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