WO2020237304A1

WO2020237304A1 - Antigen-binding molecules that bind the active conformation of platelet integrin receptor gpiib/iiia

Info

Publication number: WO2020237304A1
Application number: PCT/AU2020/050526
Authority: WO
Inventors: Karlheinz Peter; Xiaowei Wang
Original assignee: Baker Heart and Diabetes Institute
Priority date: 2019-05-27
Filing date: 2020-05-27
Publication date: 2020-12-03

Abstract

Disclosed are antigen-binding molecules that bind to activated platelets and inhibit platelet function, including platelet aggregation and thrombus formation. More particularly, antigen-binding molecules are disclosed that bind to the active conformation of platelet integrin receptor GPIIb/IIIa with greater affinity than to its inactive conformation. In specific embodiments, the antigen-binding molecules are disclosed for use alone or in combination with other agents in compositions and methods for inhibiting binding of ligands to the active conformation of GPIIb/IIIa, for inhibiting platelet aggregation and/or thrombus or embolus formation and for detecting and/or imaging of activated platelets.

Description

ANTIGEN- BINDING MOLECULES THAT BIND THE ACTIVE CONF RMATIO N

OF PLATELET INTEGRIN RECEPTOR GPIIB/IIIA

RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2019901803 entitled "Antigen-binding molecules and uses therefor" filed 27 May 2019, the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] This present disclosure relates generally to antigen-binding molecules that bind to activated platelets and inhibit platelet function, including platelet aggregation and thrombus formation. More particularly, the present disclosure relates to antigen-binding molecules that bind to the active conformation of platelet integrin receptor GPIIb/IIIa with greater affinity than to its inactive conformation. In specific embodiments, the antigen- binding molecules are used alone or in combination with other agents in compositions and methods for inhibiting binding of ligands to the active conformation of GPIIb/IIIa, for inhibiting platelet aggregation and/or thrombus or embolus formation and for detecting and/or imaging 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 OF THE DISCLOSURE

[0010] The present disclosure features antigen-binding molecules that bind to the active conformation of GPIIb/IIIa with greater affinity than to its inactive conformation.

These antigen-binding molecules do not activate platelets but are able to inhibit binding of fibrinogen to platelets and can thus be used, for example, to reduce or inhibit platelet aggregation and/or thrombus formation. In specific embodiments, the antigen-binding molecules are conjugated to a heterologous moiety for detecting activated platelets, for improving a pharmacokinetic parameter (e.g., half-life) or for targeting a therapeutic agent to a cell, tissue or microenvironment associated with activated platelets. The antigen-binding molecules may also be used as diagnostics for detecting or imaging thrombi and emboli, for detecting and imaging of activated platelets as involved in many diseases such as inflammatory diseases or cancer and its metastasis, and for isolating or separating activated platelets from a sample, as described hereafter.

[0011] Accordingly, in one aspect, the present disclosure features antigen-binding molecules that bind to activated glycoprotein Ilb/IIIa (GPIIb/IIIa). These antigen-binding molecules generally comprise:

(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

EVQLVESGGGLVQPGGSLRLSCAASGFMFSRYAMSWVRQAPGKGPEWVSGISGSGGSTYYADS VKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARIFTHRSRGDVPDQTSFDYWGQGTLVTVSS

[SEQ ID NO: 1], and a V_L that comprises, consists or consists essentially of an amino acid sequence selected from SELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKPGQAPTLVIYGLSKRPSGIPDRFSASSS GNTASLTITGAQAEDEADYYCLLYYGGGQQGVFGGGTKLTVL [SEQ ID NO: 2] and

SELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKPGQAPTLVIYGLSKRPSGIPDRFSASSS GNTASLTITGAQAEDEADYYCLLYYGGGQQGVFGGGTKLTV [SEQ ID NO: 68] ;

(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 68;

(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 68 (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 68 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 68 (e.g., in at least one framework region, such as in 1, 2, 3 or 4 framework regions, of the V_L) .

[0012] The anti-GPIIb/IIIa antigen-binding molecules of the present disclosure may be in isolated, purified, synthetic or recombinant form. Suitable antigen-binding molecules 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 molecules may be multivalent (e.g., bivalent) or monovalent. In some embodiments, the antigen binding molecules comprise an Fc domain. In other embodiments, the antigen-binding molecules lack an Fc domain. In some embodiments, the antigen binding molecules are monovalent antigen-binding molecules (e.g., Fab, scFab, Fab', scFv, one-armed antibodies, etc. ).

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

GPIIb/IIIa; (c) inhibit platelet aggregation; (d) lack platelet activation activity and (e) lack systemic inhibition of platelet function. [0014] In another aspect, the present disclosure provides a chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule described herein and a heterologous moiety. The heterologous moiety may comprise a payload. In some embodiments, the heterologous moiety is selected from a detectable moiety, a half-life extending moiety and a therapeutic moiety (e.g., an anti-cancer, immunomodulatory or anti-inflammatory moiety).

In illustrative embodiments in which the heterologous moiety is a proteinaceous molecule, the chimeric molecule may be in the form of a single chain chimeric polypeptide in which the GPIIb/IIIa antigen-binding molecule described herein is operably connected to the heterologous moiety.

[0015] In some embodiments, an antigen-binding molecule or chimeric molecule as broadly described above is 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 an anti-GPIIb/IIIa antigen-binding molecule described herein, or a chimeric molecule as described herein.

[0017] Yet another aspect of the present disclosure provides constructs comprising a nucleic acid sequence encoding an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 an anti-GPIIb/IIIa antigen-binding molecule or 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 GPIIb/IIIa-specific antigen-binding molecule or 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 GPIIb/IIIa-specific antigen-binding molecule or 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 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, 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 GPIIb/IIIa-specific antigen-binding molecule or chimeric molecule described herein. Non-limiting examples of hematologic disorders include sickle cell disease and thrombophilia.

[0029] Another aspect of the present disclosure provides methods for detecting the presence of an activated platelet. These methods generally comprise contacting the activated platelet with an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, to form a complex that comprises the activated platelet and the antigen-binding molecule or chimeric molecule, and detecting the complex to thereby detect the presence of the activated platelet.

[0030] In a related aspect, the present disclosure provides methods for detecting the presence of an activated platelet in a subject. These methods generally comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an activated platelet and the antigen-binding molecule or chimeric molecule to thereby detect the presence of the activated platelet in the subject.

[0031] In another related aspect, the present disclosure provides methods for detecting presence of a thrombus in a subject. These methods generally comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises a thrombus and the antigen-binding molecule or chimeric molecule to thereby detect the presence of the thrombus in the subject.

[0032] In a further related aspect, the present disclosure provides methods for detecting presence of an embolus in a subject. These methods generally comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an embolus and the antigen-binding molecule or chimeric molecule to thereby detect the presence of the embolus in the subject.

[0033] It is known that tumor cells activate platelets and that the activated platelets can serve as a means of detecting presence of a tumor. Accordingly, in a further related aspect, the present disclosure provides methods for detecting presence of a tumor in a subject. These methods generally comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an activated platelet and the antigen-binding molecule or chimeric molecule (e.g., adjacent to the tumor such as in the microenvironment of the tumor) to thereby detect the presence of the tumor in the subject. [0034] In a related aspect, the present disclosure provides methods for reducing or inhibiting proliferation, survival or viability of a tumor in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule described herein and an anti-cancer moiety (e.g., a chemotherapeutic agent or immunomodulating agent).

[0035] In another related aspect, the present disclosure provides methods for treating or inhibiting the development of a cancer in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule described herein and an anti-cancer moiety (e.g., a chemotherapeutic agent or immunomodulating agent).

[0036] 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.

