Factor IXa Inhibition Aptamer Technology Overview and First Results with RB006

1. Factor IXa InhibitionAptamer Technology Overview and First Results with RB006/RB007 Mauricio G. Cohen, MD Associate Professor of Medicine Director, Cardiac Cath Lab

2. Disclosure Statement of Financial Interest Within the past 12 months, I or my spouse/partner have had a financial interest/arrangement or affiliation with the organization(s) listed below. Affiliation/Financial Relationship Company Grant/Research Support REGADO BIOSCIENCES

3. Search for the Ideal Anticoagulant

4. Aptamers: Nucleic Acids and Monoclonal Antibodies Aptamers are single-stranded nucleic acids that adopt a specific shape enabling them to bind with high affinity and specificity to target proteins, in a manner very similar to the way monoclonal antibodies recognize their target proteins. In fact, the simplest way to view an aptamer is as a monoclonal antibody composed of nucleic acids. The term aptamer was chosen by Ellington and Szostak following their pioneering work published originally in Nature. A highly specific inhibitor that conforms to the surface of functional groups (on the target protein) Aptamers are single-stranded nucleic acids that adopt a specific shape enabling them to bind with high affinity and specificity to target proteins, in a manner very similar to the way monoclonal antibodies recognize their target proteins. In fact, the simplest way to view an aptamer is as a monoclonal antibody composed of nucleic acids. The term aptamer was chosen by Ellington and Szostak following their pioneering work published originally in Nature. A highly specific inhibitor that conforms to the surface of functional groups (on the target protein)

5. Aptamers Encode Their Own Control Agents For the second image: Because aptamers are nucleic acids, they encode the information necessary to create complementary oligonucleotide antidotes to them, that can specifically recognize the aptamer by simple Watson-Crick base pairing. For the second image: Because aptamers are nucleic acids, they encode the information necessary to create complementary oligonucleotide antidotes to them, that can specifically recognize the aptamer by simple Watson-Crick base pairing.

6. Aptamers Encode Their Own Control Agents Final image: Binding of an antidote oligonucleotide to the aptamer changes it shape, disabling the target binding function of the aptamer and thereby neutralizing its pharmacologic activity. Final image: Binding of an antidote oligonucleotide to the aptamer changes it shape, disabling the target binding function of the aptamer and thereby neutralizing its pharmacologic activity.

7. REG1 Anticoagulant System

8. REG1 Anticoagulation System The REG1 anticoagulant system is the first drug-antidote pair derived from this technology to be tested in humans. It is composed of the drug�a specific aptamer-based inhibitor of coagulation factor IXa�and the antidote, which was designed to neutralize the pharmacologic activity of the drug. For first image: The drug, RB006, is a competitive factor IXa inhibitor that yields an anticoagulant effect by selectively blocking the factor VIIIa/IXa-catalyzed conversion of factor X to FXa�a pivotal step in prothrombinase assembly and thrombin generation on the surface of tissue factor-bearing cells and activated platelets. Because of the availability of an antidote to reverse its activity if clinically required, the drug has been formulated to have a prolonged duration of effect. For the second image: The antidote, RB007, is an oligonucleotide complementary to the portion of the drug (highlighted in blue) that binds effectively to it, changing its conformation, and thereby neutralizing its anti-factor IXa activity. The antidote itself possesses no inherent biologic activity and is cleared rapidly from the circulation. Final image: The resultant drug-antidote complex is pharmacologically inactive.The REG1 anticoagulant system is the first drug-antidote pair derived from this technology to be tested in humans. It is composed of the drug�a specific aptamer-based inhibitor of coagulation factor IXa�and the antidote, which was designed to neutralize the pharmacologic activity of the drug. For first image: The drug, RB006, is a competitive factor IXa inhibitor that yields an anticoagulant effect by selectively blocking the factor VIIIa/IXa-catalyzed conversion of factor X to FXa�a pivotal step in prothrombinase assembly and thrombin generation on the surface of tissue factor-bearing cells and activated platelets. Because of the availability of an antidote to reverse its activity if clinically required, the drug has been formulated to have a prolonged duration of effect. For the second image: The antidote, RB007, is an oligonucleotide complementary to the portion of the drug (highlighted in blue) that binds effectively to it, changing its conformation, and thereby neutralizing its anti-factor IXa activity. The antidote itself possesses no inherent biologic activity and is cleared rapidly from the circulation. Final image: The resultant drug-antidote complex is pharmacologically inactive.

