SADEL Project:

Scaffolds for Alternative Delivery

Magali Zeisser Labouèbe1, Mathieu Cinier2, Christel Rousseaux3,

Rute Castro4, António Cunha4, Nghiep Truong Tan3, Olivier Kitten2,

Robert Gurny1, Leonardo Scapozza1,

1. School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland

2. Affilogic, Nantes, France

3. Intestinal Biotech Development, Lille, France

4. iBET, Oeiras, Portugal

11th ECCO Congress – March 16, 2016 - Amsterdam

EU Project Forum

Nanofitins : small antibody mimetics

2

Why would we need mimetics over antibodies ?

So many features in 66 amino acids

High affinity

High stability

Small size

Simple manufacturing

Orally administered

targeted therapies

in IBD

Blockade -> Inflammation

Non-i.v. -> oral

Tisue penetration -> gut

Affordable CoG

Anti-TNFα therapy in Inflammatory Bowel Diseases

Oral delivery challenge

Targeted therapies with

anti-TNFα antibodies

High efficacy in responders

Corticosteroids free remission

but ….

Only injectable drugs

Injection reaction

Systemic side effects

Immunogenicity

Expensive therapies

Patients and Clinicians

associations such as

ECCO highlight the need

for oral therapeutics.

Treating patients with Crohn’s

disease (CD) and Ulcerative

colitis (UC) with a safe orally

administered therapy.

Unmet medical need

Simple and cost-effective manufacturing

by E.coli fermentation

Fit for oral administration

Stable to T° and pH Highly resistant to human intestinal fluids Long half-life in digestive track

Affordable biologics

With demonstrated efficacy

Preclinical efficacy

Preventive and curative modes

Nanofitins, a novel family of proteins

Alternative to antibodies

Simple and cost-effective manufacturing

by E.coli fermentation

Affordable biologics

Nanofitins, a novel family of proteins

Alternative to antibodies

Opimization of the productivity up to 4 g/L

Profile of Nanofitins: Affordable biologics

Manufacturing: High Yield Bacterial Production

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Productivity (g/ L)

Complex media

Op

tim

iza

tio

ns

Expression in E. coli (30L bioreactor)

Profile of Nanofitins: Affordable biologics

Manufacturing: High Purity

100ug/ml

m/z400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 1000

%

0

100

NF1MVKKLN_lot02_March13_1 466 (3.931) MS2 ES+ 1.24e8703.27

644.47

594.91

552.54

551.75

594.65

584.48

595.57

595.97

622.76

644.67

644.87

645.40

702.28646.06687.04

703.41

773.10

703.80

704.46

705.85760.49

859.22

858.96

774.02858.56

774.35

844.90779.04

966.73

859.68

860.14

966.92

967.06

967.39

100ug/ml

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

AU

0.0

1.0e-3

2.0e-3

3.0e-3

4.0e-3

5.0e-3

6.0e-3

7.0e-3

NF1MVKKLN_lot02_March13_1 (1) PDA Ch1 280nm@4.8nmRange: 8e-33.90

0.69

0.75

NF1MVKKLN_lot02_March13_1 MS2 ES+ TIC

5.94e9

3.93

0.71

SDS page

Analysis by UPLC-MS

Simple and cost-effective manufacturing

by E.coli fermentation

Fit for oral administration

Stable to T° and pH Highly resistant to human intestinal fluids Long half-life in digestive track

Affordable biologics

Nanofitins, a novel family of proteins

Alternative to antibodies

Nanofitin (5 mg/ml) PBS

pH7.4, filter-sterilized

Storage @ 4, 25 and

40°C

Read-out : Analysis by

UPLC-UV/MS

Nanofitins are stable up to 40°C for three months.

Profile of Nanofitins: Fit for formulation

Long-term stability of PBS formulation

Nanofitin (5 mg/ml) PBS

pH7.4, filter-sterilized

Storage @ 40°C with

shaking or shearing up to

72h

Read-out : Analysis by SEC

Clear solution, no visible precipitation

No soluble aggregates visible by SEC

Profile of Nanofitins: Fit for formulation

Stability to stress

No Pepsin Pepsin 0.1 mg/mL

Fasted Simulated

Gastric Fluid

(FaSSGF) pH 1.6

0 MIN

60 MIN

40 MIN

20 MIN

Nanofitins are highly stable to very acidic pH and

protease degradation.

Profile of Nanofitins: Fit for oral treatment

Stability in biorelevant simulated fluids

No Pancreatin Pancreatin 0.1 mg/mL

Fasted simulated

Intestinal Fluid

(FaSSIF) pH 6.5

Nanofitins show a high stability profile in simulated

intestinal fluids

Profile of Nanofitins: Fit for oral treatment

Stability in biorelevant simulated fluids

0 MIN

60 MIN

40 MIN

20 MIN

Simulated Colon

Fluid

(SCoF) pH 5.8

Nanofitins show a high stability profile in colon fluids.

