ALBERT

Description: Factor XII (FXII), also called Hageman Factor, is a key component of the plasma contact system. When blood is exposed to artificial surfaces or polyanionic substances, the zymogens FXII and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and plasma kallikrein, respectively. FXIIa initiates coagulation through the intrinsic pathway by activating factor XI, while plasma kallikrein mediates generation of the potent vasodilator bradykinin. Patients lacking FXII or PK do not experience abnormal bleeding, indicating these proteins are not required for hemostasis. However, FXIIa and plasma kallikrein are required for formation of occlusive clots in animal thrombosis models, and FXIIa likely contributes to thrombus formation in humans when blood is passed through extracorporeal circuits (e.g. cardiopulmonary bypass). These observations suggest that FXIIa inhibition could be an effective antithrombotic strategy that would not have bleeding side effects associated with current approved anticoagulants. We used our human antibody phage display library to identify a highly selective and potent monoclonal antibody inhibitor of FXIIa, DX-4012. DX-4012 inhibits the proteolytic activity of FXIIa with an apparent Ki of ~15 pM, and does not inhibit closely related sequence homologs or other coagulation factors at concentrations up to 1 µM. When tested at 1 µM in human plasma, DX-4012 prolonged the activated partial thromboplastin time (aPTT) 3.4-fold, with no effect on the prothrombin time (PT). In a non-human primate pharmacokinetic study, an intravenous infusion of 10 mg/kg DX-4012 prolonged the aPTT 2.4-fold but had no effect on the PT. Given the importance of plasma kallikrein to FXII activation, we reasoned that the antithrombotic effect of DX-4012 could be augmented by combination with a kallikrein inhibitor. To test this, a variant of DX-4012 was converted into a single chain variable fragment (scFv) and combined with DX-2930, a potent and specific monoclonal antibody inhibitor of plasma kallikrein, to generate a "Morrison format" bispecific antibody. Enzyme inhibition assays determined that the apparent Ki values of the individual anti-kallikrein and anti-FXIIa components of the bispecific antibody were similar to the parent molecules (apparent Ki 389 pM and 73 pM, respectively). In contact-activated dilute plasma, the bispecific antibody was 〉 5 times more effective at preventing kallikrein generation than a 1:1 combination of DX-4012 and DX-2930, and more than 20-fold more effective than either DX-4012 or DX-2930 alone. Our data indicate that DX-4012, either as a monoclonal antibody or as a component of a bispecific antibody, shows potential as a novel antithrombotic therapy. Simultaneous inhibition of FXIIa and plasma kallikrein may be a uniquely potent method of blocking FXIIa activity through inhibition of the positive feedback loop during contact activation. Disclosures Mason: Dyax Corp: Employment. Kenniston:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Kastrapeli:Dyax Corp: Employment. Kopacz:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Kivaa:Dyax Corp: Employment. Qiu:Dyax Corp: Employment. Faucette:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Tenhoor:Dyax Corp: Employment. Wallisch:Aronora: Employment. Gruber:Aronora, Inc.: Employment, Equity Ownership, Patents & Royalties, Research Funding. Adelman:Dyax Corp: Employment. Nixon:Dyax Corp: Employment.

Description: Localized pathologic thrombin generation by the tissue factor/factor VII (TF/FVII)-dependent extrinsic pathway of blood coagulation has been suggested to be the key initiator of thrombosis following blood vessel injury and exposure of blood to the subendothelial matrix that contains coagulation/platelet activator matrix proteins, such as collagen and TF. Inherited FVII deficiency (30 days) systemic anticoagulation, as evidenced by PT increase that reached 2.0-2.6 fold baseline. Surprisingly, fibrin deposition was not affected, and platelet deposition in the thrombogenic devices (N=8) was also not inhibited on collagen-coated grafts, and only marginally inhibited on TF. Treatment with vitamin K antagonists that reduces the level of vitamin K-dependent coagulation enzymes, including FVII, also prolong the PT. Warfarin-induced doubling of the PT is antithrombotic and moderately safe. Yet, in this study where FVII ASO selectively reduced FVII levels, the observed and significant PT prolongation was not associated with anti-fibrin or anti-platelet effects on collagen, and showed an unremarkable anti-platelet effect on TF. We conclude that if the TF/FVII pathway drives the propagation of large vessel thrombi under flow in primates indeed, very low FVII activity (≈1%) must be sufficient to sustain thrombogenesis, supporting the clinical data on deep vein thrombosis, routinely observed in FVII deficiency. Since FVII activity cannot be entirely eliminated without bleeding risks, these data suggest that targeting the TF/FVII pathway with FVII ASO or inhibitors may not be an entirely sound antithrombotic treatment approach. Disclosures Wallisch: Aronora, Inc: Employment. Tucker:Aronora, Inc: Employment, Equity Ownership. Murray:Isis Pharmaceuticals, Inc.: Employment. Crosby:Isis Pharmaceuticals, Inc.: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment. Gruber:Aronora, Inc: Employment, Equity Ownership.

