Selection of safer and more effective anti-inflammatory kinase inhibitors using a platform of primary human cell

based disease models (BioMAP® systems)

Ellen L. Berg

17 April 2012

Challenges of Kinase Drug Discovery

Defining optimal target selectivity• Kinase gene family members (> 500 members) are highly related• Biochemical ≠ cellular efficacy

Limited knowledge about target biology• Individual kinases can play a role in multiple pathways - complex signaling• Is there target biology that we are missing?

Unclear significance of secondary activities• There will be secondary targets at a high enough concentration• Are secondary targets affecting safety and/or efficacy?

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Solution – Better Biology Tools

BioMAP Systems• Primary human cell-based disease & tissue models• Link in vivo complexity with in vitro reproducibility

Simultaneous evaluation of compounds across a broad range of human biology • Reveal unexpected activities• Test compounds in more “physiological” settings• Build confidence in therapeutic hypotheses, prioritized leads

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BioMAP® Technology Platform

BioMAPAssays

Human primary cells Disease-models

30+ systems

LPS

BF4T

SM3C

ReferenceProfile Database

Predictive Informatics Tools

Biomarker responses to drugs are stored in the

database

Specialized informatics tools are used to predict

clinical outcomes

Human Biology Integrated into a Robust, Scalable Platform

Compound Validation

Lead Optimization

Efficacy Biomarkers

Safety Pharmacology

Adverse Effects

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BioMAP® Systems

Human primary cell-based assays Engineered to model complex human disease biology

LPS

BF4T

HSM3C

Physiologically relevant assay conditions• Mixtures of stimulation factors, co-cultures of cells

• Complex culture conditions selected to achieve stable signaling networks that reflect in vivo tissue & disease states

Quantitative and reproducible • Robust readouts (proteins, mediators); standardized assays

• Assay formats manage disease variations among donors

Validated with known drugs• Large reference database

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Example Compounds in Development

Tofacitinib (CP-690,550)• Jak 3 kinase inhibitor – FDA review for rheumatoid arthritis

Fostamatinib (R788)• Syk kinase inhibitor – Phase III for rheumatoid arthritis

CAL-101 (GS-1101)• PI3K-d inhibitor – Phase III oncology

RN786• BTK inhibitor - Preclinical

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Tofacitinib

TofacitinibCP-690,550

Jak 3 kinase selective inhibitor Kinase activity: EC50s: Jak3 (1 nM); Jak2 (20 nM); Jak1 (112 nM); Rock-II (3.4 mM) and Lck

(3.8 mM) (Changelian, Science 2003, 302:875). Binding: JAK3 (2.2 nM) and Jak2 (5 nM) (Karaman, Nat Biotech 2008, 26:127).

Safe and effective in rheumatoid arthritis Kremer, Arthr & Rheum 2012, 60:1895; Fleischmann, Arth & Rheum 2012, 64:619 In review at the FDA for rheumatoid arthritis (Pfizer)

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BioMAP Profile of Tofacitinib

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BioMAP® SystemsBioMAP Systems

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10 primary human cell types 12 assay systems• Different cell types, co-culture combinations• Different activation conditions – disease models

BioMAP® Systems – Cell Types & Co-cultures

B cells

Endothelial Cells

Bronchial Epithelial Cells

Keratinocytes

Smooth Muscle Cells

Dermal Fibroblasts

Lung Fibroblasts

Peripheral Blood Mononuclear Cells

Macrophages

BioMAP Systems

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BioMAP® Systems – Biology Covered

Vascular Biology

Wound Healing

Th1 Inflammation

Skin Biology

Th2 Responses

Immune Biology

Th17 Biology

Lung Biology

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BioMAP Profile of Tofacitinib

95% significance envelope

Control (no drug)

DoseResponse

Readout Parameters (Protein Biomarkers)

Cytotoxicity Readouts

BioMAP Systems

Log expression ratio (Drug/DMSO control)

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BioMAP Profile of Tofacitinib (≤ 370 nM)

Key activities of Tofacitinib (CP-690,550 ) ≤ 370 nM• Inhibition of IL-4 dependent signaling in endothelial cells (4H system)• Selective inhibition of T-cell-dependent B cell activation (BT system)• Several activities consistent with clinical efficacy biomarkers (in red)

Eotaxin-3

IgG

TNFa

IL-2

IL-6

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IgG

TNFaIL-6

De Paz, 2010Lin, 2010

Kutukculer, 1998

BioMAP Profile of Tofacitinib (≥ 370 nM)

