ProFiDIΛ Profibrinolytic diabody targeting PAI-1 and TAFI for treatment of acute thrombotic disorders THROMBOLYSIS WITH IMPROVED SAFETY 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) Thrombectomy not always available Patients not eligible (time, small artery occlusion) Priming the thrombotic occlusion prior to thrombectomy with a safe profibrinolytic agent (with or without low dose of tpa) Thrombolysis for patients not eligible for thrombectomy (60% of the patients) Narrow time window for thrombolysis Bleeding risk (ICH) tpa induced neurotoxicity Improved safety (less ICH) Longer time window Treatment for patients not eligible for thrombectomy and/or thrombolysis with current agents Pulmonary thromboembolism 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) Heavy chain Light chain Antibody (150 kda) Heavy chain Light chain CL CH 1 CH 1 CL CL CH 1 CH 1 CL CH 2 CH 2 CH 2 CH 2 CH 3 anti-pai-1 Mab anti-tafi Mab MA-33H1F7 CH 3 CH 3 CH 3 MA-TCK26D6 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 vitronectinbound 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 Antibody (150 kda) Heavy chain Light chain Antibody (150 kda) Heavy chain Light chain CL CH 1 CH 1 CL CL CH 1 CH 1 CL CH 2 CH 2 CH 2 CH 2 anti-tafi Mab anti-pai-1 Mab MA-TCK26D6 CH 3 CH 3 CH 3 CH 3 MA-33H1F7 A A Anti-TAFI 9 AA linker Anti-TAFI scfv (28 kda) Anti-PAI-1 B B Anti-PAI-1 scfv (28 kda) Diabody (58 kda)
Resistence (mm) 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 Citrated whole blood + MA(s), diabody or saline 10, 37 C + CaCl 2 + Tissue factor + tpa Anti-PAI-1 MA Anti-TAFI MA Combination of MA Time (min) 7 Diabody
Strong in vitro profibrinolytic properties HUMAN BLOOD Full lysis 100 80 P < 0.05 Paired t-test L45 (%) 60 40 *** *** 20 0 MA-33H1F7 MA-TCK26D6 Combination of antibodies Diabody N = 4 *** P < 0.005 Kruskal-Wallis test No lysis
Strong in vivo profibrinolytic properties Monofilament-mediated stroke model 1 hour 24 hours MCAo 1. Reperfusion 2. Treatment 100 * Fibrin deposition Infarct size Neurological test Motor test Berressem Nutritionalneuroscience.com N =10-12/ group * P < 0.05 Lesion volume (mm 3 ) 80 60 40 20 0 Vehicle Control IgG (2 mg/kg) MA-33H1F7 (1 mg/kg) MA-TCK26D6 (1 mg/kg) combination of MA Diabody (0.8 mg/kg) Vehicle Diabody (0.8mg/kg)
Diabody vs tpa Thrombotic middle cerebral artery occlusion models FeCl 3 Thrombin pipette MCA MCA MCA MCA Karatas et al. Stroke 2011 Orset et al. Stroke 2007 FeCl 3 Thrombin Oxidative stress Platelet activation Fibrinogen Fibrin Collaboration with prof. Denis Vivien and Dr. Marina Rubio INSERM, France
Diabody vs tpa Thrombotic middle cerebral artery occlusion models FeCl 3 Thrombin pipette MCA MCA MCA MCA Karatas et al. Stroke 2011 Orset et al. Stroke 2007 Platelet-rich clot Fibrin-rich clot Resistant to tpa-mediated lysis Susceptible to tpa-mediated lysis Collaboration with prof. Denis Vivien and Dr. Marina Rubio INSERM, France
Improved efficacy compared to tpa on platelet rich clots Thrombotic middle cerebral artery occlusion models 0 15 20 60 24 hours MCAo Platelet-rich clot Db or Buffer tpa or Buffer 1. Arterial status 2. Lesion size 3. Brain perfusion Vehicle 25 * tpa Db Infarct volume (mm 3 ) 24h post stroke onset 20 15 10 5 * P < 0.05 N = 9-15/ group 0 Db 2x 12 vehicle tpa 10mg/kg Db 1.6mg/kg Db 3.6mg/kg
Improved efficacy compared to tpa on fibrin rich clots Thrombotic middle cerebral artery occlusion models 85 90 150 0 15 20 60 24 hours MCAo Fibrin-rich clot Db or Buffer tpa or Buffer 1. Arterial status 2. Lesion size 3. Brain perfusion
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 2a Phase 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 Partnering Clinical studies Elaborated POC efficacy studies Achieved Ongoing Development within ProFiDIA Partnering M1: Candidate May 2017 M2: IND application Nov 2018 M3: Safe dose in human Sep 2019 M4: Clinical POC Dec 2020
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 o To our knowledge, no other groups have applied for patent protection for a bispecific antibody that targets both PAI-1 and TAFI. 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
ProFiDIΛ Profibrinolytic diabody targeting PAI-1 and TAFI for treatment of acute thrombotic disorders THROMBOLYSIS WITH IMPROVED SAFETY