[0037] A further aspect of the present disclosure provides kits for detecting an activated platelet, thrombus and/or embolus, for detecting presence of a tumor, 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, for treating or inhibiting the development of a hematologic disorder, for reducing or inhibiting proliferation, survival or viability of a tumor, and/or for treating or inhibiting the development of a cancer. The kits generally comprises an anti-GPIIb/IIIa antigen-binding molecule, chimeric molecule or composition described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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 pM 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. [0039] Figure 2 is a schematic and photographic representation showing a vector map, generation, and purification of scFv-CD39 constructs. (A) Gene map of scFv-CD39 constructs in the pSectag2A vector for mammalian expression. The restriction enzymes used to insert the constructs are Notl, Ascl, and Xhol. (B) Electrophoresis with 1% agarose gel. Lanes 1-3: molecular cloning of constructs using PCR amplification and double digest. (1) scFv-SE, Ascl and Notl (821 bp); (2) scFvMut, Ascl and Notl (821 bp); (3) solCD39, Notl and Xhol (1326 bp). Lane 4: DNA ladder. Lanes 5 and 6: single control digests of cloned constructs in pSectag2A. (5) SE-CD39, Xhol (7247 bp); (6) Mut-CD39, Xhol (7247 bp).

Lanes 7 and 8: triple control digests of cloned constructs in pSectag2A. (7) SE-CD39, Ascl and Notl, and Xhol (821 bp for scFvSE, 1326 bp for solCD39, 5100 bp for pSectag2A); (8) Mut-CD39, Ascl and Notl, and Xhol (821 bp for scFvMut, 1326 bp for solCD39, 5100 bp for pSectag2A).

[0040] Figure 3 is a graphical representation showing flow cytometric assay of SE-CD39 (targ-CD39) and Mut-CD39 (non-targ-CD39) binding to human platelets, detected with an Alexa Fluor 488-coupled anti-Penta-His antibody that binds to the constructs' 6xHis- tag. (A) Quantitative comparison. Bar graphs depict the median fluorescence intensity values of 3 independent experiments (mean 6 standard error of the mean; ***p , .001). These assays were analyzed with 2-way repeated-measures analysis of variance with the

Bonferroni post-test. (B and C). Representative fluorescence histograms. Binding of (B) non- targ-CD39 and (C) targ-CD39 to activated platelets vs non-activated platelets is shown.

[0041] Figure 4 is a graphical representation showing a comparison of SE-CD39 (targ-CD39), Mut-CD39 (non-targ-CD39), and scFvSE (scFv control) alone in inhibition of platelet activation as assessed by P-selectin expression in flow cytometry. P-selectin expression was determined with a PE-labeled anti-P-selectin antibody. Bar graphs depict the median fluorescence intensity values of 3 independent experiments (mean 6 standard error of the mean; ***p , .001). These assays were analyzed with 2-way repeated-measures analysis of variance with the Bonferroni post-test.

[0042] Figure 5 is a graphical representation showing effects of CD39 targeting on thrombus formation and hemostasis. (A) Comparison of occlusion time measurements of ferric chloride-induced thrombosis in carotid arteries of mice administered Mut-CD39 (non- targ-CD39), SE-CD39 (targ-CD39), saline, or SE only (single-chain antibody (scFv) controls) (n=8 each) at an equimolar amount in regard to the scFv component in the SE-CD39 group. (B) Comparison of bleeding time in mice determined by tail transection between Mut-CD39, SE-CD39, and saline (n=5 each). Doses administered were activity matched: e.g., low-dose Mut -CD39 of 0.8 mg/kg corresponds to low-dose SE-CD39 of 0.4 mg/kg. Measurements were analyzed with a Gehan- Breslow-Wilcoxon survival analysis and subsequently represented as bar graphs (*P<0.05; **P<0.01).

[0043] Figure 6 is a graphical representation depicting study design for investigating in vivo action of SE-CD39 in mouse model of ischemia/reperfusion. Treatment groups shown are: PBS, SE-CD39 (Targ-CD39) and Mut-CD39 (Non-targ-CD39). [0044] Figure 7 is a graphical representation showing that SE-CD39 (Targ-CD39) treatment preserves myocardial function in a mouse model of ischaemia/ reperfusion (I/R). Treatment groups: PBS, SE-CD39 (Targ-CD39) or Mut-CD39 (Non-targ-CD39). Ejection fraction (EF) was analysed using Simpson's method from the parasternal long-axis B-mode images at baseline as well as weeks 1, 2, 3, and 4 post-I/R. Cardiac strain analysis was performed using the parasternal long-axis B-mode images at baseline and week 4 post-I/R. Statistical analyses were performed using unpaired t-test (I, J), one-way ANOVA (A, D-H) or repeated measures two-way ANOVA (B) followed by Bonferroni's multiple comparisons test. Multiple comparisons show adjusted P-values. (A) Baseline EF was similar between all groups (PBS: 65.1± 6.3 vs. SE-CD39: 62.6±3.8 vs. Mut-CD39: 63.2± 2.8; % mean± SD). The trend toward better EF was observed for the SE-CD39 treated group at week 1 post-I/R (PBS: 36.9± 12.4 vs. SE-CD39: 50.8± 11.5 vs. Mut-CD39: 39.2± 11.8). In week 2, there was significant increase in EF for the SE-CD39 group, as compared to the PBS control group (PBS: 36.5± 13.3 vs. SE-CD39: 52.5± 8.7 vs. Mut-CD39: 40.1± 11.4). Week 3 showed a significant increase in EF for the SE-CD39 mice, as compared to both PBS control group and Mut-CD39 mice (PBS: 37.4± 13.0 vs. SE-CD39: 54.4± 4.8 vs. Mut-CD39: 39.4± 8.8).

Similar results were obtained at week 4 (PBS: 38.5± 11.5 vs. SE-CD39: 55.9± 4.7 vs.Mut- CD39: 40.9± 10.5; n=6-7). (B) Individual EF is shown for each treatment group at baseline and week 4 post-I/R. (C) Representative images of radial strain curves obtained from VevoStrain analysis software showing strain measures over time. The colored lines represent the six standard myocardial regions, with a seventh black line that calculates the average (global) strain at each time point. (D) Bar charts show the significant decrease in radial strain for both PBS and Mut-CD39 treated animals, as compared to the SE-CD39 treated animals, in both global peak radial strain (PBS: 13.0± 5.2 vs. SE-CD39: 39.4± 11.1 vs. Mut- CD39: 16.5± 5.2) and infarct area (anterior apex) radial strain (PBS: 3.4± 2.5 vs. SE-CD39:

22.1± 4.1 vs. Mut-CD39: 7.9± 6.8). Maximum opposite-wall delay shows significant increases in time delay for PBS treated animals, as compared to the SE-CD39 treated animals (PBS: 74.3± 17.9 vs. SE-CD39: 13.0± 4.0; n= 3). (E) Comparison of bleeding time in mice determined by tail transection between Mut-CD39, SE-CD39, and PBS. None of the treatments led to prolonged bleeding times (PBS: 424± 98 vs. SE-CD39: 309± 90 vs.Mut- CD39: 352± 84 s; n=8).

