WO2020237307A1 - Chimeric molecules comprising an anti-coagulant agent and an anti-gpiib/iiia antigen binding molecule and uses thereof - Google Patents

Abstract

Disclosed are anti-thrombotic agents. More particularly, chimeric molecules are disclosed, which target multiple pathways in the coagulation cascade and which comprise an anti-coagulant agent and an anti-platelet antigen-binding molecule that binds to the active conformation of platelet integrin receptor GPIIb/IIIa with greater affinity than to its inactive conformation. In specific embodiments of the present disclosure, the chimeric molecules are used alone or in combination with other agents in compositions and methods for inhibiting thrombus and/or embolus formation and for treating or inhibiting the development of conditions associated with the presence of activated platelets.

Description

CHIMERIC MOLECULES COMPRISING AN ANTI-COAGULANT AGENT AND AN ANTI-GPIIB/IIIA ANTIGEN BINDING MOLECULE AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 62/853,084 entitled "Chimeric molecules and uses therefor" filed 27 May 2019, the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] This disclosure relates generally to anti-thrombotic agents. More particularly, the present disclosure relates to chimeric molecules which target multiple pathways in the coagulation cascade and which comprise an anti-coagulant agent and an anti-platelet antigen-binding molecule that binds to the active conformation of platelet integrin receptor GPIIb/IIIa with greater affinity than to its inactive conformation. In specific embodiments, the chimeric molecules are used alone or in combination with other agents in compositions and methods for inhibiting thrombus and/or embolus formation and for treating or inhibiting the development of conditions associated with the presence of activated platelets.

BACKGROUND

[0003] Ischemic complications, such as myocardial infarction and stroke, are a major cause of death and disability. Typically, these ischemic events are caused by the rupture of an unstable atherosclerotic plaque, leading to exposure of thrombogenic material and the acute formation of a vessel occluding thrombi. If circulation is not restored promptly, oxygen and nutrient deprivation, as well as the build-up of metabolic waste products will quickly lead to muscle damage and tissue death (Kalogeris et al., 2012. Int Rev Cell Mol Biol. 298: 229-317). While treatment such as percutaneous coronary intervention (PCI) is available and often successful in restoring blood flow, the risk of recurrent cardiovascular events remains high even under optimal medication (Benjamin, E.J., 2019. Circulation 139:e56-e528). Furthermore, paradoxically early restoration of blood flow causes a localized overshooting inflammatory response, thereby resulting in substantial cardiac tissue damage, described with the term ischemia/reperfusion injury (Kalogeris et al., 2012. supra).

Strategies to reduce the risk of recurrent events consist among other medications of single or combined anti-platelet and anti-coagulant therapies, however these therapies carry a substantial risk of major bleeding (Alexander et al., 2011. N Engl J Med. 365(8) :699-708).

[0004] Platelets play a key role in thrombus formation (Mackman N., 2008.

Nature 451 :914-918). They are present in the blood at a concentration of 1.5-4.0 x 10⁸ platelets per mL and are tiny blood cells that help the body to form clots in order to stop bleeding. However, this aggregation of platelets also result in ischemic and thrombotic events. It is now understood that platelets regulate coagulation and lead to thrombin generation in multiple ways, including the exposure of phosphatidylserine; by binding of other coagulation factors via the glycoprotein complexes glycoprotein (GP)Ib-V-IX,

GPIIb/IIIa and GPVI; as well as via thrombin-induced activation of the protease-activated receptors (PARs) (Jackson et al., 2011. Nature Medicine 17: 1423-1436). Once, platelets are activated, fibrin is actively formed on their surface, triggered via both the extrinsic (TF, FVII) and intrinsic (FXII, FXI) coagulation pathways (Jackson et al., 2011. supra).

[0005] The GPIIb/IIIa complex is the most abundant protein expressed on the platelet surface. It is also known as integrin allb83 or in the CD nomenclature CD41/CD61. The GPIIb/IIIa is a heterodimeric complex formed after synthesis of one lib and one Ilia subunit. The principal ligand for GPIIb/IIIa is fibrinogen, but it also binds to fibronectin, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand. The binding between GPIIb/IIIa and fibrinogen dimers leads to platelet aggregation and thrombus formation, and this is possible only when the receptor adopts its activated conformation (Armstrong et al., 2012. Thromb Haemost. 107(5) :808-814).

[0006] The integrin nature of GPIIb/IIIa, through its adoption of conformational states, is also fundamental to facilitating the interaction with potential ligands (Armstrong et al., 2012. supra). GPIIb/IIIa exists in a resting conformational state, where the integrin is bent and the headpiece in a 'closed' form, meaning the RGD binding domain is concealed and thus it has only a low affinity for many physiological ligands. Upon appropriate stimulation, an inside-out signal, a conformational change occurs with the integrin transforming from a bent to an extended form with an 'opening' of the headpiece, exposing the extracellular RGD ligand binding domain (resulting in the integrin having a much higher affinity for its ligands (Ma et al., 2007. J Thromb Haemost. 5: 1345-1352). One consequence of the induced conformational change of GPIIb/IIIa is the exposure of what have been termed ligand- induced binding sites (LIBS). This is followed by the unclasping of the tail sections of both subunits, structurally repositioning the transmembrane domains⁵.

[0007] The intracellular pathways governing inside-out signaling are both numerous and complex (Armstrong et al., 2012. supra). It is also believed that the conformational change of GPIIb/IIIa may induce further signaling, promoting actin polymerization and cytoskeletal reorganization in a process termed outside-in signaling (Armstrong et al., 2012. supra,· Bledzka et al., 2013. Circ Res. 112(8) : 1189-1200).

[0008] Targeting GPIIb/IIIa on platelets has been extensively studied for the prevention of platelet aggregation and has led to the reduction of ischemic complications (Topol et al., 1999. Lancet 353:227-231; Hagemeyer et al., 2010. Curr Pharm Des.

16:4119-4133; Armstrong et al., 2012. supra), especially in patients undergoing percutaneous coronary intervention. However, in large clinical trials, contradictory to its anticipated therapeutic effects, administration of GPIIb/IIIa receptor blockers in combination with fibrinolytic agents has shown little improvement in mortality, mainly due to excess bleeding (The GUSTO Investigators, 1993. N Engl J Med. 329:673-682; The GUSTO IV-ACS Investigators, 2001. Lancet 357: 1915-1924), limiting the broader utilization of the combination of GPIIb/IIIa inhibition and fibrinolysis. Dual anti-platelet therapy using both aspirin and clopidogrel (a P2Y12 receptor blocker) has shown to be beneficial toward reduction of cardiovascular events, however the recurrent thrombotic events cannot be completely eliminated and the combined treatment caused increased numbers of bleeding complications (Sherwood et al., 2016. JACC Cardiovasc Interv. 9(16) : 1694-1702; McFadyen et al., 2018. Nat Rev Cardiol. 15(3) : 181-191). This can be in part attributed to the fact that all currently available GPIIb/IIIa inhibitors target the receptor regardless of the activation status thereby causing complete systemic inhibition of platelet aggregation and firm adhesion. In addition, the ligand mimetic properties of clinically used GPIIb/IIIa inhibitors also can lead to paradoxical platelet activation imitating ligand-induced outside-in signaling (Peter et al., 1998. Blood 92:3240-3249; Schwarz et al., 2004. J Pharmacol Exp Ther. 308: 1002-1011). Pac-1 is the only activation-specific blocking antibody for activated GPIIb/IIIa, but it is a large multivalent IgM molecule and therefore may not be suitable for clinical use; its Fab fragments demonstrate a rather low affinity (Peter et al., 1998. supra).

[0009] Accordingly, there is an unmet medical need for improved therapeutic agents that can be used in treating conditions that require inhibition of platelet aggregation and/or thrombus formation.

SUMMARY

[0010] The present disclosure features anti-thrombotic chimeric molecules comprising an anti-platelet antigen-binding molecule that binds to the active conformation of GPIIb/IIIa with greater affinity than to its inactive conformation. Notably, the antigen binding molecule does not activate platelets but is able to inhibit binding of fibrinogen to platelets with improved potency than other anti-GPIIb/IIIa antigen-binding molecules known in the art. The chimeric molecules of the present disclosure further comprise an anti coagulant agent that suitably inhibits procoagulant activity of a coagulation factor, and/or stimulates or enhances thrombolytic activity to induce or mediate clot breakdown. These multi-pathway chimeric molecules are useful in a range of applications including in compositions and methods for inhibiting thrombus and/or embolus formation, and for treating or inhibiting the development of conditions associated with the presence of activated platelets, as described hereafter.

[0011] Accordingly, in one aspect, the present disclosure features anti-thrombotic chimeric molecules comprising an anti-coagulant agent and an antigen-binding molecule that binds to activated glycoprotein Ilb/IIIa (GPIIb/IIIa) and comprises:

(1) a heavy chain variable region (V_H) comprising the VHCDR1 amino acid sequence RYAMS [SEQ ID NO:3], the VHCDR2 amino acid sequence

GISGSGGSTYYADSVKG [SEQ ID NO:4], and the VHCDR3 amino acid sequence CARIFTHRSRGDVPDQTSFDY [SEQ ID NO: 5], and a light chain variable region (V_L) comprising the VLCDR1 amino acid sequence QGDSLRNFYAS [SEQ ID NO:6], the VLCDR2 amino acid sequence GLSKRPS [SEQ ID NO:7], and the VLCDR3 amino acid sequence LLYYGGGQQGV [SEQ ID NO:8];

(2) a V_H that comprises, consists or consists essentially of the amino acid sequence

[SEQ ID NO: 1], and a V_L that comprises, consists or consists essentially of an amino acid sequence selected from

(3) a V_H with at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a V_L with at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to the amino acid sequence of SEQ ID NO:2 or 60;

(4) a V_H as defined in (1) comprising at least 90% (including at least 91% to

99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) comprising at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ ID NO:2 or 60 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_L) ; and/or (5) a V_H as defined in (1) which is distinguished from the V_H amino acid sequence set forth in SEQ ID NO: 1 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the

V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) which is distinguished from the V_L amino acid sequence set forth in SEQ ID NO:2 or 60 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ

ID NO:2 or 60 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_L) .

[0012] Suitable antigen-binding molecule may be selected from antibodies and their antigen-binding fragments, including monoclonal antibodies (MAbs), chimeric antibodies, humanized antibodies, human antibodies, and antigen-binding fragments of such antibodies. The antigen-binding molecule may be multivalent (e.g., bivalent) or monovalent (e.g., Fab, scFab, Fab', scFv, one-armed antibody, etc.). In some embodiments, the antigen binding molecule comprises an Fc domain. In other embodiments, the antigen-binding molecule lacks an Fc domain.

[0013] The antigen-binding molecule suitably comprises any one or more of the following activities: (a) binds to the active conformation of GPIIb/IIIa with greater affinity than to the inactive conformation of GPIIb/IIIa; (b) inhibits binding of fibrinogen to

GPIIb/IIIa; (c) inhibits platelet aggregation; (d) lacks platelet activation activity and (e) lacks systemic inhibition of platelet function.

[0014] The anti-coagulant agent may be a clotting factor inhibitor or a thrombolytic agent. In representative examples, the anti-coagulant agent is a proteinaceous molecule and the chimeric molecule is in the form of a single chain chimeric polypeptide in which the GPIIb/IIIa antigen-binding molecule described herein is operably connected to the anti-coagulant agent.

[0015] In some embodiments, the chimeric molecules as broadly described above are contained in a delivery vehicle (e.g., a liposome, a nanoparticle, a microparticle, a dendrimer or a cyclodextrin).

[0016] Another aspect of the present disclosure provides isolated polynucleotides comprising a nucleic acid sequence encoding a chimeric molecule as described herein.

[0017] Yet another aspect of the present disclosure provides constructs comprising a nucleic acid sequence encoding a chimeric polypeptide described herein in operable connection with one or more control sequences. Suitable constructs are preferably in the form of an expression construct, representative examples of which include vectors such as plasmids, cosmids, phages and viruses.

[0018] In another aspect, the present disclosure provides host cells that contain constructs comprising a nucleic acid sequence encoding a chimeric molecule described herein in operable connection with one or more control sequences.

[0019] Yet another aspect of the present disclosure provides pharmaceutical compositions comprising a chimeric molecule described herein and a pharmaceutically acceptable carrier.

[0020] A further aspect of the present disclosure provides methods for inhibiting binding of a ligand to GPIIb/IIIa in its active conformation. These methods generally comprise contacting the GPIIb/IIIa with a chimeric molecule described herein, to thereby inhibit binding of the ligand to the GPIIb/IIIa.

[0021] In another aspect, the present disclosure provides methods for inhibiting binding of a ligand to an activated platelet. These methods generally comprise contacting the activated platelet with a chimeric molecule described herein, to thereby inhibit binding of the ligand to the activated platelet.

[0022] In some embodiments of the above methods, the ligand is selected from fibrinogen, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand. In preferred embodiments, the ligand is fibrinogen.

[0023] Another aspect of the present disclosure provides methods for inhibiting platelet aggregation in a subject. These methods generally comprise administering to the subject an effective amount of a chimeric molecule described herein, to thereby inhibit platelet aggregation in the subject.

[0024] A related aspect of the present disclosure provides methods for inhibiting thrombus formation in a subject. These methods generally comprise administering to the subject an effective amount of a chimeric molecule described herein, to thereby inhibit thrombus formation in the subject.

[0025] Another related aspect of the present disclosure provides methods for inhibiting embolus formation in a subject. These methods generally comprise administering to the subject an effective amount of a chimeric molecule described herein, to thereby inhibit embolus formation in the subject. [0026] Yet another aspect of the present disclosure provides methods for treating or inhibiting the development of platelet aggregation, thrombus formation and/or embolus formation in a subject having or at risk of developing a condition associated with the presence of activated platelets. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule described herein. Suitably, the condition associated with the presence of activated platelets is selected from atherosclerosis (e.g., unstable atherosclerosis), allergic disorders, autoimmune diseases, cancers, infections, neurological disorders, systemic inflammation, tissue or organ transplantation, thromboembolism-associated conditions and wounds

[0027] Still another aspect of the present disclosure provides methods for treating or inhibiting the development of a thromboembolism-associated condition in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule described herein. Illustrative thromboembolism-associated conditions can include arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart or in the peripheral circulation. The thromboembolism-associated disease or condition can also include specific disorders selected from, but not limited to, abdominal aortic aneurysm, unstable angina or other acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive a rterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis and/or embolism, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or extracorporeal circulation (ECMO, cardiopulmonary bypass) procedures in which blood is exposed to an artificial surface that promotes thrombosis. The medical implants or devices include, but are not limited to: prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices and artificial hearts or heart chambers, and vessel grafts. The procedures include, but are not limited to: cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis.

[0028] Yet another aspect of the present disclosure provides methods for treating or inhibiting the development of a hematologic disorder (e.g., a thrombosis-associated hematologic disorder) in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule described herein. Non-limiting examples of hematologic disorders include sickle cell disease and thrombophilia.

[0029] In any of the above detection embodiments, the subject suitably has or is suspected of having a condition associated with the presence of activated platelets, representative examples of which include atherosclerosis (e.g., unstable atherosclerosis), allergic disorders, autoimmune diseases, cancers, infections, neurological disorders, systemic or localized inflammation, tissue or organ transplantation, thromboembolism-associated conditions and wounds. [0030] A further aspect of the present disclosure provides kits for inhibiting binding of a ligand to GPIIb/IIIa in its active conformation, for inhibiting binding of a ligand to an activated platelet, for inhibiting platelet aggregation, for inhibiting thrombus formation, for inhibiting embolus formation, for treating or detecting conditions associated with activated platelets, for treating or inhibiting the development of a thromboembolism- associated condition, or for treating or inhibiting the development of a hematologic disorder. The kits generally comprises a chimeric molecule or composition described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a graphical representation comparing the potency of ReoPro,

SE scFv and SE5 scFv in inhibiting platelet aggregation. 96-well plate light transmission aggregometry was performed using 100 mL of platelet rich plasma (PRP). Platelet poor plasma (PPP) was obtained by centrifugation of blood at 1000xg for 10 min at room temperature. PRP was mixed with 8 mM calcium chloride, 1 : 50 thromboplastin (Siemens, USA), and 20 mM thrombin receptor activator peptide (Sigma-Aldrich, Germany), leading to platelet activation and clotting. The PRP mixture was incubated with abciximab (ReoPro), SCE5, SE or PBS (as control), then activated with 2 mM ADP. Concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL and 10 mg/mL were evaluated. Light transmission

aggregometry was measured using the Bio-Rad Benchmark Plus at wavelength 595nm. Samples were measured every 15 seconds for 10 min. Light transmission was adjusted to 0% with PPP and 100% with PRP.

[0032] Figure 2 is a graphical and photographic representation that characterizes SE-TAP antiplatelet and anticoagulant activity. Selective targeting to ADP-activated human (A-C) or mouse (D-F) platelets was assessed by flow cytometry. Construct binding to activated platelets (white histogram) or non-activated platelets (grey histogram) was detected by AlexaFluor488 anti-His antibody. SE and SE-TAP target human (A-B) or mouse (D-E) activated platelets while MUT-TAP displays no binding (C and F). Representative histograms are shown from four experiments. (G) GPIIb/IIIa blocking activity was examined through flow cytometry analysis of fibrinogen binding to human and mouse ADP-activated platelets. Fibrinogen binding was quantified as mean fluorescent intensity and % inhibition was calculated relative to vehicle control, n=4. (H-I) Anti-FXa activity was monitored using chromogenic FXa substrate in solution (H) and after targeting constructs to fibrinogen- adherent activated platelets (I). Percent inhibition of FXa was calculated relative to vehicle control with measurements performed in triplicate, n=4 experiments. (J-K) Flow chamber adhesion assay was performed with perfusion (500 s^-1) oaf whole blood over collagen coated glass capillaries (J). Phase contrast images of microthrombi formed in presence of SE (5 and 15 mg/mL), SE-TAP (5 and 15 mg/mL), MUT-TAP (15 mg/mL), or saline vehicle. Scale bars are 20 mm. (K) Microthrombi were captured at 20X and area quantified with Image J, n=4 per group. Data represent mean ± SD, n.s. (non-significant) p > 0.05, ***p < 0.001 (ANOVA and Bonferroni's multiple comparison test).

[0033] Figure 3 is a photographic and graphical representation showing that SE- TAP targets arterial thrombus and inhibits occlusion in mice. Platelet specific anti-CD42b-

Dylight 649 was infused and thrombus formation induced by laser injury was characterized over time. (A) Representative images of the fluorescence signal associated with platelet thrombus after laser injury of cremaster arterioles. Saline, MUT-TAP (0.03 mg/g), or SE-TAP (0.03 mg/g) were administered (IV) up to 30 min prior to laser injury. (B) Median integrated platelet fluorescence with administration of saline (red), MUT-TAP (blue), or SE-TAP (green). (C) Platelet accumulation was quantified as the area under the curve (AUC) as calculated for platelet RFU. Units are arbitrary and data represent mean ± SD, n=20-34 vessels in 4-5 mice/group. (D) Carotid artery thrombosis was induced with ferric chloride and construct targeting was confirmed using IVIS scan with IR800 labeled MUT-TAP or SE-TAP. (E) Ferric chloride-induced carotid artery thrombus development was monitored with a nano-flow probe. A significant increase in occlusion time was observed with SE-TAP (0.03 and 0.3 mg/g), and with reference compounds, LMWH (Enoxaparin, 10 mg/g) and Ept (Eptifibatide, 10 mg/g). Tail transection performed 1 min after IV administration revealed a significant prolongation of (F) bleeding time and (G) bleeding volume for reference agents, LMWH (Enoxaparin, 10 mg/g), and Ept (Eptifibatide, 10 mg/g), but not for SE-TAP (0.03 mg/g). Data represent mean ± SD, n = 4 per group, n.s. p > 0.05, *p £ 0.05, **p £ 0.01, and ***p £ 0.001 (ANOVA and Bonferroni's multiple comparison test).

[0034] Figure 4 is a photographic and graphical representation showing that SE- TAP targets venous platelets and inhibits venous thrombosis in vivo. (A) Systemic concentration of SE-TAP over time with IV or SC delivery. Concentrations were characterized based on circulating anti-FXa activity and compared to a standard curve. (B-H) SE-TAP targets and reduces venous thrombosis after laser injury of cremaster venules. (B) Platelet specific Dylight 649 labeled-anti-CD42b was infused with AF488-labeled SE-TAP (top row), AF488-labeled MUT-TAP (middle row), or AF488 control (bottom row) prior to laser injury of cremaster venules. Representative images illustrate that SE-TAP targets platelets within the venous thrombus. Co-localization of MUT-TAP or AF488 control was not observed. (C-D) SE- TAP efficiently reduces venous thrombus at a dose administration of 0.1 mg/g SC (solid green, thrombus inhibition characterized 4 h after SC administration). Results are compared to equivalent doses of MUT-TAP (SC) or LMWH (Enoxaparin, 4 mg/g SC), 4 h after SC administration. Results are plotted as median integrated platelet fluorescence (C) or platelet accumulation quantified as AUC (D), n = 20-34 vessels in 4-5 mice/group. (E) Circulating anti-FXa activity was measured for each agent (data collected 4 h after SC administration). Data represent mean ± SD, n = 4 per group. (F-G) SE-TAP reduces venous thrombus formation 24 h after SC administration. The dose of SE-TAP was increased to 0.5 mg/g SC (dark green) and laser injury of cremaster venules performed 24 h later. Results are compared to equivalent doses of MUT-TAP (blue, 0.5 mg/g SC), equimolar SE (yellow, 0.3 mg/g SC) or LMWH (black, 4 mg/g SC). Results are plotted as median integrated platelet fluorescence (F) or platelet accumulation quantified as AUC (G), n = 20-34 vessels in 4-5 mice/group. (H) Circulating anti-FXa activity was measured for each SC dose 4 h after administration. Data represent mean ± SD, n = 4 per group. All p values were determined by ANOVA and Bonferroni's multiple comparison test, n.s. p > 0.05, **p £ 0.01, and ***p £ 0.001. [0035] Figure 5 is a photographic and graphical representation showing that SE- TAP inhibits deep venous thrombosis without increased bleeding risk. An electrolytic inferior vena cava model (EIM) was used to generate a non-occlusive venous thrombus in the inferior vena cava. SE-TAP (0.5 mg/g SC), LMWH (4 mg/g SC), or Rivaroxaban (1 mg/g PO) were administered 4 h prior to electrolytic injury and 24 h after injury. The IVC was harvested at 48 h for thrombus characterization. (A-B) A uniform length of IVC was harvested and immediately weighed to determine vessel wall and thrombus weight. Control enrollment included uninjured IVC without thrombus induction, n = 10-35 mice/group. Data represent mean ± SD, **p £ 0.01 vs. saline control. (C-D) Thrombus area was characterized from H&E stained sections. Thrombus area is reported as area (mm²)/aortic wall thickness (mm), n = 5 mice/group, 3 sections/mouse. Data represent mean ± SD, **p £ 0.01 vs. saline control. Scale bars are 500 mm. (E-G) Systemic bleeding risk was characterized 4 h after administration of test compound by measuring circulating anti-FXa activity (E), tail transection bleeding time (F), and tail transection blood loss (G), n = 5-10 mice/group. Data represent mean ± SD, n.s. **p £ 0.01, and ***p £ 0.001. All p values were determined by ANOVA and Bonferroni's multiple comparison test.

[0036] Figure 6 is a graphical and photographic representation showing DVT associated inflammatory response. (A-D) A uniform length of IVC wall and associated thrombus were harvested 48 h after electrolytic injury and digested to a single cell suspension for flow cytometry analysis of total leukocytes (A, CD45⁺), platelets (B, CD41⁺), neutrophils (C, CD11b⁺/Ly6G⁺), and monocytes (D, CD11b⁺/Ly6G-)· Results are reported as absolute cell number relative to vessel wall weight, n = 5-10 mice/group. Data represent mean ± SD, *p £ 0.05 and **p £ 0.01 vs. saline control (ANOVA and Bonferroni's multiple comparison test). (E-G) Immunohistochemistry was performed on paraffin embedded IVC sections to characterize inflammatory cell localization. (E) CD41⁺ platelets, (F) neutrophil esterase⁺, and (G) CD68⁺ monocytes. Scale bars are 100 pm. Black arrows indicate positive staining.

[0037] Figure 7 is a graphical representation showing dose-dependent inhibition of IVC thrombosis versus tail transection bleeding time. (A-C) Antithrombotic efficacy (% inhibition) is calculated based on percent reduction of thrombus weight in treatment groups vs. saline control 48 h after electrolytic injury of the IVC. Bleeding time is reported as fold- increase in tail transection bleeding time over saline control, 4 h after administration of the test agent. (A) LMWH (enoxaparin, 2 - 6 mg/g SC). (B) Rivaroxaban (0.5 - 1.5 mg/g PO). (C) SE-TAP (0.5 - 1.5 mg/g SC). (D) Plot of thrombus reduction vs. corresponding bleeding time for LMWH, rivaroxaban, and SE-TAP. Data represent mean ± SD, n = 5-15 per treatment dose for thrombus inhibition studies and n = 4-6 per treatment dose for bleeding time studies.

[0038] Figure 8 is a photographic representation depicting a static adhesion assay showing the specificity of SCFV_SE-scuPA to CHO cells expressing activated GPIIb/IIIa receptors. Representative microscopy images showing direct fluorescence staining of scFv- scuPA on CHO cells. Direct fluorescence staining of His-tag on scFv-scuPA by anti-Penta-His AlexaFluor 488-conjugated monoclonal antibody demonstrating binding of SCFV_SE-scuPA to activated GPIIb/IIIa expressing CHO cells but neither to non-expressing nor non-activated GPIIb/IIIa expressing CHO cells. No fluorescence staining of scFv_mut-scuPA was observed on all three cells types.

[0039] Figure 9 is a graphical representation of a flow cytometry assay demonstrating preserved function of scFv-scuPA after fusion. A. Binding of scFv was shown with an anti-Penta-His AlexaFluor 488-conjugated monoclonal antibody. Bar graphs depict the median fluorescence intensity values of 3 independent experiments. Representative fluorescence histograms are shown underneath the bar graphs. Activated platelet samples were incubated with 20mM of the platelet agonist ADP. B. Competitive assays using fibrinogen-labeled FITC. Fibrinogen-FITC binds to activated GPIIb/IIIa on activated platelets when incubated with the negative control (PBS with 2mM Ca²⁺ and Mg²⁺) or the scFv_mut scuPA. However, Fibrinogen-FITC did not bind to activated platelets in the presence of scFv_SE-SCUPA. C. Competitive assays using PAC1-FITC. PAC1-FITC binds to activated GPIIb/IIIa on activated platelets when incubated with the negative control (PBS with 2mM Ca²⁺ and Mg²⁺) or scFv_mut-scuPA. However, PAC1-FITC did not bind to activated platelets in the presence of scFv_SE-SCUPA (mean ± SD; **P<0.01, ***P<0.001). These assays were analyzed with a 2-way repeated measures ANOVA with the Bonferroni post-test.

