Fibrotic Diseases

New Targets and Model Development

Kenneth Zhang, Ph.D.

Kenneth.Zhang@genscript.com

In Vivo Pharmacology

6/10/2015

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About GenScript

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Kenneth Zhang, Ph.D.

Associate Director, In vivo pharmacology

at GenScript

Tumor immunologist with expertise in

tumor microenvironment

• Published over 19 peer-reviewed

publications

Presenter Bio

3

Leads the development of:

• Hepatic, renal and pulmonary fibrosis models

• SC xenograft, orthotopic and syngeneic tumor models

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Outline

Fibrotic diseases

Anti-fibrotic therapeutic targets

GenScript fibrotic disease models

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Organ structure – parenchyma

and interstitium

Fibrotic diseases are the

replacement of organ

parenchyma by interstitium -

excessive extracellular matrix

(ECM) deposit during chronic

injury, which progressively leads

to impair or loss of organ

function.

Fibrotic Diseases

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Friedman, Sci Trans Med, 2013

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PubMed Indexed Publications on Fibrosis

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Nature

• A reversible wound-healing response characterized by the

accumulation of ECM following liver injury. Chronic injury and

inflammation leads to progressive substitution of liver parenchyma

by scar tissue (fibrosis) and finally end stage liver disease (cirrhosis).

Etiology

• The diseases can be induced by HBV/HCV infection, alcohol abuse,

nonalcoholic steatohepatitis (NASH), chronic cholestatic disorders

(including primary biliary cirrhosis) and autoimmune hepatitis etc.

Therapy

• Currently no drug approved

• Obeticholic acid designated Breakthrough Therapy by US FDA in

2015 (Farnesoid X receptor agonist).

Hepatic Fibrosis

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Development of Liver Fibrosis and Cirrhosis

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Gressner, Comparative Hepatology, 2007

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Hepatocyte injury and release of

reactive oxygen species (ROS) and

apoptotic bodies

Activation of immune-subset cells and

hepatic stellate cells (HSC), and their

cross-talk

• Immune infiltration and Kupffer cell

activation

• Hepatic stellate cell activation,

proliferation and trans-

differentiation into myofibroblasts

(α-SMA+)

Excessive ECM protein production by

myofibroblasts in the space of Disse,

blocking substance exchange between

hepatocytes and the blood

Major Cellular Events in Hepatic Fibrosis

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Bataller, J Clin Invest, 2005

Normal Liver Fibrosis

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TGF-β

• Secreted by Kupffer cells and activated HSCs

• Enhances the transition of HSCs into collagen-producing

myofibroblasts, stimulates the synthesis of ECM proteins and

over-expression of TIMP.

PDGF

• Secreted mainly by Kupffer cells

• Predominant mitogen of

activated HSCs.

MCP-1 (CCL2)

• Recruits monocytes

Key Soluble Factors Involved

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Schuppan, JCI, 2013

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Sources of Myofibroblasts in Liver Fibrosis

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Hernandez-Gea, Annu Rev Pathol Mech Dis, 2011

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The development of excessive connective tissue in the lung – most cases are

idiopathic pulmonary fibrosis (IPF)

Irreversible impairment of respiratory function

Etiology:

• Idiopathic; Secondary effect of Inhalation of pollutants or pathogens,

Cigarette smoking, Connective tissue disease, Infections, etc

Limited therapeutic:

• Pirfenidone and Nintedanib approved (Oct 2014) by US FDA

Pulmonary Fibrosis

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Wynn, Integrating mechanisms of pulmonary fibrosis, 2011

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Renal fibrosis is the common end point of virtually all

chronic (progressive) kidney diseases.