9. Rationale for Targeting FIXa Coagulation is initiated when tissue factor (TF) binds activated factor VII, a circulating coagulation factor. In general, blood is not exposed to TF, a transmembrane protein constitutively expressed on extravascular cells. However, vascular injury exposes these extravascular TF-bearing cells to blood, and thus initiates the coagulation process. In various inflammatory states, TF expression can also be upregulated on monocytes and endothelial cells by bacterial antigens,3 inflammatory cytokines,4 and tumor necrosis factor.5 The TF/VIIa complex activates factors IX and X.6 Factor IXa is relatively stable in plasma and diffuses toward activated platelets. In contrast, factor Xa is unstable in plasma and is rapidly inhibited by TF pathway inhibitor and antithrombin III.7,8 On the surface of TF-bearing cells, factor Xa binds factor Va.9 In turn, the Xa/Va complex generates a small but sufficient amount of thrombin to cause platelet activation.10,11 In the priming phase, platelets and coagulation factors are activated by thrombin.1 Thrombin binds and cleaves platelet protease-activated receptors (PAR1 and PAR4), triggering a signaling cascade that catalyzes platelet activation and release of factor V from platelet granules. In addition, thrombin activates factors V, VIII, and XI. Thrombin generation is maximized on the surface of platelets during the propagation phase. The primed, activated platelets bind the IXa/VIIIa �tenase� complex. Additional IXa is generated by factor XIa on the platelet surface.12 The IXa/VIIIa complex, in physical proximity to Va, recruits factor X to the platelet surface for activation. The Xa/Va complex on the platelet surface is protected from TF pathway inhibitor and antithrombin III.13,14 Enzymology studies have shown that activation of factor X by IXa/VIIIa is nearly 50 more efficient than activation by factor VIIa/TF.15 The platelet Xa/Va complex generates a �burst� of thrombin, resulting in a stable fibrin�platelet clot. The cell-based model of coagulation highlights the importance of the IXa/VIIIa complex in clot formation. Thus, factor IX represents an excellent target for anticoagulant therapy.Coagulation is initiated when tissue factor (TF) binds activated factor VII, a circulating coagulation factor. In general, blood is not exposed to TF, a transmembrane protein constitutively expressed on extravascular cells. However, vascular injury exposes these extravascular TF-bearing cells to blood, and thus initiates the coagulation process. In various inflammatory states, TF expression can also be upregulated on monocytes and endothelial cells by bacterial antigens,3 inflammatory cytokines,4 and tumor necrosis factor.5 The TF/VIIa complex activates factors IX and X.6 Factor IXa is relatively stable in plasma and diffuses toward activated platelets. In contrast, factor Xa is unstable in plasma and is rapidly inhibited by TF pathway inhibitor and antithrombin III.7,8 On the surface of TF-bearing cells, factor Xa binds factor Va.9 In turn, the Xa/Va complex generates a small but sufficient amount of thrombin to cause platelet activation.10,11 In the priming phase, platelets and coagulation factors are activated by thrombin.1 Thrombin binds and cleaves platelet protease-activated receptors (PAR1 and PAR4), triggering a signaling cascade that catalyzes platelet activation and release of factor V from platelet granules. In addition, thrombin activates factors V, VIII, and XI. Thrombin generation is maximized on the surface of platelets during the propagation phase. The primed, activated platelets bind the IXa/VIIIa �tenase� complex. Additional IXa is generated by factor XIa on the platelet surface.12 The IXa/VIIIa complex, in physical proximity to Va, recruits factor X to the platelet surface for activation. The Xa/Va complex on the platelet surface is protected from TF pathway inhibitor and antithrombin III.13,14 Enzymology studies have shown that activation of factor X by IXa/VIIIa is nearly 50 more efficient than activation by factor VIIa/TF.15 The platelet Xa/Va complex generates a �burst� of thrombin, resulting in a stable fibrin�platelet clot. The cell-based model of coagulation highlights the importance of the IXa/VIIIa complex in clot formation. Thus, factor IX represents an excellent target for anticoagulant therapy.