[0040] Figure 10 is a graphical representation depicting 96-well plate light transmission aggregometry demonstrating antithrombotic effects of scFv_SE-SCUPA. A. Bar chart showing % aggregometry after the addition of ADP. A) High concentrations of scFv_SE- scuPA ( 10mg/mL and 20mg/mL) and the equimolar amounts of scFv_SE alone (5mg/mL and 10mg/mL) demonstrated a strong inhibition of ADP-induced platelet activation as opposed to scFv_mut-scuPA (n=3, ***p<0.001). Lower concentrations of scFv_SE-SCUPA (0.2mg/mL and 2mg/mL) and the equimolar amounts of scFv_SE alone did not show inhibition of platelet aggregation. Platelet aggregation was not inhibited with commercial uPA. B) Aggregometry after addition of 200mM of the urokinase inhibitor amiloride further demonstrating that urokinase has no effect on thrombus formation in this assay. ScFv_SE -SCUPA (10mg/mL and 20mg/mL) and equimolar amounts of scFv_SE alone (5mg/mL and 10mg/mL) demonstrated inhibition of ADP-induced platelet activation as opposed to scFv_mut-scuPA (n=3,

***p<0.001). Lower concentrations of scFv_SE-SCUPA (0.2mg/mL and 2mg/mL) and the equimolar amounts of scFv_SE alone did not show inhibition of platelet aggregation. Platelet aggregation was not inhibited with commercial uPA.

[0041] Figure 11 is a photographic and graphical representation showing binding of scFv_SE-SCUPA to microthrombi resulting in fibrin degradation in vitro and fluorescence staining of scFv_SE-SCUPA in vivo. A. Representative microscopy images of microthrombi with scFv_SE-SCUPA. Fluorescence-labeled anti-His-tag antibody demonstrates attachment of scFv_SE-SCUPA to the microthrombi. No fluorescence was detected on microthrombi with scFv_mut-scuPA (n=3 each). B. SCFVSE-SCUPA caused fibrin degradation in vitro on microfluidics flow channels. Fibrin degradation was observed at platelet aggregation perfused with scFv_SE- scuPA but not with scFv_mut-scuPA. Fibrin degradation was also observed in platelet aggregates perfused with a high dose of commercial uPA (n=3 each). Image analysis was done with ImageJ applying a median filter ( 1.5pixel) and a "fire" false color look-up table. C. Intravital microscopy demonstrating binding of scFv_SE-SCUPA conjugated with Cy-3 fluorescence dye to thrombi in vivo. Representative images showing the binding of scFv- scuPA fusion proteins to thrombi induced by ferric-chloride injury in the mesenteric arteriole (n=3 each). ScFv-scuPA fusion proteins conjugated with Cy-3 fluorescence dye were injected into mice after the formation of stable thrombi. Increased binding of scFv_SE-SCUPA to activated platelets/thrombus was detected on the fluorescence channel. No specific fluorescence/ binding was observed using scFv_mut-scuPA.

[0042] Figure 12 is a graphical representation showing Doppler flow velocity of carotid arteries of mice for monitoring of thrombolysis showing that scFv_SE-SCUPA prevents occlusion. Thrombi were induced in the carotid artery of mice using 10% ferric chloride for 3 min. The nano Doppler flow meter was used to measure occlusion time and the baseline Doppler velocity was set to 100%. Saline was injected as negative controls and 500U/g BW of clinically used commercial uPA was used as a positive control. Analysis of velocity 10 min post injury showed occlusion for mice treated with saline and 75U/g BW commercial uPA. 20 min post injury, the Doppler velocity from mice treated with 75U/g BW targeted scFv_SE-SCUPA was significant higher than those treated with saline, 75U/g BW of non-targeted scFv_mut- scuPA, 75U/g BW of commercial uPA, the equimolar concentration of scFv_SE alone or the combination of scFv_SE with 75U/g BW of non-targeted scFv_mut-scuPA. No difference was observed in groups treated with 75U/g BW of non-targeted scFv_mut-scuPA, the equimolar concentration of scFv_SE alone or the combination of both scFv_SE and 75U/g BW of non- targeted scFv_mut-scuPA. Similar results were obtained for 30 min. The Doppler flow velocities obtained from mice treated with 75U/g targeted scFv_SE-SCUPA was similar to those treated with 500U/g BW of commercial uPA throughout the observation period. These assays were analyzed with 2-way repeated measures ANOVA with the Bonferroni post-test. Data shown as mean % ± SEM, *p<0.05, **p<0.01, ***p<0.001, n=6 each.

[0043] Figure 13 is a graphical representation showing bleeding time in mice determined by tail transection shows that there is no bleeding time prolongation at the effective dose of scFv_SE-SCUPA. Commercial uPA at 500 U/g BW demonstrated considerably longer bleeding time as compared to saline vehicle controls (*p<0.05, n=6 each). Low dose scFv_SE-SCUPA, scFv_mut-scuPA and commercial uPA at 75U/g BW did not cause prolong bleeding time. These assays were analyzed with 1-way repeated measures ANOVA with the Bonferroni post-test.

[0044] Figure 14 is a graphical and photographic representation showing monitoring of thrombolysis via molecular ultrasound imaging showed a reduction of thrombus size post administration of scFv_SE-SCUPA. A. Monitoring of thrombus area post administration of scFv-scuPA. A reduction of thrombus size was observed for animals administered with 500 U/g BW of commercial uPA (black line and B) as compared to saline (light blue line and C) as vehicle control. A reduction of thrombus size was also observed with 75 U/g BW activated platelets targeting scFv_SE-SCUPA (red line and D) as compared to 75 U/g BW non-targeted scFv_mut-scuPA (dark blue line and E). Baseline area before injection of uPA was set to 100% and areas were calculated every 5 min for 60 min. Thrombus size was traced and calculated using the VisualSonics software. The groups were compared by use of repeated measures ANOVA over time with Bonferroni post-tests at each time point (Mean % ± SEM, (*p<0.05, **p<0.01, ***p<0.001, n=3 each).

[0045] Figure 15 is a graphical and photographic representation showing reduction of thrombus size post administration of scFv_SE-SCUPA and bolus of plasminogen. A. Monitoring of thrombus area post administration of scFv-scuPA. No reduction of thrombus size was observed in mice administered with 75 U/g BW of scFv-scuPA for the first 30 min. A bolus of plasminogen was injected into the animal at the 30 min time-point. A reduction of thrombus size was observed with 75 U/g BW activated GPIIb/IIIa targeting scFv_SE-SCUPA (line with circles and B) but not with 75 U/g BW non-targeted scFv_mut-scuPA (line with squares and C). Baseline area before injection of uPA was set to 100% and areas were calculated every 5 min for 60 min. Thrombus size was traced and calculated using the VisualSonics software. The groups were compared by use of repeated measures ANOVA over time with Bonferroni post-tests at each time point (Mean % ± SEM, ***p<0.001, n=3 each).

[0046] Figure 16 is a graphical representation that Targ-scuPA preserves LV function in the context of cardiac IRI. (A) Baseline ejection fraction (EF) of mice before induction of cardiac IRI. Echocardiographic analysis of LV function 28 days after cardiac IRI in mice treated with targ-scuPA, Non-targ-scuPA and PBS control (n = 8 per group). Mice treated with targ-scuPA displayed a marked improvement in EF (B), fractional shortening (C), stroke volume (D) and cardiac output (E) compared to Non-targ-scuPA and PBS controls. *p<0.05, **p<0.01, ***p<0.001.

[0047] Figure 17 is a graphical representation showing that Targ-scuPA prevents pathological strain patterns post cardiac IRI. (A) Representative radial strain curves from VevoStrain analysis software. Colored lines represent the six standard myocardial regions, with a seventh black line that calculates the average (global) strain at each time point.. (B)Non-targ-scuPA and PBS treated mice exhibit a marked decrease in radial strain compared to targ-scuPA in the area of infarct (anterior apex) and (C) globally in the entire LV Bar chart showing radial strain. (D) Non-targ-scuPA and PBS treated mice showed significant increases in time delay for maximum opposite-wall delay as compared to targ- scuPA. *p<0.05, **p<0.01.

[0048] Figure 18 is a graphical and photographic representation showing that

Targ-scuPA treatment significantly decreases infarct size post cardiac IRI. TTC stained sections of myocardium 4 weeks post IRI demonstrate targ-scuPA treatment reduces infarct size as a percentage of (A) left ventricle (B) and area at risk.(C) No significant differences were observed in the area at risk between treatment groups. (D) Representative images displaying the significant reduction in infarct size 4 weeks post IRI in targ-scuPA mice. n = 8 mice per group, where **p<0.01, ***p<0.001, ****p<0.0001.

[0049] Figure 19 is a graphical representation showing that Targ-scuPA treatment significantly reduces platelet and fibrin deposition in the post ischemic myocardium. The extent of (A) platelet and (B) fibrin deposition in the myocardium 2 hours post IRI is markedly reduced in mice treated with targ-scuPA. Platelet and fibrin analysis was performed on sections of ischemic myocardium using multiphoton microscopy. N= 3, *p<0.05, **p<0.005.

[0050] Figure 20 is a graphical and photographic representation showing treatment with SE-TAP (referred to in this figure as "Targ-TAP") preserves myocardial function and reduces infarct size after ischemia/reperfusion (I/R). Ejection fraction (EF) was analyzed from parasternal long-axis B-mode images at baseline and 4 weeks post-I/R. (A) Echocardiographic analysis of left ventricle (LV) function 4 weeks after I/R injury. Baseline EF was similar between all groups. 4-week post-I/R EF is significantly reduced in PBS and MUT- TAP (referred to in this figure as "Non-Targ-TAP") treated hearts compared to baseline (n = 7-8 per group). Mice treated with SE-TAP demonstrated improved (B) fractional shortening; (C) Volume at systole (V;s); and (D) Volume at diastole (V;d) compared to MUT-TAP and PBS. (E) MUT-TAP and PBS treated mice exhibit a marked decrease in radial strain compared to SE-TAP treated mice in the infarcted area (anterior apex). (F) MUT-TAP and PBS treated mice exhibit a marked decrease in global peak radial strain. (G) MUT-TAP and PBS treated mice show significant increases in time delay for maximum opposite-wall delay as compared to SE-TAP. (H) Representative radial strain curves from VevoStrain analysis software.

Colored lines represent the six standard myocardial regions, with a seventh black line that calculates the average (global) strain at each time point. 4 weeks post-I/R, hearts were stained with Evans Blue/TTC and SE-TAP treated hearts show a reduced I/AaR ratio (I); and infarct size in the left ventricle (J); compared to PBS and MUT-TAP treated hearts. (K) Evans Blue/TTC representative images. Male C57BI/6J mice (20-25g), sourced from the Alfred Medical Research and Education Precinct (AMREP) Animal Services (Melbourne, Vic), were randomized into different treatment groups before they underwent I/R injury by LAD coronary artery ligation. Ultrasound imaging and all analysis were performed blinded. 1 animal from each of the 3 groups were excluded due to non-echogenic imaging and rib shadowing on ultrasound. N = 4-8 mice per group, where *p<0.05, **p<0.005,

***p<0.0001 by one-way ANOVA with Tukey's post-hoc test.

[0051] Some figures and text contain color representations or entities. Color illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

DETAILED DESCRIPTION

1. Definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0053] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. [0054] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 14, 13,

12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0055] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0056] "Affinity" or "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antigen binding molecule) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair e.g., an antigen-binding molecule). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (k_off and k_on, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0057] As used herein, the term "greater affinity" refers to the degree of binding of an antigen-binding molecule to a target antigen where an antigen-binding molecule X binds to target antigen Y more strongly and with a smaller dissociation constant than antigen-binding molecule Z binds to antigen Y, and in this context antigen-binding molecule X has a greater affinity than antigen-binding molecule Z for target antigen Y.

[0058] The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, stops, diminishes, reduces, impedes, impairs or neutralizes one or more biological activities or functions of the active form of GPIIb/IIIa such as but not limited to binding to a GPIIb/IIIa ligand including but not limited to fibrinogen, fibronectin, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand, in any setting including, in vitro, in situ, or in vivo. Likewise, the terms "antagonize", "antagonizing" and the like are used interchangeably herein to refer to blocking, inhibiting stopping, diminishing, reducing, impeding, impairing or neutralizing an activity or function as described for example above and elsewhere herein. By way of example, "antagonize" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in an activity, or function.

[0059] The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to or interacts with a particular antigen (e.g., activated GPIIb/IIIa).

The term "antibody" includes full-length immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, C_H2 and C_H3. Each light chain comprises a light chain variable region (which may be abbreviated as LCVR or V_L) and a light chain constant region. The light chain constant region comprises one domain (C_L1). The V_H and V_L regions can be further subdivided into regions of

hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the disclosure, the FRs of an antibody of the disclosure (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0060] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains,

immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0061] As used herein, the term "antigen" and its grammatically equivalents expressions (e.g., "antigenic") refer to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g.,

polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.

[0062] The terms "antigen-binding fragment", "antigen-binding portion", "antigen-binding domain" and "antigen-binding site" are used interchangeably herein to refer to a part of an antigen-binding molecule that participates in antigen-binding. These terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0063] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, one-armed antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies {e.g.

monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0064] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V_H domain associated with a V_L domain, the V_H and V_L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric a nd contain V_H-V_H, V_H-V_L or V_L-V_L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V_H or V_L domain.

[0065] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V_H- CHI ; (ii) V_H-C_H2; (iii) V_H-C_H3; (iv) V_H-C_H1-C_H2; (v) V_H-C_H1-C_H2-C_H3, (vi) V_H-C_H2-C_H3; (vii) V_H- C_L; (viii) V_L-C_H1 ; (ix) V_L-C_H2, (X) V_L-C_H3; (xi) V_L-C_H1-C_H2; (xii) V_L-C_H1-C_H2-C_H3; (xiii) V_L-C_H2- C_H3; and (xiv) V_L-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non- covalent association with one another and/or with one or more monomeric V_H or V_L domain (e.g., by disulfide bond(s)). A multispecific antigen-binding molecule will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same a ntigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art. [0066] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. Representative antigen-binding molecules that are useful in the practice of the present disclosure include antibodies and their antigen-binding fragments.

The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0067] Antigen-binding molecules can be naked or conjugated to other molecules or moieties such as toxins, radioisotopes, small molecule drugs, polypeptides, etc.

[0068] The term "bispecific antigen-binding molecule" refers to a multi-specific antigen-binding molecule having the capacity to bind to two distinct epitopes on the same antigen or on two different antigens. A bispecific antigen-binding molecule may be bivalent, trivalent, or tetravalent. As used herein, "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen-binding sites in an antigen- binding molecule. These antigen recognition sites may recognize the same epitope or different epitopes. Bivalent and bispecific molecules are described in, e.g. , Kostelny et al., 1992. J Immunol 148: 1547; Pack and Plückthun, 1992. Biochemistry 31 : 1579, Gruber et al. 1994. J Immunol 5368, Zhu et al., 1997. Protein Sci 6: 781, Hu et al., 1996. Cancer Res. 56: 3055, Adams et al., 1993. Cancer Res. 53:4026, and McCartney et al., 1995. Protein Eng. 8: 301. Trivalent bispecific antigen-binding molecules and tetravalent bispecific antigen- binding molecules are also known in the art. See, e.g. , Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199- 216 (2011). A bispecific antigen-binding molecule may also have valencies higher than 4 and are also within the scope of the present disclosure. Such antigen-binding molecules may be generated by, for example, dock and lock conjugation method. (Chang, C.-H. et al. In : Bispecific Antibodies. Kontermann RE (2011), supra).

[0069] An "antigen-binding site" refers to the site, i.e. , one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site. An antigen-binding site of an antigen-binding molecule described herein typically binds specifically to an antigen and more particularly to an epitope of the antigen.

[0070] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (V_H and V_L, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V or V_L domain may be sufficient to confer antigen-binding specificity.

[0071] The term "anti-coagulant" refers to the effect of a moiety or agent, which reduces or inhibits pro-coagulant coagulation factor activity in the blood and hence reduces or inhibits coagulation of the blood. Anti-coagulant moieties and agents may have anti- platelet and/or anti-thrombotic activity.

[0072] The term "anti-inflammatory" refers to the effect of a moiety or agent, which reduces or inhibits symptoms associated with inflammation. Representative anti- inflammatory agents include steroidal and non-steroidal anti-inflammatory agents as well as anti-inflammatory cytokines. The term "steroidal anti-inflammatory agent", as used herein, refer to any one of numerous compounds containing a 17-carbon 4-ring system and includes the sterols, various hormones (as anabolic steroids), and glycosides. Representative examples of steroidal anti-inflammatory drugs include, without limitation, corticosteroids such as hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone,

dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone,

diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate,

hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof. The term "non-steroidal anti-inflammatory agent", as used herein, refers to a large group of agents that are aspirin-like in their action, including, but not limited to, ibuprofen, naproxen sodium, and acetaminophen). Additional examples of non-steroidal anti-inflammatory agents that are usable in the context of the present disclosure include, without limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, and CP-14,304; disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone,

oxyphenbutazone, feprazone, azapropazone, and trimethazone, and mixtures thereof. The term "anti-inflammatory cytokine", as used herein, refers to a cytokine that counteracts various aspects of inflammation, for example cell activation or the production of

proinflammatory cytokines, and thus contributes to the control of the magnitude of the inflammatory response and includes, for example, interleukin-10 (IL-10) including viral IL- 10, interleukin-4 (IL-4), interleukin-13 (IL-13), a-MSII, transforming growth factor-b1 (TGF-b1), and the like. [0073] As used herein, the term "anti-platelet" refers to the effect of a moiety or agent, which inhibits activation, aggregation, and/or adhesion of platelets.

[0074] The term "anti-thrombotic" refers to the effect of a moiety or agent, which reduces the platelets ability to aggregate and adhere and interact in the clot building process and hence form thrombi.

[0075] The phrase "binds specifically" or "specific binding" refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity to that determinant. This selection may be achieved by subtracting out antigen-binding molecules that cross-react with other molecules. A variety of immunoassay formats may be used to select antigen-binding molecules (e.g., immunoglobulins)[ such that they are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods of determining binding affinity and specificity are also well known in the art (see, for example, Harlow and Lane, supra); Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (W.H. Freeman and Co. 1976)).

[0076] As used herein, a "chimeric" molecule is one which comprises one or more unrelated types of components or contain two or more chemically distinct regions which can be conjugated to each other, fused, linked, translated, attached via a linker, chemically synthesized, expressed from a nucleic acid sequence, etc. For example, a peptide and a nucleic acid sequence, a peptide and a detectable label, unrelated peptide sequences, and the like. In embodiments in which the chimeric molecule comprises amino acid sequences of different origin, the chimeric molecule includes (1) polypeptide sequences that are not found together in nature (i.e.,, at least one of the amino acid sequences is heterologous with respect to at least one of its other amino acid sequences), or (2) amino acid sequences that are not naturally adjoined. For example, a "chimeric" antibody" as used herein refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0077] The term "coagulation" or "blood clotting" as used herein refers to the process by which blood changes from a liquid to a gel. It potentially results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair.

[0078] By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene or for the final mRNA product of a gene

(e.g. the mRNA product of a gene following splicing). By contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene or for the final mRNA product of a gene.

[0079] As used herein, the term "complementarity determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined for example by Kabat (i.e. ,, about residues 24-34 (L1), 50-56 (L2) and 89- 97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological

Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (i.e. ,, about residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

[0080] As used herein, the term "complex" refers to an assemblage or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and/or indirect contact with one another. In specific embodiments, "contact", or more particularly, "direct contact" means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules. In such embodiments, a complex of molecules (e.g., a peptide and polypeptide) is formed under conditions such that the complex is thermodynamically favored (e.g., compared to a non-aggregated, or non-complexed, state of its component molecules).

[0081] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. In some embodiments, the phrase "consisting essentially of" in the context of a recited subunit sequence (e.g., amino acid sequence) indicates that the sequence may comprise at least one additional upstream subunit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids) and/or at least one additional downstream subunit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids), wherein the number of upstream subunits and the number of downstream subunits are independently selectable.

[0082] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

TABLE 1

AMINO ACID SUB-CLASSIFICATION

[0083] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions. Amino acid substitutions falling within the scope of the present disclosure, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

TABLE 2

EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS

[0084] As used herein, the terms "conjugated", "linked", "fused" or "fusion" and their grammatical equivalents, in the context of joining together of two more elements or components or domains by whatever means including chemical conjugation or recombinant means (e.g., by genetic fusion) are used interchangeably. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0085] The term "constant domains" or "constant region" as used within the current application denotes the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in binding of an antigen, but exhibits various immune effector functions.

[0086] The term "construct" refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e.,, at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present disclosure will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory

Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0087] By "control element" or "control sequence" is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a c/s-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[0088] By "corresponds to" or "corresponding to" is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence ( e.g ., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,

76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to all or a portion of the reference amino acid sequence).

[0089] By "effective amount," in the context of treating or preventing a disease or condition (e.g., a cancer) is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for the treatment or prevention of that disease or condition. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0090] The term "embolus" (plural "emboli"), as used herein, refers to a gaseous, liquid or solid (e.g., particulate) matter that acts as a traveling "clot" and usually refers to any detached intravascular matter that is capable of occluding a vessel. The occlusion can occur at a site distant from the point of origin. The composition of an embolus includes, but is not limited to, bubbles or CO₂-; oil, fat, cholesterol; debris, such as vessel debris, e.g., calcifications, tissue, or tumor fragments; coagulated blood, an organism such as bacteria or a parasite, or other infective agent; or foreign material. The term "bubbles" includes an embolus formed of air or other gas, or in certain instances, a liquid that is not blood or coagulated blood. A bubble may be spherical or non-spherical in shape. The term

"microembolus" is encompassed by the term "embolus" as used herein, and refers to an embolus of microscopic size and may be comprised of the same materials as an embolus as defined above. A common example of an embolus is a platelet aggregate dislodged from an atherosclerotic lesion. The dislodged platelet aggregate is transported by the bloodstream through the cerebrovasculature until it reaches a vessel too small for further propagation.

The clot remains there, clogging the vessel and preventing blood flow from entering the distal vasculature. Emboli can originate from distant sources such as the heart, lungs, and peripheral circulation, which may eventually travel within the cerebral blood vessels, obstructing flow and causing stroke. Other sources of emboli include atrial fibrillation and valvular disease.

[0091] As used herein, the terms "encode", "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode", "encoding" and the like include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0092] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to a region of an antigen that is bound by an antigen-binding molecule or antigen-binding fragment thereof. Epitopes can be formed both from contiguous amino acids (linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Morris G. E., Epitope Mapping Protocols, Meth Mol Biol, 66 (1996)). A preferred method for epitope mapping is surface plasmon resonance. Bispecific antibodies may be bivalent, trivalent, or tetravalent. When used herein in the context of bispecific antibodies, the terms "valent", "valence", "valencies", or other grammatical variations thereof, mean the number of antigen binding sites in an antibody molecule. These antigen recognition sites may recognize the same epitope or different epitopes. Bivalent and bispecific molecules are described in, for example, Kostelny et al., (1992) J Immunol 148: 1547; Pack and Plückthun (1992) Biochemistry 31 : 1579;

Hoi linger et al. , 1993, supra, Gruber et al., (1994) J Immunol 5368, Zhu et al. , (1997) Protein Sci 6: 781 ; Hu et al., (1996) Cancer Res 56: 3055; Adams et al., (1993) Cancer Res 53:4026; and McCartney et al., (1995) Protein Eng 8: 301. Trivalent bispecific antibodies and tetravalent bispecific antibodies are also known in the art (see, e.g. , Kontermann R E (ed.), Springer Heidelberg Dordrecht London New York, 199-216 (2011)). A bispecific antibody may also have valencies higher than 4 and are also within the scope of the present disclosure. Such antibodies may be generated by, for example, dock and lock conjugation method (see, Chang, C.-H. et al. In : Bispecific Antibodies. Kontermann R E (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011)).

[0093] As used herein, the terms "function", "functional" and the like refer to a ligand-binding, multimerizing, activating, signaling, biologic, pathologic or therapeutic function.

[0094] "Framework regions" (FR) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 36- 49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the CDRs comprise amino acid residues from hypervariable loops, the light chain FR residues are positioned about at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain and the heavy chain FR residues are positioned about at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues. In some instances, when the CDR comprises amino acids from both a CDR as defined by Kabat and those of a hypervariable loop, the FR residues will be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.

[0095] "Glycoprotein Ilb/IIIa" (GPIIb/IIIa, also known as integrin allb33 and CD41/CD61) refers to a polypeptide that is an integrin complex found on platelets. It is a receptor for several ligands including fibrinogen, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand, and aids platelet activation. The complex is formed via calcium-dependent association of GPIIb and GPIIIa, a required step in normal platelet aggregation and endothelial adherence. Platelet activation by ADP leads to the

conformational change in platelet GPIIb/IIIa receptors to their active form, which induces binding to fibrinogen (factor I). This results in many platelets "sticking together" as they may connect to the same strands of fibrinogen, resulting in a clot. The coagulation cascade then follows to stabilize the clot, as thrombin (factor Ila) converts the soluble fibrinogen into insoluble fibrin strands. These strands are then cross-linked by factor XIII to form a stabilized blood clot.

[0096] The terms "GPIIb/IIIa antigen-binding molecule", "anti-GPIIb/IIIa antigen-binding molecule", "anti-GPIIb/IIIa", "antigen-binding molecule that binds to GPIIb/IIIa" and any grammatical variations thereof refer to an antigen-binding molecule that binds specifically to the active conformation of GPIIb/IIIa receptor with sufficient affinity such that the antigen-binding molecule is useful as a therapeutic agent or diagnostic reagent in targeting GPIIb/IIIa in its active conformation (also referred to herein as "activated GPIIb/IIIa"). The extent of binding of an anti-GPIIb/IIIa antigen-binding molecule disclosed herein to GPIIb/IIIa protein in its inactive conformation (also referred to herein as "non- activated GPIIb/IIIa" or "resting GPIIb/IIIa") or to an unrelated, non-GPIIb/IIIa protein, is less than about 10% of the binding to GPIIb/IIIa in its active conformation as measured, e.g., by a radioimmunoassay (RIA), BIACORE™ (using recombinant GPIIb/IIIa in its active conformation as the analyte and antigen-binding molecule as the ligand, or vice versa), or by platelet aggregation assays as described for instance in Example 1, or other binding assays known in the art. In certain embodiments, an antigen-binding molecule that binds to activated GPIIb/IIIa has a dissociation constant (K_D) of £ 1 mM, £ 750 nM, £ 500 nM, £ 250 nM, £ 200 nM, £ 150 nM, £ 100 nM, £ 75 nM, £ 50 nM, £ 10 nM, £ 1 nM, £ 0.1 nM, £ 10 pM, £ 1 pM, or £ 0.1 pM. The anti-GPIIb/IIIa antigen-binding molecule can comprise a V_H and V_L domain. Representative examples of anti-GPIIb/IIIa antigen-binding molecules include an antigen-binding molecule comprising, consisting or consisting essentially of one or more amino acid sequences selected from SEQ ID NOs: 1-10, 12 and 60-63.