Renal fibrosis includes

• Glomerulosclerosis

• Tubulointerstitial fibrosis -> Renal failure

• Excessive production of ECM between microtubules

and peritubular vasculature

Renal Fibrosis

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Renal Fibrosis in Response to

Epithelial Injury

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Friedman, Sci Trans Med, 2013

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Epithelial injury and dysfunction

Accumulation of myofibroblasts

Deposition of fibrotic ECM

Inflammation and recruitment of immune infiltrate

Pro-fibrotic macrophages

Common Features of Fibrotic Diseases

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Target Agent

TGF-β expression and activation Pirfenidone; αvβ6 antibody; ATI and ATII receptor blockers; ACE inhibitors; CAT-192 (anti-TGFβ1 mAb)

TGF-β signaling pathways

TGFβR1 (ALK5) SM305

SMAD3 SIS3

CTGF Antibody

Tyrosine kinase inhibitors of non-canonical TGF-β and other signaling pathways

c-Abl, c-kit, PDGFR Imatinib

PDGFR, c-Abl, src Dasatinib

PDGFR, c-Abl, c-kit Nilotinib

PDGFR, VEGFR, FGFR Nintedanib (BIBF1120)

VEGFR, PDGFR Sorafenib

Transcription factors

FXR Agonist (Obeticholic acid)

PPARγ Agonist (Rosiglitazone)

AP-1 T-5524

Anti-fibrotic Therapeutic Targets

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Adapted from Rosenbloom, Biochimica et Biophysica Acta, 2013

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Target Agent

Recruitment of fibrocytes and monocytes/macrophages

CXCL12 (SDF-1) Antibody

CXCR4 AMD3100

MCP-1/CCR2 Inhibitors (PF-04136309)

Immune response regulator

IL-13 Antibody

IL-6 receptor Tocilizumab

TLR TLR inhibitors (E5564, TAK-242)

Other intracellular pathways

JAK2 TG101209

Wnt Dkk-1

RhoA Statins, GGT inhibitor

JNK CC-930

NOX4 (production of ROS) GKT136901

Endothelin-1 Bosentan, other ET receptor blockers

Matrix stiffness, collagen crosslinking D-penicillamine, clostridial collagenase

Anti-fibrotic Therapeutic Targets (cont’d)

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Adapted from Rosenbloom, Biochimica et Biophysica Acta, 2013

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Hepatic fibrosis

• CCl4-induced hepatic injury and fibrosis in rats

• CCl4-induced hepatic injury and fibrosis in mice

• BDL-induced hepatic injury and fibrosis in rats

• BDL-induced hepatic injury and fibrosis in mice

• TAA-induced hepatic fibrosis in mice (in development)

Renal fibrosis

• UUO-induced renal fibrosis in rats

• UUO-induced renal fibrosis in mice

Pulmonary fibrosis

• Bleomycin-induced pulmonary fibrosis in mice

Note:

BDL, Bile duct ligation

TAA, Thioacetamide

UUO, Unilateral ureteral obstruction

GenScript Fibrotic Disease Models

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CCl4-induced Liver Fibrosis in Mouse

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Animal: Balb/c mice

CCl4-olive oil mixed solution; i.p. injection

Induction of hepatocyte damage, necrosis, inflammation

and fibrosis

CCl4-induced Fibrosis (Intoxication)

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• Liver and body weight

• Serum biochemistry

• HE staining

• Sirius Red staining

• Masson trichrome staining

• IHC for myofibroblasts & macrophages

• Quantitative analysis of histol. images

• Hydroxyproline assay

• Profiling of markers by RT-qPCR

Available endpoints of measurement

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Disease Development at 6 Weeks

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Olive oil only

CCl4

H&E Sirius Red Myofibroblasts Macrophages

(α-SMA) (F4/80)

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A B

C

Disease Development at 6 Weeks (continued)

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O liv e o il C C l4

0

2 0 0

4 0 0

6 0 0

8 0 0A L T

* *

IU/L

O liv e o il C C l4

0

5 0 0

1 0 0 0

1 5 0 0A S T

* *

IU/L

Olive oil CCl4

0

100

200

300

400

500

Hydroxyproline

**

g

/gOlive oil CCl4

0

2

4

6

8Acta2

**

Fo

ld c

ha

ng

e

(mR

NA

/-a

cti

n)

