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The added value of cellular analysis in Early (SAD/MAD) Clinical Trials
Exciting possibilities…Roger Greathead
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04/13/232
Fast Track to Proof of ConceptChoices in Early Clinical Development: Maximal effect, Minimal costs
Discovery Toxicology Phase I/IIa Phase IIb Phase III
Phase I/IIaPhase I/IIa
# Compounds
AttritionAttrition
Costs per Compound
RiskRisk
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Cellular Analysis
• What is Flow cytometry (short)• Results of Studies at PRA
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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
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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
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Basic FACS protocol
Blood(fresh)
Stimulation(37 °C, 30min)
Lysis of redblood cells /Wash cells
Incubation withFACS antibodies
Wash cells(remove excess
antibodies)
Analyze sampleby FACS
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How to ‘read’ FACS plots?
Forward scatter
Side
sca
tter
Unstimulated:
IL-2 stimulated:
Lysed whole blood
“dot-plots” “histogram-plots”
0.6%
62.3%
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FACS Studies at PRA
• Inhibition of PI3K• Toll-Like Receptor 2 blockade• Inhibition of JAK1/3 signaling pathway
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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
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Principle of Basophil activation
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Inhibition of CD63 is dose-dependentEx-vivo dose response in basophils
after stimulation with FcR1
1.0×10
-01
1.0×
1000
1.0×
1001
1.0×
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1003
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1004
1.0×
1005
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1007
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1008
0
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100Donor 1Donor 2Donor 3
Concentration compound (µM)
% C
D63
of
CC
R3/
SS
C_l
ow
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CD63 expression in dosed volunteers
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Integration of PK/PD results:PK vs PD
2 4 6
0.1
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1000
10 20
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1.0Cohort 1 PKCohort 2 PKCohort 3 PKCohort 4 PKCohort 5 PKCohort 6 PK
Cohort 1 PD
Cohort 3 PDCohort 4 PDCohort 5 PDCohort 6 PD
Cohort 2 PD
time (h)
Co
nce
ntr
atio
nco
mp
ou
nd
(n
g/m
l)
% C
D63 o
f CC
R3/S
SC
_low
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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
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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 %
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Results – Cohort 1 – starting dose
OPS444EC-111133-H cohort 1
0
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0 h 3 h 24 h Day 7 Day28 Day 56 Day 90
tijd
% o
ccu
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time
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Cohort 2
OPS444EC-111133-H cohort 2
0
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0 h 3 h 24 h Day 7 Day28 Day56 Day90
tijd
% o
ccu
pan
cy
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time
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Cohort 3
OPS444EC-111133-H cohort 3
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0 h 3 h 24 h Day 7 Day28 day 56 Day90
tijd
% o
ccu
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time
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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 chronicinflammatory reactions:
• Autoimmunity• Allograft rejection
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Inhibition of pSTAT5 is Dose-Dependent after stimulation of IL-2
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IL-2 induced p-Stat5 in lymphocytes
0.0
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40.0
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0 2 4 6 8 10 12 14 16 18 20 22 24
sample time (hours)
% A
ctivi
ty (r
elati
ve t
o pr
edos
e, t
=0)
Subject1005Subject1006Subject1007Subject1008
Cohort 2
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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
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