[0097] As used herein, the term "hematological disease" or "hematological disorders" is used interchangeable herein, and refers to disorders that primarily affect the cells of hematological origin, in common language denoted as cells of the blood.

[0098] The terms "host", "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the antigen binding molecules of the present disclosure. Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.

[0099] A "human" antibody is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody encoding sequences. This definition of a human anti body specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0100] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0101] By "linker" is meant a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a desirable configuration. In specific embodiments, a "peptide linker" refers to an amino acid or an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function (e.g., compatible with the spacing of antigen-binding fragments so that they can bind specifically to their cognate epitopes). In certain embodiments, a linker is comprised of about 1 to about 35 amino acids, about 2 to about 35 amino acids; for instance, about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.

[0102] The term "microparticle" refers to a particle having a characteristic dimension of less than about 1 millimeter and at least about 1 micrometer, where the characteristic dimension of the particle is the smallest cross-sectional dimension of the particle.

[0103] As used herein, the term "moiety" refers to a portion of a molecule, which may be a functional group, a set of functional groups, and/or a specific group of atoms within a molecule, that is responsible for a characteristic chemical, biological, and/or medicinal property of the molecule.

[0104] The term "monoclonal antibody" (MAb), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), and as modified by the somatic hybridization method as set forth above; or may be made by other recombinant DNA methods (such as those described in U.S. Patent No. 4,816,567).

[0105] The term "monospecific antigen-binding molecule" as used herein refers to an antigen-binding molecule that has one or more antigen-binding sites each of which bind to the same epitope of the same antigen.

[0106] The term "multispecific antigen-binding molecule" is used in its broadest sense and specifically covers an antigen-binding molecule with specificity for at least two

(e.g., 2, 3, 4, etc.) different epitopes (i.e. ,, is capable of specifically binding to two, or more, different epitopes on one antigen or is capable of specifically binding to epitopes on two, or more, different antigens).

[0107] The term "monovalent antigen-binding molecule" refers to an antigen- binding molecule that binds to a single epitope of an antigen. Monovalent antigen-binding molecule are typically incapable of antigen-crosslinking.

[0108] The term "multivalent antigen-binding molecule" refers to an antigen- binding molecule comprising more than one antigen-binding site. For example, a "bivalent" antigen-binding molecule has two antigen-binding sites, whereas a "tetravalent" antigen- binding molecule has four antigen-binding sites. The terms "monospecific", "bispecific", "trispecific", "tetraspecific", etc. refer to the number of different antigen-binding site specificities (as opposed to the number of antigen-binding sites) present in a multivalent antigen-binding molecule. For example, a "monospecific" antigen-binding molecule's antigen- binding sites all bind the same epitope. A "bispecific" or "dual specific" antigen-binding molecule has at least one antigen binding site that binds a first epitope and at least one antigen binding site that binds a second epitope that is different from the first epitope. A "multivalent monospecific" antigen-binding molecule has multiple antigen-binding sites that all bind the same epitope. A "multivalent bispecific" antigen-binding molecule has multiple antigen-binding sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

[0109] The term "nanoparticle" refers to a particle having a characteristic dimension of less than about 1 micrometer and at least about 1 nanometer, where the characteristic dimension of the particle is the smallest cross-sectional dimension of the particle.

[0110] The term "noble metal" as used herein refers to a metallic element that is resistant to corrosion in moist air. Non-limiting examples of noble metals include Copper (Cu), Ruthenium (Ru), Rhodium (Rh), Palladium (Pd), Silver (Ag), Rhenium (Re), Osmium (Os), Iridium (Ir), Platinum (Pt), Gold (Au), Mercury (Hg), or combinations thereof.

[0111] The term "operably connected" or "operably linked" as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) "operably linked" to a nucleotide sequence of interest (e.g., a coding and/or non-coding sequence) refers to positioning and/or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non-coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Likewise, "operably connecting" a first antigen-binding fragment to a second antigen-binding fragment encompasses positioning and/or orientation of the antigen-binding fragments in such a way as to permit binding of each antigen-binding fragment to its cognate epitope.

[0112] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0113] The term "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.

The term includes single and double stranded forms of DNA.

[0114] The terms "polypeptide," "proteinaceous molecule", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

[0115] As used herein, "recombinant" antigen-binding molecule means any antigen-binding molecule whose production involves expression of a non-native DNA sequence encoding the desired antibody structure in an organism, non-limiting examples of which include tandem scFv (taFv or scFv₂), diabody, dAb₂/VHH₂ , knob-into-holes derivatives, SEED-lgG, heteroFc-scFv, Fab-scFv, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, DNL- F(ab)₃, scFv₃- C_H1/C_L, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-lgG, scFv₂-Fc, F(ab')₂- scFv₂, scDB-Fc, scDB- C_H3, Db-Fc, SCFV₂- H/L, DVD-lg, tandAb, scFv-dhlx-scFv, dAb₂-lgG, dAb-lgG, dAb-Fc-dAb, CrossMAbs, MAb₂, FIT-Ig, and combinations thereof.

[0116] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. ,, the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0117] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Tables 1 and 2 supra.

Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984. Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0118] Illustrative calculations of sequence similarity or sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, usually at least 40%, more usually at least 50%, 60%, and even more usually at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at

corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. For amino acid sequence comparison, when a position in the first sequence is occupied by the same or similar amino acid residue (i.e.,, conservative substitution) at the corresponding position in the second sequence, then the molecules are similar at that position.

[0119] The percent identity between the two sequences is a function of the number of identical amino acid residues shared by the sequences at individual positions, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. By contrast, the percent similarity between the two sequences is a function of the number of identical and similar amino acid residues shared by the sequences at individual positions, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0120] The comparison of sequences and determination of percent identity or percent similarity between sequences can be accomplished using a mathematical algorithm. In certain embodiments, the percent identity or similarity between amino acid sequences is determined using the Needleman and Wünsch, (1970. J Mol Biol 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In specific embodiments, the percent identity between nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. An non-limiting set of parameters (and the one that should be used unless otherwise specified) includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0121] Alternatively, the percent identity or similarity between amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989. Cabios 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0122] Nucleic acid and protein sequences can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., 1990. J Mol Biol 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997. Nucleic Acids Res 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0123] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window", "sequence identity," "percentage of sequence identity" and "substantial identity". A

"reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two

polynucleotides may each comprise (1) a sequence (i.e.,, only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e.,, gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. ,, resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997. Nucleic Acids Res 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current

[0124] The terms "subject", "patient", "host" or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the present disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomolgus monkeys such as Macaca fascicularis, and/or rhesus monkeys ( Macaca mulatta )) and baboon ( Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees ( Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. A preferred subject is a human in need of inhibiting binding of a ligand to GPIIb/IIIa in its active conformation, inhibiting binding of a ligand to an activated platelet, inhibiting platelet aggregation, inhibiting thrombus formation, inhibiting embolus formation, treating or detecting conditions associated with activated platelets, treating or inhibiting the

development of a thromboembolism-associated condition, and/or treating or inhibiting the development of a hematologic disorder. However, it will be understood that the

aforementioned terms do not imply that symptoms are present.

[0125] The term "thrombosis" as used herein refers to the formation of a blood clot inside a blood vessel that obstructs the flow of blood through the circulatory system.

[0126] The term "thrombus" (plural "thrombi") or "blood clot" as used herein refers to a solid or semi-solid mass formed from the constituents of blood within the vascular system that is the product of blood coagulation. There are two components to a thrombus, aggregated platelets that form a platelet plug, and a mesh of cross-linked fibrin protein.

[0127] By "treatment", " "treat", "treated" and the like is meant to include both prophylactic and therapeutic treatment, including but not limited to preventing, relieving, altering, reversing, affecting, inhibiting the development or progression of, ameliorating, or curing (1) a disease or condition associated with the presence or aberrant expression of a target antigen, or (2) a symptom of the disease or condition, or (3) a predisposition toward the disease or condition, including conferring protective immunity to a subject.

[0128] By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0129] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0130] The following abbreviations are used throughout the application:

2. Abbreviations

3. GPIIb/IIIa-specific antigen-binding molecules

[0131] The present disclosure features anti-thrombotic chimeric constructs that comprise an anti-coagulant agent and an antigen-binding molecule that bind to the active conformation of GPIIb/IIIa (also referred to herein as "activated GPIIb/IIIa") with greater affinity than to its inactive conformation.

3.1 Anti-GPIIb/IIIa antigen-bindina molecules

[0132] The anti-GPIIb/IIIa anti-antigen-binding molecule suitably antagonizes a function of activated GPIIb/IIIa, including inhibiting or reducing binding of activated GPIIb/IIIa to a GPIIb/IIIa ligand such as fibrinogen.

[0133] In specific embodiments, the antigen-binding molecule disclosed herein comprises:

(1) a heavy chain variable region (V_H) comprising the VHCDR1 amino acid sequence RYAMS [SEQ ID NO: 3], the VHCDR2 amino acid sequence

GISGSGGSTYYADSVKG [SEQ ID NO:4], and the VHCDR3 amino acid sequence

CARIFTHRSRGDVPDQTSFDY [SEQ ID NO: 5], and a light chain variable region (V_L) comprising the VLCDR1 amino acid sequence QGDSLRNFYAS [SEQ ID NO: 6], the VLCDR2 amino acid sequence GLSKRPS [SEQ ID NO: 7], and the VLCDR3 amino acid sequence LLYYGGGQQGV [SEQ ID NO: 8] ;

(2) a V_H that comprises, consists or consists essentially of the amino acid sequence

[SEQ ID NO: 1], and a V_L that comprises, consists or consists essentially of an amino acid sequence selected from

(3) a V_H with at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a V_L with at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to the amino acid sequence of SEQ ID NO: 2 or 60;

(4) a V_H as defined in (1) comprising at least 90% (including at least 91% to

99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) comprising at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ ID NO:2 or 60 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_L) ; and/or (5) a V_H as defined in (1) which is distinguished from the V_H amino acid sequence set forth in SEQ ID NO: 1 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) which is distinguished from the V_L amino acid sequence set forth in SEQ ID NO:2 or 60 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ ID NO:2 or 60 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_L) .

[0134] Representative antigen-binding molecules contemplated by the present disclosure include full-length immunoglobulins and antigen-binding fragments, including recombinant antigen-binding molecules, which may be monovalent or multivalent, monospecific or multispecific.

[0135] In certain embodiments, the anti-GPIIb/IIIa antigen-binding molecule has an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region can be a wild-type human Fc region, or a human Fc region that includes one or more amino acid substitutions. The antibodies can have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al., 1993. Mol. Immunol., 30: 105-08). See also, e.g., U.S. 2005/0037000. The heavy chain constant region can also have substitutions that modify the properties of the antigen-binding molecule (e.g., decrease one or more of: Fc receptor binding, antigen-binding molecule glycosylation, deamidation, binding to complement, or methionine oxidation). In some instances, the antigen-binding molecules may have mutations such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, the antigen-binding molecule is modified to reduce or eliminate effector function. The heavy chain constant region can be chimeric, e.g., the Fc region can comprise the C_H1 and C_H2 domains of an IgG antibody of the IgG4 isotype, and the C_H3 domain from an IgG antibody of the IgG1 isotype (see, e.g., U.S. Patent Appl. No. 2012/0100140A1).

[0136] In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule is a monovalent antigen-binding molecule. Non-limiting monovalent antigen-binding molecules include: a Fab fragment consisting of V_L, V_H, CL and CHI domains; a Fab' fragment consisting of V_L, V_H, CL and CHI domains, as well as a portion of a C_H2 domain; an Fd fragment consisting of V_H and CHI domains; an Fv fragment consisting of V_L and V_H domains of a single arm of an antibody; a single-chain antibody molecule (e.g., scFab and scFv); a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341 : 544-546), which consists of a V_H domain; and a one-armed antibody, such as described in US20080063641 (Genentech) or other monovalent antibody, e.g., such as described in W02007048037 (Amgen).

[0137] In specific embodiments, a monovalent anti-GPIIb/IIIa antigen-binding molecule comprises an Fv fragment. The Fv fragment is the smallest unit of an

immunoglobulin molecule with function in antigen-binding activities. An antigen-binding molecule in scFv (single chain fragment variable) format consists of variable regions of heavy (VH) and light (VL) chains, which are joined together by a flexible peptide linker that can be easily expressed in functional form in an expression host such as E. coli and mammalian cells, allowing protein engineering to improve the properties of scFv such as increase of affinity and alteration of specificity (Ahmed et al., 2012. Clin Dev Immunol. 2012:980250). Representative examples of linker sequences are described in Section 4.5 infra. In the scFv construction, the order of the domains can be either VH-linker- V_L or V_L-linker-V_H and both orientations can applied.

[0138] In some embodiments, the linker sequences used in scFvs are multimers of the pentapeptide GGGGS [SEQ ID NO: 58] (or G4S or Gly4Ser). Those include the 15-mer (G4S)3 (Huston et al., 1988. Proc Natl Acad Sci USA. 85(16), 5879-83), the 18-mer GGSSRSSSSGGGGSGGGG [SEQ ID NO: 59] (Andris-Widhopf et al., "Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences." Cold Spring Harbor Protocols, 2011(9)) and the 20-mer (G4S)4 (Schaefer et al., "Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly." In: Antibody Engineering, R. Kontermann and S. Dübel, Springer Verlag, Heidelberg, Germany (2010) pp. 21-44). Many other sequences have been proposed, including sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing a Cre-Lox recombination site or sequences improving scFv properties, often in the context of particular antibody sequences.

[0139] Cloning of the scFv is usually done by a two-step overlapping PCR (also known as Splicing by Overlap Extension or SOE-PCR), as described (Schaefer et al., 2010, supra). The VH and VL domains are first amplified and gel-purified and secondarily assembled in a single step of assembly PCR. The linker is generated either by overlap of the two inner primers or by adding a linker primer whose sequence covers the entire linker or more (three-fragment assembly PCR).

[0140] In some embodiments, the anti-GPIIb/IIIa scFv molecule comprises CDR sequences derived from the from the V_H and V_L sequences of the anti-GPIIb/IIIa scFv clone SE described herein, as set out in Table 3. TABLE 3

[0141] In representative examples of this type, an anti-GPIIb/IIIa scFv comprises a V_H comprising, consisting or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1 and a V_L comprising, consisting or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 2 or 60.

[0142] In some embodiments, the anti-GPIIb/IIIa scFv comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• X₁ is a linker that is suitably a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; and

• X₂ is an optional linker that is suitably a flexible linker.

[0143] In non-limiting examples, the anti-GPIIb/IIIa scFv comprises or consist essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

•

is a flexible linker; and · Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE. [0144] In some embodiments, the a nti -GPIIb/IIIa scFv comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• X₁ is an amino acid sequence that suitably comprises a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; and

• X₂ is an optional amino acid sequence that suitably comprises a flexible linker.

[0145] In an illustrative example of this type, the a nti -GPIIb/IIIa scFv may comprise or consists essentially of the following amino acid sequence:

wherein :

Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

is a flexible linker; and

Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE.

[0146] In a specific embodiment, the anti-GPIIb/IIIa scFv comprises the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE

•

is a flexible linker; • Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a V5 epitope tag;

• is a sortase conjugation tag ;

.

is a C-myc tag ; and

.

is a His tag.

[0147] In another specific embodiment, the anti-GPIIb/IIIa scFv comprises, consists or consists essentially of the following amino acid sequence :

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE

• G

is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a V5 epitope tag;

•

is a sortase conjugation tag ;

. is a C-myc tag ; and

.

is a His tag.

[0148] Single chain Fv (scFv) antigen-binding molecules may be recombinantly produced for example in E. coli, insect cells or mammalian host cells upon cloning of the protein coding sequence for the scFv in the context of appropriate expression vectors with appropriate translational, transcriptional start sites and, in the case of mammalian expression, a signal peptide sequence.

[0149] In other embodiments, the monovalent anti-GPIIb/IIIa antigen-binding molecule comprises an Fab fragment. In an illustrative example of this type, the monovalent anti-GPIIb/IIIa antigen-binding molecule is a one-armed antibody consisting or consisting essentially of a single antigen-binding fragment (Fab) and a Fc region, wherein the Fc region comprises a first and a second Fc polypeptide, and wherein the first and second Fc polypeptides are present in a complex.

[0150] Recombinant expression of Fc-containing monovalent antigen-binding molecules can often lead to undesirable bivalent, homodimer contaminants. Strategies to inhibit formation of homodimers are known including methods that introduce mutations into immunoglobulin constant regions to create altered structures that support unfavorable interactions between polypeptide chains and suppress unwanted Fc homodimer formation. Non-limiting examples of this strategy to promote heterodimerization include the

introduction of knobs-into-holes (KIH) structures into the two polypeptides and utilization of the naturally occurring heterodimerization of the C_L and C_H1 domains (see, Kontermann, supra, pp. 1 -28 (2011) Ridgway et al., 1996. Protein Eng. 9(7) :617-21; Atwell et al., 1997.

J Mol Biol. 270(l) :26-35; as described in WO 2005/063816). These KIH mutations promote heterodimerization of the knob containing Fc and the hole containing heavy chain, improving the assembly of monovalent antibody and reducing the level of undesired bivalent antibody.

[0151] Modifications in the Fc domain of an anti-GPIIb/IIIa antigen-binding molecules may also be desirable to reduce Fc receptor binding and therefore reduce the potential for FcgRIIa-mediated activation of platelets. For example, the so-called 'LALA' double mutation (Leu234Ala together with Leu235Ala) in human IgG (including IgG1) is known to significantly impair Fc receptor binding and effector function (Lund et al., 1991, J. Immunol. 147, 2657-2662; Lund et al., 1992, Mol. Immunol. 29: 53-59). For human IgG4, engineering mutations S228P/L235E variant (SPLE) has previously demonstrated minimal FcgR binding (Newman et al., 2001, Clin. Immunol. 98, 164-174). Mutations in IgG1 or IgG4 Fc domains can be combined, for instance combining the LALA mutations in human IgG1 with a mutation at P329G or combining the SPLE mutation in human IgG4 with a mutation at P329G, completely abolished FcgR and C1q interactions (Schlothauer et al., 2016, Protein

Eng Des. Sel. 29, 457-466).

[0152] In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule (e.g., a MAb or an antigen-binding fragment thereof), in which each of the IgG1 Fc chains of the antibody carries P329G, L235A, L234A (P329G LALA) mutations or each of the IgG4 Fc chains carries P329G, S228P, L235E mutations, in order to reduce or abolish any undesired cross-linking, platelet activation, or immune effector function (e.g., antibody-dependent cell- meditated cytotoxicity (ADCC), phagocytosis (ADCP) and complement dependent cytotoxicity (CDC)) of the antigen-binding molecule.

[0153] Thus, in some embodiments, the present disclosure contemplates monovalent anti-GPIIb/IIIa antigen-binding molecules produced by co-expression of a light chain, heavy chain and a truncated Fc domain. Suitably, the heavy chain incorporates hole mutations and P329G LALA mutations, while the truncated Fc domain incorporates knob mutations and P329G LALA mutations. In some embodiments, the monovalent anti- GPIIb/IIIa antigen-binding molecule comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (SE V_H sequence), a CHI sequence and a first Fc polypeptide and (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:2 (SE V_L sequence), and a CLI sequence. In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule further comprises (c) a third polypeptide comprising a second Fc polypeptide.

[0154] In a representative example of constructing a monovalent anti-GPIIb/IIIa antigen-binding molecule, three constructs are made. First, the heavy chain (V_H) domains of SE are directly or indirectly fused in tandem with a truncated heavy chain (C_H1-C_H2-C_H3) of a human IgG1 molecule (e.g., atezolizumab) at the NH₂-terminus, in which the heavy chain C_H3 domain is suitably altered at position 407 (Y407A), termed the "hole" to promote knobs- into-holes (KiH) heterodimerization of the heavy chains. The second construct comprises V_L of SE directly or indirectly fused in tandem with a C_L of a human IgG1 molecule (e.g., atezolizumab) and the third construct is a truncated heavy chain (C_H2-C_H3) of a human IgG1 molecule (e.g., atezolizumab) in which one of the heavy chain C_H3 domain is suitably altered at position 366 (T366W), termed the "knob" to promote KiH heterodimerization of the heavy chains. Both heavy chain constructs may include L234A, L235A, P329G substitutions for reduced FcgR and C1q interactions.

[0155] In non-limiting examples:

[0156] The first construct comprises heavy chain (V_H) domains of SE directly fused in tandem with the truncated heavy chain (C_H1-C_H2-C_H3) of atezolizumab, in which the heavy chain C_H3 domain is altered at position 407 (Y407A), termed the "hole" to promote KiH heterodimerization of the heavy chains, comprises the following amino acid sequence:

wherein:

mature amino acid sequence of the anti-GPIIb/IIIa SE V_H sequence is shown in capital letters;

the constant region (C_H1-C_H2-C_H3) of atezolizumab is shown in lowercase letters; and

the L234A, L235A, P329G substitutions for reduced FcgR and C1q interactions and the Y407A "hole" substitution are in bold uppercase text.

[0157] The second construct comprises V_L of SE directly fused in tandem with C_L of atezolizumab and comprises the following amino acid sequence:

wherein:

· mature amino acid sequence of the anti-GPIIb/IIIa SE V_L sequence is shown in capital letters; and

• the constant region (C_L) of atezolizumab light chain is shown in lowercase letters.

[0158] The third construct comprises truncated heavy chain (C_H2-C_H3)of atezolizumab in which the heavy chain C_H3 domain is altered at position 366 (T366W), termed the "knob" to promote KiH heterodimerization of the heavy chains and comprises the following amino acid sequence:

wherein:

• mature amino acid sequence of the constant region (C_H2-C_H3) of atezolizumab is shown in capital letters; and

· the L234A, L235A, P329G substitutions for reduced FcγR and C1q interactions and the T366W "knob"" substitution are in bold uppercase text.

[0159] Another strategy that avoids cross-linking of a monovalent binding interaction includes the generation of Fc variants in the context of an Fc/scFv-Fc agent. Heterodimeric Fc-based monospecific antibodies (mAbs) with monovalent antigen binding have been generated by fusion of the scFv to the N-terminus of only one Fc chain (Fc/scFv- Fc, also referred to as a "hetero Fc scFv") (Moore et al., 2011. MAbs. 3(6) : 546-557; Ha et al., 2016. Front Immunol. 7: 394). In order to produce a heterodimeric, monovalent Fc/scFv- Fc agent, DNA constructs are designed encoding two different immunoglobulin polypeptides: (i) an Fc (Hinge-C_H2-C_H3") and (ii) an scFv-Fc (VH-linker-VL-Hinge-C_H2-C_H3'). Here the two different C_H3 domains, C_H3' and C_H3", represent asymmetric changes to generate "Knobs- into-holes" structures, which facilitate heterodimerization of polypeptide chains by introducing large amino acids (knobs) into one chain of a desired heterodimer and small amino acids (holes) into the other chain of the desired heterodimer. Both constructs include L234A, L235A, P329G substitutions for reduced FcγR and C1q interactions.

[0160] In one embodiment of generating a monovalent, heterodimeric Fc/scFv-Fc anti-GPIIb/IIIa antigen-binding molecule, two constructs encoding two different

immunoglobulin polypeptides are designed. The first construct comprises a truncated heavy chain (Hinge-C_H2-C_H3) of a human IgG1 (e.g., atezolizumab), in which the heavy chain C_H3 domain is altered at position 407 (Y407A), termed the "hole" to promote KiH

heterodimerization of the heavy chains and includes the L234A, L235A, P329G substitutions. In representative examples of this type, the first construct comprises or consists essentially of the following amino acid sequence:

wherein:

the C_H2-C_H3 sequence of atezolizumab is shown in lowercase letters; • the hinge region AA sequence of atezolizumab is shown in underlined, capital letters; and

• the L234A, L235A, P329G substitutions for reduced FcγR and C1q interactions and the Y407A "hole" substitution are in bold uppercase text.

[0161] The second construct comprises a scFv portion (V_H-linker-V_L) derived from the V_H and V_L sequences of the anti-GPIIb/IIIa SE scFv directly or indirectly fused in tandem with a truncated heavy chain (Hinge-C_H2-C_H3' ) sequences of a human IgG1 (e.g., atezolizumab), in which the heavy chain C_H3 domain is suitably altered at position 366 (T366W), termed the "knob" to promote KiH heterodimerization of the heavy chains and includes the L234A, L235A, P329G substitutions. In illustrative examples of this type, the second construct comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa SE scFv;

• AAA is a flexible linker;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

• the amino acid sequence of the hinge and constant region (C_H2-C_H3) of atezolizumab is shown in underlined capital letters; and

• the L234A, L235A, P329G substitutions for reduced FcγR and C1q interactions and the T366W "knob"" substitution are in bold uppercase text.

[0162] Expression of the anti-GPIIb/IIIa antigen-binding molecule disclosed herein can be achieved for example in bacterial (e.g., Escherichia coli), yeast, insect or mammalian host cells upon cloning of the protein coding sequences of the constructs in the context of appropriate expression vectors with appropriate translational, transcriptional start sites, and, where appropriate, signal peptide sequences.

[0163] In other embodiments, the anti-GPIIb/IIIa antigen-binding molecule is a multivalent antigen-binding molecule, non-limiting examples of which include:

immunoglobulins, F(ab')2, tandem scFv (taFv or scFv₂), scFv-Fc, diabody, dAb₂/V_HH₂, minibodies, ZIP miniantibodies, barnase-barstar dimer, knobs-into-holes derivatives, SEED- IgG, heteroFc-scFv, Fab-scFv, Fab)2/sc(Fab)2, scFv-(TNFa)3, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, trimerbody, tribi-minibody, barnase-barstar trimer, collabody, DNL-F(ab)3, scFv₃- C_H1/CL, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, scFv₂-Fc, F(ab')2-scFv₂, scDB-Fc, scDb- C_H3, Db-Fc, SCFV₂- H/L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb₂-IgG, dAb-IgG, dAb-Fc-dAb, tetrabody, streptabody (scFv-streptavidin)₄, (scFv-p53)₄, [sc(Fv)₂]2; tandem diabody (tandab) and combinations thereof.