[0045] Figure 8 is a graphical representation showing that treatment with SE- CD39 resulted in continuous improvement of echocardiographically determined ejection fraction and fractional shortening. Treatment groups shown are: PBS, SE-CD39 (Targ-CD39) or Mut-CD39 (Non-targ-CD39). (A) Baseline FS was similar between all groups (PBS: 15.4 ± 3.1 vs SE-CD39: 16.8 ± 3.3 vs Mut-CD39: 15.5 ± 3.6; % mean ± SD; n=6-7). At week 1 post-LAD ligation, there was a decrease of FS in all three groups (PBS: 8.9 ± 3.4 vs SE- CD39: 10.9 ± 4.4 vs Mut-CD39: 7.2 ± 3.6; n=6-7). Similar results were obtained at week 2 (PBS: 8.1 ± 3.7 vs SE-CD39: 11.6 ± 2.7 vs Mut-CD39: 7.9 ± 3.0; n=6-7). Week 3 showed a significant increase in FS for the SE-CD39 group, as compared to both PBS and Mut-CD39 groups (PBS: 8.1±2.0 vs SE-CD39: 12.9±4.0 vs Mut-CD39: 7.4±3.5; n=6-7). Similar results were obtained at week 4 (PBS: 8.5 ± 3.6 vs SE-CD39: 13.8 ± 4.5 vs Mut-CD39: 8.1 ± 2.4; n=6-7). (B) Individual FS is shown for each treatment group at baseline and week 4 post- I/R; n = 6-7.

[0046] Figure 9 is a schematic and graphical representation of a radial and longitudinal strain analysis showing that cardiac performance is retained 4 weeks post-I/R for the SE-CD39 treated animals. (A) longitudinal and radial strain measurements. (B)

Representative images of longitudinal strain curves obtained from VevoStrain analysis software showing strain measurements over time. The colored lines represent the 6 standard myocardial regions, with a seventh black line that calculates the average (global) strain at each time point. Representative images of longitudinal strain time to peak showed delays (pink and red sections) in the PBS control and Mut-CD39 treated animals, but not the SE- CD39 treated animals or baseline analysis. (C) Longitudinal strain analysis obtained similar differences as the radial strain analysis, n=3.

[0047] Figure 10 is a graphical representation showing that Targ-CD39 treatment ameliorates cardiac remodeling in a mouse model of ischemia/reperfusion (I/R). Treatment groups: PBS, SE-CD39 (Targ-CD39) and Mut-CD39 (Non-targ-CD39). (A) Heart-type fatty acid binding protein (H-FABP) plasma levels were analyzed 2 h post-I/R. SE-CD39 treated mice showed a significant decrease in H-FABP, as compared to PBS and Mut-CD39 treated animals (PBS: 51.3 ± 16.6 vs. SE-CD39: 33.1 ± 13.5 vs. Mut-CD39: 51.3 ± 11.5 ng/mL; n = 8). (B) Four weeks post-I/R, cardiac sections were stained using Evans blue/TTC.

Quantitative analysis of infarct size (I) per area at risk (AaR) is shown. SE-CD39 treated mice showed a significant decrease I/AaR ratio 4 weeks post-I/R, as compared to PBS and Mut-CD39 treated groups (PBS: 47.5 ± 10.3 vs. SE-CD39: 22.9 ± 13.0 vs. Mut-CD39:

48.1 ± 7.4%I/AaR; n = 6-7, scale bar 1 mm). (C) Four weeks post-I/R, cardiac sections were immunostained with anti-CD31 antibody. Blood vessels were stained brown and cell nuclei were counterstained with hematoxylin (blue). Quantitative analysis of neovascularization in a certain tissue area (capillaries/mm²) is shown. Treatment of SE-CD39 significantly increased neovascularization, as compared to PBS or Mut-CD39 treated mice (PBS: 290 ± 32 vs. SE- CD39: 604 ± 90 vs. Mut-CD39: 276 ± 56 capillaries/mm²; n = 6-7, scale bar 50 mm, magnification 200x). (D) At 2 h post-reperfusion, expression of CD41 was significant decreased in the SE-CD39 treated animals (n = 10-11). (E) Two hours post-I/R, cardiac sections were immunostained with anti-CD41 antibody (clone MWReg30). Platelets were stained brown and cell nuclei were counterstained with hematoxylin (blue). Quantitative analysis of platelets per high power field (platelet surface coverage/HPF) is shown.

Treatment of SE-CD39 significantly decreased platelet surface coverage, as compared to Mut-CD39 treated mice (SE-CD39: 671 ± 49 vs. Mut-CD39: 279 ± 79 platelet surface coverage/HPF; n = 8, scale bar 20 mm, magnification 400x).

[0048] Figure 11 is a graphical representation showing that SE-HSA-CD39 binds to activated platelets. (A) Binding of SE-HSA-CD39 to non-activated human platelets using method described in Example 4. (B) Binding of SE-HSA-CD39 to activated human platelets using method described in Example 4. [0049] Figure 12 is a graphical representation demonstrating that SE-HSA-CD39 strongly inhibits ADP-induced platelet activation. (A) No platelet activation without ADP: Binding of PAC-1 FITC to non-activated human platelets using method described in Example 5. (B) Platelet activation with 20nM ADP: Binding of PAC-1 FITC to activated human platelets (by 20nM ADP) using method described in Example 5. (C) No platelet activation with 20mM ADP that was retreated with SE-HSA-CD39: Binding of PAC-1 FITC to human platelets in presence of 20nM ADP, that was preincubated with SE-HSA-CD39, using method described in Example 5, demonstrates that CD39 is effectively hydrolyzing ADP to AMP, thereby preventing platelet activation.

[0050] Figure 13 is a graphical representation showing deconvoluted mass spectrum of SE prior to conjugation, showing molecular weight of approximately 40,500 Daltons. B. Deconvoluted mass spectrum of SE after conjugation with MeCOSar identifies SE- MeCOSarn species with n = 1, 2, 3, 4 corresponding to mass increments of about 461 Daltons.

[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 present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, 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 (/.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] The terms "administration concurrently" or "administering concurrently" or "co-administering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By "contemporaneously" it is meant that the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term

"sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.

[0056] 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).

[0057] "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).

[0058] 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.

[0059] 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. [0060] 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, C_H1, 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 present 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 and 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.

[0066] 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- C_H1 ; (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 antigen. 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.

[0067] 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.

[0068] Antigen-binding molecules can be naked or conjugated to other molecules or moieties such as toxins, radioisotopes, small molecule drugs, polypeptides, etc.

[0069] 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 ai. 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).

[0070] 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.

[0071] 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_H or V_L domain may be sufficient to confer antigen-binding specificity.

[0072] The term "anti-cancer" refers to the effect of a moiety or agent, which reduces or inhibits proliferation, survival or viability of a cancer cell. Anti-cancer moieties or agents include, but are not limited to, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents and immunomodulating agents. In some embodiments, an anti-cancer agent moiety or agent is a chemotherapeutic agent.

[0073] 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.

[0074] The term "anti-inflammatory" refers to the effect of a moiety or agent, which reduces or inhibits symptoms associated with inflammation.

[0075] 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.

[0076] The term "anti-thrombotic" refers to the effect of a moiety or agent, which reduces the ability of platelets to aggregate and adhere and interact in the clot building process and hence form thrombi.

[0077] 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)). [0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 (LI), 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 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987, J. Mol. Biol. 196:901- 917). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

[0082] 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).

[0083] 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.

[0084] The term "conjugate" as used herein refers to a covalent or non-covalent association of an antigen-binding molecule or chimeric molecule of the present disclosure and another molecule or payload regardless of the method of association.

[0085] 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

[0086] 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

[0087] 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.

[0088] 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.

[0089] 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 present 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.

[0090] 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.

[0091] 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).

[0092] "Detectable moiety" as used herein refer to a moiety attached to or otherwise associated with an antigen-binding molecule disclosed herein to render the reaction between the antigen-binding molecule and the analyte to which it binds detectable. A detectable moiety can produce a signal that is detectable by visual or instrumental means. Various detectable moieties include signal-producing substances, such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, metal- containing nanoparticles and the like.

[0093] 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.

[0094] The term "embolus" (plural "emboli"), as used herein, refers to a gaseous or particulate matter that acts as a traveling "clot". 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.

[0095] 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.

[0096] 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 ai., (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)).

[0097] As used herein, the terms "function", "functional" and the like refer to a ligand-binding, multimerizing, activating, signaling, biologic, pathologic or therapeutic function.