O liv e o il C C l4

0

1 0

2 0

3 0

4 0

5 0C o l1 a 1

* *

Fo

ld c

ha

ng

e

(mR

NA

/-a

cti

n)

O liv e o il C C l4

0

1

2

3

4

5T g fb 1

* * *

Fo

ld c

ha

ng

e

(mR

NA

/-a

cti

n)

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A

B C

Case Study

Therapeutic Effect of Pirfenidone at 6 Weeks

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Olive oil CCl4 CCl4+PFD

S iriu s R e d

O liv e o il C C l4 C C l4 + P F D

0

5

1 0

1 5

* * ** * *

Re

lati

ve

Sir

ius

Re

d+

Are

a

Col1a1

Olive oil CCl4 CCl4+PFD0

50

100

150

200***

Fo

ld c

ha

ng

e

(mR

NA

/-a

cti

n)

N.S.

Tgfb1

Olive oil CCl4 CCl4+PFD0

1

2

3

4

5 *** *

Fo

ld c

ha

ng

e

(mR

NA

/-a

cti

n)

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Bile-duct Ligation (BDL)-induced Liver Fibrosis in Mouse and Rat

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Animal: C57BL/6 mice; Wistar rats

Surgery: Ligation of common bile duct (BDL)

Study conclusion at 2-4 weeks after surgery.

Bile-duct Ligation-induced Fibrosis

(Cholestasis)

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• Liver and body weight

• Serum biochemistry

• HE staining

• Sirius Red staining

• Masson trichrome staining

• IHC for myofibroblasts & macrophages

• Quantitative analysis of histol. images

• Hydroxyproline assay

• Profiling of markers by RT-qPCR

Available endpoints of measurement

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Hepatocellular injury and proliferation

Portal inflammatory response

• Neutrophils, B/T cells, Kupffer cells

Cholangiocellular proliferation

Fibrosis development

Development of the Experimental

Disease in Mouse

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Georgiev, Br J Surg., 2008

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Model Development in Mouse

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Sham

BDL

2 wks 4 wks 8 wks

ALT AST A

B

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Model Development in Rat – Serum Biochemistry

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A S T

2 3 14 280

5 0 0

1 0 0 0

1 5 0 0S h a m

B D L

* *

* *

N SN S

D a y s a f t e r s u r g e r y

AS

T

(I

U/

L)

A L T

2 3 14 280

5 0 0

1 0 0 0

1 5 0 0S h a m

B D L

* *

*

N SN S

D a y s a f t e r s u r g e r y

AL

T

(I

U/

L)

T B I L

2 3 14 280

5 0

1 0 0

1 5 0S h a m

B D L* * *

* * *

N S

* * *

D a y s a f t e r s u r g e r y

TB

IL

(¦

Ìm

ol

/L

)

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Model Development in Rat - Histology

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100x

Sham Day 14 Day 28 40x

100x

40x

H&

E

S

iriu

s R

ed

Hydroxyproline content

14 280

100

200

300

400

500 Sham

BDL

**

Days after surgery

Hyp

co

nte

nt

( g

/g)

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Unilateral Ureteral Obstruction (UUO)-induced Renal Fibrosis in Rat

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Animal: SD rats

Surgery: Ligation of the ureter of left kidney, leaving the

contralateral kidney as a control

Induction of chronic epithelial injury, inflammation and

tubulointerstitial fibrosis in left kidney

Method

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• Kidney and body weight

• Serum biochemistry

• HE staining

• Sirius Red staining

• Masson trichrome staining

• IHC staining

• Quantification analysis of histological

images

• Hydroxyproline assay

• RT-qPCR

Available endpoints of measurement

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A B

C

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Model Development

Hydroxyproline content

Sham 3 7 14 21 280

500

1000

1500

Days after surgery

****

***

Hyp

co

nte

nt

( g

/g)