[0164] In specific embodiments, the multivalent antigen-binding molecules are selected from IgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech; knobs-into-holes, Genentech; CrossMAbs, Roche; electrostatically matched antibodies,

AMGEN; LUZ-Y, Genentech; strand exchange engineered domain (SEED) body, EMD Serono; biolonic, Merus; and Fab-exchanged antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig, GSK/Domantis; two-in-one antibody, Genentech; crosslinked MAbs, karmanos cancer center; MAb₂, F-star; and Coy X-body, Coy X/Pfizer), IgG fusions (e.g., dual variable domain (DVD)-Ig, Abbott; IgG-like bispecific antibodies, Eli Lilly; Ts2Ab,

Medimmune/AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idee; TvAb, Roche) Fc fusions (e.g., scFv/Fc fusions, Academic Institution; SCORPION, Emergent BioSolutions/Trubion, ZymoGenetics/BMS; dual affinity retargeting technology (Fc-DART), MacroGenics; dual (ScFv)₂-Fab, National Research Center for Antibody Medicine) Fab fusions (e.g., F(ab)₂, Medarex/AMGEN; dual-action or Bis-Fab, Genentech; Dock-and-Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs), Micromet; tandem diabodies (Tandab), Affimed; DARTs, MacroGenics; Single-chain diabody, Academic; TCR-like antibodies, AIT, Receptor Logics; human serum albumin scFv fusion, Merrimack; and COMBODIES, Epigen Biotech), IgG/non-IgG fusions (e.g., immunocytokins, EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantigen fusion protein, Active Biotech; and immune mobilizing mTCR Against Cancer, ImmTAC) and oligoclonal antibodies (e.g., Symphogen and Merus).

[0165] Linkers may be used to covalently link antigen-binding domains of an antigen-binding molecule. The linkage between may provide a spatial relationship to permit binding of individual antigen-binding domains to their corresponding cognate epitopes. In this context, an individual linker serves to join two distinct functional antigen-binding domains. Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers.

[0166] The linker may be chemical and include for example an alkylene chain, a polyethylene glycol (PEG) chain, polysuccinic anhydride, poly-L-glutamic acid,

poly(ethyleneimine), an oligosaccharide, an amino acid chain, or any other suitable linkage.

In certain embodiments, the linker itself can be stable under physiological conditions, such as an alkylene chain, or it can be cleavable under physiological conditions, such as by an enzyme (e.g., the linkage contains a peptide sequence that is a substrate for a peptidase), or by hydrolysis (e.g., the linkage contains a hydrolyzable group, such as an ester or thioester). The linker can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain, or can be biologically active, such as an oligo- or polypeptide that, when cleaved from the moieties, binds a receptor, deactivates an enzyme, etc. The linker may be attached to the antigen-binding domains by any suitable bond or functional group, including carbon- carbon bonds, esters, ethers, amides, amines, carbonates, carbamates, sulfonamides, etc.

[0167] In certain embodiments, the linker represents at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) derivatized or non-derivatized amino acid. In illustrative examples of this type, the linker is preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences or repeats of Ala-Ala-Ala. Depending on the particular construct, the linkers may be long (e.g., greater than 12 amino acids in length) or short (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids in length). For example, to make a single chain diabody, the first and the third linkers are preferably about 3 to about 12 amino acids in length (and more preferably about 5 amino acids in length), and the second linker is preferably longer than 12 amino acids in length (and more preferably about 15 amino acids in length).

[0168] Representative peptide linkers may be selected from: [AAA]_n, [SGGGG]_n, [GGGGS]_n, [GGGGG]_n, [GGGKGGGG]_n, [GGGNGGGG]_n, [GGGCGGGG]_n, wherein n is an integer from 1 to 10, suitably 1 to 5, more suitably 1 to 3.

[0169] The present disclosure also encompasses multivalent antigen-binding molecules including bivalent, trivalent, quadrivalent, pentavalent, hexavalent, octavalent etc. antigen-binding molecules, in which at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 etc. valence(s)) has specificity for activated GPIIb/IIIa. Accordingly, multivalent antigen-binding molecules encompassed in the present disclosure can be monospecific or multispecific, wherein at least one specificity is for activated GPIIb/IIIa.

[0170] In some embodiments, an anti-GPIIb/IIIa multivalent antigen-binding molecule is a DART™ diabody molecule that comprises at least two polypeptide chains which form at least two epitope binding sites, at least one of which specifically binds to activated GPIIb/IIIa. Exemplary DART™ diabody molecules are disclosed in US20100174053,

US20090060910, US20070004909, EP2158221, EP1868650, W02010080538,

WO2008157379, and WO2006113665.

[0171] In representative examples, the DART™ diabody molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises: (i) a domain (A) comprising a light chain variable domain of a first

immunoglobulin (V_L1) specific for an epitope (1); (ii) a domain (B) comprising a heavy chain variable domain of a second immunoglobulin (V_H2) specific for an epitope (2); and (iii) a domain (C), and wherein the second polypeptide chain comprises: (i) a domain (D) comprising a light chain variable domain of the second immunoglobulin ( V_L2) specific for epitope (2); (ii) a domain (E) comprising a heavy chain variable domain of the first immunoglobulin (V_H1) specific for epitope (1); and (iii) a domain (F). The DART™ diabody domains (A) and (B) do not associate with one another to form an epitope binding site. Similarly, the DART™ diabody domains (D) and (E) do not associate with one another to form an epitope binding site. Rather, the DART™ diabody domains (A) and (E) associate to form a binding site that binds epitope (1); the DART™ diabody domains (B) and (D) associate to form a binding site that binds said epitope (2) and domains (C) and (F) are covalently or non-covalently associated together (e.g., domains (C) and (F) may be connected by a disulfide bridge, ionic interaction between oppositely charged amino acid sequences such as coils of opposite charge, illustrative examples of which include E-coils and K-coils). Epitopes (1) and (2) can be the same or different, wherein at least one is an epitope that is characteristic of activated GPIIb/IIIa. In some embodiments, one of epitopes (1) and (2) is an epitope present on activated GPIIb/IIIa and the other is present on a heterologous antigen. In other embodiments, both epitopes (1) and (2) are present on activated

GPIIb/IIIa, which can be the same or different.

[0172] Each polypeptide chain of the DART™ diabody molecule comprises a V_L domain and a V_H domain, which are covalently linked such that the domains are constrained from self-assembly. Interaction of two of the polypeptide chains will produce two V_L-V_H pairings, forming two epitope binding sites, i.e., a bivalent molecule. Neither the V_H or V_L domain is constrained to any position within the polypeptide chain, i.e., restricted to the amino (N) or carboxy (C) terminus, nor are the domains restricted in their relative positions to one another, i.e., the V_L domain may be N-terminal to the V_H domain and vice-versa. The only restriction is that a complementary polypeptide chain be available in order to form functional DART™ diabodies. Where the V_L and V_H domains are derived from the same antigen-binding molecule, the two complementary polypeptide chains may be identical. For example, where the binding domains are derived from an antigen-binding molecule specific for epitope A (i.e., the binding domain is formed from a V_LA-V_HA interaction), each polypeptide will comprise a V_HA and a V_LA- Homodimerization of two polypeptide chains of the antigen-binding molecule will result in the formation two V_LA-V_HA binding sites, resulting in a bivalent monospecific antigen-binding molecule. Where the V_L and V_H domains are derived from antigen-binding molecules specific for different antigens, formation of a functional bispecific DART™ diabody requires the interaction of two different polypeptide chains, i.e., formation of a heterodimer. For example, for a bispecific DART™ diabody, one polypeptide chain will comprise a V_LA and a V_LB; homodimerization of the chain will result in the formation of two V_LA-V_HB binding sites, either of no binding or of unpredictable binding. In contrast, where two different polypeptide chains are free to interact, e.g., in a recombinant expression system, one comprising a V_LA and a V_HB and the other comprising a V_LB and a V_HA, two different binding sites will form: V_LA-V_HA and V_LB-V_HB. For all DART™ diabody polypeptide chain pairs, the misalignment or mis-binding of the two chains is possible, i.e., interaction of V_L-V_L or V_H-V_H domains; however, purification of functional diabodies is easily managed based on the immunospecificity of the properly dimerized binding site using any affinity based method known in the art, e.g., affinity chromatography.

[0173] One or more of the polypeptide chains of the DART™ diabody may optionally comprise at least one Fc domain or portion thereof (e.g. a C_H2 domain and/or C_H3 domain). The Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In specific

embodiments, the Fc domain (or portion thereof) is derived from IgG. In representative examples of this type, the IgG isotype is IgG1, IgG2, IgG3 or IgG4 or an allotype thereof. In one embodiment, the diabody molecule comprises an Fc domain, which Fc domain comprises a C_H2 domain and C_H3 domain independently selected from any immunoglobulin isotype (i.e. , an Fc domain comprising the C»2 domain derived from IgG and the C_H3 domain derived from IgE, or the C_H2 domain derived from IgG1 and the C_H3 domain derived from IgG2, etc.). The Fc domain may be engineered into a polypeptide chain comprising a diabody molecule of the present disclosure in any position relative to other domains or portions of said polypeptide chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the V_L and V_H domains of the polypeptide of the chain; may be N-terminal to both the V_L and V_H domains; or may be N-terminal to one domain and C-terminal to another (i.e. , between two domains of the polypeptide chain)).

[0174] The Fc domains in the polypeptide chains of the DART™ diabody molecules preferentially dimerize, resulting in the formation of a DART™ molecule that exhibits immunoglobulin-like properties, e.g., Fc-FcgR, interactions. Fc comprising diabodies may be dimers, e.g., comprised of two polypeptide chains, each comprising a V_H domain, a V_L domain and an Fc domain. Dimerization of the polypeptide chains results in a bivalent DART™ diabody comprising an Fc domain, albeit with a structure distinct from that of an unmodified bivalent antibody. Such DART™ diabody molecules may exhibit altered phenotypes relative to a wild-type immunoglobulin, e.g. , altered serum half-life, binding properties, etc. In other embodiments, DART™ diabody molecules comprising Fc domains may be tetramers. Such tetramers comprise two 'heavier' polypeptide chains, i.e., a polypeptide chain comprising a V_L, a V_H and an Fc domain, and two 'lighter' polypeptide chains, i.e., polypeptide chain comprising a V_L and a V_H. The lighter and heavier chains interact to form a monomer, and said monomers interact via their unpaired Fc domains to form an Ig-like molecule. Such an Ig-like DART™ diabody is tetravalent and may be monospecific, bispecific or tetraspecific.

[0175] In one embodiment of generating a bivalent, monospecific a nti-GPIIb/IIIa antigen-binding molecule, first and second constructs encoding two different polypeptides are designed. The first construct comprises V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a C-terminal E-coil, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and a linker that suitably comprises a flexible linker interposed between the V_H sequence and the E-coil. In illustrative examples, the first construct comprises or consists essentially of the following amino acid sequence:

wherein :

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

.

is a flexible linker; Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa SE scFv; and

the amino acid sequence of the E-coil is shown in underlined capital letters.

[0176] The second construct comprises V_L and V_H sequences of the anti- GPIIb/IIIa SE scFv, a C-terminal K-coil, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and a linker that suitably comprises a flexible linker interposed between the V_H sequence and the K-coil. In non-limiting examples, the second construct comprises the following amino acid sequence:

wherein :

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

. is a flexible linker;

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa SE scFv; and

• the amino acid sequence of the K-coil is shown in underlined capital letters.

[0177] In another embodiment of generating a bivalent, monospecific anti-

GPIIb/IIIa antigen-binding molecule, first and second constructs encoding two different polypeptides are designed. The first construct comprises V_L and V_H sequences of the anti- GPIIb/IIIa SE scFv, a C-terminal first disulfide bond-forming moiety, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and a linker that suitably comprises a flexible linker interposed between the V_H sequence and the first disulfide bond-forming moiety, a representative example of which comprises the following amino acid sequence:

wherein :

Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

is a flexible linker;

Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa SE scFv; and the amino acid sequence of the first disulfide-bond forming moiety is shown in underlined capital letters.

[0178] The second construct comprises V_L and V_H sequences of the anti- GPIIb/IIIa SE scFv, a C-terminal second disulfide bond-forming moiety, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and a linker that suitably comprises a flexible linker interposed between the V_H sequence and the second disulfide-bond forming moiety, a representative example of which comprises the following amino acid sequence:

wherein :

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

. is a flexible linker;

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa SE scFv; and

• the amino acid sequence of the second disulfide-bond forming moiety is shown in underlined capital letters.

[0179] In another embodiment of generating a bivalent, monospecific anti- GPIIb/IIIa antigen-binding molecule, a single construct is designed, comprising V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a C-terminal truncated heavy chain (Hinge- C_H2-C_H3 ) sequences of a human IgG1 (e.g., atezolizumab), in which the heavy chain C_H3 domain suitably includes the L234A, L235A, P329G substitutions, and a linker separating the V_H sequence and the C-terminal truncated heavy chain. A non-limiting example of this construct comprises the following amino acid sequence:

wherein : • Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa SE scFv;

• AAA is a flexible linker;

.

is a flexible linker;

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa SE scFv;

•

is a linker sequence; and

• the amino acid sequence of the hinge and constant region (C_H2-C_H3) of atezolizumab is shown in underlined capital letters.

3.2 Anti-coaaulant agents

[0180] In some embodiments, the chimeric molecule comprises at least one anti coagulant agent. The anti-coagulant agent may inhibit clotting factor activity or stimulate thrombolytic activity. The anti-coagulant agent may be conjugated directly or indirectly to the anti-GPIIb/IIIa antigen-binding molecules of the present disclosure at any suitable position. For example, the anti-coagulant agent may be conjugated to the N-terminus or C- terminus of the anti-GPIIb/IIIa antigen-binding molecule.

[0181] In some embodiments, the anti-coagulant agent is a proteinaceous anti coagulant moiety. For example, tissue factor pathway inhibitor (TFPI) is known to inhibit the function of an active complex which is normally formed between tissue factor, factor Vila, and factor Xa. TFPI is a 276-residue soluble polypeptide whose positively charged C-terminus binds to heparin sulfate in the proteoglycan layer of endothelial cells. It has been notionally divided into "Kunitz" domains, in which, for example, Kunitz domain I binds tissue factor and factor Vila and domain II binds factor Xa.

[0182] Another proteinaceous anticoagulant moiety is tick anticoagulant peptide (TAP), which is a specific and potent inhibitor of factor Xa. This 60-amino acid polypeptide has been purified from the soft tick Ornithodoros moubata.

[0183] Anti-coagulant peptides have also been isolated from nematodes. For example, a 78-amino acid peptide, designated NAP5, that inhibits factor Xa (see, e.g., Rios- Steiner et al. , 2007. J. Mol. Biol. 371(3) : 774-786; GenPept Accession No. 2P3F_N ), an 84- amino acid peptide, designated NAP6, that inhibits factor Vila and tissue factor (TF) (see, e.g., Stassens et al., 1996. Proc. Natl. Acad. Sci. USA 93(5) : 2149-2154; GenPept Accession No. ICOU_A), and a 90-amino acid peptide, designated NAP10, that inhibits factor XVIIa and factor VIIa/TF see, e.g. , Li et al. , 2010. Biochim. Biophys. Res. Commun. 392(2) : 155-159; GenPept Accession No. ABP88128) have been isolated from the blood-feeding nematode Ancylostoma caninum. Other nematode A. caninum anti-coagulant peptides that inhibit coagulation factor XIa are disclosed for example in US 2014/0323404.

[0184] Many snake venoms also contain anti-coagulant polypeptides. For instance, a 231-amino acid protein C activator has been purified from the venom of the snake Agkistrodon contortrix contortrix (McMullen et al. , 1989. Biochemistry 28: 674-679; Kisiel et al., 1987. J. Biol. Chem. 262: 12607-12613) and Agkistrodon piscivorus leucostoma (Sukkapan et al., 2011. Toxicon 58(2) : 168-178).

[0185] Hirudin is the anti-coagulant protein utilized by the leech Hirudo medicinalis when extracting blood from its victim. It is highly potent and binds to thrombin at a 1 : 1 ratio with a dissociation constant in the femtomolar range. The active site of thrombin is masked in the stable complex and so the hirudin prevents fibrinogen breakdown, thus inhibiting clot formation.

[0186] In some embodiments, the anti-coagulant agent is a thrombolytic agent. The thrombolytic agent is generally capable of inducing reperfusion by dissolving, dislodging or otherwise breaking up a clot, e.g., by either dissolving a fibrin-platelet clot, or inhibiting the formation of such a clot. Reperfusion occurs when the clot is dissolved and blood flow is restored. Exemplary thrombolytic agents include, but are not limited to, tissue-type plasminogen activator (t-PA), streptokinase (SK), prourokinase, urokinase-type plasminogen activator (uPA), alteplase (also known as ACTIVASE, Genentech, Inc.), reteplase (also known as r-PA or RETAVASE, Centocor, Inc.), tenecteplase (also known as TNK, Genentech, Inc.), STREPTASE (AstraZeneca, LP), lanoteplase (Bristol-Myers Squibb Company), monteplase (Eisai Company, Ltd.), saruplase (also known as r-scu-PA and RESCUPASE, Grunenthal GmbH, Corp.), staphylokinase, and anisoylated plasminogen-streptokinase activator complex (also known as APSAC, Anistreplase and EMINASE, SmithKIine Beecham Corp.). Thrombolytic agents also include other genetically engineered plasminogen activators. The present disclosure can additionally employ hybrids, physiologically active fragments or mutant forms of the above thrombolytic agents. The term "tissue-type plasminogen activator" as used herein is intended to include such hybrids, fragments and mutants, as well as both naturally derived and recombinantly derived tissue-type plasminogen activator.

3.3 Peptide linkers

[0187] In some embodiments, the chimeric molecule comprises one or more peptide linkers. Examples of peptide linkers are well known in the art, for example peptide linkers according to the formula [(Gly)_x-Ser_y]_z where x is from 1 to 4, y is 0 or 1, and z is from 1 to 50. In certain embodiments z is from 1 to 6. In one embodiment, the peptide linker comprises the sequence G_n, where n can be an integer from 1 to 100. In a specific embodiment, the sequence of the peptide linker is GGGG [SEQ ID NO:29]. The peptide linker can comprise the sequence (GA)_n. The peptide linker can comprise the sequence (GGS)_n. In other embodiments, the peptide linker comprises the sequence (GGGS)_n. In still other embodiments, the peptide linker comprises the sequence (GGS)_n (GGGGS)_n. In these instances, n can be an integer from 1-100. In other instances, n can be an integer from 1- 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS [SEQ ID NO:30],

GGSGGSGGSGGSGGG [SEQ ID NO:31], GGSGGSGGGGSGGGGS [SEQ ID NO:32],

GGSGGSGGSGGSGGSGGS [SEQ ID NO:33], or GGGGSGGGGSGGGGS [SEQ ID NO:34] In other embodiments, the linker is a poly-G sequence (GGGG)_n, where n can be an integer from 1-100. [0188] Peptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.

[0189] It will be understood that various constructs disclosed herein comprise specific linkers and the present disclosure extends to the substitution of these with other suitable linkers known in the art, as for example described above and in Chen et al. (2013, Adv Drug Deliv Rev. 65(10) : 1357-1369).

[0190] When multiple linkers are present in a chimeric molecule of the present disclosure, each of the linkers can be the same or different. Generally, linkers provide flexibility to the chimeric molecule. Linkers are not typically cleaved; however in certain embodiments, such cleavage can be desirable. Accordingly, in some embodiments a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the sequence of the linker.

[0191] In some embodiments, the linker is specifically cleaved by an enzyme such that the anti-coagulant agent is released in the presence of the enzyme. Such linkers are typically peptide-based or include peptidic regions that act as substrates for enzymes. Peptide based linkers tend to be more stable in plasma and extracellular milieu than chemically labile linkers. Peptide bonds generally have good serum stability, as lysosomal proteolytic enzymes have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. Release of a drug from an antibody occurs specifically due to the action of lysosomal proteases, e.g., cathepsin and plasmin. These proteases may be present at elevated levels in certain tumor cells.

[0192] In exemplary embodiments, the cleavable peptide is selected from tetrapeptides such as Gly-Phe-Leu-Gly [SEQ ID NO:35], Ala-Leu-Ala-Leu [SEQ ID NO:36] or dipeptides such as Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp 5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, AM Met- (D)Lys, Asn-(D)Lys, AW Met-(D)Lys, and Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides due to hydrophobicity of the longer peptides.

4. Exemplary anti-thrombotic chimeric molecules

[0193] In specific embodiments, the anti-coagulant agent comprises a TAP that comprises, consists or consists essentially of the amino acid sequence:

YNRLCIKPRDWIDECDSNEGGERAYFRNGKGGCDSFWICPEDHTGADYYSSYRDCFNACI [SEQ ID NO:37] Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-

GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a TAP sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the TAP sequence. In representative examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• AAA is a flexible linker;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; and

• Uppercase underlined text corresponds to the TAP amino acid sequence.

[0194] In other representative examples, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• AAA is a flexible linker;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; and

• Uppercase underlined text corresponds to the TAP amino acid sequence.

[0195] In still other representative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• AAA is a flexible linker;

· is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

•

is a flexible linker;

. is a V5 epitope tag;

·

is a Factor Xa recognition site.

• Uppercase underlined text corresponds to the TAP amino acid sequence; and

. HHHHHH [SEQ ID NO: 16] is a His tag.

[0196] In other representative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• AAA is a flexible linker;

·

is a f!exibie linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

•

is a flexible linker;

.

is a V5 epitope tag;

·

is a Factor Xa recognition site.

• Uppercase underlined text corresponds to the TAP amino acid sequence; and .

is a His tag.

[0197] In specific embodiments, the anti-coagulant agent comprises a nematode anti-coagulant peptide corresponding to the A. caninum NAP5 peptide, which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a NAP5 peptide sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the NAP5 peptide sequence. In

representative examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a flexible linker; and

• Uppercase underlined text corresponds to the A. caninum NAP5 peptide

amino acid sequence.

[0198] In other embodiments, the anti-coagulant agent comprises a nematode anti-coagulant peptide corresponding to the A. caninum NAP6 peptide, which comprises or consists essentially of the following amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a NAP6 peptide sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the NAP6 peptide sequence. In non-limiting examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a flexible linker; and

• Uppercase underlined text corresponds to the A. caninum NAP6 peptide amino acid sequence.

[0199] In still other embodiments, the anti-coagulant agent comprises a nematode anti-coagulant peptide corresponding to the A. caninum NAP10 peptide, which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of

the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a NAP10 peptide sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the NAP10 peptide sequence. In illustrative examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

•

is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

.

is a flexible linker; and

• Uppercase underlined text corresponds to the A. caninum NAP10 peptide amino acid sequence.

[0200] In other embodiments, the anti-coagulant agent comprises an anti coagulant peptide corresponding to hirudin, which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-

GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a hirudin sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the hirudin sequence. In illustrative examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker,

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a flexible linker; and

• Uppercase underlined text corresponds to the hirudin amino acid sequence.

[0201] In other embodiments, the anti-coagulant agent comprises an anti- coagulant peptide corresponding to the mature chain of the Agkistrodon piscivorus leucostoma protein C activator, which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a protein C activator sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the protein C activator sequence. In non-limiting examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

. is a flexible linker; and

• Uppercase underlined text corresponds to the protein C activator amino acid sequence.

[0202] In some embodiments, the anti-coagulant agent comprises a thrombolytic polypeptide corresponding to ACTIVASE, which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-

coagulant agent comprises V_L and V_H sequences of the anti-GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, an ACTIVASE sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the ACTIVASE sequence.

[0203] In illustrative examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

.

is a flexible linker; and

• Uppercase underlined text corresponds to the amino acid sequence of

ACTIVASE.

[0204] In other embodiments, the anti-coagulant agent comprises a thrombolytic polypeptide corresponding to single chain urokinase-type plasminogen activator (scuPA), which comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the anti-coagulant agent comprises V_L and V_H sequences of the anti-

GPIIb/IIIa SE scFv, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences, a scuPA sequence downstream of the V_L and V_H sequences and an optional linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and the scuPA sequence.

[0205] In non-limiting examples of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; AAA is a flexible linker; and

• Uppercase underlined text corresponds to the amino acid sequence of scuPA.

[0206] In other non-limiting examples, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• AAA is a flexible linker; and

• Uppercase underlined text corresponds to the amino acid sequence of scuPA.

[0207] In other non-limiting examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• is a flexible linker;

Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

AAA is a flexible linker;

Uppercase underlined text corresponds to the amino acid sequence of scuPA;

is a FLAG tag ;

is a sortase conjugation tag ; and

.

is a His tag.

[0208] In still other non-limiting examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

Uppercase regular text corresponds to variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

is a flexible linker;

Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

AAA is a flexible linker;

Uppercase underlined text corresponds to the amino acid sequence of scuPA;

is a FLAG tag ;

is a sortase conjugation tag ; and

is a His tag.

[0209] In some embodiments, the chimeric construct comprises a half-life extending moiety, representative examples of which include: XTEN polypeptides; Fc;

albumin, albumin binding polypeptide or fatty acid, the C-terminal peptide (CTP) of the 13 subunit of human chorionic gonadotropin, PAS; HAP; transferrin; polyethylene glycol (PEG) ; hydroxyethyl starch (HES), polysialic acids (PSAs) ; a clearance receptor or fragment thereof which blocks binding of the chimeric molecule to a clearance receptor; low complexity peptides; or any combinations thereof. [0210] In representative embodiments of this type, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• X₁ is an optional linker that is suitably a flexible linker (e.g., [GGGGS]_n, wherein n is an integer from 1 to 10, suitably 1 to 5, more suitably 1 to 3) ;

• Uppercase bold text corresponds to HSA amino acid sequence;

• X2 is an optional linker that is suitably a flexible linker (e.g., [GGGGS]_n, wherein n is an integer from 1 to 10, suitably 1 to 5, more suitably 1 to 3) ; and

Uppercase underlined text corresponds to the TAP amino acid sequence.

[0211] In illustrative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase italic text corresponds to a leader sequence used for expression of the construct;

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• aaaas is a flexible linker;

• Uppercase bold text corresponds to HSA amino acid sequence; and

• Uppercase underlined text corresponds to the TAP amino acid sequence;

[0212] In other illustrative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase italic text corresponds to a leader sequence used for expression of the construct;

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• Lower case underlined and italicized text correspond to linker sequences;

• Uppercase bold text corresponds to HSA amino acid sequence;

• Uppercase underlined text corresponds to the TAP amino acid sequence; and is a His tag.