[0098] "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.

[0099] "Glycoprotein Ilb/IIIa" (GPIIb/IIIa, also known as integrin allb83 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.

[0100] 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, 17, 22 and 68-71.

[0101] 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.

[0102] 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, NSO 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.

[0103] 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 antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0104] 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.

[0105] The term "immunomodulating agent" refers to a chemical or biological substance that can enhance an immune response of a mammal. Immunomodulating agents include a diverse array of recombinant, synthetic and natural preparations, examples of which include, but are not limited to, interleukins such as IL-2, IL-7, IL-12; cytokines such as granulocyte colony-stimulating factor (G-CSF), interferons; various chemokines such as CXCL13, CCL26, CXCL7; antagonists of immune checkpoint blockades such as anti-CTLA-4, anti-PD1 or anti-PD-L1 (ligand of PD-1), anti-LAG3, anti-B7-H3, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans; and modulators of regulatory T cells (Tregs) such as cyclophosphamide.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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).

[0110] 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.

[0111] 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).

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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. [0116] 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.

[0117] The term "payload" as used herein refers to any agent that can be conjugated to the antigen-binding molecule or chimeric molecule of the present disclosure.

The payload can be selected from a label, a dye, a polymer, a water-soluble polymer, polyethylene glycol, a derivative of polyethylene glycol, a photocrosslinker, a cytotoxic compound, a radionuclide, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a

polynucleotide, a DNA, a RNA, an antisense polynucleotide, a peptide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a heterologous functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a

photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, or any combination thereof. In an embodiment, the payload is a label, a dye, a polymer, a cytotoxic compound, a radionuclide, a drug, an affinity label, a resin, a protein, a polypeptide, a polypeptide analog, an antibody, antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, a peptide, a fluorophore, or a carbon-linked sugar. In another embodiment, the payload is a label, a dye, a polymer, a drug, an antibody, antibody fragment, a DNA, an

RNA, or a peptide. (e.g., protein, peptide, small molecule, drug or composition of matter) for delivery to a target cell, tissue or microenvironment. In certain embodiments, the payload has a biological, diagnostic, pharmacological or therapeutic activity or beneficial effect that can be demonstrated in an in vitro assay or when administered to a subject, representative examples of which include cytokines, enzymes, hormones, blood coagulation factors, and growth factors, antiviral compounds, toxins, anti-cancer agents, anti-inflammatory agents, chemotherapeutic agents, immunomodulating agents, cytotoxic drugs, radioactive compounds, tracers and contrast agents.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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/CL, 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.

[0122] 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, Ile, 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. [0123] "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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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 at., 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.

[0129] 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 [0130] 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 detecting an activated platelet, thrombus and/or embolus, detecting presence of a tumor, 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, treating or inhibiting the development of a hematologic disorder, reducing or inhibiting proliferation, survival or viability of a tumor, and/or treating or inhibiting the development of a cancer. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0131] The term "therapeutic moiety" as used herein refers to an atom, molecule, or compound that confers a therapeutic benefit (e.g., prevention, eradication, amelioration of the underlying disorder being treated). The term "therapeutic moiety" includes within its scope proteinaceous molecules (e.g., peptides, polypeptides, lipoproteins, glycoproteins), nucleic acids, carbohydrates and small molecules. Therapeutic moieties include, but are not limited to, antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNA (siRNA), chelators, boron compounds, photoactive agents, dyes, and radioisotopes. In various embodiments, therapeutic moieties/agents such as cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes or other agents may be used as adjunct therapies to the anti- thrombotic constructs described herein. Drugs of use may, e.g., possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating,

antimetabolite, antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents and combinations thereof.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] The term "tumor," as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term "precancerous" refers to a condition or a growth that typically precedes or develops into a cancer. By "non-metastatic" is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer. By "early stage cancer" is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, I, or II cancer. The term "late stage cancer" generally refers to a Stage III or Stage IV cancer, but can also refer to a Stage II cancer or a substage of a Stage II cancer. One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer. In an exemplary embodiment, the cancer is selected from prostate, lung, pancreatic, breast, ovarian and bone cancer.

[0136] 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.

[0137] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0138] The following abbreviations are used throughout the application:

2. Abbreviations

3. GPIIb/IIIa-specific antigen-binding molecules

[0139] The present disclosure features antigen-binding molecules 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. These antigen-binding molecules suitably antagonize a function of activated GPIIb/IIIa, including inhibiting or reducing binding of activated GPIIb/IIIa to a GPIIb/IIIa ligand such as fibrinogen. These antagonist antigen binding molecules can be used alone, or in combination with other agents, in a range of applications including in the treatment or prophylaxis of conditions associated with activated platelets, representative examples of which include thromboembolism-associated conditions, hematologic disorders and cancers.

[0140] In specific embodiments, the antigen-binding molecules disclosed herein comprise: (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] ;

EVQLVESGGGLVQPGGSLRLSCAASGFMFSRYAMSWVRQAPGKGPEWVSGISGSGGSTYYADS

VKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARIFTHRSRGDVPDQTSFDYWGQGTLVTVSS

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

SELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKPGQAPTLVIYGLSKRPSGIPDRFSASSS GNTASLTITGAQAEDEADYYCLLYYGGGQQGVFGGGTKLTVL [SEQ ID NO: 2] and

[0141] 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.

[0142] 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).

[0143] 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 C_H1 domains; a Fab' fragment consisting of V_L, V_H, CL and C_H1 domains, as well as a portion of a C_H2 domain; an Fd fragment consisting of V_H and C_H1 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).

[0144] 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.

[0145] In some embodiments, the linker sequences used in scFvs are multimers of the pentapeptide GGGGS [SEQ ID NO: 66] (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: 67] (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.

[0146] 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).

[0147] In some embodiments, the anti-GPIIb/IIIa scFv molecule comprises CDR sequences derived 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

[0148] 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 68.

[0149] In some embodiments, the anti-GPIIb/IIIa scFv comprises or consists essentially of the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFMFSRYAMSWVRQAPGKGPEWVSGISGSGGSTYYAD SVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARIFTHRSRGDVPDQTSFDYWGQGTLVTV SSXiseltqdpavsvalgqtvritcqgdslrnfyaswyqqkpgqaptlviyglskrpsgipdrfsasssgntasltitgaqa edeadyycllyygggqqgvfgggtkltvlX₂ [SEQ ID NO:9],

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.

[0150] In non-limiting examples, the anti-GPIIb/IIIa scFv may comprise 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;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker; and · Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE.

[0151] In some embodiments, the anti-GPIIb/IIIa scFv comprises or consists essentially of the following amino acid sequence:

wherein :

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

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

[0152] In an illustrative example of this type, the anti-GPIIb/IIIa scFv may comprise or consists essentially of the following amino acid sequence:

wherein :

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker; and

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

[0153] In a 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

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

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

. GKPIPNPLLGLDST [SEQ ID NO: 13] is a V5 epitope tag;

• LPETGG [SEQ ID NO: 14] is a sortase conjugation tag ;

. EQKLISEEDL [SEQ ID NO: 15] is a C-myc tag ; and

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

[0154] In another specific embodiment, the anti-GPIIb/IIIa scFv comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; . GKPIPNPLLGLDST [SEQ ID NO: 13] is a V5 epitope tag;

• LPETGG [SEQ ID NO: 14] is a sortase conjugation tag ;

. EQKLISEEDL [SEQ ID NO: 15] is a C-myc tag ; and

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

[0155] In yet another specific embodiment, the anti-GPIIb/IIIa scFv 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 scFv;

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

. GKPIPNPLLGLDST [SEQ ID NO: 13] is a V5 epitope tag;

• LPETGG [SEQ ID NO: 14] is a sortase conjugation tag ;

. DYKDDDDK [SEQ ID NO: 18] is a FLAG tag ;

• ENLYFOG [SEQ ID NO: 19] is a TEV protease sequence;

. GGGGSGGGGSGGGGS [SEQ ID NO: 20] is a flexible linker;

• Lowercase regular text corresponds to a SpyCatcher tag;

. HHHHHHHH [SEQ ID NO: 21] is a His tag ; and

• Lowercase bold text corresponds to a streptavidin-binding peptide.