Profile of Nanofitins: Fit for oral treatment

Stability in biorelevant simulated fluids

0 MIN

60 MIN

40 MIN

20 MIN

Nanofitins, a novel family of proteins

Alternative to antibodies

Simple and cost-effective manufacturing

by E.coli fermentation

Fit for oral administration

Stable to T° and pH Highly resistant to human intestinal fluids Long half-life in digestive track

Affordable biologics

With demonstrated efficacy

Preclinical efficacy

Preventive and curative modes

Screening 9 anti-TNFα Nanofitin candidates

Gold standard Model of colitis induced by TNBS

Preventive effect / Intrarectal > Oral

1 Lead 1 Back-up

9 Candidates

Curative effect / Oral

TNBS

Nanofitins

5-ASA control

D-5

†

D-4 D-3 D-2 D-1 D0 D1 D2 Daily intrarectal instillation / oral gavage

TNBS

Nanofitins

Prednisolone

†

D0 D1 D2 D3 D4 Daily oral gavage

Mortality rate Macroscopic score Histologic score Collection of samples

Preventive set-up intrarectally Nanofitins in PBS pH7.4 @ 10 mg/kg

Macroscopic evaluation : wallace’s score

Histologic evaluation : Ameho’s score

C57bl6 mice n = 20 mice/group

* p<0.05

Nanofitins locally reduce inflammation Efficacy confirmed in a DSS-induced model Good correlation between in vivo data and data obtained by Surface Plasmon Resonance (SPR)

Comparison intrarectal vs. oral effect

Wallace’s score

Nanofitin N9 in PBS pH7.4 @ 10 mg/kg

Macroscopic evaluation

Similar anti-inflammatory properties of N9

after intrarectal and oral administration

Histologic evaluation

Ameho’s score

C57bl6 mice n = 20 mice/group

Preventive set-up orally: dose response

Nanofitin N9 in PBS pH7.4 @ 10, 100 and 400 mg/kg

Higher preventive effect @100 mg/kg

by oral administration

C57bl6 mice n = 20 mice/group

30 mM

Macroscopic evaluation: Wallace’s score

Histologic evaluation : Ameho’s score

* p<0.05** p<0.01

3226

37

20

0

5

10

15

20

25

30

35

40

Vehicle N1 N4 N9 Torental IP200mg/kg

Effi

cacy

to

re

du

ce le

sio

ns

(%)

3226

37

20

0

5

10

15

20

25

30

35

40

Vehicle N1 N4 N9 Torental IP200mg/kg

0

43

30

43

33

0

5

10

15

20

25

30

35

40

45

50

Vehicle N1 N4 N9 Torental IP200mg/kg

3226

37

20

0

5

10

15

20

25

30

35

40

Vehicle N1 N4 N9 Torental IP200mg/kg

Curative set-up orally

Wallace’s score

Nanofitins in PBS pH7.4 @ 100 mg/kg

Macroscopic evaluation

Efficacy confirmed orally in a curative model -> Very good stability of Nanofitins without any formulation optimization

Histologic evaluation

Ameho’s score

C57bl6 mice n = 20 mice/group

* p<0.05

Effi

cacy

to

re

du

ce le

sio

ns

(%) *

*

Effi

cacy

to

re

du

ce le

sio

ns

(%) * *

Effi

cacy

to

re

du

ce le

sio

ns

(%)

0

43

30

43

33

0

5

10

15

20

25

30

35

40

45

50

Vehicle N1 N4 N9 Torental IP200mg/kg

0

43

30

43

33

0

5

10

15

20

25

30

35

40

45

50

Vehicle N1 N4 N9 Torental IP200mg/kg

0

35

5444

70

52 52

0

10

20

30

40

50

60

70

80

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

0

4336

43

80

39 43

0

10

20

30

40

50

60

70

80

90

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

0

4336

43

80

39 43

0

10

20

30

40

50

60

70

80

90

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

Eff

ica

cy t

o r

ed

uce

le

sio

ns

(%)

0

4336

43

80

39 43

0

10

20

30

40

50

60

70

80

90

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

**

Second generation Nanofitins

Wallace’s score

Nanofitins in PBS pH7.4 @ 10 mg/kg

Macroscopic evaluation

Comparison of efficacy in preventive mode

Histologic evaluation

Ameho’s score

C57bl6 mice n = 20 mice/group

* p<0.05 ** p<0.01

Effi

cacy

to

re

du

ce le

sio

ns

(%)

Effi

cacy

to

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ce le

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ns

(%) *

*

*

**

**

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* ** **

**

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cacy

to

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ce le

sio

ns

(%)

0

35

5444

70

52 52

0

10

20

30

40

50

60

70

80

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

0

35

5444

70

52 52

0

10

20

30

40

50

60

70

80

TNBS +vehicle

TNBS + N1 TNBS + N9 TNBS + N10 TNBS + N11 TNBS + N12 TNBS +Pentasa

**

The SADEL talent chain

Joao V. Rodrigues

Antonio Cunha

Manuel Carrondo

Christel Rousseaux

Nghiep Truong Tan

Ilaria Bonetti Magali Zeisser Labouèbe

Robert Gurny

Leonardo Scapozza

Raquel Fortunato

Teresa Alves

Mathieu Cinier

Justine Picot

Simon Huet

Olivier Kitten

Georges Kolias

Piyi Papadaki

Manolis Roulis

Julia Gabriel

Johannes Meier

Andrew Sadler

Thomas Jorgensen

Daniel Plaksin

SADEL is a project supported by the European Commission through the Seventh Framework

Programme (FP7)

Next steps – SADEL and Beyond

23

After Oral administration - Bioavailability ? - Towards oral antibody-mimetics

Beyond TNF - Other IBD-related cytokines - Oncology targets - Microbiome modulation

Document MoA/Tox - Move towards the clinic