[0213] In other representative embodiments, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• X₁ is an optional linker that is suitably a flexible linker (e.g., [GGGGS]_n, wherein n is an integer from 1 to 10, suitably 1 to 5, more suitably 1 to 3) ;

• Uppercase bold text corresponds to HSA amino acid sequence;

• X₂ is an optional linker that is suitably a flexible linker (e.g., [GGGGS]_n, wherein n is an integer from 1 to 10, suitably 1 to 5, more suitably 1 to 3) ; and

Uppercase underlined text corresponds to the amino acid sequence of scuPA.

[0214] In illustrative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase italic text corresponds to a leader sequence used for expression of the construct;

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• aaaas is a flexible linker; and

• Uppercase bold text corresponds to HSA amino acid sequence;

Uppercase underlined text corresponds to the amino acid sequence of scuPA;

[0215] In other illustrative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• Uppercase italic text corresponds to a leader sequence used for expression of the construct;

• Uppercase regular text corresponds to scFv SE amino acid sequence;

• Lower case underlined and italicized text correspond to linker sequences;

• Uppercase bold text corresponds to HSA amino acid sequence;

• Uppercase underlined text corresponds to the amino acid sequence of scuPA;

.

is a C-myc tag ; and

. is a His tag.

5. Methods of Preparation

[0216] The present disclosure also provides a nucleic acid molecule or a set of nucleic acid molecules encoding a chimeric molecule disclosed herein.

[0217] Also provided is a nucleic acid construct or a set of nucleic acid constructs comprising such nucleic acid molecule or a set of the nucleic acid molecules or a complement thereof, operably connected to a regulatory sequence, as well as a host cell comprising the construct or set of constructs.

[0218] The instant disclosure also provides methods for producing a chimeric molecule disclosed herein, such methods comprising culturing the host cell disclosed herein and recovering the chimeric molecule from the host cell or culture medium.

[0219] A variety of methods is available for recombinantly producing a chimeric molecule disclosed herein. It will be understood that because of the degeneracy of the code, a variety of nucleic acid sequences will encode the amino acid sequence of the polypeptide. The desired polynucleotide can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared polynucleotide.

[0220] In some embodiments an expression vector or set of expression vectors from which a nucleic acid sequence encoding the amino acid sequence of a chimeric molecule disclosed herein is expressible, is transfected into a host cell (e.g., 293, CHO, COS) and the host cell is cultured under conditions that allow for the expression of the chimeric molecule. The chimeric polypeptide may be recovered from the cell or culture medium.

[0221] Oligonucleotide-mediated mutagenesis is one method for preparing a substitution, in-frame insertion, or alteration (e.g., altered codon) to introduce a codon encoding an amino acid substitution such as a conservative or non-conservative substitution (e.g., into an anti-GPIIb/IIIa antigen-binding molecule and/or anti-coagulant polypeptide). For example, the starting polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer. In one embodiment, genetic engineering, e.g., primer-based PCR mutagenesis, is sufficient to incorporate an alteration, as defined herein, for producing a polynucleotide encoding a chimeric molecule disclosed herein.

[0222] For recombinant production, a polynucleotide sequence encoding a chimeric polypeptide disclosed herein will generally include a translation start-site encoding an N-terminal methionine to facilitate recombinant expression of the polypeptide. Optionally, the coding sequence of the polypeptide may encode a purification moiety that facilitates purification of the polypeptide. Purification moieties typically comprise a stretch of amino acids that enables recovery of the polypeptide through affinity binding. Numerous purification moieties or 'tags' are known in the art, illustrative examples of which include biotin carboxyl carrier protein-tag (BCCP-tag), Myc-tag (c-myc-tag), Calmodulin-tag, FLAG- tag, HA-tag, His-tag (Hexahistidine-tag, His6, 6H), Maltose binding protein-tag (MBP-tag), Nus-tag, Chitin-binding protein-tag (CBP-tag) Glutathione-S-transferase-tag (GST-tag), Green fluorescent protein-tag (GFP-tag), Polyglutamate-tag, Amyloid beta-tag, Thioredoxin- tag, S-tag, Softag 1, Softag 3, SpyCatcher tag, Spy tag, Strep-tag, Streptavidin-binding peptide-tag (SBP-tag), biotin-tag, streptavidin-tag and V5-tag.

[0223] The polypeptide-encoding polynucleotide is typically inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The expression vector is then transfected into a suitable target cell which will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al., 1978. Cell 14:725) and electroporation (Neumann et al., 1982. EMBO J.

1 :841). A variety of host-expression vector systems can be utilized to express the polypeptides described (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein) in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including mammalian cells (e.g., 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When the polypeptide is expressed in a eukaryotic cell, the DNA encoding the polypeptide (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein) can also code for a signal sequence that will permit the polypeptide to be secreted. One of skill in the art will understand that while the polypeptide is translated, the signal sequence is cleaved by the cell to form the mature polypeptide. Various signal sequences are known in the art, e.g., native GPIIb signal sequence, native GPIIIa signal sequence, and the mouse IgK light chain signal sequence. Alternatively, where a signal sequence is not included, the chimeric molecule disclosed herein can be recovered by lysing the cells.

[0224] Numerous expression vector systems can be employed. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors can include expression control sequences including, but not limited to, promoters (e.g., naturally-associated or

heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Suitably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors can also utilize DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites.

[0225] Commonly used expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362). Cells which have integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of transfected host cells. The marker can provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation.

[0226] An exemplary expression vector is NEOSPLA (U.S. Pat. No. 6,159,730).

This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in cells, followed by selection in G418 containing medium and methotrexate amplification. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., >30 pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

[0227] In other embodiments, chimeric polypeptides of the present disclosure (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein) can be expressed using polycistronic constructs. In these expression systems, multiple gene products of interest such as multiple polypeptides of multimer binding protein can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides of the present disclosure in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed in the instant application.

[0228] More generally, once the vector or DNA sequence encoding the polypeptide has been prepared, the expression vector can be introduced into an appropriate host cell. That is, the host cells can be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian

Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Suitably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, immunohistochemistry, and the like.

[0229] Polynucleotides encoding the chimeric polypeptides disclosed herein can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e., those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella,· Bacillaceae, such as Bacillus subtilis,· Pneumococcus,· Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides typically become part of inclusion bodies. The polypeptides must be isolated, purified and then assembled into functional molecules.

[0230] In addition to prokaryotes, eukaryotic microbes can also be used.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., 1979. Nature 282:39; Tschemper et al., 1980. Gene 10: 157) is commonly used. Other yeast hosts such Pichia can also be employed. Yeast expression vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization. Insect host cells may also be used for recombinant expression in combination with expression vectors that are operable in such cells (e.g., baculovirus expression vectors). Representative examples of insect host cells include Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells).

[0231] In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein. An affinity tag sequence (e.g. a His(6) tag can optionally be attached or included within the polypeptide sequence to facilitate downstream purification.

[0232] Once expressed, the chimeric polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). Substantially pure proteins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.

6. Pharmaceutical Compositions

[0233] The present disclosure also provides pharmaceutical compositions comprising an agent of the disclosure and a pharmaceutically acceptable carrier. The agent is suitably selected from: (i) a chimeric molecule disclosed herein; (ii) a nucleic acid molecule or the set of nucleic acid molecules disclosed herein; or (iii) a construct or set of constructs disclosed herein. In some embodiments, administering a pharmaceutical composition comprising an agent of the disclosure can be used, for example, to reduce or inhibit the development of platelet aggregation or thrombosis in a subject in need thereof.

[0234] Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated.

[0235] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, sublingually, nasally, rectally, vaginally or via pulmonary route. In specific embodiments, the compositions are in the form of injectable or infusible solutions. A preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In specific embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In other embodiments, the pharmaceutical composition is administered by intramuscular or subcutaneous injection.

[0236] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0237] Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0238] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin and/or by the maintenance of the required particle size. In specific embodiments, an agent of the present disclosure may be conjugated to a vehicle for cellular delivery. In these embodiments, the agent may be encapsulated in a suitable vehicle to either aid in the delivery of the agent to target cells, to increase the stability of the agent, or to minimize potential toxicity of the agent. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering an agent of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating agents of the present disclosure into delivery vehicles are known in the art. Although various embodiments are presented below, it will be appreciate that other methods known in the art to incorporate an antigen binding molecule or chimeric molecule of the disclosure into a delivery vehicle are contemplated.

[0239] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, a continuous infusion is administered over time, i.e., without interruption. An antigen-binding molecule or chimeric molecule of the present disclosure can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly.

Intervals can also be irregular as indicated by measuring blood levels of modified polypeptide or antigen in the patient. Alternatively, the antigen-binding molecule or chimeric molecule can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0240] It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0241] Dosages and therapeutic regimens of the chimeric molecule can be determined by a skilled artisan. In certain embodiments, the chimeric molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 0.01 to 40 mg/kg, e.g., 0.01 to 0.1 mg/kg, e.g. , about 0.1 to 1 mg/kg, about 1 to 5 mg/kg, about 5 to 25 mg/kg, about 10 to 40 mg/kg, , or about 0.4 mg/kg. The dosing schedule can vary from e.g. , once a week to once every 2, 3, or 4 weeks. In one embodiment, the chimeric molecule is administered at a dose from about 10 to 20 mg/kg every other week. An exemplary, non-limiting range for an effective amount of a chimeric molecule of the present disclosure is 0.01-5 mg/kg, more suitably 0.1-2 mg/kg.

[0242] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0243] The pharmaceutical compositions of the present disclosure may include an effective amount of agent of the present disclosure. The effective amount may be a

"therapeutically effective amount" or a "prophylactically effective amount". A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent is outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably inhibits a measurable parameter, e.g., platelet aggregation platelet aggregation, thrombus formation or embolus formation by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of an agent to inhibit a measurable parameter, e.g., platelet aggregation, thrombus formation or embolus formation can be evaluated in an animal model system predictive of efficacy in human condition associated with the presence of activated platelets (e.g., atherosclerosis (e.g., unstable atherosclerosis), allergic disorders, autoimmune diseases, cancers, infections, neurological disorders, systemic inflammation, tissue or organ transplantation,

thromboembolism-associated conditions and wounds) . Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, for example in in vitro by assays known to the skilled practitioner.

[0244] By contrast, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

7. Methods of Treatment

[0245] The agents of the disclosure (e.g., a chimeric molecule disclosed herein, a nucleic acid molecule or the set of nucleic acid molecules disclosed herein, or a construct or set of constructs disclosed herein) can be useful in methods of treating or inhibiting the development of platelet aggregation, thrombus formation or embolus formation in a subject having or at risk of developing a condition associated with the presence of activated platelets. The methods generally involve administering to a subject (e.g., a mammalian subject such as a human) in need thereof an effective amount of the agent.

[0246] Conditions associated with the presence of activated platelets include a range of inflammatory conditions including for example abdominal aortic aneurysm, acid reflux/heartburn, acne, acne vulgaris, allergies and sensitivities, Alzheimer's disease, anaphylaxis, asthma, asthma, atherosclerosis (e.g., unstable atherosclerosis) and vascular occlusive disease, dementia, ischaemic heart disease, myocardial infarction, stroke, peripheral vascular disease, or vascular stent restenosis, autoimmune diseases {e.g. multiple sclerosis), bronchitis, cancer and its various metastases, carditis, cataracts, celiac disease, chronic inflammation, optionally type IV delayed hypersensitivity associated for example with infection or systematic inflammatory response syndrome, or multiple organ failure, chronic pain, chronic prostatitis, cirrhosis, colitis, connective tissue diseases, systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, or Sjogren's syndrome, corneal disease, Crohn's disease, crystal Arthropathies, optionally gout, pseudogout, calcium pyrophosphate deposition disease, dementia, dermatitis, diabetes, dry eyes, eczema, edema, emphysema, fibromyalgia, gastroenteritis, gingivitis, glomerulonephritis, graft vs. host disease, heart disease, hepatitis, high blood pressure, hypersensitivities, inflammatory bowel diseases, inflammatory conditions associated with trauma or ischemia, insulin resistance, interstitial cystitis, iridocyclitis, iritis, joint pain, arthritis, Lyme disease, metabolic syndrome (syndrome x), multiple sclerosis, myositis, nephritis, obesity, ocular diseases including uveitis, osteopenia, osteoporosis, Parkinson's disease, pelvic inflammatory disease, periodontal disease, polyarteritis, polychondritis, polymyalgia rheumatica, psoriasis, reperfusion injury, rheumatic diseases, rheumatic arthritis, osteoarthritis, or psoriatic arthritis, sarcoidosis, scleroderma, sepsis, rhinitis, sinusitis, Sjogren's syndrome, spastic colon, spondyloarthropathies, optionally ankylosing spondylitis, reactive arthritis, or Reiter's syndrome, systemic candidiasis, tendonitis, transplant rejection, thyroiditis, UTIs, vaginitis, vascular diseases including atherosclerotic vascular disease, vasculitides, polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, vasculitis, or wounds including chronic wounds.

[0247] In some embodiments, the condition associated with the presence of activated platelets arterial is a thromboembolism-associated condition including, for example, cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart or in the peripheral circulation. The thromboembolism-associated condition can also include specific disorders selected from, but not limited to, unstable angina or other acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis and/or embolism, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, extracorporeal circulation (ECMO, cardiopulmonary bypass) procedures in which blood is exposed to an artificial surface that promotes thrombosis. The medical implants or devices include, but are not limited to: prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices and artificial hearts or heart chambers, and vessel grafts. The procedures include, but are not limited to: cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis.

In another embodiment, the disease or condition associated with thromboembolism includes acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism.

[0248] The agents of the present disclosure can optionally be administered in combination with ancillary agents (e.g., prophylactic or therapeutic) that are effective in treating the condition associated with the presence of activated platelets. As used herein, concurrent administration of the agents in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed

polypeptides. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen can be timed to enhance the overall effectiveness of the treatment. A skilled artisan (e.g., a physician) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.

8. Kits

[0249] A further embodiment of the present disclosure is a kit for inhibiting binding of a ligand to GPIIb/IIIa in its active conformation, for inhibiting binding of a ligand to an activated platelet, for inhibiting platelet aggregation, for inhibiting thrombus formation, for inhibiting embolus formation, for treating or detecting conditions associated with activated platelets, or for treating or inhibiting the development of a thromboembolism- associated condition, or for treating or inhibiting the development of a hematologic disorder, or for reducing or inhibiting proliferation, survival or viability of a tumor, or for treating or inhibiting the development of a cancer. This kit comprises any active agent disclosed herein (e.g., anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein) or pharmaceutical composition disclosed herein, and optionally instructions for detecting activated platelets, thrombi or emboli, or for treating or detecting conditions associated with activated platelets. The kits may also include suitable storage containers (e.g., ampules, vials, tubes, etc.), for each active agent or pharmaceutical composition and other included reagents (e.g., buffers, balanced salt solutions, labeling reagents, etc.) for use in administering the active agents or pharmaceutical compositions to subjects. The active agents or pharmaceutical compositions and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the active agents or pharmaceutical compositions and other optional reagents.

[0250] In order that the present disclosure may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1

SE SCFV

[0251] An scFv has been generated, designated SE, which has the amino acid sequence set out in SEQ ID NO: 12. The platelet aggregation inhibitory activity of SE was compared to that of another scFv with specificity to activated GPIIb/IIIa, designated SCE5 (U.S. Patent No. 7,812,136) and of ReoPro (Abciximab, Janssen Biologies BV) which lacks this specificity and which is currently used in a clinical setting. The results presented in Figure 1 clearly show that SE binds to activated platelets and inhibits platelet activation with significantly higher potency than SCE5 and with similar potency to ReoPro.

Materials and Methods

Purification of endotoxin free DNA for transfection

[0252] DNA of both scFvs (SCE5 and SE) constructs in the pSectag2A vector was purified using the endotoxin free plasmid maxiprep kit (Promega Corporation, USA), according to the manufacturer's instruction manual. The concentration of the DNA was measured using a NanoDrop 2000 spectrophotometer (ThermoFisher, USA). DNA was filtered through a 0.22mM sterile syringe filter prior to its use for transfection.

Expression in mammalians cells and purification of scFv constructs

[0253] Production of mammalian cells was performed using the human embryonic kidney cells (H293F) suspension culture transfection with polyethyleneimine (PEI)

(Polyscience Inc., Germany). This system is used for the production of proteins from pSectag vectors. DNA plasmid for transfection was diluted to a ratio of 1 :4 with PEI. 24 hours prior to transfection, H293F cells were diluted with Freestyle 293 expression medium (Invitrogen, USA) to a concentration of 1 x 106 cells/mL. The cell density was approximately 2 × 106 cells/mL at time of transfection and the viability was greater than 95%. A ratio of 9: 1 was used for the amount of Freestyle 293 expression medium to the PBS mixture of DNA and PEI. Appropriate amount of cell culture medium was transferred into a shaker flask and placed in a CO2 incubator at 37°C, shaking at 110 rpm. 1mg/mL of DNA plasmid was added to pre-warmed (37°C) PBS and vortexed gently. PEI was added at a concentration of 3mg/mL, and vortexed three times for three seconds. The mixture was incubated for 15 min at room temperature (RT). The cell culture medium was removed from the incubator. The DNA/PEI mixture was added to the medium while swirling gently. Glucose was added to a final concentration of 6g/L. The flask was returned to the incubation and cultured at 37°C, with 5% C02, shaking at 110 - 140 rpm. The culture was supplemented with 5 g/L Lupin after one day. At day 3, 5 and 7 after transfection, the culture was supplemented with 2 mmol/L glutamine. At day 5, the culture was supplemented with 5 g/L Lupin. The glucose level was maintained at a final concentration of 5 - 6 g/L. The cells were harvested when viability was 40 - 50%. The cells were centrifuged at 3000xg for 15 min at 4°C and supernatant was collection for protein purification. All purified single-chain antibodies carry a 6x His-tag at the C-terminal end of their amino acid sequence for purification by IMAC and for FACS analysis. Proteins were purified with a nickel-based metal affinity chromatography column, Ni-NTA column (Invitrogen, USA), according to the manufacturer's instruction manual. Fractions of lmL were collected and dialyzed against PBS.

Evaluation of the scFv proteins

[0254] Purity of the proteins was analyzed using SDS-PAGE. 30 mL of each purified protein and 6 mL of 5X reducing SDS loading buffer were added to 1.5 mL tubes and denatured at 96°C for 5 min. The samples were run on SDS-PAGE gel in SDS running buffer at 30mA for 2 hours. The gel was then stained with Coomassie Brilliant Blue for 1 hour and subsequently destained for at least 12 hours with Coomassie destaining solution. The gel was visualized and analyzed using a Bio-Rad Gel-Doc system with Quantity One software.

[0255] After SDS-gel electrophoresis and Western blotting, the membrane was blocked with 1% BSA and hybridized with a specific horseradish peroxidase (HRP). Anti-6x His-tag® antibody HRP was used to detect the fusion proteins. Secondary hybridization was performed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific Inc, USA), an enhanced chemiluminescent (ECL) substrate for the HRP enzyme.

Blood collection and platelet preparation

[0256] Blood was collected from healthy volunteers who had taken no medication for at least 10 days. In an attempt to minimize platelet activation during blood collection, blood was obtained by venipuncture from an antecubital vein through a 21 gauge needle with no tourniquet. The first 2mL of blood were discarded. The collected blood was anticoagulated with 10% citric acid. Platelet rich plasma (PRP) was obtained by

centrifugation at 180xg for 10 min at room temperature. 96 well light aggregation assay

[0257] 96-well plate light transmission aggregometry was performed using 100mI of PRP. Platelet poor plasma (PPP) was obtained by centrifugation of blood at 1000xg for 10min at room temperature. PRP was mixed with 8 mM calcium chloride, 1 : 50

thromboplastin (Siemens, USA), and 20 mM thrombin receptor activator peptide (Sigma- Aldrich, Germany), leading to platelet activation and clotting. The PRP mixture were incubated with abciximab (ReoPro), SCE5, SE or PBS (as control), then activated with 2mM ADP. Concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL, and 10 mg/mL were evaluated. Light transmission aggregometry was measured using the Bio-Rad Benchmark Plus at wavelength 595nm. Samples were measured every 15 seconds for 10 min. Light transmission was adjusted to 0% with PPP and 100% with PRP.

EXAMPLE 2

SE-TAP DEMONSTRATES ANTI-PLATELET AND ANTI-COAGULANT ACTIVITY

[0258] The objective of this example was to develop a dual pathway therapeutic capable of concentrating anti-platelet and anti-coagulant activity at the site of clot formation. A recombinant fusion protein, SE-TAP was engineered, which consists of a SE single-chain antibody (comprising the amino sequence set out in SEQ ID NO: 12) that targets the activated GPIIb/IIIa complex, and TAP, a potent direct inhibitor of FXa. TAP DNA was synthesized (Waxman et al.1,990. Science 248(4955) : 593-596 [SEQ ID NO: 37]) and cloned in frame to the C-terminus of SE scFv to provide a chimeric polypeptide comprising the sequence set forth in SEQ ID NO:39. In addition, a control construct was generated, MUT- TAP, which consists of a mutated scFv (MUT), generated by alanine substitution mutagenesis of heavy-chain CDR3, which displays no platelet binding activity, fused with active TAP. Recombinant expression was performed in Drosophila pMT/BiP/V5-His and constructs were purified using metal affinity and size exclusion chromatography.

[0259] Selective binding to activated platelets was confirmed using flow cytometry. SE, SE-TAP, or MUT-TAP were incubated with resting or 20 mM ADP-activated human or mouse platelets and binding was assessed using anti-His-mAb-AF488. Activation- specific binding of SE to both human and mouse platelets was observed, with no binding to resting platelets (Figure 2, A and D). Fusion construct SE-TAP also displayed activation- specific binding (Figure 2, B and E) while platelet binding was not observed with the MUT- TAP control construct (Figure 2, C and F). These results confirm that C-terminal TAP fusion does not impede scFv targeting to activated GPIIb/IIIa receptors and, thus, to activated platelets. SE-TAP selective binding to activated platelets was further confirmed with additional platelet agonists, including collagen related peptide (CRP) and thrombin receptor- activating peptide (TRAP). Because SE also serves as a conformation-specific inhibitor of GPIIb/IIIa, the present inventors examined the ability of fusion constructs to inhibit fibrinogen binding to activated platelets. Human or mouse PRP (± 20 mM ADP) was incubated with SE, SE-TAP, MUT-TAP, or vehicle control and fibrinogen binding was detected using flow cytometry with FITC-labeled anti-fibrinogen antibody. Maximum fibrinogen binding was defined with respect to the fluorescent shift detected in 20 mM ADP-activated vehicle control with results plotted as percent inhibition. A significant inhibition of fibrinogen binding with both SE and SE-TAP was observed, confirming activation-specific blockade of GPIIb/IIIa (Figure 2G). There was no significant difference between SE and SE-TAP indicating no impairment of the SE GPIIb/IIIa blocking function by C-terminal TAP fusion. No inhibition was observed with MUT-TAP.

[0260] The present inventors characterized anti-FXa activity to confirm functional integrity of the TAP fusion. The soluble activity of constructs was assessed by incubating SE- TAP with purified FXa and a chromogenic, Xa specific substrate. Results are reported as percent inhibition of FXa relative to vehicle control. SE-TAP and MUT-TAP inhibited FXa equally, while inhibition was not observed with scFv SE (Figure 2H). Additionally, retention of anti-FXa activity was confirmed when SE-TAP was bound to a fibrinogen-adherent platelet covered surface (Figure 21). Finally, a flow chamber adhesion assay was employed to examine the effect of dual pathway inhibition in whole blood. To mimic initial platelet adhesion and consequent platelet aggregation, blood was perfused over collagen fibers in the presence of SE, SE-TAP, MUT-TAP, or saline control and platelet adhesion/aggregation assessed using bright field microscopy. As compared to saline control, SE at 5 mg/mL produced a slight reduction of platelet aggregates, while an equivalent dose of SE-TAP nearly fully eliminated platelet aggregate formation (Figure 2, J and K). Only when blood was perfused with SE at a three-fold higher concentration (15 mg/mL), was a nearly full reduction of platelet microthrombi observed. The enhanced efficacy of the fusion construct highlights the advantage of TAP fusion to SE with dual pathway blockade. Significantly, the present inventors continued to observe platelet adhesion to collagen fibers in the presence of both SE and SE-TAP, suggesting that primary platelet attachment to collagen was not impeded.

EXAMPLE 3

SE-TAP TARGETS ARTERIAL THROMBUS AND INHIBITS OCCLUSION IN MICE

[0261] The capacity of SE-TAP to target and limit arterial thrombus formation was investigated using intravital microscopy (IVM) of localized laser-induced injury to murine cremaster arterioles (Figure 3, A-C). To characterize targeting, SE-TAP and MUT-TAP were incubated with anti-His AF488 to label construct His tags. Mice were infused with platelet- specific Dylight 649 anti-CD42b and AF488-SE-TAP, AF488-MUT-TAP, or anti-His AF488 control and subject to laser injury. Platelet thrombi targeting, as indicated by fluorescent co localization, was only observed with AF488-SE-TAP infusion. Next the capacity of SE-TAP to limit arterial thrombus formation was investigated. Laser injury was performed with quantitative analysis of Dylight 649-labeled platelets. Intravenous infusion of MUT-TAP (0.03 mg/g) resulted in little change in platelet accumulation as compared to the saline vehicle. However, infusion of SE-TAP (0.03 mg/g) resulted in significant reduction in platelet deposition at the site of vessel wall injury (Figure 3, A-C).

[0262] To examine efficacy in a larger diameter artery, constructs were studied in a ferric chloride (FeCl₃)-induced arterial thrombosis model of the left common carotid artery (29). Thrombus targeting was confirmed using IVIS in vivo imaging with IR800-labeled SE-

TAP (Figure 3D). Vessel flow was monitored for a maximum of 30 min and both enoxaparin (LMWH) and eptifibatide (Ept) were included as clinically relevant standards that block FXa or platelet GPIIb/IIIa receptors, respectively. Carotid artery occlusion was noted within ~10 min (10.2 ± 1.6 min) for those animals treated with saline alone, while administration of SE- TAP at a dose that proved efficacious in the IVM laser injury model (0.03 mg/g) significantly delayed arterial occlusion (24.8 ± 3.3 min) (Figure 3E). In contrast, administration of MUT- TAP (0.03 mg/g) did not affect occlusion time. At a 10-fold higher dosing regimen (0.3 mg/g) of SE-TAP, the present inventors continued to observe a significant prolongation in occlusion time, while the effect of MUT-TAP (0.3 mg/g) and SE (0.3 mg/g) were similar to saline control (Figure 3E). Tail transection bleeding time was evaluated to examine whether hemostasis is maintained or impaired by the administered agents. At a therapeutic dose of 0.03 mg/g, SE- TAP did not increase either bleeding time or bleeding volume when compared to saline vehicle (Figure 3, F and G). Reference agents, enoxaparin and eptifibatide, administered at clinically relevant dosing levels, demonstrated both significant thrombus inhibition and marked impairment in hemostasis. A smaller, but statistically significant increase in bleeding time and bleeding volume were observed at the highest dose (0.3 mg/g) of SE-TAP and MUT- TAP, while increased bleeding was not observed with administration of SE (Figure 3, F and G). Collectively, these data demonstrate the potential to target a dual pathway inhibitor to activated platelets using a lower, yet efficacious dosing regimen, which limits the risk of increased bleeding associated with conventional anticoagulants.