10. Rationale for Targeting Factor IXa FVIIIa/FIXa activation of FX is the rate limiting step in thrombin generation FIX knockout mice lack occlusive clot formation following vascular injury due to insufficient generation of thrombin to form platelet aggregates. FIXa concentration is lower than Xa and thrombin, making high levels of target inhibition more readily achievable High Factor IX levels are associated with increase in ACS and venous thromboembolism Transgenic mice overexpressing FIXa have a shorter lifespan and develop arterial thrombosis and myocardial fibrosis with vascular distribution patterns similar to those of ischemic cardiomyopathy in humans Hemophilia B Carriers-Reduced CHD Mortality Foreign materials (eg. catheters and guidewires) lead directly to FIX activation There is already evidence that activation FX by the complex FVIIIa/FIXa is the rate limiting step for thrombin generation. As an example FIX knock out mice lack occlusive clot formation after vascular injury due to insufficient generation of thrombin to generate platelet aggregates. Compared to other targets, such as Xa and thrombin, the concentrations of FIXa are substantially lower, making FIXa a more efficient target for inhibition. There is already evidence that activation FX by the complex FVIIIa/FIXa is the rate limiting step for thrombin generation. As an example FIX knock out mice lack occlusive clot formation after vascular injury due to insufficient generation of thrombin to generate platelet aggregates. Compared to other targets, such as Xa and thrombin, the concentrations of FIXa are substantially lower, making FIXa a more efficient target for inhibition.

11. CLIN101 � Phase 1A Study Study Design 85 healthy volunteers Multi-center, randomized, subject blinded, placebo controlled Evaluate safety of dose escalation of REG1 system or components and pharmacokinetic/pharmacodynamic relationships Single-dose, dose escalation through 4 dose levels RB006, RB007, and REG1 treatment arms at each dose level Results 1 SAE but no trend of safety signals RB006 produced dose dependent aPTT levels RB007 neutralized observed pharmacodynamic effects of RB006

12. CLIN101 � REG1 Activity in Healthy Volunteers

13. Stable CAD � Phase 1B Study Study Design 50 patients w/ stable CAD on aspirin/clopidogrel 56-68 years of age Multi-center, randomized, double-blind, placebo-controlled Evaluate safety of dose escalation of the REG1 system and pharmacokinetic/pharmacodynamic relationships Single-dose, dose escalation through 4 dose levels RB006 and REG1 treatment arms at each dose level Results No SAE�s or safety signals RB006 produced dose-dependent aPTT levels RB007 neutralized observed pharmacodynamic effects of RB006

14. Stable CAD � Phase 1B StudyResults

15. Repeat-dose � Phase IC Study Study Design 38 healthy volunteers Single-center, randomized, double-blind, placebo controlled Primary endpoints: Evaluate safety and tolerability of repeated doses of REG1 system Determine the optimal dose ratio for RB007 and RB006 3 consecutive weight-adjusted, drug-antidote treatment cycles or double placebo Fixed doses of RB006 followed by titrated dose of RB007 (2:1 � 0.125) Results Achieved highly reproducible aPTT levels with repeat doses of RB006 Reversed aPTT levels with RB007 dose-dependently and reproducibly

16. Repeat-dose � Phase IC StudyAntidote Dose Response

17. Repeat-dose � Phase IC StudyActivity In Healthy Volunteers Activated partial thromboplastin time (APTT) response with alternate-day injections of 0.75 mg kg)1 RB006. RB006, active drug. Means with upper 95% confidence intervals are displayed. Subjects in groups 1, 2 and 3 received 0.75 mg/kg RB006 on all 3 days. Fold change refers to the number of times that the APTT was elevated over baseline levels. Only the pharmacodynamic population (subjects who completed per protocol treatment) was included in this analysis. Time weighted average APTT values were determined using the 5-min, 15-min, 30-min and 59-min observations for RB006.Activated partial thromboplastin time (APTT) response with alternate-day injections of 0.75 mg kg)1 RB006. RB006, active drug. Means with upper 95% confidence intervals are displayed. Subjects in groups 1, 2 and 3 received 0.75 mg/kg RB006 on all 3 days. Fold change refers to the number of times that the APTT was elevated over baseline levels. Only the pharmacodynamic population (subjects who completed per protocol treatment) was included in this analysis. Time weighted average APTT values were determined using the 5-min, 15-min, 30-min and 59-min observations for RB006.

