And Now For SomethingCompletely Different

Lee Landeen, Ph.D.Associate Director, Research

Advanced BioHealing

Advanced BioHealing• Regenerative medicine company

– Not wound care company• Celaderm

– Keratinocyte-based, tissue-engineered product• Purchased rights to Advanced Tissue Sciences / Smith

& Nephew dermal products– May 2006– Dermagraft and TransCyte

• Acquired by Shire – June 2011 ($750M)

The History• 1986: Marrow-Tech incorporates; tissue-engineering platform technology• 1990: Markets skin2 for cytotoxicity testing (pre-cursor to Dermagraft)• 1991: Marrow-Tech renamed Advanced Tissue Sciences; began

development of Dermagraft (burns)• 1993: Began development of TransCyte (burns)• 1996: ATS / Smith & Nephew form joint venture• 1996: Dermagraft clinical trials for ulcers initiated• 1997: TransCyte approved for partial and full-thickness burns• 1998: ATS / S&N joint venture expanded to include TransCyte• 2001:Dermagraft approved for diabetic foot ulcers• 2002: ATS files Chapter 11; S&N acquires the JV• Dec 2005: S&N decided to exit the business, put assets related to

Dermagraft and TransCyte up for sale• May 2006: ABH purchased assets• Feb 2007: ABH revalidated Manufacturing facility and shipped first

Dermagraft to customers

Why Has ABH Succeeded(When Others Have Failed)?

• To date, ABH has generated > $400MM in sales revenue on Dermagraft• Principles of Jim Collins’ “Good to Great”

– Narrow focus, culture of discipline, hire the right people, flywheel concept

• Selling price doubled– Dermagraft was off market for >6 mo

• Positioned as the “Standard of Care”– Close the wound vs. manage the wound– Non-healing wounds after 4 weeks need an advanced therapy

• Commercially focused Sales team– Multiple disciplines including pharma, biologics and device– Single product portfolio

• Dedicated reimbursement staff and expertise– Heal2Gether program to work with patient compliance issues– Health economics of using vs. not using Dermagraft– Physicians get reimbursed

Case StudiesBefore After

Case 1(plantar surface)(ulcer > 2 yr)(healed at 8 wk)

Case 2(heel)(healed at 11 wk)

Case 3(toe)(ulcer 3 mo)(healed at 7 wk)

Tissue Engineering

PolymerPolymerScienceScience

BiochemistryBiochemistryCell BiologyCell Biology BioengineeringBioengineering

TransplantationTransplantation

SEM of Fibroblasts on Nylon

SEM of Vicryl SEM of Dermagraft

Dermagraft• Human neonatal dermal fibroblasts (MCB, WCB)• Resorbable knitted mesh scaffold (poly[glycolide-co-L-lactide])• Class III medical device (CDRH, 2001) (biologic, cell therapy)• Treatment of diabetic foot ulcers (1 x 8 wks)• Cryopreserved dermal replacement (-75oC ± 10oC)• Past/present evaluation in venous leg ulcers, epidermolysis bullosa,

gingival resectioning, epicardial revascularization, rotator cuff repair

Dermagraft Manufacturing

Cell Expansion

Dermagraft Bioreactor Manifold

Product

Manifold(up to 12 per lot)

(up to 1,152 units)

8-cavity bag

HEMOSTASIS PLATELET AGGREGATION

REMODELING

SECOND MINUTE DAY WEEK MONTH YEAR

SCAR FORMATION

WOUND CLOSURE

RE-EPITHELIALIZATION

GRANULATION TISSUE

MONOCYTE IMMIGRATION

NEUTROPHIL IMMIGRATION

Normal Wound Healing

HEMOSTASIS PLATELET AGGREGATION

REMODELING

SECOND MINUTE DAY WEEK MONTH YEAR

SCAR FORMATION

WOUND CLOSURE

RE-EPITHELIALIZATION

GRANULATION TISSUE

MONOCYTE IMMIGRATION

NEUTROPHIL IMMIGRATION

Ulcers

Proposed Mechanisms of Action of Dermagraft®

• Delivery of normal, non-senescent fibroblasts• Delivery of growth factors

– Extracellular matrix-bound– Secreted

• Substrate for re-epithelialization– Extracellular matrix molecules

• Influence on inflammatory processes– Neutrophils

Proposed Mechanisms of Action of Dermagraft®

• Delivery of normal, non-senescent fibroblasts• Delivery of growth factors

– Extracellular matrix-bound– Secreted

• Substrate for re-epithelialization– Extracellular matrix molecules

• Influence on inflammatory processes– Neutrophils

Normal Fibroblast Replicative Senescence• Hayflick’s proposed 50 cell doublings

Reactive Oxygen Species

ROS

Pressure Ulcers

Diabetic Ulcers

Venous Stasis Ulcers

Chronic ischemia/reperfusion injury

High mitochondrial potentialChronic neutrophil activationChronic inflammation

Iron from extravasated red cellsChronic neutrophil activationChronic Inflammation

Protein

DNA

Lipid

Senescence-likePhenotype

Role of Senescence

• Fibroblasts with a stress-induced premature senescent phenotype fail to respond appropriately to injury