EXAMPLE 4

SE INHIBITS MOUSE MODELS OF VENOUS THROMBOSIS

[0263] Prior to initiating enrollment of SE-TAP in a model of venous thrombosis, we explored the influence of the administration route on circulating drug half-life. SE-TAP was administered via subcutaneous (SC) or IV routes and circulating anti-FXa in plasma was monitored over time. Results were compared to an in vitro standard curve of anti-FXa activity as a function of SE-TAP concentration. SC delivery significantly extended the circulating half-life of SE-TAP from 2.75 ± 0.15 h (IV) to 10.13 ± 1.07 h (SC) (Figure 4A). Intravital microscopy of localized laser-induced injury to cremaster venules was used to investigate targeting and the antithrombotic profile. Construct targeting to activated platelets at sites of venule wall injury was confirmed with infusion of Dylight 649 anti-CD42b and AF488-SE-TAP, AF488-MUT-TAP, or anti-His AF488 control. Time-lapse analysis

demonstrates delayed binding of AF488-SE-TAP to an established Dylight 649 platelet surface, suggesting that SE does not inhibit the initial sealing layer of platelets. Moreover, fluorescence co-localization of platelet Dylight 649 and construct AF488 signal was only observed with AF488-SE-TAP administration (Figure 4B). Noting peak circulating anti-FXa activity 4 h after SC administration of SE-TAP (Figure 4A), inhibition of venule thrombus formation was assessed at this time point. Studies were initially performed to verify a therapeutic effect of SE-TAP and to determine the lowest effective IV (0.05 mg/g) or SC (0.1 mg/g) dose when laser injury was performed immediately or 4 h after drug administration, respectively. Significant reduction in venous platelet accumulation was achieved with SE-TAP (0.1 mg/g SC) while MUT-TAP (0.1 mg/g SC) and equimolar SE (0.06 mg/g SC) displayed no inhibitory effect (Figure 4, C and D). LMWH (4 mg/g SC) was dosed according to recommended clinical guidelines for peak anti-FXa activity (0.4 - 0.6 IU/mL), as required for prevention of VTE (Figure 4E) (McRae et al., 2004. Circulation 110(9 suppl 1) : I-3-I-9) .

Notably, the antithrombotic activity of SE-TAP was equivalent to that observed for LMWH, but at a significantly lower peak level of circulating anti-FXa (Figure 4, D and E). Next, a SE- TAP dosing strategy was explored that would sustain antithrombotic activity up to 24 h post administration. Significant reduction in venous platelet accumulation was achieved at a five fold increased concentration of SE-TAP (0.5 mg/g SC), while MUT-TAP (0.5 mg/g SC) and equimolar SE (0.3 mg/g SC) displayed no inhibitory activity (Figure 4, F and G). Peak circulating anti-FXa activity remained low relative to LMWH (Figure 4H) suggesting that therapeutic SE-TAP would be associated with reduced bleeding risk.

[0264] The present inventors employed this prophylactic administration strategy to evaluate the capacity of SE-TAP to limit murine deep venous thrombosis using an electrolytic inferior vena cava model (EIM). EIM was specifically selected to achieve reproducible and nonocclusive IVC thrombus, formed in the presence of blood flow. The present inventors administrated SE-TAP (0.5 mg/g SC) or the clinically relevant anti-FXa therapeutics, LMWH (4 mg/g SC) or rivaroxaban (1 mg/g PO), 4 h prior to electrolytic injury and 24 h post-injury. Controls included saline vehicle, uninjured IVC, and surgical sham with needle insertion into the IVC but without induction of current (sham no current). A uniform length of IVC was harvested 48 h post-injury and immediately weighed to measure vessel wall and thrombus weight. SE-TAP displayed 43.24 ± 4.09% thrombus inhibition as compared to saline vehicle, which was comparable to the effect observed for LMWH (47.31 ± 3.58%) and rivaroxaban (42.09 ± 2.50%) (Figure 5, A and B). Harvested IVC cross sections were stained and thrombus area measured. Treatment groups displayed a ~45% reduction in thrombus area without a significant difference between SE-TAP, LMWH, and rivaroxaban (Figure 5, C and D). Bleeding risk was assessed by measuring peak circulating anti-FXa activity and by determining tail transection bleeding time and blood loss, 4 h after drug administration. Significantly, at therapeutic dosing, SE-TAP displayed minimal circulating anti-FXa activity and no increase in bleeding time or blood loss volume as compared to saline vehicle. Therapeutic dosing of LMWH and rivaroxaban, consistent with clinical guidelines for prevention of VTE, was associated with increased bleeding (Figure 5, E and G). Of note, a ten-fold increase in MUT-TAP (5 mg/g SC) was necessary to achieve a therapeutic effect comparable to SE-TAP (0.5 mg/g SC), but with significant impairment in hemostasis.

Collectively, these results highlight the advantage of selective targeting to reduce the dose and limit bleeding risk associated with conventional dual pathway blockade through combined anticoagulation and antiplatelet regimens.

[0265] To analyze the corresponding inflammatory response 48 h post-EIM, a uniform length of IVC and thrombus was harvested and digested to a single cell suspension for flow cytometry analysis of total leukocytes (CD45⁺), platelets (CD41⁺), neutrophils (CD11b⁺/Ly6G⁺), and monocytes (CD11b⁺/Ly6G-). The present inventors observed significant reductions in total leukocytes, neutrophils, and monocytes with SE-TAP, LMWH, and rivaroxaban relative to saline vehicle (Figure 6, A and C-D). Results were reported as absolute cell number relative to vessel wall weight to reflect the concentration of leukocytes per thrombus. These results suggest that reduced leukocyte content in treatment groups is not simply a function of thrombus trapping, but rather may be a downstream benefit of anti- FXa treatment and reduced production of pro-inflammatory mediators, such as thrombin.

The reduction of neutrophils (Figure 6C) is consistent with their dominant and early role in vessel wall inflammation after thrombus formation. A significant reduction was also observed in platelet accumulation across all treatment groups (Figure 6B). Standard

immunohistochemistry was performed on paraffin embedded IVC sections to characterize inflammatory cell localization. Leukocyte and platelet staining were localized to the luminal thrombus in all groups (Figure 6, E-G).

EXAMPLE 5

SE-TAP PROVIDES AN IMPROVED THERAPEUTIC WINDOW FOR TREATMENT OF DEEP VENOUS

THROMBOSIS

[0266] To define a therapeutic index for SE-TAP, LMWH, and rivaroxaban, the present inventors investigated the dose-dependent inhibition of IVC thrombosis in relation to tail transection bleeding time. Agents were administered 4 h prior to and 24 h after injury with thrombus weight assessed 48 h after injury. Circulating anti-FXa activity confirmed peak anti-FXa activity 4 h post-administration. Dose relationship studies were initiated to identify maximum achievable thrombus inhibition with the reference therapeutics, LMWH (SC) or rivaroxaban (PO). For both agents, a maximum inhibition of 60% was observed, with higher dosing regimens limited by increased bleeding risk. 60% thrombus inhibition was achieved at 1.5 mg/g SE-TAP (SC) with a corresponding 1.71 ± 0.17-fold increase in bleeding time (Figure 6C). Equivalent inhibition with LMWH (6 mg/g SC) or rivaroxaban (1.5 mg/g PO) resulted in a 3.16 ± 0.31-fold and 2.98 ± 0.33-fold increase in bleeding time, respectively (Figure 7, A and B). For antithrombotic agents, a smaller dose response slope of thrombus inhibition related to bleeding time is favorable because it offers reduced risk of excessive bleeding. SE-TAP administration demonstrated a dose response slope of 6.11 while the slopes for LMWH and rivaroxaban were 10.47 and 10.05, respectively. Despite dual pathway inhibition, statistical analysis confirmed a significantly lower dose response slope for SE-TAP as compared to LMWH (p = 0.0098) or rivaroxaban (p = 0.001136) with an efficacy-to- bleeding profile suggesting a greater safety margin for SE-TAP treatment of venous thrombosis (Figure 7D). Following the characterization of percent thrombus inhibition and bleeding time for SE-TAP doses ranging between 0.5 and 1.5 mg/g, an equimolar dose escalation study was performed of the single chain antibody alone (SE) and non-targeted TAP (MUT-TAP). Limited impact on bleeding time was observed for both agents. Significantly, these results substantiate the independent contribution of both SE and TAP towards inhibition of venous thrombus formation with maximum potency observed with

administration of the SE-TAP fusion construct (Figure 7C).

Discussion of Examples 2-5

[0267] The risk of recurrent coronary or cerebrovascular ischemia or venous thromboembolism remains unacceptably high, but strategies that utilize combined anti- platelet and anti-coagulant therapy for these or other conditions carry a substantial risk of major bleeding with incomplete protection of thrombotic risk. In Examples 2-5, the present inventors disclose a strategy to target activated platelets and enrich anti-platelet and anti coagulant agents at the site of thrombus formation, but after initial platelet adhesion has been established. The present inventors have engineered a dual pathway inhibitor, SE-TAP, which consists of a single-chain antibody, SE, that targets and blocks the activated

GPIIb/IIIa complex and TAP, a potent direct inhibitor of coagulation factor Xa. SE-TAP, administered through intravenous or subcutaneous routes, demonstrated selective platelet targeting and inhibition of thrombosis in murine models of both carotid artery and inferior vena cava thrombosis, without significant impact on hemostasis. Clinical effectiveness following subcutaneous administration represents a significant advantage.

[0268] Despite advances in antithrombotic therapy, recurrent thromboembolic events involving the arterial and venous circulation remain unacceptably high. At the same time, current clinical strategies that combines antiplatelet and anticoagulant therapy to achieve increased anti-thrombotic efficacy carries a substantial risk of major bleeding with incomplete protection of thrombotic risk. Selective targeting to activated platelets provides a means to enrich anti-platelet and anti-coagulant drug activity at the site of thrombus formation, but after the initial sealing platelet layer has been established and at low systemic concentrations that do not impair hemostasis. This approach represents an attractive strategy to potentially break the link between high antithrombotic efficacy and bleeding complications.

Methods for Examples 2-5

Cloning, expression, and purification

[0269] The SE scFv was prepared comprising the amino acid sequence set forth in SEQ ID NO: 11. A non-binding control scFv, MUT, was generated through alanine substitution mutagenesis of the scFv heavy-chain complexity determining region (CDR3) mutation (RND to AND), effectively eliminating GPIIb/IIIa binding (Schwarz et al., 2006. Cic Res. 99(1) :25-33. TAP was designed according to published sequence information (Waxman et al., 1990, supra ) with inclusion of restriction sites Bglll and Xbal for ligation into pHOG21. To generate fusion constructs, TAP was cloned in frame to the C-terminus of SE or MUT in pHOG21 to generate SE-TAP and MUT-TAP. Both SE and MUT were subcloned from pHOG21 into a Drosophila expression vector pMT/BiP/V5-His (Invitrogen), using restriction sites Ncol and Apal. Constructs were housed in E. coli BL21. All recombinant constructs were expressed in Drosophila melanogaster Schneider 2 (S2) cells using the expression vector pMT/BiP/V5- His with a copper-inducible metallothionein promoter for expression, a signal sequence for secretion, and a 6x His tag for purification. S2 cells were cultured in Express Five® media to a density of 1 x 10⁶ cells/mL with a viability greater than 95%. Transfection was performed with 400 ng/mL of DNA and 100 mg/mL of dimethyldioctadecylammonium bromide (DDAB, Sigma). The DNA/DDAB mixture was incubated 20 min prior to addition to S2 culture. At day 4 post-transfection, cells were supplemented with 500 mM copper sulfate to induce expression. Three days later, cells were centrifuged (8000 g, 15 min, 4°C) and supernatant was collected for protein purification. Chelating Sepharose chromatography (20 mL bed volume, 5 mL/min flow rate, GE Healthcare) was used to collect the copper sulfate with the attached recombinant fusion molecule from the supernatant )Lehr et al., Protein Expr Purif. 19(3):362-368. The column was washed to baseline with PBS, 0.5 M NaCI and 10 mM imidazole in 50 mM Tris, pH 8.0 to remove non-specifically bound proteins. Elution was carried out with 50 mM imidazole in 50 mM Tris, pH 8.0. Fractions were collected and those containing significant amounts of product were pooled and dialyzed against PBS. Proteins were re- purified using nickel-based affinity chromatography (BioLogic DuoFlow FPLC). As a final polishing step, size exclusion chromatography was performed using a HiPrep 16/60

Sephacryl S-200 HR column (GE Healthcare) with a flow rate of 1 mL/min with PBS as eluent. Purified constructs were characterized via SDS-PAGE with Coomassie blue total protein stain and Western blot was performed with horseradish peroxidase-conjugated His Tag monoclonal antibody (PentaHis, Qiagen). Final yield was approximately 3 mg/L.

Platelet preparation

[0270] Mouse blood was collected in citrate via cardiac puncture under the approval of the Beth Israel Deaconess Medical Center (BIDMC) Animal Care and Use

Committee. Normal human blood draws were collected in citrate under approval of the

BIDMC Institutional Review Board. Platelet-rich plasma (PRP) was isolated by centrifugation at 300 g at room temperature for 10 min. Washed platelets were prepared by diluting PRP (1 :20) in citrate wash buffer (11 mM glucose, 128 mM NaCI, 4.3 mM NaH₂PO₄ , 7.5 mM Na₂HPO₄ , 4.8 mM sodium citrate, 2.4 mM citric acid, 0.35% w/v bovine serum albumin (BSA), pH 6.5). Samples were centrifuged at 1,200 g for 5 min and washed in citrate wash buffer. The washed platelets were re-suspended in modified Tyrode's modified buffer (134 mM NaCI, 0.34 mM Na₂HPO₄ , 2.9 mM KCI, 12 mM NaHCO₃, 20mM HEPES, 5 mM glucose, 0.35% w/v BSA, pH 7.0) to obtain a final concentration of 100,000 platelets/mL and 1 mM CaCI₂.

Platelet flow cvtometrv and aaareaometrv

[0271] Flow cytometry was performed on the LSR II (BD) or the FACS Calibur (BD) with 50 mL PRP diluted 1 :20 in modified Tyrode's buffer. scFv (10 mg/mL) targeting was characterized to resting platelets and to platelets activated with 20 mM ADP, 5 mg/mL CRP, or 30 mmol/L TRAP. Secondary staining was performed using 10 mL/mL Alexa-Fluor 488 (AF488) anti-His tag antibody (PentaHis, Qiagen) directed against the His-tag of the scFv. In some assays, platelet activation was confirmed by PAC1-FITC (BD Biosciences 340507) or CD62P- PE (BD Biosciences 555524) staining. Fibrinogen binding to activated platelets in the presence or absence of an scFv was determined with a polyclonal FITC-labeled rabbit anti- fibrinogen antibody (Emfret, catalogue P140-1). Platelets were activated with 20 mM ADP in the presence of 3 mg/mL anti-fibrinogen antibody and SE, SE-TAP, or MUT-TAP added at a concentration of 15 mg/mL. Fibrinogen binding was recorded as mean fluorescence intensity (MFI) and data reported as percent inhibition relative to maximum binding observed to 20 mM ADP-activated platelets in buffer control samples. Where indicated, fibrinogen binding was also characterized using Alexa Fluor 488-labeled fibrinogen (Molecular Probes F13191), as previously reported (Wang et al., 2014. Circ Res. 114(7) : 1083-1093. Data were analyzed using Flowjo software and all experiments were performed in triplicate. [0272] Light transmission aggregometry was performed as previously reported (58) with 100 mL PRP, incubated with SE, SE-TAP, or MUT-TAP and then activated with 10 mM ADP. Platelet poor plasma (PPP) was obtained after centrifugation of PRP (1000 x g, 10 min). Samples were measured for 10 min.

FXa activity assay

[0273] Purified TAP fusion constructs were characterized for their ability to inhibit FXa in solution using chromogenic substrate Spectrozyme^®FXa (Sekisui Diagnostics). Briefly, 165 mL of recombinant construct (100 nM) was incubated with 10 mL of 500 pM FXa and 10 mL of 5 mM Spectrozyme^®FXa in 50 mM Tris buffer, at pH 8.4 and 37°C for 10 min.

Reactions were stopped with the addition of 20% acetic acid to a final concentration of 5% and absorbance was measured at 405 nm. Absorbance values generated in vehicle control were considered to have maximum FXa activity and results presented as percent inhibition of this activity. Anti-FXa activity was also assessed after targeting constructs to fibrinogen- adherent platelets. Microwell plates were coated overnight with 100 mg/mL of purified fibrinogen (Sigma-Aldrich), blocked with 1% BSA, and gently washed three times with modified Tyrode's buffer. Citrate washed platelets were resuspended in modified Tyrode's buffer containing 1 mM CaCl₂ and incubated for 30 min on fibrinogen-coated wells. SE, SE- TAP, or MUT-TAP (0.2 mg/mL) were added to fibrinogen-adherent platelets and incubated for 30 min. Following incubation, supernatant was removed and surfaces were gently washed with modified Tyrode's buffer. FXa activity was measured using Spectrozyme^®FXa, as described above. Absorbance values generated with vehicle control were considered to have maximum FXa activity and results presented as percent inhibition of this activity. Circulating anti-FXa activity, including peak circulating anti-FXa activity, was measured in mouse plasma using the Actichrome^® Heparin Anti-FXa kit (Sekisui Diagnostics). A standard curve was generated with the low molecular weight heparin (LMWH), Lovenox (enoxaparin sodium injection, Sanofi), with defined anti-FXa units of activity. Results were reported as anti-FXa units (U) of activity/mL.

Platelet adhesion to collagen under flow

[0274] Flow-chamber adhesion assays were performed in a rectangular glass capillary (10 mm length x 2 mm width x 0.2 mm height) flow chamber (Vitrotubes, New

Jersey, USA). The glass capillaries were coated with collagen fibers by overnight incubation in a collagen (100 mg/mL) solution (Takeda, Austria), followed by a blocking step in 1% BSA. Whole blood was collected in sodium citrate and recalcified. A total of 5 or 15 mg/mL of SE, SE-TAP, MUT-TAP, or PBS control was added to calcified whole blood, followed by perfusion through collagen coated glass capillaries to form microthrombi at a shear-rate of 500 s^-1 (5 dyn/cm²) for 5 min (PHD 2000, Harvard Apparatus). PBS was then perfused through the glass capillaries for 10 min to remove unbound cells and microthrombi were visualized with bright field microscopy (20X objective) using the IX81 Olympus microscope (Olympus, USA) and Cell^R 1692 software. Images were analyzed with Image J and results reported as percent area covered by platelets. Ferric chloride-induced carotid artery occlusion

[0275] C57BL/6 mice (22 - 25 grams) were anesthetized with intraperitoneal administration of ketamine (100 mg/kg) and xylazine (5 mg/kg) and placed under a dissecting microscope. The left common carotid artery was dissected from connective tissue. Experimental reagents were administered intravenously (100 mL, IV jugular) 5 min prior to initiation of thrombosis, including saline vehicle control, SE (0.3 mg/g), MUT-TAP (0.03 and 0.3 mg/g), SE-TAP (0.03 and 0.3 mg/g), LMWH (10 mg/g), and eptifibatide (10 mg/g). To induce thrombosis, filter paper (1 × 2 mm), saturated with 10% ferric chloride, was placed under the left common carotid artery for 3 min. Following a 3 min exposure period, the area was flushed with saline and a nano Doppler-flow probe (0.5 VB, Transonic) was positioned to record flow (T106, Transonic). Flow speed was recorded for up to 30 min and occlusion was defined as a decrease in flow to 0.0 ± 0.2 mL/min. To assess bleeding risk, agents were administered (100 mL, IV jugular) 1 min prior to tail transection for determination of bleeding time and bleeding volume measurements. A detailed protocol for tail transection and bleeding risk assessment is described below. Animal care followed national guidelines with procedures approved by the Alfred Medical Research and Education Precinct - Baker Heart and Diabetes Institute.

In vivo imaging

[0276] SE-TAP and MUT-TAP were labeled with NHS-IR800 dye (1 : 5 molar ratio, Pierce) for 1 h at room temperature, according to manufacturer's instructions, and purified over a desalting column. Agent administration and initiation of carotid artery thrombosis were performed as described above. Animals were imaged 2 hours after the administration of the labeled agent (1 mg/g, IV) using an IVIS® Lumina Series II Imaging System (Caliper Life Sciences). Fluorescent images were obtained by a charge-coupled device (CCD) camera using the XFO-12 fluorescence equipment (excitation filter 710 - 760 nm, emission filter 810 - 875 nm) on automated exposure time. Photographic pictures were also obtained during illumination. Overlays of fluorescence and photographic images, as well as processing and analysis were performed using Living Image 4.4 software.

Electrolytic inferior vena cava model

[0277] The electrolytic inferior vena cava model (EIM) was used to determine the effectiveness of test agents to prevent non-occlusive venous thrombosis, as previously described (33, 59). Saline, SE-TAP (0.5-1.5 mg/g SC), LMWH (2-6 mg/g SC), or rivaroxaban (0.5-1.5 mg/g PO) were administrated 4 h prior to electrolytic injury and 24 h post-injury. C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA), weighing 22 to 25 grams, were anesthetized with 2% isoflurane and the IVC exposed via a midline laparotomy. Venous side branches were either ligated using 7-0 Prolene suture (Ethicon, Inc, Somerville, NJ) or cauterized using Change-a-tip^® (Bovie medical, Clearwater, FL), while posterior branches were left patent. A 25-gauge stainless-steel needle, attached to a silver coated copper wire (KY-30-1-GRN, Electrospec, Dover, NJ, USA) was inserted into the exposed caudal IVC and positioned against the anterior wall (anode). A second wire was implanted subcutaneously to complete the circuit (cathode). A current of 250 mAmps over 15 min was applied using a

Grass S48 square wave stimulator and a constant current unit (Grass Technologies, Astro- Med, Inc., West Warwick, RI, USA). In sham animals, the needle was placed into the IVC for 15 min without application of current. After 15 min, the needle was removed and a cotton swab was placed in contact with the puncture site to prevent bleeding. The laparotomy was closed with 5-0 vicryl suture (Ethicon, Inc, Somerville, NJ) and skin approximated using a tissue adhesive glue (Vetbond, 3M, Maplewood, MN). Forty-eight hours after injury, the IVC was excised from below the renal veins to just above the bifurcation to determine the wet thrombus weight (TW), as well as to characterize the thrombus using flow cytometry and histology. The IVC was imaged using a stereoscope Zeiss Axio Zoom V16 with 8x

magnification. All procedures were approved by the BIDMC Animal Care and Use Committee.

Ex vivo thrombus flow cvtometrv

[0278] The IVC and thrombus were finely minced and shaken for 60 min at 37°C in 1 mL of RPMI-1640 supplemented with 10% FBS, 62.5 U/mL collagenase VII (Sigma), and 0.625 U/mL Dispase (BD Bioscience). Isolated cells were passed through a 70-micron cell strainer to remove debris. Collected cells were separated by centrifugation (300 x g for 10 min) at room temperature. The supernatant was removed and erythrocytes were lysed in 9 mL of DI-water for 7 seconds followed by 1 mL of 10x PBS. Cells were counted and stained according to standard protocol. Antibodies included APC rat anti-mouse CD45 (Clone 30-F11, BD Bioscience), Pacific Blue rat anti-mouse CD11b (Clone M1/70.15, eBioscience), PE rat anti-mouse Ly6G (Clone 1A8, BD Bioscience), and PE rat anti-mouse CD41 (Clone MWReg30, BD Bioscience). Isotype IgG was included as a negative control for each marker. Cell suspensions were analyzed using a BD LSRII four-laser benchtop analyzer.

Bleeding time and blood loss measurement

[0279] Animals were anesthetized with intraperitoneal administration of ketamine HCI (125 mg/kg) and xylazine (12.5 mg/kg). Bleeding time and volume were assessed by transection of the tail, 10 mm from its end, using a blade (60). The tail was immediately immersed in a 50 mL Falcon tube containing warm saline (37°C). The tail was positioned vertically with the tip 2 cm below the body horizon. The time at which the tail first stopped bleeding for 30 seconds was recorded as bleeding time. Blood loss was determined by measuring hemoglobin concentration after erythrocyte lysis. Collected blood cells were separated by centrifugation (300 g ) for 10 min at room temperature. The supernatant was removed and erythrocytes lysed in 9 mL of DI-water for 7 seconds followed by the addition of 1 mL of 10x PBS. The concentration of hemoglobin was determined spectrophotometrically by measuring absorbance at 550 nm. Blood volume was calculated using a hemoglobin standard curve. Measurements were performed to correlate with agent administration in the ferric chloride-induced carotid artery occlusion model, 1 min after IV administration, and in the electrolytic inferior vena cava model, 4 h after SC or PO administration, as indicated.

Immunohistochemistrv

[0280] Specimens were fixed overnight in 10% neutral buffered formalin, processed for paraffin embedding, and 5 pm sections stained with hematoxylin and eosin (H8iE). Additional staining was performed for platelets (rabbit anti-mouse CD41, clone

MWReg30, AbCam), neutrophils (Naphthol AS-D Chloroacetate Esterase, Sigma Aldrich), and monocytes (rabbit anti-mouse CD68, AbCam, catalogue abl25212). Images were taken from 3 to 5 representative sections from each group (n = 4) using Olympus BX41 at lOx and 40x magnification. To characterize thrombus area, the IVC (n = 5) was sectioned 48 hour after injury, stained with H8iE, and examined under light microscopy (Olympus BX41). The area of the venous thrombus was analyzed using Image J 1.46r (NIH, Bethesda, MD) from H8iE stained specimens (3 sections/mouse) at the point of greatest luminal stenosis. To eliminate variability between groups, thrombus area was normalized to aortic wall thickness and reported as thrombus area (mm²)/aorta wall thickness (mm) (Diaz et al ,013. Thrombosis and Haemostasis 109(6) : 1158-1169.