[0156] In still 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;

· GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

. GKPIPNPLLGLDST [SEQ ID NO: 13] is a V5 epitope tag;

• LPETGG [SEQ ID NO: 14] is a sortase conjugation tag ;

· DYKDDDDK [SEQ ID NO: 18] is a FLAG tag ;

• ENLYFOG [SEQ ID NO: 19] is a TEV protease sequence;

. GGGGSGGGGSGGGGS [SEQ ID NO: 20] is a flexible linker;

• Lowercase regular text corresponds to a SpyCatcher tag;

. HHHHHHHH [SEQ ID NO: 21] is a His tag ; and

· Lowercase bold text corresponds to a streptavidin-binding peptide.

[0157] In another specific embodiment, the anti-GPIIb/IIIa scFv comprises, consists or consists essentially of the following amino acid sequence:

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

• Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; . GKPIPNPLLGLD [SEQ ID NO: 13] is a V5 epitope tag;

• LPETGG [SEQ ID NO: 14] is a sortase conjugation tag ;

. DYKDDDDK [SEQ ID NO: 18] is a FLAG tag ;

• ENLYFOG [SEQ ID NO: 19] is a TEV protease sequence;

. GGGGS [SEQ ID NO: 23] is a flexible linker;

• Lowercase regular text corresponds to a Spy tag;

. HHHHHHHH [SEQ ID NO: 21] is a His tag ; and

• Lowercase bold text corresponds to a streptavidin-binding peptide.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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).

[0162] 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.

[0163] 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: l (SE V_H sequence), a C_H1 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 C_L1 sequence. In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule further comprises (c) a third polypeptide comprising a second Fc polypeptide.

[0164] 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 CL 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.

[0165] In non-limiting examples:

[0166] 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.

[0167] The second construct comprises a V_L of SE directly fused in tandem with C_L of atezolizumab and comprises or consists essentially of 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.

[0168] The third construct comprises a 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 has 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 FcgR and C1q interactions and the T366W "knob'"' substitution are in bold uppercase text.

[0169] 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 FcgR and C1q interactions.

[0170] 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

[0171] 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;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] 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

[0172] 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.

[0173] 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/C_L, 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.

[0174] 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).

[0175] 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.

[0176] 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.

[0177] 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). Reducing the linker length to below three residues can force single chain antibody fragments into the present disclosure allowing the bispecific antibody to become bivalent, trivalent, or tetravalent, as desired. [0178] Representative peptide linkers may be selected from: [AAA]_n, [8GGGG]_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.

[0179] 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.

[0180] 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.

[0181] 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 2) 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.

[0182] 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 pairings, forming two epitope binding sites, i.e., a bivalent molecule. Neither the V 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.

[0183] 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_H2 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)).

[0184] 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.

[0185] In one 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 E-coil, a linker that suitably comprises a flexible linker interposed between the V_L and V_H sequences and an optional 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 :

. GGGSGGGG [SEQ ID NO: 30] 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.

[0186] 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 an optional 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 or consists essentially of the following amino acid sequence:

wherein :

. GGGSGGGG [SEQ ID NO: 30] 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.

[0187] 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 an optional 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 or consists essentially of the following amino acid sequence:

wherein :

. GGGSGGGG [SEQ ID NO: 30] is a flexible linker;

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

[0188] 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 an optional 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 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;

. GGGSGGGG [SEQ ID NO: 30] 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.

[0189] 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 this construct comprises or consists essentially of the following amino acid sequence:

wherein :

• AAA is a flexible linker;

. GGGSGGGG [SEQ ID NO: 30] is a flexible linker;

• Uppercase regular text corresponds to variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa SE scFv;

• LGGC [SEQ ID NO: 35] 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.

4. Chimeric molecules

[0190] The present disclosure also provides chimeric molecules that comprise an anti-GPIIb/IIIa antigen-binding molecule described herein and at least one heterologous moiety. The heterologous moiety may comprise a payload. In some embodiments, heterologous moieties are selected from detectable moieties, half-life extending moieties and therapeutic moieties (e.g., anti-inflammatory moieties, immunomodulating moieties, anti- cancer moieties, etc.). 4.1 Detectable moieties

[0191] Detectable moieties contemplated by the present disclosure include for example any species known in the art that is appropriate for diagnostic detection, including in vitro detection and in vivo imaging. The detectable moiety may be, for example, a fluorophore, a radionuclide reporter, a metal-containing nanoparticle or microparticle, an ultrasound contrast agent (e.g., a nanobubble or microbubble) or an optical imaging dye. This also includes contrast particles visible in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Fluorophores can be detected and/or imaged, for example, by fluorescence polarization, fluorescence-activated cell sorting and fluorescence microscopy, which may or may not be in combination with electrospray ionization-mass spectrometry (ESI-MS) detection, as well as fluorescence emission computed tomography (FLECT) imaging. Radionuclide reporters can be detected and imaged by radionuclide (nuclear) detection, such as, for example, single-photon emission computed tomography (SPECT), positron emission tomography (PET) or scintigraphic imaging. Metal-containing nanoparticles or microparticles may be detected using optical imaging, including MRI, which is typically used with paramagnetic nanoparticles or microparticles, and MPI, which is generally used with superparamagnetic particles. Ultrasound contrast agents can be detected using ultrasound imaging including contrast-enhanced ultrasound (CEU),

[0192] In general, the anti-GPIIb/IIIa antigen-binding molecules of the present disclosure can be bound directly or covalently to the detectable moiety, or it can be coupled or conjugated to the detectable moiety using a linker, which can be, without limitation, amide, urea, acetal, ketal, double ester, carbonyl, carbamate, thiourea, sulfone, thioester, ester, ether, disulfide, lactone, imine, phosphoryl, or phosphodiester linkages; substituted or unsubstituted saturated or unsaturated alkyl chains; linear, branched, or cyclic amino acid chains of a single amino acid or different amino acids (e.g., extensions of the N- or C- terminus of the anti-GPIIb/IIIa antigen-binding molecule) ; derivatized or underivatized polyethylene glycols (PEGs), polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted polyamide chains; derivatized or underivatized polyamine, polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol), polyglycerol, or oligosaccharide (e.g., dextran) chains; alternating block copolymers; malonic, succinic, glutaric, adipic and pimelic acids; caproic acid; simple diamines and dialcohols; any of the other linkers disclosed herein; or any other simple polymeric linkers known in the art (see, for example, WO 98/18497 and WO 98/18496). Preferably the molecular weight of the linker can be tightly controlled. The molecular weights can range in size from less than 100 to greater than 1000. Preferably the molecular weight of the linker is less than 100. In addition, it can be desirable in some embodiments to utilize a linker that is biodegradable in vivo to provide efficient routes of excretion for the imaging reagents of the present disclosure. Depending on their location within the linker, such biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities.

