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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 [email protected]: 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
re
du
ce le
sio
ns
(%) *
*
*
**
**
**
* ** **
**
**
Effi
cacy
to
re
du
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