Intravital microscopy

[0281] Surgical preparation of the mouse cremaster muscle microcirculation for intravital microscopy was performed as previously described (61, 62). Briefly, C57BL/6 mice were anesthetized with intraperitoneal administration of ketamine HCI (125 mg/kg), xylazine (12.5 mg/kg), and atropine (0.25 mg/mL) and placed on a 37°C surgical blanket. The jugular vein was cannulated with PE 10 tubing to allow introduction of reagents, including anti-CD42b-Dylight 649 (Emfret Analytics, catalogue M040-3), SE-TAP, MUT-TAP, LMWH, or saline vehicle control. In selected studies, therapeutics were delivered subcutaneously (SC) 4 h or 24 h prior to cremaster exteriorization and laser injury, as indicated. For colocalization studies, SE-TAP and MUT-TAP were pre-incubated (10 mg/mL, 10 min) with penta-His Alexa Fluor 488 Conjugate (Qiagen, catalogue 35310). The cremaster muscle was exteriorized, pinned to the stage, and superfused with therma-controlled bicarbonate buffered saline equilibrated with 5% CO₂ in N₂. Between 5 and 30 min after infusion of the platelet label (anti-CD42b-Dylight 649), injury to a cremaster arteriolar or venular vessel wall was induced with a Micropoint Laser System (Photonic Instruments) focused through the microscope objective parfocal with the focal point and tuned to 440 nm. Microvessel data were obtained using an Olympus AX microscope with a 60x water immersion objective recorded with a Hamamatsu C11440 Digital Camera. Image acquisition and data analysis were performed using SlideBook software (Intelligent Imaging Innovations). For each treatment group, platelet accumulation was characterized as median integrated fluorescence plotted over 3 min from thrombi (n = 25-35) generated in 4 to 5 mice. In addition, platelet signals were quantified as area under the curve for each individual thrombus plotted against time (Falati et al., 2002. Nat Med. 8(10) : 1175-1180. All experiments were performed in the BIDMC Center for Hemostasis and Thrombosis Research Core. All procedures were approved by the BIDMC Animal Care and Use Committee.

Statistics

[0282] Data are presented as mean ± SD for the indicated experimental groups. Statistical comparisons were performed with one-way ANOVA and post hoc testing performed using Bonferroni's modification of Student's t-test for multiple comparisons or two-tailed t- test for paired comparisons using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA). Significance was indicated as *p £ 0.05, **p £ 0.01, and ***p £ 0.001. Study approval

[0283] Human blood samples were obtained from healthy volunteers, who provided written informed consent. Studies were approved the by the BIDMC Institutional Review Board. All animal experiments were approved by the Animal Care and Use Committee of BIDMC and The Alfred Medical Research and Education Precinct - Baker Heart and

Diabetes Institute. All investigators adhered to NIH guidelines for the care and use of laboratory animals.

EXAMPLE 6

CLONING AND PURIFICATION OF SCFV-SCUPA CONSTRUCTS

[0284] Two different scFvs (activated GPIIb/IIIa-targeted SCFV_SE and non- targeted scFv_mut, which is also referred to herein as mutated scFv (MUT), were fused with active scuPA to produce scFv_SE-SCUPA (comprising the amino acid sequence set forth in SEQ ID NO: 56) and scFv_mut-scuPA and cloned into the pSectag2A vector system. Briefly, both scFv-scuPA plasmid constructs were then transformed into BL21 Star E. coli cells (Invitrogen, USA) and produced using mammalian cells via the human embryonic kidney cells (H293F) suspension culture. All scFv-scuPAs contain a 6x His-tag, which was used for purification with nickel-based metal affinity chromatography (Invitrogen, USA).

[0285] The success of DNA amplification and restriction digest of scFv-scuPA fragments was evaluated by electrophoresis. Both constructs were visualized between the 1.5kbp and 2kbp marker after amplification with PCR and restriction digest. The pSectag2A plasmid was visualized at around 5kbps after single cut restriction digest. After the respective constructs were cloned into the pSectag2A plasmid, transformed and purified, they were analyzed by gel electrophoresis. The sequences of both fusion constructs were confirmed via DNA sequencing. After production of the scFvs, SDS-PAGE and Western blot were used to prove successful purification. Western blot was also used to demonstrate digestion of scuPA after the addition of plasmin.

EXAMPLE 7

BINDING OF TARGETED SCUPA TO CHO CELLS IN VITRO

[0286] The specificity of scFv_SE-SCUPA (comprising the amino acid sequence set forth in SEQ ID NO: 56) was also observed under static adhesion conditions. Direct fluorescence staining using anti-His-488 demonstrated binding of scFv_SE-SCUPA to the activated GPIIb/IIIa expressing CHO cells but not on either non-expressing or non-activated GPIIb/IIIa expressing CHO cells. No fluorescence staining was observed for all three cell types with scFv_mut-scuPA (Figure 8). In flow cytometry, incubation of scFv_mut-scuPA with the CHO cells resulted in an increase in fluorescence intensity for samples with activated GPIIb/IIIa expressing CHO cells but not on either non-expressing or non-activated GPIIb/IIIa expressing CHO cells. EXAMPLE 8

EVALUATION OF THE FUNCTIONALITY OF SCFV-SCUPA BY FLOW CYTOMETRY

[0287] To confirm retained binding capacity of the scFv to ADP-activated platelets, the functionality of scFv-scuPA was evaluated with anti-His-488 (Figure 9). No binding was observed for control non-targeting scFv_mut to neither activated nor non-activated platelets, (2.69 ± 0.18 vs 2.84 ± 0.25 arbitrary units [AU] ; mean ± SD; ns). Incubation of activated platelets with SCFV_SE resulted in an increase in fluorescence intensity as compared to non-activated platelets (2.69±0.16 vs 23.88±8.22 AU; mean ± SD; p<0.01). Competitive assays were performed using FITC-labeled fibrinogen to demonstrate the binding of scFv_SE- scuPA. After incubation with scFv_SE-SCUPA fibrinogen was not able to bind to activated platelets anymore. However after incubation with scFv_mut-scuPA, fibrinogen binding was not inhibited (57.11±5.82 vs 75.54±23.2 vs 4.67±2.92 AU; p<0.001). Competitive assays using PAC1 showed similar results as with FITC-labeled fibrinogen (39.03±6.5 vs 41.74±6.45 vs 3.03±0.21 AU; p<0.01). Binding of scFv_SE was also confirmed with CRP and TRAP activated platelets. Platelet activation by these platelet agonists was demonstrated using PAC1 and anti-CD62P fluorescence staining. Specificity of scFv_mut-scuPA binding to activated GPIIb/IIIa on activated platelets was demonstrated via competition with abciximab. Specificity of scFv_mut-scuPA towards the fibrinogen binding sites on activated GPIIb/IIIa was demonstrated by decreased binding of fibrinogen upon increased concentrations of scFv_mut-scuPA in flow cytometry.

EXAMPLE 9

IN VITRO EVALUATION OF SCFV-SCUPA ACTIVITY IN PLATELET AGGREGATION

[0288] Light transmission aggregometry in a 96-well plate assay was performed to determine the ability of the recombinant fusion proteins to inhibit platelet aggregation. High concentrations of the SCFV_SE alone (5 mg/mL and 10 mg/mL) and the equimolar amounts of scFv_SE-SCUPA (10 mg/mL and 20 mg/mL) demonstrated a strong inhibition of ADP-induced platelet aggregation as opposed to scFv_mut-scuPA, which showed no inhibitory effect (Figure 10A; n=4; p<0.001). At lower concentrations, SCFV_SE alone (0.1 mg/mL and 1 mg/mL) and the equi-molar amounts of scFv_SE-SCUPA (0.2 mg/mL and 2 mg/mL) would not inhibit platelet aggregation. Platelet aggregation also was not inhibited with 100U or 200U of commercial uPA (p<0.001). Similar results were obtained when 200 mM of amiloride was used to block the function of scuPA (Figure 10B), demonstrating that urokinase has no effect on thrombus formation in this assay.

EXAMPLE 10

IN VITRO EVALUATION OF THE UROKINASE ACTIVITY OF SCFV-SCUPA

[0289] Urokinase activity was monitored by incubating scFv-scuPA with urokinase substrate S2444L in comparison to commercial uPA. Both scFv-scuPAs and standards using commercial uPA at different concentrations resulted in linear enzymatic activity over 60 min. EXAMPLE 11

IN VITRO EVALUATION FOR THE CONVERSION OF PLASMINOGEN TO PLASMIN USING SCFV-SCUPA

[0290] Conversion of plasminogen to plasmin was monitored using the S2251 amidolytic assay. Both scFv-scuPA versions and the commercial uPA at 10 nmol/L generated plasmin activity. uPA-dependent plasmin generation was blocked in the presence of 200 mM of the urokinase inhibitor, amiloride. SDS-PAGE fibrin zymography was also performed to demonstrate the direct digestion of fibrin. Commercial uPA produced a lytic zone as expected at around 55kD. SCFV_SE-SCUPA and ScFv_mut-scuPA produced a lytic zone at around 70kD.

EXAMPLE 12

BINDING TO ACTIVATED PLATELETS AND FIBRIN DEGRADATION WITH TARGETED SCUPA TO

MICROTHROMBI IN VITRO

[0291] Targeting of the scFv_SE-SCUPA was determined by binding performance in vitro in a flow chamber adhesion experiment with microthrombi. Fluorescence staining using anti-His-488 demonstrated binding of scFv_SE-SCUPA but not with scFv_mut-scuPA (Figure 11A). Fibrin degradation was observed when scFv_SE-SCUPA and a high dose of commercial uPA was used but not with scFv_mut-scuPA (Figure 11B). Using 2 mg/mL of SCFV_SE-SCUPA, fibrin degradation was observed specifically around the platelet aggregates.

EXAMPLE 13

EVALUATION OF SCFV-SCUPA BINDING TO THROMBI IN VIVO

[0292] Binding of scFv-scuPA was determined by intravital microscopy in a ferric chloride-induced thrombosis model in the mesenteric arterioles of mice. Binding of scFv_SE- scuPA conjugated with the fluorescent dye Cy3 to developing thrombi could be

demonstrated, while no fluorescence was observed with scFv_mut-scuPA (Figure 11C).

EXAMPLE 14

IN VIVO EVALUATION OF SCFV-SCUPA FOR PROPHYLACTIC FIBRINOLYSIS

[0293] Thrombi were induced in the carotid artery of mice using 10% ferric chloride for 3 min. Blood flow was measured by a nano Doppler-flow probe and was used as an indicator of an occlusive thrombus (Figure 12). Saline was injected as negative control and 500 U/g of commercial uPA was used as a positive control. The baseline Doppler velocity was set to 100%. At 20 min, the Doppler flow velocities obtained from mice treated with 75 U/g targeted SCFV_SE-SCUPA was significantly higher than those treated with saline, the equimolar concentration of SCFV_SE alone, 75 U/g of non-targeted scFv_mut-scuPA, the combination of SCFV_SE and 75 U/g of non-targeted scFv_mut-scuPA, or 75 U/g of commercial uPA (84.0±9.4 vs 5.4±2.7 vs 23.8± 11.8 vs 45.3± 13.9 vs 38.5± 11.8 vs 21.6± 11.4, respectively, mean %±SEM, p<0.05, n=6). There was no difference observed in groups treated with 75 U/g BW of non-targeted scFv_mut-scuPA, the equimolar concentration of SCFV_SE alone or the combination of both SCFV_SE and 75 U/g BW of non-targeted scFv_mut-scuPA. The Doppler flow velocities obtained from mice treated with 75 U/g targeted SCFV_SE-SCUPA were similar to those treated with 500 U/g of commercial uPA throughout the observation period. Similar results were obtained at 30 min. EXAMPLE 15

IN VIVO ASSESSMENT OF BLEEDING TIME OF SCFV-SCUPA

[0294] Bleeding times were evaluated by surgical tail transection (Figure 13). Commercial uPA at 500 U/g considerably prolonged bleeding compared to vehicle control (saline). In contrast, a lower dose of 75 U/g of SCFV_SE-SCUPA, scFv_mut-scuPA and commercial uPA minimized bleeding time. The lower dose of scFv_SE-SCUPA (75 U/g) had an anticoagulant effect without prolonging bleeding time.

EXAMPLE 16

IN VIVO MOLECULAR ULTRASOUND IMAGING OF THROMBOLYSIS

[0295] Imaging of the mouse carotid artery on ultrasound typically shows luminal blood as black or dark color and microbubbles appear as a bright white color in the lumen. The thrombus was visualized as a white and bright signal after injection with platelet targeted ultrasound contrast (LIBS-MB) on real time ultrasound imaging. The baseline area before injection of uPA was set to 100% and the area was calculated every 5 min for 60 min (Figure 14). The targeting ability of 75 U/g of SCFV_SE-SCUPA is demonstrated by ultrasound imaging as such a reduction in thrombus size was observed following its administration. Treatment with scFv_SE-SCUPA (75 U/g) significantly reduced thrombus size after 60 min, while no significant difference was observed in the scFv_mut-scuPA (75 U/g) treatment group (36.8±4.6 vs 81.1±2.6, mean % ± SEM, p<0.001, n=3) (Figure 14, Video 1 and 2).

Thrombolysis was observed via ultrasound imaging using 500 U/g of commercial uPA. The ability of SCFV_SE-SCUPA to target and dissolve the thrombus was compared against mice injected with a higher dose of commercial uPA. There were no significant differences between groups of mice treated with 500 U/g commercial uPA and those treated with 75 U/g of scFv_SE-SCUPA over a period of 60 min (40.05±9.2 vs 36.8±4.6, ns, n=3). The thrombolytic ability of scFv_SE-SCUPA was also compared with the control group where saline was administrated over a period of 60 min. SCFV_SE-SCUPA caused a reduction in thrombus size at 60 min post administration, compared to control (36.8±4.6 vs 99.2± 1.3, p<0.001, n=3).

This control group was also compared against the non-targeted treatment using 75 U/g of scFv_mut-scuPA. Although post administration of scFv_mut-scuPA showed some reduction in the thrombus area, it was not significantly smaller than those injected with saline (81.1±2.6 vs 99.2± 1.3, ns, n=3).

EXAMPLE 18

IN VIVO ULTRASOUND MOLECULAR IMAGING OF CAROTID ARTERY THROMBOLYSIS ON PLASMINOGEN-

DEFICIENT MICE

[0296] Plg^-/- mice were subjected to ferric chloride induced thrombosis. Mice were then administered either scFv_SE-SCUPA (75 U/g) or scFv_mut-scuPA (75 U/g). Over a 30 min period, there was no change in thrombus size in both groups of animals (96.0±0.1 vs 98.55±0.1, mean % ± SEM, ns, n=3). However, when Plg^-/- mice were then reconstituted with human plasminogen (100 mg/mL, 150 mL bolus at 30 min time-point) mice treated with scFv_SE-SCUPA developed a significant thrombus size reduction, while no significant difference was observed in plg^-/- mice treated with scFv_mut-scuPA (23.1± 1.5 vs 92.1±2.2, mean % ± SEM, p<0.001, n=3) (Figure 15).

Discussion of Examples 6-18

[0297] In Examples 6-18, the present inventors have generated a dual pathway inhibitor SE-scuPA, in which the highly potent anti-coagulant and highly specific targeting SE scFv that binds to the activated platelet integrin receptor GPIIb/IIIa is fused to recombinant scuPA. The data presented herein demonstrate that scuPA delivery to activated platelets allows local enrichment of the fibrinolytic agents at the site of the developing or existing thrombus, thereby increasing fibrinolytic potency without increasing side effects. Through in vitro assays the present inventors provide evidence that both the scFv and the scuPA retain their individual function in the fusion molecule. In vivo evaluation of these targeted fibrinolytics both as a prophylactic and therapeutic agent showed plasminogen-dependent inhibition of thrombus growth as well as reduction in thrombus size via molecular ultrasound imaging. A low dose of 75 U/g of platelet-targeted scuPA was sufficient for localized thrombolysis, which was not achieved using non-targeted scuPA or commercial uPA at the same dose. The same effects were only achieved using urokinase at 500 U/g. Increased bleeding was not observed at the effective dose of the novel targeted scuPA, compared to an equally effective higher dose of non-targeted scuPA, which resulted in a significantly prolonged bleeding time. This antithrombotic effect of the low dose of 75 U/g of platelet- targeted scuPA was more potent than the combination of equimolar non-targeted scuPA and the activation-specific anti-GPIIb/IIIa scFv_SE. This indicates that the superior anti-thrombotic effect of the fusion protein scFv_SE-SCUPA can be attributed to the antibody-targeting of scuPA to activated GPIIb/IIIa on activated platelets and the resulting local enrichment of scuPA at the thrombus.

Methods for Examples 6-18

Evaluation of the scFv-scuPA constructs

[0298] The purity of the proteins was analyzed using SDS-PAGE and Western blotting. Anti-6x His-tag® antibody horseradish peroxidase was used to detect the purified scFv-scuPA constructs. The scuPA in the fusion protein was converted to the active form using plasmin to cleave the Lys-158 to lie- 159 bond.

Static adhesion assay

[0299] The specificity of the scFvSE targeting activated GPIIb/IIIa was demonstrated using Chinese Hamster Ovary (CHO) cells that were either expressing activated GPIIb/IIIa integrin, non-activated GPIIb/IIIa or not expressing the GPIIb/IIIa integrin. Cells were grown to confluency in 6-well plates (BD Bioscience, USA), incubated with purified scFv-scuPAs, followed by anti-Penta-His AlexaFluor 488-conjugated monoclonal antibody (anti-His-488; Qiagen, Germany). The cells were visualized with the IX81 Olympus microscope (Olympus, Japan) and Cell^P 1692 (ANALysis Image Processing) software. Flow cvtometrv

[0300] Platelet rich plasma (PRP) was obtained by centrifugation of blood was collected from healthy volunteers. Diluted PRP was either not activated or activated with 20mM adenosine diphosphate (ADP), 5 mg/mL collagen-related peptide (CRP) or 30 mM thrombin receptor activating peptide (TRAP) before incubation with the purified scFv constructs, followed by anti-His-488 for detection. Activity of platelets was determined by FITC-labeled fibrinogen, PAC1-FITC and CD62P-PE. The specificity of scFvSE targeting activated platelets was analyzed using FITC-labeled fibrinogen and PAC1-FITC. Competitive assays were performed using abciximab (ReoPro®) and FITC-labeled fibrinogen. Samples were fixed with 1× Cellfix (BD Bioscience, USA) and analyzed by FACS Calibur (BD

Bioscience, USA). In addition, CHO cells were also used for flow cytometry.

Urokinase activity assay

[0301] Urokinase activity was determined with a chromogenic substrate assay. Comparison between clinically used uPA (Medac GmbH, Germany) and scFv-scuPA was made on the basis of equal urokinase activity. lOOnmol/L of scFv-scuPA was monitored against urokinase standards (0-100 U/mL) used as positive controls. Plasmin was added to activate the scuPA. S2444 (Chromogenix, Italy) was added and samples were measured on a Victor3V Multi-label counter (PerkinElmer, USA) at a wavelength of 405 nm every 5 min over a period of 60 min.

Plasmin activity assay

[0302] The conversion of plasminogen to plasmin using commercial uPA or the two scFv-scuPA versions was determined in microtiter plates using a chromogenic substrate. 10 nmol/L of commercial uPA and scFv-scuPAs were incubated with 400 nmol/L of human glu-plasminogen (Sigma-Aldrich, Germany) and 1 mmol/L of S2251 (Chromogenix, Italy). Samples were measured using the Bio-Rad Benchmark Plus (Bio-Rad, USA) at a wavelength of 405nm every 30 seconds over a period of 60 min.

Fibrin zvmoaraphv

[0303] SDS-PAGE-based fibrin zymography (Granelli-Piperno et al. , Exp Med. 1978; 148: 223-234) was performed to evaluate plasminogen dependent fibrinolytic activity of the targeted and non-targeted scFv-scuPA. Briefly, the commercial uPA and scuPA were subjected to SDS-PAGE. After electrophoresis, gels were washed in 2.5% Triton X-100 for 1.5 hour, then placed on top of a fibrin/agarose: plasminogen matrix. The washed SDS-PAGE gel was then overlaid onto the exposed agarose gel and incubated in a humidified 37°C oven until lytic zones were evident. Images were captured at various incubation times using a flatbed document scanner.

Light transmission aaareaometrv

[0304] 96-well plate light transmission aggregometry was performed using 100 mL of PRP. PRP were incubated with abciximab, scFv-scuPA, scFvSE alone or commercial uPA, then activated with 10 mM ADP. Platelet poor plasma (PPP) was obtained by

centrifugation of blood at 1000xg for 10 min at room temperature. Light transmission was adjusted to 0% with PRP and 100% with PPP. In order to differentiate the effects of scFv from those of urokinase, 200 mM of the urokinase blocker amiloride (Vassalli et al., 1987. FEBS Lett. 214: 187-191) was added. Light transmission aggregometry was measured using the Bio-Rad Benchmark Plus at wavelength 595nm. Samples were measured every 30 seconds for 60 min.

Flow chamber adhesion assay

[0305] Flow chamber in vitro adhesion assays were performed with glass capillaries or microfluidics flow channels, which were coated overnight with collagen. Whole blood was perfused through the capillaries or channels to form microthrombi. Binding of scFv-scuPAs were observed via staining with anti-His-488. Fibrin degradation was demonstrated using Oregon-Green Fibrinogen (Invitrogen, USA). The microthrombi were visualized with the IX81 Olympus microscope and Cell^P 1692 software, using bright field, DIC and fluorescence imaging.

In vivo mouse experiments

[0306] Male C57BL/6 mice and plasminogen knockout mice (plg-/- mice, Jackson Laboratories, USA) were maintained at the Alfred Medical Research and Education Precinct Animal Services and assigned randomly to the different groups. The amount of the targeted and non-targeted scFv-scuPA for injection was calculated according to units per gram (U/g) body weight (BW) of the animals. The animals were anaesthetized, shaved and placed on a 37°C heater mat to prevent hypothermia. All experiments involving animals were approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee

(E/ 1160/2011/B) .

Femoral vein catheterization and ferric chloride injury model for Doppler flow velocity measurement

[0307] A catheter was placed into the femoral vein to facilitate injection. A small filter paper saturated with 10% ferric chloride was placed under the carotid artery of the animal for 3 min to induce an occlusive thrombus (Stoll et al., 2007. Arteriosder Thromb Vase Biol. 27: 1206-1212). Animals were injected with either commercial uPA, targeted scuPA (scFvSE-scuPA), non-targeted scuPA (scFvmut-scuPA), scFvSE alone or saline as vehicle control 1 min before the injury. The nano-Doppler flow-probe (0.5VB, Transonic, Japan) was placed under the carotid artery post injury to measure the thrombotic occlusion.

Intravital microscopy of the mesenteric arterioles in mice

[0308] Intravital microscopy was performed as previously described (Hohmann et al., 2013. Blood 121 :3067-3075). Briefly, the mesentery was exteriorized through a midline abdominal incision. 6% ferric chloride was used to induce thrombus formation on the mesenteric arterioles. Binding of the scFv-scuPAs conjugated with Cy3 fluorescence dye (Lumiprobe, USA) was monitored using the fluorescence channel on the Nikon Air confocal microscope (Nikon, Japan). Assessment of tail bleeding time

[0309] An incision to reveal the left jugular vein was made in order to insert a catheter to facilitate injections. 1 min after injecting commercial uPA, scFv-scuPAs or vehicle, the tail was transected 5mm from the tip and immediately submersed in saline at 37°C. The bleeding time was monitored and recorded as the time needed for the cessation of visible blood stream, for 1 min.

In vivo ultrasound molecular imaging of carotid artery thrombolysis

[0310] Ultrasound of animals was performed with a Vevo770 high-resolution imaging system (VisualSonics Inc. Canada) using a 40 MHz RMV704 transducer. Animals were placed on the imaging station after 6% ferric chloride injury was performed to the left carotid artery. Videos and images were acquired before, during and at several time points after injecting 1.5×10⁷ targeted microbubbles (LIBS-MBs) specific for activated platelets (targeting the ligand induced binding site on activated GPIIb/IIIa) in a total volume of 100 mL. The present inventors have established this ultrasound imaging methodology for the assessment of thrombosis and thrombolysis (Wang et al., 2012. Circulation. 125:3117- 3126). 500 U/g of urokinase plasminogen activator (uPA) (Medac, Germany), 75U/g of scFv- scuPA or saline as vehicle control were injected into the animals. Repetitive ultrasound imaging sequences were performed every 5 min for an hour after thrombolysis. Analysis was performed using a linear contrast agent imaging software (VisualSonics Inc.).

In vivo ultrasound molecular imaging of carotid artery thrombolysis using plasminogen- deficient (plg-/-) mice

[0311] Plg-/- mice were placed on the VisualSonics imaging station after 6% ferric chloride injury was performed to the left carotid artery. Images were acquired before injection of LIBS-MBs. Thereafter, 75 U/g of scFvSE-scuPA or scFvmut-scuPA was injected. Repetitive ultrasound imaging sequences were performed every 5 min for 30 min. A 150 mL bolus of 100 mg/mL human plasminogen (Sigma-Aldrich, Germany) was injected at the 30 min time-point and repetitive imaging sequences continued for another 30 min. Analysis was performed using a linear contrast agent imaging software.

Statistical analysis

[0312] Unless otherwise specified, data are expressed as mean ± standard error of the mean (SEM). Flow cytometry, flow chamber and data for thrombolysis were analyzed with two-way ANOVA repeated measures analysis using Bonferroni's multiple-comparison post-test. All analyses containing more than two groups were corrected by post hoc analysis and the corrected p values are given. Statistical analyses were performed using Graphpad Prism 5.0.

EXAMPLE 19

PLATELET TARGETED THROMBOLYSIS PRESERVES MYOCARDIAL FUNCTION FROM CARDIAC ISCHEMIA

REPERFUSION INJURY (IRI)

[0313] To investigate the effects of the SE-scuPA construct (also referred to herein as "Targ-scuPA construct"), the present inventors utilized the temporary LAD occlusion model of cardiac IRI. Briefly, mice underwent ischaemia induction via ligation of the LAD for 60 minutes and were then randomized to receive either 75 units/g body weight of Targ-scuPA or Non-targ-scuPA (scuPA fused with a non-binding scFv) immediately upon reperfusion. Strikingly, analysis of cardiac function using echocardiography 4 weeks post IRI demonstrated that the targ-scuPA treatment group demonstrated the marked preservation of ejection fraction (52%) vs the Non-targ-scuPA (30%) treatment group (Figure 16).