4.1.1 Fluorophores

[0193] In some embodiments, the detectable moiety is a fluorophore (e.g., an organic fluorophore). The fluorophore can be, for example, a charged ((i.e., ionic) molecule ( e.g ., sulfonate or ammonium groups), uncharged (i.e., neutral) molecule, saturated molecule, unsaturated molecule, cyclic molecule, bicyclic molecule, tricyclic molecule, polycyclic molecule, acyclic molecule, aromatic molecule, and/or heterocyclic molecule (i.e., by being ring-substituted by one or more heteroatoms selected from, for example, nitrogen, oxygen and sulfur). The unsaturated fluorophores may contain one or more carbon-carbon and/or carbon-nitrogen double and/or triple bonds. In some embodiments, the fluorophore is a fused polycyclic aromatic hydrocarbon (PAH) containing at least two, three, four, five, or six rings (e.g., naphthalene, pyrene, anthracene, chrysene, triphenylene, tetracene, azulene, and phenanthrene) wherein the PAH can be optionally ring-substituted or derivatized by one, two, three or more heteroatoms or heteroatom-containing groups.

[0194] The fluorophore may also be a xanthene derivative, such as fluorescein, rhodamine, or eosin; cyanine, or its derivatives or subclasses, such as the streptocyanines, hemicyanines, closed chain cyanines, phycocyanins, allophycocyanins, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanins, and phthalocyanines; naphthalene derivatives, such as the dansyl and prodan derivatives; coumarin and its derivatives;

oxadiazole and its derivatives, such as the pyridyloxazoles, nitrobenzoxadiazoles, and benzoxadiazoles; pyrene and its derivatives; oxazine and its derivatives, such as Nile Red, Nile Blue, and cresyl violet; acridine derivatives, such as proflavin, acridine orange, and acridine yellow; arylmethine derivatives, such as auramine, crystal violet, and malachite green; and the tetrapyrrole derivatives, such as the porphyrins and bilirubins. Some examples of such dyes include the Cy family of dyes (e.g., Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7), the Alexa family of dyes, the ATTO family of dyes, and the Dy family of dyes. The fluorophore may also be a dipyrromethene dye, such as boron-dipyrromethene (BODIPY). The fluorophore may alternatively be an inorganic type of fluorophore, such as a quantum dot nanoparticle. Some examples of quantum dot compositions include sulfides, selenides, and tellurides of gallium, indium, or cadmium.

[0195] In specific embodiments, the anti-GPIIb/IIIa antigen-binding molecule described herein is labeled with a fluorophore (e.g., a fluorescent dye such as a Cy dye) and imaged using a fluorescence imaging system (e.g., fluorescence emission computed tomography; "FLECT") to image activated platelets in vivo, as described for example in Yap et al. (2017. Theranostics 7(10) : 2565-2574; 2019. Theranostics 9(4) : 1154-1169).

4.1.2 Metal-containing particles

[0196] The detectable moiety may be a metal-containing nanoparticle or microparticle. The metal in the metal-containing nanoparticle or microparticle may be in its zerovalent state, which is typically a noble metal. In illustrative examples of this type, the metal-containing nanoparticle or microparticle includes or is completely composed of, for example, gold, silver, palladium, or platinum. Such particles are particularly suited for detection and imaging by microscopy or surface plasmon resonance (SPR) spectroscopy.

[0197] In other embodiments, the anti-GPIIb/IIIa antigen-binding molecules described herein can advantageously be conjugated with a paramagnetic metal chelate in order to form a contrast agent for use in MRI. In representative examples of this type, the metal in the metal-containing nanoparticle or microparticle is a paramagnetic metal ion. Exemplary paramagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83, which includes, for example, the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton. Non-limiting examples of paramagnetic metal ions include chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III), chromium (III), iron (III), and gadolinium (III).

[0198] The paramagnetic metal ions are typically conjugated with or without a linker to a paramagnetic metal chelator. Paramagnetic metal chelators are a molecules having one or more polar groups that act as a ligand for, and complex with, a paramagnetic metal ion. Suitable chelators are known in the art and include, for example, acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups,

carboxyethylidene groups, or carboxymethylene groups. Representative examples of chelators include diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10- tetraazacyclotetradecane- 1,4,7,10-tetraacetic acid (DOTA), 1-substituted 1,4,7- tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-CI-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (0 and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is 1,4,7-triazacyclononane N,N',N''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7, 10-tetraazacyclotetradecane- 1,4,7, 10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11- tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid); derivatives of 1,3-propylene- diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTNA); derivatives of 1,5,10-N,N',N"-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and 1,3,5-N,N',N"- tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). In specific embodiments, the chelator is DTPA or DO3A. Illustrative examples of representative chelators and chelating groups contemplated by the present disclosure are described in WO 98/18496, WO

86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274.

[0199] In general, known methods can be used to couple the metal chelate and the anti-GPIIb/IIIa antigen-binding molecule using such linkers (WO 95/28967, WO

98/18496, WO 98/18497 and discussion therein). The anti-GPIIb/IIIa antigen-binding molecules can be linked through an N- or C-terminus via an amide bond, for example, to a metal coordinating backbone nitrogen of a metal chelate or to an acetate arm of the metal chelate itself. The present disclosure contemplates linking of the chelate on any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity.

[0200] MRI contrast reagents prepared according to the disclosure herein can be used in the same manner as conventional MRI contrast reagents. Certain MR techniques and pulse sequences can be preferred to enhance the contrast of the site to the background blood and tissues. These techniques include (but are not limited to), for example, black blood angiography sequences that seek to make blood dark, such as fast spin echo sequences (Alexander et al., 1998. Magn. Reson. Med. 40: 298-310) and flow-spoiled gradient echo sequences (Edelman et al., 1990. Radiology 177: 45-50). These methods also include flow independent techniques that enhance the difference in contrast, such as inversion -recovery prepared or saturation-recovery prepared sequences that will increase the contrast between activated GPIIb/IIIa-expressing platelets, thrombi or emboli and background tissues. Finally, magnetization transfer preparations also can improve contrast with these agents (Goodrich et al., 1996. Invest. Radia 31 : 323-32).

[0201] The contrast agent is administered to the patient in the form of an injectable composition. The method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarily. For imaging activated GPIIb/IIIa-expressing platelets, thrombi or emboli, or tumors, intravenous or intraarterial administration is preferred. For MRI, it is contemplated that the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the site of activated GPIIb/IIIa expression by at least 10%. After injection with the activated GPIIb/IIIa imaging agent containing MRI reagent, the patient is scanned in the MRI machine to determine the location of any sites of activated GPIIb/IIIa expression. In therapeutic settings, upon identification of a site of activated GPIIb/IIIa expression (e.g., fluid or tissue), a therapeutic agent (as described, for example, in Section 4.3 and Section 6, infra) can be administered, if necessary, and the patient can be subsequently scanned to visualize the presence of activated GPIIb/IIIa-expressing platelets, thrombi or emboli, or tumors.

[0202] In still other embodiments, the anti-GPIIb/IIIa antigen-binding molecules described herein can be conjugated with a superparamagnetic particle in order to form a tracer for use in MPI. In representative examples of this type, the superparamagnetic particle is a superparamagnetic iron oxide (SPIO) nanoparticle or an ultra-small

superparamagnetic iron oxide (USPIO) nanoparticle. The SPIO or USPIO nanoparticle may be doped with magnesium, zinc, manganese, nickle, cobalt, cadmium, gold, platinum, silver or the combination thereof. The SPIO or USPIO nanoparticle may comprise maghemite (y- Fe₂O₃) or magnetite (Fe₃O₄), or nanoparticles composed of both phases. Superparamagnetic particles can be synthesized with a suitable method and dispersed as a colloidal solution in organic solvents or water. Methods to synthesize the superparamagnetic iron oxide nanoparticles particles are known in the art (see, for example, Morteza Mahmoudi et al., Superparamagnetic Iron Oxide Nanoparticles: Synthesis, Surface Engineering, Cytotoxicity and Biomedical Applications, published by Nova Science Pub Inc, 2011). In some embodiments, the superparamagnetic iron oxide nanoparticles can be made through wet chemical synthesis methods which involve co-precipitation of Fe and Fe salts in the presence of an alkaline medium. During the synthesis, nitrogen may be introduced to control oxidation, surfactants and suitable polymers may be added to inhibit agglomeration or control particle size, and/or emulsions (such as water-in-oil microemulsions) may be used to modulate the physical properties of the superparamagnetic iron oxide nanoparticle (see, for example, Jonathan W. Gunn, The preparation and characterization of superparamagnetic nanoparticles for biomedical imaging and therapeutic application, published by ProQuest, 2008). In other embodiments, the superparamagnetic iron oxide nanoparticles can be generated by thermal decomposition of iron pentacarbonyl, alone or in combination with transition metal carbonyls, optionally in the presence of one or more surfactants (e.g., lauric acid and oleic acid) and/or oxidants (e.g., trimethylamine-N-oxide), and in a suitable solvent (e.g., dioctyl ether or hexadecane) (see, for example, US 2006/0093555).

