Nanobodies® -

Inspired by nature

9th RSV Symposium

Stellenbosch –South Africa

13th November 2014

Laurent Detalle

Delivery of ALX-0171 by inhalation greatly reduces disease burden in a neonatal lamb RSV infection model

www.ablynx.com

Disclosure

Laurent Detalle

• employee at Ablynx

Study performed at Iowa State University

• paid for by Ablynx

• Ablynx as study monitor

2

www.ablynx.com

Ablynx – Facts and figures

• Drug discovery and development company in Ghent, Belgium

• >300 employees

3

Corporate

Technology

Partners

Products

• Pioneer in next generation biologics – Nanobodies®

• >500 granted and pending patents

• >30 programmes – six in clinical development

• Three clinical proof-of-concepts (POC)

• >900 healthy volunteers and patients treated with Nanobodies

• AbbVie, Boehringer Ingelheim, Eddingpharm, Merck & Co,

Merck Serono and Novartis

www.ablynx.com

Camelid heavy-chain only antibodies are stable and fully functional

Nanobodies represent the next generation of antibody-derived biologics

Nanobodies – derived from heavy-chain only antibodies

Conventional

antibodies

Heavy chain only

antibodies

Ablynx’s Nanobody

• small

• robust

• sequence homology comparable

to humanised/human mAbs

• easily linked together

• nano- to picomolar affinities

• intractable targets

• multiple administration routes

• manufacturing in microbial cells

CH2

CH3

CH1

CL

VL

VH 12-15kDa

CH2

CH3

VHH

VHH

4

www.ablynx.com

ALX-0171 – designed to treat infant RSV infection

5

First-in-class treatment of RSV infection

Being developed to treat hospitalised and out-patient infants

Trivalent Nanobody: demonstrated in vitro efficacy against A

and B serotypes

anti-RSV

Nanobody

anti-RSV

Nanobody anti-RSV

Nanobody

ALX-0171

42kD

Pulmonary delivery: fast onset of action and high concentration at

infection site

In vivo efficacy demonstrated in cotton rat model and neonatal lambs

Three phase I studies in man with inhaled ALX-0171 successfully

completed

First-in-infant phase IIa study expected to start in Nov/Dec 2014

www.ablynx.com

Preclinical

In vivo studies Efficacy,

Safety and PK in

Neonatal setting with

relevant administration

route

Clinical

In vivo studies Safety and PK in

Human adults

Preclinical

In vivo studies Efficacy in cotton rats

Safety and PK

in adult/juvenile rats

Preclinical

In vitro studies ALX-0171

characterisation

ALX-0171 – translational strategy

6

Bridging the translational gap between adults and infants

• Requirement for a fit-for-purpose model

Intranasal/whole

body delivery

Stability/

nebulisation

studies

Oral inhalation

Breath

actuation

Nasal

inhalation with

facemask AL

X-0

17

1

delivery

AL

X-0

17

1

Eff

icacy/s

afe

ty

www.ablynx.com

Neonatal lambs as model system for safety and efficacy testing of inhaled ALX-0171 upon RSV infection

7

Age/lung development

• 2-5 days old

• Colostrum-deprived

• Alveolarisation starts preterm1

• Immature immune system

Size

• Allows relevant administration route (facemask)

• Enables multiple assessments

• Similar breathing patterns

Respiratory tract anatomy

• Size of nasal cavity and airways2

• Organisation of local lymphoid tissue2

• Airway branching pattern2

• Distributon of epithelial cells, mast cells and airway smooth muscle3

• ...

RSV disease

• Lower respiratory tract infection

• Develop mild clinical symptoms4

• Histologic lesions4

• Enhanced disease post-vaccination5

1ATS American journal of respiratory and critical care medicine, 2004. 170(3): p. 319-43 2Scheerlinck, J.P., et al., Trends in biotechnology, 2008. 26(5): p. 259-66. 3Meeusen, E.L., et al., Drug Discovery Today, 2009. 6(4): p. 101-106

Safety Lung deposition/

Pharmacokinetics Efficacy

Similar characteristics to human infants 4Derscheid, R.J. and M.R. Ackermann, Viruses, 2012. 4(10): p. 2359-78. 5Derscheid, R.J., et al. PloS one, 2013. 8(12): p. e81472.