Description: Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin’s procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (

Description: Thrombin-activatable fibrinolysis inhibitor (TAFI) activation during thrombolysis may limit the effectiveness of tissue plasminogen activator (tPA) treatment. Our data suggest that an antithrombotic human thrombin analog, E-WE thrombin (ProCase), which is a selective protein C activator under development for treating acute thrombotic emergencies, also promotes tPA-induced fibrinolysis. We found that E-WE thrombin is a poor activator of TAFI compared to wild-type (WT) thrombin. In the presence of thrombomodulin (TM), WT thrombin induced TAFI activation within 5 min. In contrast, detectable amounts of activated TAFI were not generated by E-WE thrombin in the presence of TM until 60 min. Further studies using a quantitative TAFI activity assay showed that E-WE thrombin inhibited thrombin/thrombomodulin-mediated TAFI activation in a concentration-dependent manner. The ability of E-WE to enhance tPA induced clot lysis was also evaluated in vitro. When both E-WE thrombin and tPA were incorporated within plasma clots, lysis was accelerated by up to 74% compared with the addition of tPA alone. We then tested the ability of E-WE thrombin to interrupt arterial-type experimental thrombus formation in baboons when combined with a standard interventional dose of tPA (1mg/kg). Thrombosis was initiated in the baboons by interposing 4mm internal diameter collagen coated ePTFE vascular grafts within an arterio/venous shunt. Thrombus formation was monitored by real-time gamma camera imaging of autologous 111In-labelled platelet accumulation in the grafts for a total of 90 min. Fibrin deposition was determined by direct endpoint measurement of incorporated 125I-labelled fibrinogen. Antithrombotic interventions were injected intravenously at 30 min after graft deployment into the shunt. Treatment with tPA (1mg/kg, iv) reduced fibrin deposition by 57%, but did not reduce graft-associated platelet accumulation compared with controls (n=8 and 9, respectively). E-WE thrombin, at doses ranging from 2-10µg/kg (n=8), interrupted thrombus growth within 10 min of treatment, and reduced platelet and fibrin deposition by 53-70% and 45-58% at 90 min, respectively, compared with controls. When E-WE thrombin (2µg/kg) was co-administered with tPA (1mg/kg), a profound 91% reduction in thrombus fibrin content was observed (n=1), with platelet deposition being reduced by 34%. Bleeding time and volume were also assessed during the studies, with E-WE treated animals showing no increased bleeding compared with controls. However, tPA caused ecchymoses that were not observed with E-WE thrombin treatment. The combination therapy showed no overt anti-hemostatic effects beyond tPA administration alone. These data suggest that E-WE thrombin can inhibit TAFI activation, and co-administration of E-WE thrombin with tPA may improve the efficacy of thrombolysis without additional hemostasis impairment. Disclosures Tucker: Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Markway:Aronora, Inc: Employment, Equity Ownership. Wallisch:Aronora, Inc: Employment, Equity Ownership. Verbout:Aronora, Inc: Employment, Equity Ownership. Lorentz:Aronora, Inc: Employment, Equity Ownership. Carris:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: A hemostatically safe antithrombotic treatment with rapid onset could significantly improve the chances for survival during acute thrombotic events such as heart attack and ischemic stroke. Current thrombolytic treatment with tissue plasminogen activator (tPA) is effective at halting and reversing the progression of arterial thrombi, but bleeding side-effects significantly limit its usefulness. EWE thrombin (ProCase) is an investigational selective protein C activator thrombin analog under development for treating acute thrombotic emergencies. We therefore tested the ability of EWE thrombin to interrupt arterial-type thrombus formation in baboons compared to a standard interventional dose of tPA (1mg/kg, iv). Thrombosis was initiated by interposing 2mm or 4mm internal diameter (ID) collagen coated ePTFE vascular grafts within an arterio/venous shunt. Platelet thrombus formation was monitored by gamma camera imaging of autologous 111In-labelled platelets for a total of 90 min in the 4mm grafts, or until occlusion time in the 2mm grafts. Fibrin deposition was determined by direct measurement of 125I-labelled fibrinogen. All interventions were given systemically, starting 30 min or 15 min after thrombus initiation for the 4mm grafts and 2mm grafts, respectively. In the 4mm devices, treatment with tPA reduced graft-associated platelet accumulation by 17% and fibrin deposition by 61% compared with controls (n=6 each). A negative platelet accumulation rate, which is an indication of thrombolysis, occurred between 40-50 min after initiating tPA treatment. EWE thrombin, at doses ranging from 2-10µg/kg iv bolus rapidly interrupted thrombus development and reduced platelet deposition by 43-65% (n=4). Fibrin deposition was also reduced by 36-49% compared with controls. A negative platelet accumulation rate occurred between 10-15 min after EWE thrombin treatment, compared with continuous platelet accumulation in all control experiments. In the 2mm ID grafts, 0/6 control group devices remained patent with an average occlusion time of 25±2 min. By comparison, EWE thrombin (10µg/kg iv bolus given at 15 min) successfully interrupted occlusive thrombus formation in 40% of the devices (2/5) during the 60 min study, and significantly prolonged the occlusion time to an average of 43±8 min. These data suggest that EWE thrombin can effectively interrupt occlusive arterial-type thrombus development at very low doses, and appears to be more effective and act more rapidly than tPA at limiting experimental platelet-rich thrombus accumulation. We conclude that EWE thrombin may be a more effective and safer alternative to tPA for treating acute thrombotic emergencies. Disclosures: Tucker: Aronora, Inc: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Wallisch:Aronora, Inc: Employment. Leung:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Verbout:Aronora, Inc: Employment, Equity Ownership.