Many additional activities at higher doses (≥ 370 nM):• Most are clinical efficacy biomarkers for RA (Kuan, 2010; Klimiuk, 2002; Kutukcular,

1998; Dolhain, 1998; Metawi, 2011) (in red) Activities are consistent with clinical effects and dosing

• Van Gurp, Transpl. 2009, 87:79 • Cmax in one clinical study was ~1 mM (Cohen, BJCP 2010, 69:143)

Higher dose (less selective) profile is more “efficacious” in BioMAP and in the clinic

Eotaxin-3

IgG

TNFa

IL-2

MIGHLA-DR

VCAM CD38

MIG

CD40IL-17A, F

MIG

HLA-DRMIG

HLA-DRICAM

IP-10

MIGIP-10IP-10

VCAMIL-6

CD69

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IgG

TNFa

MIG

VCAM CD38

MIG IL-17A, FMIG

MIGIP-10IP-10

VCAMIL-6 ICAM

IP-10MIG

Fostamatinib (R788)

Syk kinase inhibitor Kinase activity: IC50= 41 nM (active form). Braselmann, JPET 2006, 319:998

Well tolerated in Phase II studies for rheumatoid arthritis Genovese, Arth & Rheum 2011, 63:337 Currently in Phase III clinical trials for RA (Rigel/AstraZeneca)

R788Fostamatinib

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BioMAP Profile of Fostamatinib

Key activities of Fostamatinib (R788) • Broadly active in endothelial cells, leukocytes, epithelial cells and SMC• Inhibition of monocyte activation, T cell activation, and T-dependent B cell activation• Many activities (in red) consistent with clinical efficacy biomarkers (Szekanecz, 1997;

Klimiuk, 2002, 2005; Kutukculer, 1998; Kaun, 2010) Consistent with Clinical effects

• Peak serum concentrations of > 800 ng/ml (1.4 mM) reported in clinical studies (Podolanczuk, Blood 2009, 113:154)

Eotaxin-3 TNFa

TF uPAR

MIG

uPAR

IL-6

IL1auPAR

MCP-1

MIP1auPAIL-8IL-1a

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MMP3

TNFa

uPAR

MIG

uPAR

IL-6

uPAR

MCP-1

IL-8 MMP3

Expression of Syk Kinase in BioMAP Systems

Expression of Syk mRNA in BioMAP Systems (AU units)

Fostamatinib is active in systems where syk kinase mRNA is not

expressed

Fostamatinib is not a selective syk inhibitor at clinically relevant doses

3C 4H LPS SAg BT BF4T BE3C CASM3C HDF3CGF KF3CT MyoF /Mphg

100.4 98.5 133 137.3 550 251.3 165.3 179.1 108.3 87 108.1 733.7

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PI3-Kd and BTK Inhibitors

Cal-101 (GS-1101) – PI3-K delta inhibitor• Lannutti, Blood 2011, 117:591• In clinical trials for CLL (Phase 3) and NHL (Phase 2)

RN486 – BTK inhibitor• Active in CIA and AIA models of RA • Xu, JPET 2012, 341:90

PI3Kd and BTK inhibitors are highly selective • Is this sufficient for efficacy in rheumatoid arthritis??18

IgG

IL-6IL-2

B cellProliferation

TNFa

Clinical Standards of Care

E-sel

IgG

TNFaIL-8

E-sel

IL-17

IL-2

TNFa

IP-10IL-8

MMP-9IL-6

MCP-1 VCAM

IL-8

MCP-1VCAM

E-sel

IL-8

SAA

Rheumatoid Arthritis - Clinical Standards of Care• Methotrexate – inhibitor of DHFR

• Selective inhibitor of T cell dependent B cell activation

• Prednisolone – corticosteroid• Inhibition of macrophage > monocyte activation; T cell activation

• Remicade – TNF antagonist• Inhibition of monocyte > macrophage activation; myofibroblast activation

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Summary

Clinically effective kinase inhibitors for rheumatoid arthritis are not selective• Tofacitinib and fostamatinib are broadly acting agents• Selective PI-3K delta inhibitors are progressing in oncology

indications, but not arthritis• Selective BTK inhibitors remain to be tested in patients

BioMAP systems of primary human cell based assays can be useful for characterizing novel kinase inhibitors• Comparison to standards of care• Testing combination therapies

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Acknowledgements

BioSeek• Alison O’Mahony• Mark A. Polokoff• Dat Nguyen

Roche• Daigen Xu• Dinesh Srinivasan• Jay Fine• Julie DeMartino

Cellzome• Oliver Rausch

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