cell are
analyzed by FACS

5. What does PRA do with flow cytometry
• Quantitative assessment of white blood cell subsets
• Assessment of cell surface antigens (characterization
of white blood cell subsets)
• Assessment of the activation status of various cell
types (with effect of compound)
• Assessment of cell functions: intracellular cytokine
production and apoptosis (with effect of compound)
• Receptor occupancy assessment

6. Basic FACS protocol
Stimulation Incubation with Analyze sample
(37 °C, 30min) FACS antibodies by FACS
1
6
3
5
Lysis of red Wash cells
2 4
blood cells / (remove excess
Wash cells antibodies)
Blood
(fresh)

7. How to ‘read’ FACS plots?
Lysed whole blood
0.6%
Unstimulated:
Side scatter
62.3%
IL-2 stimulated:
Forward scatter
“dot-plots” “histogram-plots”

8. FACS Studies at PRA
• Inhibition of PI3K
• Toll-Like Receptor 2 blockade
• Inhibition of JAK1/3 signaling pathway

9. FACS Studies at PRA
• Inhibition of PI3K signaling
– PI3K signaling, involved in cell activation/survival and
differentiation
– Targeting PI3K may provide opportunities to develop
therapies against inflammatory diseases as well as
hematologic cancers
– Read-out: expression of CD63 on the membrane of
activated basophils

10. Principle of Basophil activation

11. Inhibition of CD63 is dose-dependent
Ex-vivo dose response in basophils
after stimulation with FcεR1
100
% CD63 of CCR3/SSC_low
Donor 1
80 Donor 2
Donor 3
60
40
20
0
1. 0 -01
1 . 0 00
1. 0 01
1. 0 02
1. 0 03
1. 0 04
1. 0 05
1. 0 06
07
08
10
10
1
1
1
1
1
1
1
1
0×
0×
0×
0×
0×
0×
0×
0×
0×
0×
1.
1.
Concentration compound (µM)

12. CD63 expression in dosed volunteers

13. Integration of PK/PD results:
PK vs PD
Cohort 1 PK
1.0
% CD63 of CCR3/SSC_low
1000 Cohort 2 PK
Cohort 3 PK
compound (ng/ml)
0.8
Concentration
100 Cohort 4 PK
0.6 Cohort 5 PK
10 Cohort 6 PK
0.4
Cohort 1 PD
1 0.2 Cohort 2 PD
2 4 6 10 20
Cohort 3 PD
0.1 time (h) 0.0 Cohort 4 PD
Cohort 5 PD
Cohort 6 PD

14. FACS Studies at PRA
• Toll-Like Receptor 2 blockade
– TLR2 is an important receptor of the immune system,
involved in detection of pathogens (recognizes bacterial
cell component peptidoglycan)
– Can also detect endogenous ‘danger-molecules’
– TLR2 blockade may be beneficial after transplantation
because this significantly reduces the influx of immune
cells
– The compound is an antibody (IgG4) that binds to TLR2
– FACS: detection of bound antibody

15. FACS Studies at PRA
• Toll-Like Receptor 2 blockade
– Method:
• Tube 1: blood from subject + Candidate Drug + anti-
IgG4(FITC) 
determination of the max fluorescence (all receptors
occupied)
• Tube 2: blood from subject + anti-IgG4(FITC) 
determination of receptor occupancy
– Ratio tube2/tube1 = receptor occupancy %

16. Results – Cohort 1 – starting dose
OPS444EC-111133-H cohort 1
160
140
120
101
% occupancy
100
102
103
80
104
105
60
106
40
20
0
0h 3h 24 h Day 7 Day28 Day 56 Day 90
tijd
time

17. Cohort 2
OPS444EC-111133-H cohort 2
140
120
100 201
% occupancy
202
80
203
204
60
205
40 206
20
0
0h 3h 24 h Day 7 Day28 Day56 Day90
tijd
time

18. Cohort 3
OPS444EC-111133-H cohort 3
200
180
160
140 301
% occupancy
120 302
303
100
304
80 305
60 306
40
20
0
0h 3h 24 h Day 7 Day28 day 56 Day90
tijd
time

19. FACS Studies at PRA
• Inhibition of JAK1/3 signaling pathway:
– Read-out: phosphorylation of STAT-5
• Transcription Factor
• Gene transcription
• Cell activation/proliferation
– Target for chronic
inflammatory reactions:
• Autoimmunity
• Allograft rejection

20. Inhibition of pSTAT5 is Dose-Dependent
after stimulation of IL-2

21. Cohort 2
IL-2 induced p-Stat5 in lymphocytes
200.0
180.0
% Activity (relative to predose, t=0)
160.0
Subject
140.0 1005
Subject
120.0
1006
100.0 Subject
1007
80.0 Subject
60.0 1008
40.0
20.0
0.0
0 2 4 6 8 10 12 14 16 18 20 22 24
sample time (hours)

22. Summary
• Flow Cytometry can be used to demonstrate
efficacy of Drugs as early as Phase I
(SAD/MAD)
• Data used for Phase II dose selection and trial
duration without the time and expense of
recruiting patients as well as validation of
mechanism of action