Description: Background: End stage renal disease (ESRD) patients on chronic hemodialysis (HD) repeatedly have their blood exposed to artificial surfaces within the hemodialysis circuit, which triggers contact system-initiated coagulation. Clot formation within the HD circuit results in blood loss, decreased HD efficiency, and device circuit failure. Heparin reduces circuit clotting but it is not tolerated by all patients. Mounting experimental data suggest that contact system inhibition is antithrombotic without significantly compromising hemostasis. AB023 is a unique recombinant anti-factor (F) XI antibody that interferes with the interactions of FXI and FXII without inhibiting FXI activation by thrombin or the activation of FIX by activated FXI. This study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of AB023 in patients with ESRD on chronic HD. Methods: In this phase 2, randomized, double-blind, placebo-controlled, single-administration study, AB023 (0.25 or 0.5 mg/kg) or placebo was injected into the HD circuit of 24 ESRD patients at the start of heparin-free HD (NCT03612856). All patients underwent three pre-dose HD procedures and four monitored post-dose HD sessions. In total, the study encompassed 168 individual HD sessions, 72 of which occurred pre-dose and 96 of which occurred post-dose. Safety parameters, including time to hemostasis at HD access site, prothrombin time, physical examinations, vital signs, electrocardiograms, clinical chemistry, immunogenicity and other adverse events (AEs), were recorded. Clotting within the hemodialyzer circuit was assessed using a visual clotting scale by investigators who were blinded to treatment. The frequency of circuit clot obstructions requiring dialyzer exchange was also recorded. Pharmacokinetic and pharmacodynamic parameters, including activated partial thromboplastin times, were also assessed. The study was approved by an ethics review board and all subjects provided written informed consent. Results: AB023 demonstrated a favorable safety profile in ESRD patients. No drug-related AEs were noted. Clinically relevant bleeding did not occur in any patient, and the time to hemostasis at the HD access site was unchanged after AB023 administration. As expected, the aPTT was prolonged after AB023 administration, reaching saturation at both dose levels (about 2-fold prolongation) immediately after dosing, and remaining prolonged for up to 9 days with the high dose. Compared with pretreatment, the frequency of occlusive events requiring circuit exchange decreased by 68% and 50% in subjects given the 0.25 and 0.5 mg/kg AB023 respectively, while the number of occlusive events was numerically unchanged in the placebo arm. Likewise, the number of saline flushes required to maintain circuit patency decreased by 44% and 85%, and the incidence of high-grade clotting in the hemodialyzer as assessed by visual scoring decreased by 15% and 43% on day 1, 8% and 35% by day 3, and 0% and 19% by day 5 in the 0.25 and 0.5 mg/kg cohorts, respectively. Conclusions: Anticoagulation with AB023 was well-tolerated and reduced clot formation within the HD circuit of ESRD patients. These findings suggest that inhibiting contact system activation could reduce medical device-initiated blood clot formation in patients. Disclosures Lorentz: Aronora, Inc.: Current Employment. Verbout:Aronora, Inc.: Current Employment. Shatzel:Aronora, Inc.: Consultancy. Wallisch:Aronora, Inc.: Current Employment. Markway:Aronora, Inc.: Current Employment. Tucker:Aronora, Inc.: Current Employment. Gruber:Aronora, Inc.: Current Employment, Current equity holder in private company.