Profibrinolytic diabody targeting PAI-1 and TAFI
for treatment of acute thrombotic disorders
THROMBOLYSIS WITH IMPROVED SAFETY
ProFiDIΛ
A valorisation project of
Unmet Medical Need = Safe Thrombolysis
• 1 out of 4 deaths due to cardiovascular thrombosis
N° 1 Acute coronary syndromes
N° 2 Stroke
N° 3 Pulmonary embolism
• Significant innovation in mechanical thrombectomy,
but not in pharmacological strategies for clot lysis
• Risk of bleeding precludes the use of plasminogen
activators in the patients with stroke and pulmonary
embolism
Opportunities for thrombolytics with
improved safety profile
Indications Standard-of-Care Limitations Unmet Needs
Acute Ischemic
Stroke
• Thrombectomy
preferred (40%
of the patients)
• Thrombolysis for
patients not
eligible for
thrombectomy
(60% of the
patients)
• Thrombectomy not
always available
• Patients not eligible
(time, small artery
occlusion)
• Narrow time window
for thrombolysis
• Bleeding risk (ICH)
• tPA induced
neurotoxicity
• Priming the thrombotic occlusion
prior to thrombectomy with a safe
profibrinolytic agent (with or without
low dose of tPA)
• Improved safety (less ICH)
• Longer time window
• Treatment for patients not eligible
for thrombectomy and/or
thrombolysis with current agents
Pulmonary
thrombo-
embolism
• Thrombolysis
(shown to
reduce
cardiovascular
collapse)
• Bleeding risk (ICH) • Reduce residual clot burden
• Improved safety
• Preventing right ventricular failure
in patients
• Decrease risk of recurrence
Dual TAFI/PAI-1 inhibition improves the
efficacy of endogenous tPA
Parental antibody generation
Antibody
(150 kDa)
CH3
CH2 CH2
CH1CH1
CLCL
VHVH
VL VL
Heavy chain
Light chain
anti-TAFI Mab
MA-TCK26D6
CH2 CH2
CH3
CH1CH1
CLCL
VHVH
VL VL
Heavy chain
Light chain
Antibody
(150 kDa)
anti-PAI-1 Mab
MA-33H1F7CH3 CH3
• MA-33H1F7 was raised by immunizing
PAI-1 knock-out mice with the human PAI-
1/tPA complex.
• MA-33H1F7 crossreacts and binds murine
PAI-1 with almost a similar degree.
• MA-33H1F7 neutralizes murine PAI-1 to a
similar extent as human PAI-1.
• MA-33H1F7 does not inhibit vitronectin-
bound PAI-1
• MA-TCK26D6 was raised by immunizing
TAFI knock-out mice with human TAFI.
• MA-TCK26D6 binds both human and
mouse TAFI with a similar affinity.
• MA-TCK26D6 does not inhibit
Thrombin/Thrombomodulin activation of
TAFI
Diabody construction
scFv
(28 kDa)
Diabody
(58 kDa)
VH VL
VHA VLA
VHBVLB
Antibody
(150 kDa)
CH3
CH2 CH2
CH1CH1
CLCL
VHVH
VL VL
Heavy chain
Light chain
anti-TAFI Mab
MA-TCK26D6
CH2 CH2
CH3
CH1CH1
CLCL
VHVH
VL VL
Heavy chain
Light chain
Antibody
(150 kDa)
anti-PAI-1 Mab
MA-33H1F7
9 AA linker
VH
Anti-TAFI
VL
Anti-TAFI
VL
Anti-PAI-1
VH
Anti-PAI-1 scFv
(28 kDa)
VH VL
CH3CH3
Strong in vitro profibrinolytic properties
• In a thromboelastometric assay, we confirmed that simultaneous
inhibition of TAFI and PAI-1 causes an enhancement of in vitro
fibrinolysis in human blood
Anti-TAFI MA
Anti-PAI-1 MA
Combination of MA
Diabody
Resis
tence (
mm
)
Time (min)7
Citrated whole blood
+ MA(s), diabody or saline
10’, 37°C
+ CaCl2+ Tissue factor
+ tPA
Strong in vitro profibrinolytic properties
Full
lysis
No
lysisM
A-3
3H1F
7
MA-T
CK26
D6
Com
binat
ion o
f antib
odies
Dia
body
0
20
40
60
80
100
******
L
45 (
%)
HUMAN BLOOD
N = 4
*** P < 0.005
P < 0.05
Paired t-test
Kruskal-Wallis test
Strong in vivo profibrinolytic properties
Monofilament-mediated stroke model
MCAo 1. Reperfusion
2. Treatment
• Fibrin deposition
• Infarct size
• Neurological test
• Motor test
1 hour 24 hours
© Berressem
Nutritional-
neuroscience.com
Veh
icle
Contr
ol IgG
(2 m
g/kg)
MA-3
3H1F
7 (1
mg/k
g)
MA-T
CK26
D6
(1 m
g/kg)
com
binat
ion o
f MA
Dia
body (0
.8 m
g/kg)
0
20
40
60
80
100*
Le
sio
n v
olu
me
(m
m3)
VehicleDiabody
(0.8mg/kg)N =10-12/ group
* P < 0.05
Collaboration with prof. Denis Vivien and Dr. Marina Rubio INSERM, France
Orset et al. Stroke 2007
MCA
Thrombin
MCA
pipette