Superparamagnetic iron oxide nanoparticles can also be made through gas deposition methods, which involves laser vaporization of iron in a helium atmosphere containing different concentrations of oxygen (see, Miller J. S. et al., Magnetism: Nanosized magnetic materials, published by Wiley-VCH, 2002). In certain embodiments, the superparamagnetic iron oxide nanoparticles are those disclosed in US 2010/0008862. Superparamagnetic iron oxide are also available commercially and include, for example, FERAHEME (also known as ferumoxytol), FERIDEX I.V. (also known as ENDOREM and ferumoxides), RESOVIST (also known as CLIAVIST), SINEREM (also known as COMBIDEX), LUMIREM (also known as GASTROMARK), and C LARI SCAN (also known as PEG-FERO, Feruglose, and NC100150).

[0203] In general, known methods can be used to couple the anti-GPIIb/IIIa antigen-binding molecule to a superparamagnetic particle, which include for example, functionalizing the particle with amine, aldehyde, hydroxyl or carboxyl groups and covalently coupling the antigen-binding molecule to the functionalized particle. Representative methods for such couplings are disclosed in Arruebo et al. (2007. Adv Funct Mater. 17: 1473-1479), Arruebo et at. (2009. J Nanomat. Article ID 438839), Shamsipour et at. (2009. Avicenna J Med Biotech 1(1) : 27-31), Chapa Gonzalez et al. (2014. J Nanomat. Article ID 978284) and Mu et al. (2015. Mol Imaging May 2015: 1-12).

[0204] Superparamagnetic particles can be imaged using MPI, which is a tomographic or volumetric imaging technique that directly detects the magnetization from such particles. The basic principle of MPI involves applying a magnetic field to the superparamagnetic particles in a selected region (e.g., superparamagnetic particles injected into the blood stream or labeled into or on cells) and detecting the magnetic fields generated by the superparamagnetic particles. Similar to tomographic reconstruction, the data acquired from MPI can be processed using algorithms to produce images of the superparamagnetic particles in the sample or subject.

[0205] Superparamagnetic particles are typically administered to a subject in the form of an injectable composition. The method of administering the superparamagnetic particles is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarily. For imaging activated GPIIb/IIIa-expressing platelets, thrombi or emboli, or tumors, intravenous or intraarterial administration is preferred. After injection with the activated GPIIb/IIIa imaging agent containing superparamagnetic particles, the subject is scanned in an MPI machine to determine the location of any sites of activated GPIIb/IIIa expression. In therapeutic settings, upon identification of a site of activated

GPIIb/IIIa expression (e.g., fluid or tissue), a therapeutic agent (as described, for example, in Section 4.3 or Section 6 infra) can be administered, if necessary, and the patient can be subsequently scanned to visualize the presence of activated GPIIb/IIIa-expressing platelets, thrombi or emboli, or tumors.

4.1.3 Radionuclide reporters

[0206] The anti-GPIIb/IIIa antigen-binding molecules of the disclosure can be conjugated with a radionuclide reporter appropriate for scintigraphy, SPECT, or PET imaging and/or with a radionuclide appropriate for radiotherapy. Constructs in which the anti- GPIIb/IIIa antigen-binding molecules are conjugated with both a chelator for a radionuclide useful for diagnostic imaging and a chelator useful for radiotherapy are within the scope of the present disclosure.

[0207] For use as a PET agent, a disclosed imaging agent may be complexed with one of the various positron emitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As, ⁹⁴mTc, or ¹¹⁰In. The antigen-binding molecules of the present disclosure can also be labeled by halogenation using radionuclides such as ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides for scintigraphy or radiotherapy include "mTc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ^min, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu,

⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹⁷mSn, ¹⁴⁹Pm,

¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, "mTc, and ^{i n}In. For therapeutic purposes, the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ^{i n}In, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Ln, ^{186/ 188}Re, a nd ¹⁹⁹Au. ^99mTc is usefu l for diagnostic application because of its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of ^99mTc make this isotope an ideal scintigraphic imaging agent. This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from a ⁹⁹Mo-^99mTc generator. ¹⁸F, 4-[¹⁸F]fluorobenzaldehyde ¹⁸FB), AI[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are typical radionuclides for conjugation to anti-GPIIb/IIIa antigen-binding molecules of the disclosure for diagnostic imaging.

[0208] The metal radionuclides may be chelated, for example, by linear, macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S. Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, DO3A, TETA, NOTA and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No. 5,720,934). For example, N.sub.4 chelators are described in U.S. Pat. No. 6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No. 5,608,110; U.S. Pat. No. 5,665,329; U.S. Pat. No.

5,656,254; and U.S. Pat. No. 5,688,487. Certain N. sub.35 chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495; and U.S. Pat. No. 5,780,006. The chelator also can include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N₃S, and N2S2 systems such as MAMA (monoamidemonoaminedithiols), DADS (N2S

diaminedithiols), CODADS and the like. These ligand systems and a variety of others are described in, for example, Liu, S, and Edwards, D., 1999. Chem. Rev., 99:2235-2268, and references therein.

[0209] The chelator also can include complexes containing ligand atoms that are not donated to the metal in a tetradentate array. These include the boronic acid adducts of technetium and rhenium dioximes, such as are described in U.S. Pat. No. 5,183,653; U.S.

Pat. No. 5,387,409; and U.S. Pat. No. 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety.

[0210] The chelators can be covalently linked directly to the anti-GPIIb/IIIa antigen-binding molecule via a linker, as described previously, and then directly labeled with the radioactive metal of choice (see, WO 98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261).

[0211] Anti-GPIIb/IIIa antigen-binding molecules comprising ¹⁸F, 4- [¹⁸F]fluorobenzaldehyde (¹⁸F FB), AI[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are of preferred interest for diagnostic imaging. Complexes of radioactive technetium are also useful for diagnostic imaging, and complexes of radioactive rhenium are particularly useful for radiotherapy. In forming a complex of radioactive technetium with the reagents of the present disclosure, the technetium complex, preferably a salt of ^99mTc pertechnetate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride. Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the disclosure to be labeled and a sufficient amount of reducing agent to label the reagent with ^99mTc. Alternatively, the complex can be formed by reacting an antigen-binding molecule of the present disclosure, which is conjugated with an appropriate chelator, with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex can be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among the ^99mTc pertechnetate salts useful with the present disclosure are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.

[0212] Preparation of the complexes of the present disclosure where the metal is radioactive rhenium can be accomplished using rhenium starting materials in the +5 or +7 oxidation state. Examples of compounds in which rhenium is in the Re(VII) state are NH₄ReO₄ or KReO₄ Re(V) is available as, for example, [ReOCI₄](NBu₄), [ReOCI₄](AsPh₄), ReOC_L3(PPh₃)2 and as ReO₂(pyridine)⁴⁺, where Ph is phenyl and Bu is n-butyl. Other rhenium reagents capable of forming a rhenium complex also can be used. [0213] Radioactively labeled PET, SPECT, or scintigraphic imaging agents provided by the present disclosure are encompassed having a suitable amount of radioactivity. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL. It is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 mCi to 100 mCi per mL.

