The added value of cellular analysis in Early (SAD/MAD) Clinical Trials

Exciting possibilities…Roger Greathead

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

Cellular Analysis

• What is Flow cytometry (short)• Results of Studies at PRA

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

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

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%

FACS Studies at PRA

• Inhibition of PI3K• Toll-Like Receptor 2 blockade• Inhibition of JAK1/3 signaling pathway

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

Principle of Basophil activation

Inhibition of CD63 is dose-dependentEx-vivo dose response in basophils

after stimulation with FcR1

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CD63 expression in dosed volunteers

Integration of PK/PD results:PK vs PD

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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

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 %

Results – Cohort 1 – starting dose

OPS444EC-111133-H cohort 1

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Cohort 2

OPS444EC-111133-H cohort 2

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Cohort 3

OPS444EC-111133-H cohort 3

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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

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

IL-2 induced p-Stat5 in lymphocytes

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ctivi

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Subject1005Subject1006Subject1007Subject1008

Cohort 2

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