• Neutrophil activation fails• Bacterial colonization occurs• Keratinocyte closure of the wound is

delayed

Proposed Mechanisms of Action of Dermagraft®

• Delivery of normal, non-senescent fibroblasts• Delivery of growth factors

– Extracellular matrix-bound– Secreted

• Substrate for re-epithelialization– Extracellular matrix molecules

• Influence on inflammatory processes– Neutrophils

Growth Factor Expression in Dermagraft• IGF-II• IGFBP-4• IGF-BP-5• IGFBP-6• IL-l• IL-1 RA• Pro-IL-1• IL-6• IL-8• IL-11• TNF-• MCP-1• MCP-3• SDF-1• Interferon-• Gro-• Gro-• Gro-• CSF-1• G-CSF• GM-CSF• MAC25

• PDGF A chain• KGF• TGF-1• TGF-1 BP• TGF--related• TGF- latency protein• CTGF• Wnt 4• Wnt 6• Wnt 11• WISP-2• VEGF• VEGF-B• HGF• PLGF• bFGF• Cyr61• FGF-1BP• FGF-5• HSFGF• AAMP • Angiopoietin-1

ECM-bound Growth Factors

Immunohisto for bFGF ELISA for bFGF

Secretion of VEGF by Dermagraft® After Thawing

00.20.40.60.8

11.21.41.61.8

0 1 2 3 4 5 6

Time in culture, days

VE

GF

in m

ed

ium

, ng

/mL

Chick Chorioallantoic Membrane Assayfor Angiogenesis

Control, Scaffold Dermagraft®

Chick Chorioallantoic Membrane Assayfor Angiogenesis

0

5

10

15

20

25

ScaffoldControl

3-D culture + anti-VEGF + anti-HGF + anti-VEGF+ anti-HGF

bra

nc

h p

oin

ts/5

mm

dis

c

Laser-Doppler Showing Blood Supplyin a Treated Diabetic Ulcer

Pre-treatment

2 weeks

5 weeks

Proposed Mechanisms of Action of Dermagraft®

• Delivery of normal, non-senescent fibroblasts• Delivery of growth factors

– Extracellular matrix-bound– Secreted

• Substrate for re-epithelialization– Extracellular matrix molecules

• Influence on inflammatory processes– Neutrophils

Extracellular Matrix Composition• Biglycan• Collagen I• Collagen III• Collagen V• Collagen VI• Collagen VII• Decorin• Elastin• Fibronectin• Glycosaminoglycans (HSPG)• Lumican• SPARC• Tenascin• Thrombospondin II

Keratinocyte Migration

On Wound bed On Dermagraft

Krejci-Pappa, et al. 1999

Proposed Mechanisms of Action of Dermagraft®

• Delivery of normal, non-senescent fibroblasts• Delivery of growth factors

– Extracellular matrix-bound– Secreted

• Substrate for re-epithelialization– Extracellular matrix molecules

• Influence on inflammatory processes– Neutrophils

Factors Induced in DERMAGRAFT®

Merge of prenormalized data Prenormalized data0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

26.0

28.0

30.0

32.0

34.0

36.0

38.0

40.0

42.0

44.0

46.0

48.0

50.0NormalizedIntensity

Moerge of normalized DG and ML data

Dermagraft® Monolayer

IL-8

IL-8 secretion by Dermagraft

0

10

20

30

40

50

60

70

0 2 4 6

Time after thawing, days

IL-8

, n

g/1

06 c

ell

s

Chemokines

0

5000

10000

15000

20000

25000

CCL2

CCL7

CCL13

CCL20

CXCL1

CXCL2

CXCL3

CXCL5

CXCL6 IL8

CXCL12

CXCL12

CXCL14

CXCL16

Sig

na

l Monolayer fibroblasts3D fibroblastsKeratinocytes

Macrophage attractants

Neutrophil attractants

Stem cellattractants

Dendritic cellattractants

Chemokine Message in Dermagraft

CXCL-1, 5, 6, 8(Gro-, ENA-78, GCP-2, IL-8)

• Secreted by fibroblasts, neutrophils, keratinocytes, and other cells

• Chemoattractants and activators for neutrophils• Primes neutrophil respiratory burst• Antibacterial/antimicrobial activity• Heparan sulphate-binding• Activated by MMP-9

Overall Hypothesis

Ulcer Fibroblasts(Senescence-like Phenotype)

BacterialColonization

No Closure

InappropriateResponse to

Injury

ChronicWound

HEMOSTASIS PLATELET AGGREGATION

Dermagraft®

Cells G-CSF IL-8

Gro-ENA-78GCP-2

TGF1

IL-6IL-11

VEGF ECM Substrate

HGF

KGF

Dermagraft®

SECOND MINUTE DAY WEEK MONTH YEAR

Proposed Dermagraft MOA

REMODELING

SCAR FORMATION

WOUND CLOSURE

RE-EPITHELIALIZATION

GRANULATION TISSUE

MONOCYTE IMMIGRATION

NEUTROPHIL IMMIGRATION

HSPs, necrotic cells,scaffold

MCP-1MCP-3MCP-4

bFGF

“Doctor, my brain hurts”