18. REG1 Phase 1 Studies- Overview Demonstrated safety of 006, controlling agent (007) and REG1 system Demonstrated controlling agent efficacy No known drug interactions with common anti-platelet therapy Demonstrated RB006 and RB007 PK and excretion Understanding of relationship between RB006 PK and PD Identifed optimal dose of RB006 for maximal FIXa inhibition Determined optimal dosing ratio of controlling agent to drug Demonstrated ability to titrate the controlling agent Ability to proceed to Phase II development of REG1 in PCI and ACS Demonstrated safety of 006, controlling agent (007) and REG1 system Demonstrated controlling agent efficacy No known drug interactions with common anti-platelet therapy Demonstrated RB006 and RB007 PK and excretion Understanding of relationship between RB006 PK and PD Identifed optimal dose of RB006 for maximal FIXa inhibition Determined optimal dosing ratio of controlling agent to drug Demonstrated ability to titrate the controlling agent Ability to proceed to Phase II development of REG1 in PCI and ACS

19. Phase 1 Program Summary Phase 1 Program Highlights: Demonstrated safety of RB006, controlling agent (RB007) and the overall REG1 system Demonstrated controlling agent efficacy No known drug interactions with common anti-platelet therapy Demonstrated RB006 and RB007 PK and elimination Understanding of relationship between RB006 PK and PD Identified optimal dose of RB006 for maximal FIXa inhibition Demonstrated ability to titrate the controlling agent Ability to proceed to Phase II development of REG1 in PCI and ACS Together, these studies demonstrated several favorable pharmacodynamic properties for RB006- it achieved anticoagulant activity through factor IXa inhibition within minutes of administration; possessed a long duration of effect, and was well tolerated in both healthy volunteers and subjects with stable coronary artery disease receiving aspirin with or without clopidogrel. In addition, these studies demonstrated that RB007 could rapidly (within 1 to 5 minutes) and either partially, in a graded fashion, or fully neutralize the pharmacologic activity of RB006. Reversal of RB006 activity by RB007 administration yielded durable neutralization of the drugs activity, with no signs of rebound anticoagulation. There were no major hemorrhagic events, liver or kidney toxicity, or evidence of complement activation-a concern with early generation oligonucleotides. Together, these studies demonstrated several favorable pharmacodynamic properties for RB006- it achieved anticoagulant activity through factor IXa inhibition within minutes of administration; possessed a long duration of effect, and was well tolerated in both healthy volunteers and subjects with stable coronary artery disease receiving aspirin with or without clopidogrel. In addition, these studies demonstrated that RB007 could rapidly (within 1 to 5 minutes) and either partially, in a graded fashion, or fully neutralize the pharmacologic activity of RB006. Reversal of RB006 activity by RB007 administration yielded durable neutralization of the drugs activity, with no signs of rebound anticoagulation. There were no major hemorrhagic events, liver or kidney toxicity, or evidence of complement activation-a concern with early generation oligonucleotides.

20. PCI Pilot � Reversal PCI Feasibility study comparing the REG1 system with UFH in stable CAD patients undergoing elective PCI Multi-center, open-label, randomized (10/07�10/08) Black Hills Cardiology (Rapid City, SD) Henry Ford Hospital (Detroit, MI) University of North Carolina at Chapel Hill (NC) The Care Group (Indianapolis, IN) Hospital Italiano (Buenos Aires, Argentina) Central Coordination: Duke Clinical Research Institute Primary Endpoint Major Bleeding using the ACUITY bleeding criteria until hospital discharge or 48�hours whichever occurs first. Composite of death, nonfatal myocardial infarct (MI), and urgent target vessel revascularization (TVR) through Day�14 Our primary objectives were to determine whether RB006-mediated factor IXa inhibition could achieve a consistent and readily measurable level of anticoagulation and support PCI. We also wanted to assess the feasibility and initial safety of active reversal with RB007 upon completion of the procedure in anticipation of femoral arterial sheath removal.Our primary objectives were to determine whether RB006-mediated factor IXa inhibition could achieve a consistent and readily measurable level of anticoagulation and support PCI. We also wanted to assess the feasibility and initial safety of active reversal with RB007 upon completion of the procedure in anticipation of femoral arterial sheath removal.