R e n a l w e i g h t / b o d y w e i g h t

Sham 3 7 14 21 280

1 0

2 0

3 0

4 0

D a y s a f t e r s u r g e r y

* *

* * *

* ** *

*

le

ft

k

id

ne

y w

eig

ht

/b

od

y w

eig

ht

(

¡ë

)

CREA

Sham 3 7 14 21 280

10

20

30

40

50

Days after surgery

** ** *****

CR

EA

(u

mo

l/L

)

BUN

Sham 3 7 14 21 280

2

4

6

8

10

Days after surgery

** *** **

BU

N(m

mo

l/L

) *

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Model Development Shown by

Sirius Red Staining (100x)

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Sham Day 3 Day 7

Day 14 Day 21 Day 28

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A

B C

Case Study

Therapeutic Effect of Pirfenidone at 4 Weeks

Sham UUO UUO+PFD

Sirius

Red

100x

H&E

200x

Sirius Red

0

5

10

15

20 ** *

Sir

ius R

ed

+ A

rea (

%)

Sham

UUO

UUO+PFD

H y d ro x y p ro lin e c o n te n t

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0* * *

g

/g*

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Bleomycin-induced Lung Fibrosis in Mouse

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Animal: C57BL/6 mice

Disease induction: Intratracheal instillation of bleomycin (BLM)

• Induction of lung epithelial injury, inflammation and fibrosis

Model Development

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• Lung and body weight

• BALF cell count

• HE staining

• Masson trichrome staining

• IHC staining

• Quantification analysis of histological

images

• Ashcroft score

• Hydroxyproline assay

• RT-qPCR

Available endpoints of measurement

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Histopathological Changes 3 Days after

Bleomycin Instillation (HE Staining)

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Saline Bleomycin dose gradient

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A B C

D E F

Model Development

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BAL total cells

Vehicle BLM0

20

40

60

80

*

To

tal cells (

×10

4)

3 days after BLM treatment

Animal Survival

0 10 20 300

20

40

60

80

100

BLM

Vehicle

Days after BLM treatment

Perc

en

t su

rviv

al (%

)

Body weight

0 5 10 15 20 25 3015

20

25

30

35 Vehicle

BLM

Days after BLM treatment

Bo

dy w

eig

ht

(g)

Mean

SE

M

L u n g w e ig h t/b o d y w e ig h t

V e h ic le 3 7 1 4 2 1

0

5

1 0

1 5

2 0

2 5

D a y s a f te r B L M tre a tm e n t

*

* * *

* * * * * *

Lu

ng

we

igh

t/b

od

y w

eig

ht(

‰)

Me

an

±S

EM

Hydroxyproline content

Vehicle 7 14 21 280

100

200

300

400

Days after BLM treatment

*

*** ******

Hyp

co

nte

nt

( g

/lu

ng

)

A s h c ro ft s c o re

V e h ic le 7 1 4 2 1 2 8

0

2

4

6

8

D a y s a f te r B L M tre a tm e n t

As

hc

ro

ft s

co

re

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Model Development - Histology

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H&E staining (40x)

Masson’s trichrome staining (40x)

Vehicle Day 7 Day 14 Day 21 Day 28

Bleomycin

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There is huge unmet clinical need for effective and potent

therapeutics of fibrotic diseases.

Properly developed animal models that mimic diseases in

human are critical for efficacy evaluation of leads.

GenScript has developed multiple fibrotic disease models

and will develop more to facilitate drug discovery and

development for fibrotic diseases.

Concluding Remarks

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Discovery Biology Services

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Antibody and Protein Engineering

•Single domain antibody generation

•Antibody sequencing

•Affinity maturation and humanization

In-vitro Pharmacology

•CellPower™ custom stable cell line for assays

•GenCRISPR™ custom knock-out or knock-in cell lines

•Cell-based assays and Ion channel and GPCR assays

In-vivo Pharmacology

•Tumor models including SC xenograft, orthotopic and syngeneic

•Bioluminescence imaging of tumors

•Fibrosis models

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Thank you for your participation

We wish you all success in your research

Email me: Maxine.Chen@GenScript.com

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