Concurrently, a significant improvement was observed in fractional shortening (11% vs 4%), stroke volume (41 mL vs 26 mL) and cardiac output (21 vs 14 mL/min) in the Targ-scuPA treated mice (Figure 16). In addition, strain analysis was performed since this better reflects myocardial contractility compared to standard echocardiography and is a stronger clinical predictor of mortality than EF. In contrast to Non-targ-scuPA, pathological changes in strain were prevented by treatment with Targ-scuPA, which is consistent with clinical data where patients with early reperfusion had less myocardial deformation and damage compared to those with delayed reperfusion (Figure 17).

EXAMPLE 20

PLATELET TARGETED FIBRINOLYSIS REDUCES INFARCT SIZE POST CARDIAC IRI

[0314] To examine the effects of treatment with Targ-scuPA on the size of myocardial infarction post IRI, and correlate with the echocardiography evaluation, cardiac sections were taken 4 weeks post IRI and stained with TTC. Treatment with Targ-scuPA was associated with an approximately 50% reduction in infarct size and infarct size/area at risk ratio compared to Non-targ-scuPA treated mice (Figure 18).

EXAMPLE 21

TARG-SCUPA TREATMENT REDUCES PLATELET AND FIBRIN DEPOSITION POST CARDIAC IRI

[0315] The present inventors have shown herein that the Targ-scuPA construct dramatically reduces arterial thrombus formation in a ferric chloride model of thrombosis. Accordingly, they postulated that a major mechanism by which the Targ-scuPA could preserve left ventricle (LV) function post IRI was by its ability to inhibit the formation of thrombi in the microcirculation which are well known to contribute to the no reflow phenomenon and exacerbate myocardial injury. To investigate the effects of Targ-scuPA on microthrombi formation, mice were injected before the induction of LAD occlusion with

BV421-CD31 antibody, Alexa 546 anti-fibrin antibody and Dylight 647 GPIb antibody to label endothelial cells, fibrin and platelets respectively. Upon reperfusion, mice were treated with Targ-scuPA or Non-targ-scuPA. After 1 hour of reperfusion, the post ischemic myocardium was harvested and imaged using multiphoton microscopy. Strikingly, the percentage of CD31 surface area with platelets (5.89% vs 23.03%) and fibrin (1.87% vs 11.13%) was markedly reduced in Targ-scuPA treated mice compared to Non-targ-scuPA treatment, thus highlighting the potency of Targ-scuPA at reducing the burden of microthrombi in cardiac IRI (Figure 19). Methods for Examples 19-21

Generation, expression and purification of single-chain antibodies and single-chain urokinase plasminogen activator

[0316] The generation of two different scFvs (activated GPIIb/IIIa-targeted scFvTarg and non-targeted scFvmut), both fused with active scuPA, were cloned into the pSectag2A vector system. Briefly, for both scFv-scuPAs, polymerase chain reaction (PCR) was performed with a sense primer that anneals at the beginning of the scFv sequence and an antisense primer that anneals directly to the 6x His-tag region at the end of the scFv. The sense strand includes the Ascl restriction site and the antisense strand includes the Xhol restriction site. The scFvTarg -scuPA was generated with the following primers: sense strand : 5'- ATC TTA GGC GCG CCA TGG CGG AGG TGC AGC TGG T -3', antisense strand : 5'- GCC CGT CTC GAG TAC CGG TAC GCG TAG AAT CGA GAC C -3'. The scFvmut-scuPA was generated with the following primers: sense strand : 5'- ATC TTA GGC GCG CCA TGG CGG AAG TGC AGC TGG TG -3', antisense strand : 5'- GCC CGT CTC GAG TAC CGG TAC GCG TAG AAT CGA GAC C -3'. After amplification by PCR, the constructs were digested with the restriction enzymes Ascl and Xhol (both NEB, USA), and cloned into pAC6. Electrophoresis on a 0.8% agarose gel with SYBR® Safe DNA gel stain (Invitrogen, USA) was utilized to analyze DNA amplified by PCR and restriction digests. Ligation of the plasmids was performed with T4 ligase (NEB, USA) at 16°C overnight. The resulting plasmid constructs were then transformed into BL21 Star E.coli cells (Invitrogen, USA).

Expression in mammalians cells and purification of scFv-scuPA fusion constructs

[0317] Production of mammalian cells was performed using the human embryonic kidney cells (H293F) suspension culture transfection with polyethyleneimine (Polyscience Inc., Germany). This system is used for the production of proteins from pSectag vectors.

DNA plasmid for transfection was diluted to a ratio of 1 :4 with polyethyleneimine (PEI). 24 hours prior to transfection, H293F cells were diluted with Freestyle 293 expression medium (Invitrogen, USA) to a concentration of 1 x 106 cells/mL. The cell density was approximately 2 x 10⁶ cells/mL at time of transfection and the viability was greater than 95%. A ratio of 9: 1 was used for the amount of Freestyle 293 expression medium to the PBS mixture of DNA and PEI. Appropriate amount of cell culture medium was transferred into a shaker flask and placed in a C02 incubator at 37°C, shaking at 110 rpm. 1 mg/mL of DNA plasmid was added to pre-warmed (37°C) PBS and vortexed gently. PEI was added at a concentration of 3mg/mL, and vortexed three times for three seconds. The mixture was incubated for 15 min at room temperature (RT). The cell culture medium was removed from the incubator. The DNA-PEI mixture was added to the medium while swirling gently. Glucose was added to a final concentration of 6g/L. The flask was returned to the incubation and cultured at 37°C, with 5% CO2, shaking at 110 - 140rpm. The culture was supplemented with 5 g/L Lupin and 0.2 mM butyric acid after one day. At day 3, 5 and 7 after transfection, the culture was supplemented with 2mmol/L glutamine. At day 5, the culture was supplemented with 5 g/L Lupin. The glucose level was maintained at a final concentration of 5 - 6 g/L. The cells were harvested when viability was 40 - 50%. The cells were centrifuged at 3000xg for 15 min at 4°C and supernatant was collection for protein purification. All purified single-chain antibodies carry a 6x His-tag at the C-terminal end of their amino acid sequence for purification by IMAC and for FACS analysis. Proteins were purified with a nickel-based metal affinity chromatography column, Ni-NTA column (Invitrogen, USA), according to the manufacturer's instruction manual. Fractions of lmL were collected and dialyzed against PBS.

Evaluation of the scFv-scuPA fusion proteins

[0318] Purity of the proteins was analyzed using SDS-PAGE. 30ml of each purified protein and 6mI of 5X reducing SDS loading buffer were added to 1.5mL tubes and denatured at 96°C for 5 min. The samples were run on SDS-PAGE gel in SDS running buffer at 30 mA for 2 hours. The gel was then stained with Coomassie Brilliant Blue for 1 hour and subsequently destained for at least 12 hours with Coomassie destaining solution. The gel was visualized and analyzed using a Bio-Rad Gel-Doc system with Quantity One software.

[0319] After SDS-gel electrophoresis and Western blotting, the membrane was blocked with 1% BSA and hybridized with a specific horseradish peroxidase (HRP). Anti-6x His-tag® antibody HRP was used to detect the fusion proteins. Secondary hybridization was performed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific Inc, USA), an enhanced chemiluminescent (ECL) substrate for the HRP enzyme. Plasmin was added to digest the scuPA to the two chain urokinase by cleaving the Lys-158 and Ile-159 bond.

Flow cvtometrv

[0320] Blood was collected from healthy volunteers who had taken no medication for at least 10 days. In an attempt to minimize platelet activation during blood collection, blood was obtained by venipuncture from an antecubital vein through a 21-gauge needle with no tourniquet. The first 2mL of blood were discarded. The collected blood was anticoagulated with 10% citric acid. Platelet rich plasma (PRP) was obtained by

centrifugation at 180xg for 10 min at room temperature. The PRP was collected and diluted 1 :20 with PBS containing 100 mg/L calcium chloride and 100 mg/L magnesium chloride. Diluted PRP was either not activated or activated with 20 mM ADP, 5mg/mL collagen-related peptide (CRP) or 30mM thrombin receptor-activating peptide (TRAP) for 5 min. Activity of platelets was determined by FITC-labelled fibrinogen, PAC1-FITC and anti-CD62P-PE. As described in details below, the platelets were analyzed by flow cytometry using either a single fluorochrome (single staining) or two fluorochromes (dual staining). For single staining, incubation with 10 mg/mL of purified scFv for 10 min at 37°C was followed by 1 mL of anti-Penta-His AlexaFluor 488-conjugated monoclonal antibody for 15 min. Samples were fixed with 1x Cellfix (BD Bioscience, USA) and analyzed by FACS Calibur (BD Bioscience,

USA). In addition, the specificity of scFvTarg targeting activated platelets was analyzed in a competitive flow cytometry assay, using FITC-labelled fibrinogen and PAC1-FITC. Both FITC- labelled fibrinogen and PAC1-FITC bind to the activated GPIIb/IIIa receptors on activated platelets. For dual staining, incubation with both 10 mg/mL of purified scFv and CD62P-PE for 10 min at 37oC was followed by ImI of anti-Penta-His AlexaFluor 488-conjugated monoclonal antibody for 15 min. Competitive assays were also performed using 10 mg/mL of abciximab (ReoPro®). Further dose dependent competitive assays were performed using the scFvTarg and FITC-labelled fibrinogen. Samples were fixed with lx Cellfix and analyzed by FACS Calibur.

[0321] Samples were analyzed using a Becton Dickinson FACS Calibur flow cytometer. The platelets were distinguished using the forward and sideward light scatter profile. A gate was set around the platelets and 10,000 cells were analyzed. For single staining, the AlexaFluor 488 fluorescence is used to quantitate the amount of platelet-bound scFv. The scFv binding was expressed as mean fluorescence intensity. For dual staining, the AlexaFluor 488 fluorescence and CD62P-PE fluorescence is used to quantitate the amount of activated platelet-bound scFv.

Urokinase activity assay

[0322] Urokinase activity was determined in microtiter plates with a chromogenic substrate assay. Comparison between clinically used uPA (Medac GmbH, Hamburg,

Germany) and scFv-scuPA was made on the basis of equal urokinase activity. A volume of 50 mL scFv-scuPA, each with a final concentration of 100 nmol/L, was monitored against 50mI urokinase standards (0-100U/mL) used as positive control. Single-chain urokinase was converted to the active form using 0.1 U/L plasmin in assay buffer (38 mmol/L of NaCI, 5mmol/L of Tris-HCI, 0.1% bovine serum albumin, pH 8.8). After incubation for 2 hours at room temperature under shaking, 0.5mmol/L of S2444 (Chromogenix, Milano, Italy) in 125 mL assay buffer was added. Samples were measured on a Victor3V Multi-label counter (PerkinElmer, Massachusetts, USA) at wavelength 405 nm every 5 min over a period of 60 min.

Animal experiments

[0323] C57BL/6 mice were acquired from Jackson Laboratories and bred by the

Alfred Medical Research and Education Precinct (AMREP) Animal Services in Melbourne, VIC. All experiments involving mice were approved by the AMREP Animal Ethics Committee (E/1627/2016/B).

Myocardial ischemia-reperfusion injury in mice

[0324] The ligation of the left coronary artery was performed as described previously.2 Briefly, 20 - 25g male, C57BL/6 mice were anaesthetized using a combination of ketamine HCI (100 mg/kg body weight (wt); Lyppard, Australia), xylazine HCI (5 mg/kg BW; Lyppard, Australia) and atropine (1 mg/kg body wt; Pfizer, Australia) via intraperitoneal (ip.) injection. Mice were orally intubated and ventilated throughout the procedure using a rodent ventilator (Model 687, Harvard Apparatus, USA), with a tidal volume of 0.18 mL at 120 breaths/min. Mice underwent myocardial ischemia-inducing surgery by a left anterior descending (LAD) coronary artery ligation for 60 min. Immediately after reperfusion mice were randomly injected via tail-vein with either PBS, Targ-scuPA (75 units/g body wt), or Non-targ-scuPA (75 units/g body wt). Ultrasound and echocardioaraphic analysis

[0325] Ultrasound of animals was performed with a Vevo2100 high-resolution small animal scanner (VisualSonics Inc. Toronto, Canada) using a 22-55 MHz MS550D transducer. Animals were placed under light sedation (range of 1.0% to 2% isoflurane), on the VisualSonics imaging station. The imaging station was heated to prevent hypothermia. Electrode gel was applied to the limbs of the animals and secured using tape to the imaging platform to obtain electrocardiogram recording. The temperature of the animal, its heart rate, electrocardiogram as well as its breathing was monitored. During each

echocardiographic examination, the parasternal long-axis and parasternal short-axis views of the heart were obtained. Imaging was performed at baseline (before LAD ligation), as well as at weeks 1, 2, 3 and 4 post-I/R injury. Videos and images were analyzed by a blinded investigator using the VisualSonics imaging software (VisualSonics Inc. Toronto, Canada).

[0326] The parasternal long-axis view was obtained by placing the transducer in a vertical fashion, with the marker of transducer pointing towards the head of the animal.

The transducer is then rotated approximated 30° counterclockwise so that the marker 10 and 11 o'clock. The parasternal short-axis view was obtained by rotating the transducer 90° clockwise so that the marker was positioned between 1 and 2 o'clock. The Y axis was slightly adjusted to include both papillary muscles.

[0327] The ejection fraction (EF) and fractional shortening (FS) were calculated using the Simpson method from the parasternal long-axis B-mode images. The dimensions of the LV interventricular septal (IVS), left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) at end-diastole (d) and end-systole (s) were measured using the parasternal short-axis B-mode images.

[0328] Radial and longitudinal strain analyses were conducted using a speckle tracking algorithm provided by VevoStrain, VisualSonics imaging software (VisualSonics Inc, Canada) by the same experienced investigator. Prior to analysis, all the B-mode images were reviewed to be of good echogenicity (based on the visualization of the endocardial border) and without any image artifacts (such as shadowing of the rib cage). The endocardial and epicardial borders were traced using the semi-automated tracing mode provided by the VevoStrain imaging software for at least three consecutive cycles. In order to obtain the strain measurements, the software analyzed the tracked images in a frame-by-frame manner. The regional speckle-tracking based strain analyzed the LV by dividing the myocardium into 6 standard anatomic segments. The anterior apex section is the infarcted area and the posterior base and post mid are the remote, non-infarcted area. For the global assessment, all sections (posterior base, mid, apex and anterior base, mid, apex) were included. The maximum opposite wall delay was also measured as a marker to LV dyssynchrony.

Evans Blue/TTC staining

[0329] Mice were anaesthetized 4 weeks post-I/R and the ischemic area (area at risk (AaR)) and infarcted area (infarct size (I)) was assessed by Evans

Blue/triphenyltetrazolium chloride (TTC) staining. The LAD was re-ligated with the original suture and 4% Evans Blue (AppliChem) was injected to stain the perfused regions blue. The heart was then cut into 6 transversal slices and stained with 1% TTC (Sigma) for 10 min at 37°C. TTC turns the metabolically active areas red while the infarcted, necrotic myocardial tissue remains white (I). Thereafter, the heart slices are photographed on both sides using a digital camera. A blinded researcher determined the infarct sizes by quantitative

morphometric planimetry using an image analysis software program.

Statistical analysis

[0330] All quantitative data is reported as mean ± standard deviation (SD). Statistical analyses were performed using unpaired t-test, one-way ANOVA or repeated measures two-way ANOVA followed by Bonferroni's multiple comparisons test. Statistical analyses were performed using GraphPad Prism Software, with p<0.05 considered statistically significant.

EXAMPLE 22

COMBINING ANTIPLATELET AND ANTICOAGULANT POTENCY IN AN ACTIVATED PLATELET-TARGETED

DRUG PREVENTS CARDIAC ISCHEMIA/ REPERFUSION INJURY

[0331] The present inventors investigated whether SE-TAP can be used to prevent cardiac I/R injury. Using a murine model of transient ischemia with occlusion of the left anterior descending artery (LAD) for 60 min (Ziegler, M. et al., 2018. Eur. Heart J. 39: 111-116), the clinical scenario was recapitulated where a patient presents with myocardial infarction (MI) and undergoes recanalization of the occluded coronary artery. SE-TAP was administered at the timepoint of reperfusion to mimic its potential clinical application. Mice were randomized to receive PBS as a control, MUT-TAP, which is a mutant that has lost GPIIb/IIIa binding ability, or SE-TAP (both @ 0.03 mg/g body weight, i.v. Echocardiography was performed at baseline and 4 weeks post-I/R (Ziegler, M. et al., 2018. supra). A significant decline in ejection fraction (EF) was observed in the MUT-TAP and PBS treatment groups, while mice treated with SE-TAP exhibited preserved cardiac function. Overall, SE-TAP protected against loss of cardiac function, whereas a significant decline was observed in animals treated with MUT-TAP or PBS (Figure 20A & B). Significant increases in diastolic and systolic left ventricular volume were noted in MUT-TAP and PBS treatment groups, whereas the mice treated with SE-TAP were protected from ventricular dilatation (Figure 20C 8i D). Strain analysis, which more accurately reflects myocardial contractility compared with conventional echocardiography, was employed for assessment of left ventricle function (Kalam, K et al., 2014. Heart Br. Card. Soc. 100: 1673-1680). Strain analysis showed that animals treated with MUT-TAP or PBS had significantly more regional function abnormalities as compared to SE-TAP-treated mice (Figure 20E-H). This was reflected in the radial strain of the infarct area, as well as the global readout. Furthermore, the opposing wall showed a significant increase in time delay. Importantly, this protection of cardiac function by SE-TAP correlated with a significantly reduced infarct size in comparison with the MUT-TAP and PBS treated mice, as assessed by Evans Blue/triphenyltetrazolium chloride (TTC) staining 4 weeks post-I/R (Figure 20I-K). [0332] Despite enormous advances in achieving rapid and complete coronary reperfusion, cardiac I/R injury so far remains an untreatable consequence of MI. To reduce the morbidity/mortality of patients with MI, it is therefore crucial to develop novel therapeutics that reduce I/R injury. Targeted drug delivery is a means to direct drugs to the area where their action is needed without the effect of systemically active drug

concentrations. This approach promises particular clinical benefits, if side effects such as bleeding complications can be avoided. Activated platelets, which accumulate early in ischemic and reperfused myocardium, provide an immediate and unique cellular target for site-directed delivery of drugs to the area of risk for I/R injury. No other such cellular or molecular target has yet been described. In addition, the ligand-binding pocket of GPIIb/IIIa on platelets offers an ideal molecular epitope for targeted drug delivery, because it is highly abundant, platelet-specific, and only expressed on activated platelets. SE-TAP possesses this targeting capability and thereby allows local delivery/enrichment of both antiplatelet potency, by blocking fibrinogen binding to GPIIb/IIIa, and anticoagulant effects, by TAP's factor Xa inhibitory effects. SE-TAP can be applied i.v. as an acute application in the catherization laboratory or subcutaneously for chronic application. The recombinant antibody drug format allows optimization in size and consequently in pharmacokinetic/dynamic characteristics.

[0333] With SE-TAP, a highly effective anti-thrombotic drug is provided herein, which uniquely combines localized antiplatelet and anticoagulant effects while preserving hemostasis. Its flexible drug format supports broad application and includes prophylaxis and treatment of arterial and venous thrombosis and, importantly, prevention of cardiac I/R injury.

[0334] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0335] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0336] Throughout the specification the aim has been to describe the preferred embodiments of the present disclosure without limiting the disclosure to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A chimeric molecules comprising an anti-coagulant agent and an antigen-binding molecule that binds to activated glycoprotein Ilb/IIIa (GPIIb/IIIa) and comprises:

(1) a heavy chain variable region (V_H) comprising the VHCDR1 amino acid sequence RYAMS [SEQ ID NO: 3], the VHCDR2 amino acid sequence

GISGSGGSTYYADSVKG [SEQ ID NO:4], and the VHCDR3 amino acid sequence CARIFTHRSRGDVPDQTSFDY [SEQ ID NO: 5], and a light chain variable region (V_L) comprising the VLCDR1 amino acid sequence QGDSLRNFYAS [SEQ ID NO: 6], the VLCDR2 amino acid sequence GLSKRPS [SEQ ID NO: 7], and the VLCDR3 amino acid sequence LLYYGGGQQGV [SEQ ID NO: 8] ;

(2) a V_H that comprises, consists or consists essentially of the amino acid sequence

[SEQ ID NO: 1], and a V_L that comprises, consists or consists essentially of an amino acid sequence selected from

(3) a V_H with at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a V_L with at least 90% (including at least 91% to 99% and all integer percentages therebetween) seq uence identity to the amino acid sequence of SEQ ID NO: 2 or 60;

(4) a V_H as defined in (1) comprising at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g. , to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) comprising at least 90% (including at least 91% to 99% and all integer percentages therebetween) sequence identity to at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ ID NO: 2 or 60 (e.g., to at least one framework region, such as 1, 2, 3 or 4 framework regions, of the V_L) ; and/or

(5) a V_H as defined in (1) which is distinguished from the V_H amino acid sequence set forth in SEQ ID NO: 1 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the V_H amino acid sequence set forth in SEQ ID NO: 1 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_H), and a V_L as defined in (1) which is distinguished from the V_L amino acid sequence set forth in SEQ ID NO: 2 or 60 by a deletion, substitution or addition of one or more (e.g., 1, 2, 3, 4 or 5) amino acids in at least one region other than a CDR of the V_L amino acid sequence set forth in SEQ ID NO: 2 or 60 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_L) .

2. The chimeric molecule of claim 1, wherein the antigen-binding molecule is a monovalent antigen-binding molecule.

3. The chimeric molecule of claim 2, wherein the monovalent antigen-binding molecule is selected from Fab, scFab, Fab', scFv and one-armed antibodies.

4. The chimeric molecule of claim 1, wherein the antigen-binding molecule is a multivalent antigen-binding molecule.

5. The chimeric molecule of claim 4, wherein the multivalent antigen-binding molecule is a diabody.

6. The chimeric molecule of any one of claims 1 to 5, comprising one or more of the following activities: (a) binds to the active conformation of GPIIb/IIIa with greater affinity than to the inactive conformation of GPIIb/IIIa; (b) inhibits binding of fibrinogen to GPIIb/IIIa; (c) inhibits platelet aggregation; (d) lacks platelet activation activity and (e) lacks systemic inhibition of platelet aggregation.

7. The chimeric molecule of any one of claims 1 to 6, wherein the anti-coagulant agent is a clotting factor inhibitor or a thrombolytic agent.

8. The chimeric molecule of any one of claims 1 to 7, wherein the anti-coagulant agent is a proteinaceous molecule and the chimeric molecule is in the form of a single chain chimeric polypeptide.

9. The chimeric molecule of any one of claims 1 to 8, which is contained in a delivery vehicle.

10. The chimeric molecule of claim 9, wherein the delivery vehicle is a liposome, a nanoparticle, a microparticle, a dendrimer or a cyclodextrin.

11. An isolated polynucleotide comprising a nucleic acid sequence encoding the chimeric molecule of any one of claims 1 to 8.

12. A construct comprising a nucleic acid sequence encoding the chimeric molecule of any one of claims 1 to 8 in operable connection with one or more control sequences.

13. A host cell that contains the construct of claim 12.

14. A pharmaceutical composition comprising the chimeric molecule of any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

15. A method for inhibiting binding of a ligand to GPIIb/IIIa in its active

conformation, the method comprising contacting the GPIIb/IIIa with the chimeric molecule of any one of claims 1 to 8, to thereby inhibit binding of the ligand to the GPIIb/IIIa.

16. A method for inhibiting binding of a ligand to an activated platelet, the method comprising contacting the activated platelet with the chimeric molecule of any one of claims 1 to 8, to thereby inhibit binding of the ligand to the activated platelet.

17. The method of claim 15 or claim 16, wherein the ligand is selected from fibrinogen, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand.

18. The method of claim 15 or claim 16, wherein the ligand is fibrinogen.

19. A method for inhibiting platelet aggregation in a subject, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 8, or the composition of claim 14, to thereby inhibit platelet aggregation in the subject.

20. A method for inhibiting thrombus formation in a subject, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 10, or the composition of claim 14, to thereby inhibit thrombus formation in the subject.

21. A method for inhibiting embolus formation in a subject, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 10, or the composition of claim 14, to thereby inhibit embolus formation in the subject.

22. A method for treating or inhibiting the development of platelet aggregation, thrombus formation or embolus formation in a subject having or at risk of developing a condition associated with the presence of activated platelets, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 10, or the composition of claim 14.

23. The method of claim 22, wherein the condition is selected from atherosclerosis (e.g., unstable atherosclerosis), allergic disorders, autoimmune diseases, cancers, infections, neurological disorders, systemic inflammation, tissue or organ transplantation,

thromboembolism-associated conditions and wounds.

24. A method for treating or inhibiting the development of a thromboembolism- associated condition in a subject, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 10, or the composition of claim 14.

25. The method of claim 24, wherein the thromboembolism-associated conditions is selected from arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders and thromboembolic disorders in the chambers of the heart or in the peripheral circulation.

26. The method of claim 24, wherein the thromboembolism-associated conditions is selected from unstable angina or other abdominal aortic aneurysm, acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from a medical implant, device or procedure in which blood is exposed to an artificial surface that promotes thrombosis.

27. The method of claim 26, wherein the medical implant or device is selected from prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices and artificial hearts or heart chambers, and vessel grafts.

28. The method of claim 26, wherein the procedure is selected from

cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis.

29. The method of claim 24, wherein the thromboembolism-associated conditions is acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism.

30. A method for treating or inhibiting the development of a thrombosis-associated hematologic disorder in a subject, the method comprising administering to the subject an effective amount of the chimeric molecule of any one of claims 1 to 10, or the composition of claim 14.

31. The method of claim 30, wherein the hematologic disorder is sickle cell disease or thrombophilia.

32. The method of any one of claims 19 to 31, wherein subject has or is suspected of having a condition associated with the presence of activated platelets.

33. The method of claim 32, wherein the condition is selected from atherosclerosis (e.g., unstable atherosclerosis), allergic disorders, autoimmune diseases, cancers, hematologic disorders, infections, neurological disorders, systemic inflammation, tissue or organ transplantation, thromboembolism-associated conditions and wounds.

34. A kit for inhibiting binding of a ligand to GPIIb/IIIa in its active conformation, for inhibiting binding of a ligand to an activated platelet, for inhibiting platelet aggregation, for inhibiting thrombus formation, for inhibiting embolus formation, for treating or detecting conditions associated with activated platelets, for treating or inhibiting the development of a thromboembolism-associated condition, or for treating or inhibiting the development of a hematologic disorder, the kit comprising a chimeric molecule of any one of claims 1 to 10, or the composition of claim 14.