21. PCI Pilot � Reversal PCI

22. Timeline of Procedures

23. Baseline Characteristics

24. Procedural Characteristics

25. Primary Outcomes Measures Ischemic events were infrequent and there were no deaths. The composite of death, MI, and urgent TVR occurred in 2 of the 20 patients treated with REG1 and in one of the 4 patients treated with heparin (MI). One patient in the partial reversal arm experienced a periprocedural MI after two-vessel PCI without angiographic complications, electrocardiographic changes or clinical instability. The patient was discharged the following day without any subsequent events. A patient in the complete REG1 reversal group was rehospitalized for chest pain 6 days after the index procedure. Repeat coronary angiography demonstrated no evidence of complications at the stented site. However, intravascular ultrasound evaluation demonstrated the presence of atherosclerotic disease in a segment proximal to the initially placed stent. Therefore an additional stent was placed. There were no thrombotic complications such as major dissection, abrupt or threatened closure, deterioration of flow, no-reflow, side-branch closure, distal embolization, or clot formation in catheter and/or wires. There was no need for bail-out use of heparin in any of the study patients and no vascular access complications were recorded. Bleeding events were infrequent. One patient in the unfractionated heparin arm experienced a major bleeding within 48 hours of randomization, consisting of a groin hematoma larger than 5 cm in diameter at the puncture site. Non-serious bleeding occurred in three patients treated with REG1 and in one treated with unfractionated heparin, all related to vascular access site. None of these events met criteria for TIMI major or minor bleeding.Ischemic events were infrequent and there were no deaths. The composite of death, MI, and urgent TVR occurred in 2 of the 20 patients treated with REG1 and in one of the 4 patients treated with heparin (MI). One patient in the partial reversal arm experienced a periprocedural MI after two-vessel PCI without angiographic complications, electrocardiographic changes or clinical instability. The patient was discharged the following day without any subsequent events. A patient in the complete REG1 reversal group was rehospitalized for chest pain 6 days after the index procedure. Repeat coronary angiography demonstrated no evidence of complications at the stented site. However, intravascular ultrasound evaluation demonstrated the presence of atherosclerotic disease in a segment proximal to the initially placed stent. Therefore an additional stent was placed. There were no thrombotic complications such as major dissection, abrupt or threatened closure, deterioration of flow, no-reflow, side-branch closure, distal embolization, or clot formation in catheter and/or wires. There was no need for bail-out use of heparin in any of the study patients and no vascular access complications were recorded. Bleeding events were infrequent. One patient in the unfractionated heparin arm experienced a major bleeding within 48 hours of randomization, consisting of a groin hematoma larger than 5 cm in diameter at the puncture site. Non-serious bleeding occurred in three patients treated with REG1 and in one treated with unfractionated heparin, all related to vascular access site. None of these events met criteria for TIMI major or minor bleeding.

26. Pharmacodynamics � WB PTT POC

27. Pharmacodynamics � ACT ACT in the 50% reversal group was 172 sec.ACT in the 50% reversal group was 172 sec.

28. Conclusions: Reversal PCI All procedures were successfully completed. No signs of catheter or guidewire thrombosis. Monitoring by ACT, POC aPTT and plasma aPTT demonstrated measurable differences in both partial and total RB007 reversal doses. The REG1 System was well tolerated. RB007 facilitated early sheath removal. RB006 (1mg/kg) demonstrated rapid onset with consistent coagulation measures during PCI.

29. In Summary� The REG 1 System offers the possibility of balancing the bleeding-ischemia risk in PCI patients This concept can be expanded to other clinical scenarios in which anticoagulation can be tailored to individual patient needs The Phase 2b RADAR trial will evaluate the safety and efficacy of the REG1 System in 800 acute coronary syndrome patients undergoing cardiac catheterization.