www.ablynx.com

Lamb study design

Objectives

• Assess efficacy/safety in neonatal setting

- therapeutic setting starting at viral peak

• Pharmacokinetics in target tissue

- epithelial lining fluid (ELF)

Study design

8

Group RSV

status

Treatment Nebuliser

filling dose

Time taken

for dose to

be delivered

Endpoints

1 Mock-

infected

Vehicle - - • Viral titers (qPCR, FFU)

• Viral antigen expression in lungs (IHC)

• Gross lung viral lesions

• Histopathology

• General health status

• ELF Ctrough ALX-0171 levels

2 Mock-

infected

ALX-0171 11 mg

(Low dose)

~2 minutes

3 RSV Vehicle - -

4 RSV ALX-0171 11 mg

(Low dose)

~2 minutes

Note: Pilot study was performed with similar schedule

Day -1 0 1 3 4 6 2 5 8

Onset of

RSV infection

Treatment

ALX-0171 or formulation buffer

Necropsy

7 5-6 animals/group

Nebulisation of

RSV M37 strain or

culture medium

www.ablynx.com

Nebulisation and PK

Nebuliser

• Mesh nebuliser

• MMAD: 3.27 ± 0.13 μm

• 2L/min airflow

9

ALX-0171 concentrations in ELF were far above the IC90 following 3 daily

inhalations and 2-3 log higher than systemic concentrations

Veh

icle

AL

X-0

171

Lo

w d

ose

RS

V V

eh

icle

RS

V A

LX

-0171

Lo

w d

ose

0 .0 1

0 .1

1

1 0

1 0 0

A L X -0 1 7 1 c o n c e n tr a t io n s in

lu n g s

[AL

X-0

17

1]

EL

F(µ

g/m

L)

IC 9 0

0 2 0 4 0 6 0 8 0

1 0

1 0 0

1 0 0 0

N o m in a l t im e (h o u rs p o s t f irs t d o s e )

[AL

X-0

17

1]

pla

sm

a (

ng

/mL

)

L o w d o s e A L X -0 1 7 1

H ig h d o s e A L X -0 1 7 1

L L O Q

A L X -0 1 7 1 c o n c e n tr a t io n s

in p la s m a fro m p ilo t s tu d y

IC 9 0

www.ablynx.com

Improved health status in RSV infected lambs

General health status of lambs was scored on a daily basis

10

-1 0 1 2 3 4 5 6

2 0

4 0

6 0

8 0

1 0 0

D a y s p o s t in fe c tio n

% l

am

bs

wit

h s

co

re

1

R S V V e h ic le

R S V A L X -0 1 7 1

M o c k V e h ic le

M o c k A L X -0 1 7 1

G e n e r a l i l ln e s s s c o r e

Day 1

Day 3

Day 4

Day 6

Day 8

0

1

2

3

4

5

6

V ira l k in e tic s in

n e o n a ta l la m b s

Vir

al

tite

rs

in

BA

LF

(L

og

10 F

FU

/mL

) (

se

)

Decline in general health status was observed in all lambs

starting on day 3

Treatment with ALX-0171 treatment improved the health

status

Poster number 120

Score General illness score

0 No clinical signs

1 Reluctant to move

2 Reluctant to move, head down, depressed, not

interested in eating

3 Down, unwilling to get up or difficulty standing, not

eating

4 Down and should be euthanised, probably cannot eat

www.ablynx.com

Antiviral effect of ALX-0171

ALX-0171 decreases viral load in the lungs of infected lambs

• 4 log10 on cultivatable virus

• 0.5 log10 on viral RNA copies (>1 Log10 in pilot study)

11

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

V ira l t ite r s

Lo

g1

0 F

FU

/mL

BA

L (

Rt

Cd

lo

be

) (

se

)

4 .1 L o g

D L

Viral RNA

Viral Titers

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

8

Lo

g1

0 M

37

RN

A c

op

ies

/mL

(B

AL

) (

se

) V ira l R N A in B A L F

0 .5 5 L o g

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

Lo

g1

0 M

37

RN

A c

op

ies

/mg

lu

ng

(

se

)

V ira l R N A in lu n g t is s u e

0 .4 7 L o g

www.ablynx.com

Antiviral effect of ALX-0171

ALX-0171 decreases viral load in the lungs of infected lambs

• 4 log10 on cultivatable virus

• 0.5 log10 on viral RNA copies (>1 Log10 in pilot study)