Karatas et al. Stroke 2011
MCA
FeCl3
MCA
Thrombotic middle cerebral artery occlusion models
Thrombin
Fibrinogen Fibrin
FeCl3
Oxidative stress
Platelet activation
Diabody vs tPA
Collaboration with prof. Denis Vivien and Dr. Marina Rubio INSERM, France
Orset et al. Stroke 2007
MCA
Thrombin
MCA
pipette
Karatas et al. Stroke 2011
MCA
FeCl3
MCA
Thrombotic middle cerebral artery occlusion models
Fibrin-rich clot
Susceptible to tPA-mediated lysis
Platelet-rich clot
Resistant to tPA-mediated lysis
Diabody vs tPA
Improved efficacy compared to tPA on
platelet rich clots
12
Thrombotic middle cerebral artery occlusion models
* P < 0.05
N = 9-15/ group
Platelet-rich clot
MCAo 1. Arterial status
2. Lesion size
3. Brain perfusion
24 hours60’15’ 20’0’
Db
or
Buffer
tPA
or
Buffer
Vehicle
tPA
Db
Db 2xve
hicle
tPA 1
0mg/k
g
Db 1
.6m
g/kg
Db 3
.6m
g/kg
0
5
10
15
20
25 *
Infa
rct
vo
lum
e (
mm
3)
24h
po
st
str
oke o
nset
Improved efficacy compared to tPA on
fibrin rich clotsThrombotic middle cerebral artery occlusion models
Fibrin-rich clot
MCAo 1. Arterial status
2. Lesion size
3. Brain perfusion
24 hours60’15’ 20’0’
Db
or
Buffer
tPA
or
Buffer
90’ 150’85’
Improved safety compared to tPA
Tail vein bleeding model in mice:
tPA increases hemoglobin loss, whereas the diabody does not
Summary
• Db-TCK26D6x33H1F7 has strong profibrinolytic properties in vitro and in
vivo:
- More effective than the combined addition of antibodies
- More effective in lysing a thrombus than tPA
- Beneficial effect in thrombo-embolism model and in all MCAo models
tested
- No increased tail bleeding times
• Db-TCK26D6x33H1F7 accumulates in the thrombus
• Db-TCK26D6x33H1F7 has a circulating half-life of 121 min in mice (tPA = 5
min)
ProFiDIA development plan
Antibody discovery and diabody generation
Diabody optimization
Candidate selection
Non clinical development
Phase 1 Phase 2aPhase 2b/3
• Antibody screening
• Antibody engineering
• In vitro testing• In vivo POC
efficacy
• Humanization• Optimizing
antigen binding potency, expression, stability and biophysical properties
• In vivo POC efficacy
• Extensive testing of humanized diabody in preclinical models
• t-PA comparison studies
• Bleeding studies• PK/PD relationship
• CMC• Scale-up, process &
analytical development
• Non-clinical batch development
• Non clinical studies• Safety
pharmacology (mice, cynomolgus)
• Single/2 week dose toxicity in mice
• 2 week dose toxicity in cynemolgus
• Human TCR
• CMC• Clinical batch
production• Clinical study
• Dose escalation
• Dose finding• Drug-drug
interaction study
• CMC• Clinical batch
production• Clinical study
• Feasibility POC efficacy study in stroke patients
M1: CandidateMay 2017
M2: IND applicationNov 2018
M3: Safe dose in humanSep 2019
M4: Clinical POCDec 2020
Achieved
Development within ProFiDIA
Partnering
Ongoing
• Partnering
• Clinical studies• Elaborated
POC efficacy studies
IP & FTO
• IP
o DUAL TARGETING OF TAFI AND PAI-1 (US 61/937323 &
PCT/EP2015/052624): Priority date 07/02/2014;
• Use claims alone or in combination with tPA for treatment of acute
thrombotic disorders
• Composition claims
o Claims approved for N, IS and IA; entering national phase 08/2016
• FTO
o To our knowledge, no other groups have applied for patent protection for a
bispecific antibody that targets both PAI-1 and TAFI.
o No hits were identified in keyword searches conducted in Jan 2014, prior to
filing the earliest priority patent application, in a commercial patent
database (Thomson Innovation).
Conclusion
• The ProFiDIA diabody is a first-in class fibrinolysis enhancer for
treatment of stroke and submassive PE
• The ProFiDIA diabody is protected by granted patent (national phase
aug/sept 2016)
• The (non) clinical development roadmap is feasible
Profibrinolytic diabody targeting PAI-1 and TAFI
for treatment of acute thrombotic disorders
THROMBOLYSIS WITH IMPROVED SAFETY
ProFiDIΛ