12

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

V ira l t ite r s

Lo

g1

0 F

FU

/mL

BA

L (

Rt

Cd

lo

be

) (

se

)

4 .1 L o g

D L

Viral RNA

Viral Titers

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

8

Lo

g1

0 M

37

RN

A c

op

ies

/mL

(B

AL

) (

se

) V ira l R N A in B A L F

0 .5 5 L o g

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

2

4

6

Lo

g1

0 M

37

RN

A c

op

ies

/mg

lu

ng

(

se

)

V ira l R N A in lu n g t is s u e

0 .4 7 L o gV

eh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

5

1 0

1 5

2 0

2 5

8 0

1 0 0

1 2 0

1 4 0

IH C s c o r e s

V ir a l a n t ig e n e x p r e s s io n

Me

an

nu

mb

er o

f a

ffe

cte

d

bro

nc

hi/

bro

nc

hio

les

or a

lve

oli

pe

r f

ield

(

se

)

B ro n c h i/b ro n c h io le s

A lveo li

Decrease in virus load is reflected

by a strong decrease in viral antigen

expression

Daily inhalation of ALX-0171 markedly reduced viral titres and lung

lesions in RSV-infected lambs

RSV Vehicle RSV ALX-0171

www.ablynx.com

Effect of ALX-0171 on viral lung lesions

Plum red RSV lesions seen in lungs of RSV infected lambs on day 6

post-infection

• present on all lung lobes assessed

13

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

1 0

2 0

3 0

4 0

5 0

G ro s s v ir a l le s io n s

Me

an

% i

nv

olv

me

nt/

lob

e (

se

) R ig h t C ra n ia l

R ig h t M id d le

R ig h t C a u d a l

L e ft C a u d a l

A c c e s s o ry

L e ft C ra n ia l

L e ft M id d le

Daily inhalation of ALX-0171 markedly reduced gross lung viral lesions

www.ablynx.com

Histological scoring of lamb lung

Alveolar consolidation was scored

14

Degenerate/Necrotic epithelial cells

Syncytial cell

Accumulation of degenerate neutrophils

Observed lesions

inflammatory infiltrate in bronchiolar lumen, alveolar spaces, and alveolar septa

Bronchioles and alveolar spaces variably filled with degenerate neutrophils and

sloughed epithelial cells with occasional large epithelial syncytial cells.

alveolar spaces variably filled with degenerate neutrophils and sloughed epithelial

cells

alveolar septa mildly to moderately thickened by hyperplasia of type II cells

0% consolidation = 0

1%-9% consolidation = 1

10%-39% consolidation = 2

40%-69% consolidation = 3

70%-100% consolidation = 4

Daily inhalation of ALX-0171 markedly reduced microscopic lung viral

lesions

Veh

icle

AL

X-0

171

RS

V V

eh

icle

RS

V A

LX

-0171

0

1

2

3

4

His

tolo

gic

al

co

ns

oli

da

tio

n s

co

re

www.ablynx.com

ALX-0171 – Conclusions

15

Neonatal lambs proves to be a relevant model for treatment of RSV infection

Administrations of nebulized ALX-0171 resulted in effective target lung

concentrations and low systemic exposure

ALX-0171 administration was well tolerated with no adverse events noted in a

neonatal setting

ALX-0171 decreased RSV disease burden in neonatal lambs when treatment

started at peak of viral load and when symptoms were apparent

• Beneficial influence shown on:

- general health status

- viral titers and lung viral antigen expression

- gross and microscopic lung viral lesions

First-in-infant study expected to start in Nov/Dec 2014 using a custom-

developed infant inhalation device based on a vibrating mesh

www.ablynx.com

Acknowledgements

16

Iowa State University • Mark R Ackermann

• Jack M Gallup

• Albert Van Geelen

• Alejandro Larios-Mora

• Shannon Jones Hostetter

IWT, Belgium • Grant 130562

Ablynx, Gent, Belgium • Thomas Stöhr

• Kjell Mortier

• Linde Duprez

• Lieselot Bontinck

• Massimiliano Germani

• Judith Baumeister

• Sandy Jacobs

• Donata Thuy

• Toon Wauman

• Gregory Daelman

• Koen Allosery

• Erik Depla

• Catelijne Stortelers

• Stephanie Staelens

• Francis Descamps