[0214] Typical doses of a radionuclide-labeled anti-GPIIb/IIIa antigen-binding molecule according to the disclosure provide 10-20 mCi. After injection of the radionuclide- labeled anti-GPIIb/IIIa antigen-binding molecules into the patient, a gamma camera calibrated for the gamma ray energy of the nuclide incorporated in the imaging agent is used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site. Imaging of the site in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after the radiolabeled peptide is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos.

[0215] Proper dose schedules for the radiotherapeutic compounds of the present disclosure are known to those skilled in the art. The compounds can be administered using many methods including, but not limited to, a single or multiple IV or IP injections, using a quantity of radioactivity that is sufficient to cause damage or ablation of the targeted activated GPIIb/IIIa-expressing platelets, thrombi or emboli, but not so much that substantive damage is caused to non-target (normal tissue). The quantity and dose required is different for different constructs, depending on the energy and half-life of the isotope used, the degree of uptake and clearance of the agent from the body and the mass of the activated GPIIb/IIIa-expressing platelets, thrombi or emboli. In general, doses can range from a single dose of about 30-50 mCi to a cumulative dose of up to about 3 Ci.

[0216] The radiotherapeutic compositions of the present disclosure can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection. Radiation stabilizers are known to those skilled in the art, and can include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.

[0217] A single, or multi-vial kit that contains all of the components needed to prepare the complexes disclosed herein, other than the radionuclide, is an integral part of this disclosure.

[0218] A single-vial kit preferably contains a chelating ligand, a source of stannous salt, or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9. The quantity and type of reducing agent used would depend on the nature of the exchange complex to be formed. The proper conditions are well known to those that are skilled in the art. It is preferred that the kit contents be in lyophilized form. Such a single vial kit can optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or a, b, or y cyclodextrin that serve to improve the radiochemical purity and stability of the final product. The kit also can contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.

[0219] A multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical. For example, one vial can contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent). Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the ligand, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present disclosure are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized. As above, reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. can be present in either or both vials.

[0220] Also provided herein is a method to incorporate an ¹⁸F radiolabeled prosthetic group onto an anti-GPIIb/IIIa antigen-binding molecule. In one embodiment, 4- [¹⁸F]fluorobenzaldehyde (¹⁸FB) is conjugated onto an imaging agent bearing an aminooxy moiety, resulting in oxime formation. In another embodiment, [¹⁸F]fluorobenzaldehyde is conjugated onto an imaging agent bearing an acyl hydrazide moiety, resulting in a hydrazone adduct. 4-Fluorobenzaldehyde, can be prepared in ¹⁸F form by displacement of a leaving group, using ¹⁸F ion, by known methods.

[0221] ¹⁸F-labeled imaging agents can also be prepared from imaging agents possessing thiosemicarbazide moieties under conditions that promote formation of a thiosemicarbazone, or by use of a ¹⁸F-labeled aldehyde bisulfite addition complex.

[0222] The above methods are particularly amenable to the labeling of imaging agents, e.g., the imaging agents described herein, which can be modified during synthesis to contain a nucleophilic hydroxylamine, thiosemicarbazide or hydrazine (or acyl hydrazide) moiety that can be used to react with the labeled aldehyde. The methods can be used for any imaging agent that can accommodate a suitable nucleophilic moiety. Typically the nucleophilic moiety is appended to the N-terminus of the peptide, but the skilled artisan will recognize that the nucleophile also can be linked to an amino acid side chain or to the peptide C-terminus. Methods of synthesizing a radiolabeled peptide sequence are provided in which 4-[¹⁸F]fluorobenzaldehyde is reacted with a peptide sequence comprising either a hydroxylamine, a thiosemicarbazide or a hydrazine (or acyl hydrazide) group, thereby forming the corresponding oximes, thiosemicarbazones or hydrazones, respectively. The 4- [¹⁸F]fluorobenzaldehyde typically is generated in situ by the acid-catalyzed decomposition of the addition complex of 4-[¹⁸F]fluorobenzaldehyde and sodium bisulfite. The use of the bisulfite addition complex enhances the speed of purification since, unlike the aldehyde, the complex can be concentrated to dryness. Formation of the complex is also reversible under acidic and basic conditions. In particular, when the complex is contacted with a peptide containing a hydroxylamine, a thiosemicarbazide or a hydrazine (or acyl hydrazide) group in acidic medium, the reactive free 4-[¹⁸F]fluorobenzaldehyde is consumed as it is formed in situ, resulting in the corresponding ¹⁸F radiolabeled polypeptide sequence.

[0223] In the instances when the oxime, thiosemicarbazone or hydrazone linkages present in vivo instability, an additional reduction step may be employed to reduce the double bond connecting the peptide to the ¹⁸F bearing substrate. The corresponding reduced peptide linkage would enhance the stability. One of skill in the art would appreciate the variety of methods available to carry out such a reduction step. Reductive amination steps as described in Wilson et ai. (1990. Journal of Labeled Compounds and

Radiopharmaceuticals XXVIII (10), 1189-1199) may also be used to form a Schiffs base involving a peptide and 4-[¹⁸F]fluorobenzaldehyde and directly reducing the Schiff's base using reducing agents such as sodium cyanoborohydride.

[0224] The 4-[¹⁸F]fluorobenzaldehyde may be prepared as described in Wilson etal. (1990, supra), Iwata et ai. (2000, Applied Radiation and Isotopes 52, 87-92); Poethko etal. (2004. The Journal of Nuclear Medicine 45, 892-902), and Schottelius et al. (2004.

Clinical Cancer Research 10, 3593-3606). The Na¹⁸F in water may be added to a mixture of Kryptofix and K₂CO₃. Anhydrous acetonitrile may be added and the solution is evaporated in a heating block under a stream of argon. Additional portions of acetonitrile may be added and evaporated to completely dry the sample. The 4-trimethylammoniumbenzaldehyde triflate may be dissolved in DMSO and added to the dried F-18. The solution may then be heated in the heating block. The solution may be cooled briefly, diluted with water and filtered through a Waters. Oasis HLB LP extraction cartridge. The cartridge may be washed with 9: 1 water: acetonitrile and water to remove unbound ¹⁸F and unreacted 4- trimethylammoniumbenzaldehyde triflate. The 4-[¹⁸F]fluorobenzaldehyde may then be eluted from the cartridge with methanol in fractions.

4.1.4 Ultrasound contrast agents

[0225] The anti-GPIIb/IIIa antigen-binding molecules of the disclosure can be conjugated with an ultrasound contrast agent (UCA) for ultrasound imaging. Constructs in which the anti-GPIIb/IIIa antigen-binding molecules are conjugated with a UCA are within the scope of the present disclosure.

[0226] UCAs are suitably imaged using contrast-enhanced ultrasound (CEU) molecular imaging, which is a technique that relies on the ultrasound detection of encapsulated gas microbubbles (MBs) or nanobubbles (NBs) or other acoustically active micro- or nanoparticles that are retained in tissue on the basis of their ability to bind to molecules or cells of interest. CEU has several unique characteristics that differentiate it from other molecular imaging techniques. It relies on receiving signal generated by encapsulated gas MBs or NBs when exposed to ultrasound. In the pressure fluctuations of an ultrasound field, MBs and NBs undergo volumetric oscillation. Steady expansion and contraction (stable cavitation) occurs at low pressure which can be non-linear with regard to the relationship between pressure and volume. Destruction of MBs or NPs can occur from exaggerated oscillation at high pressures, known as inertial cavitation. Both inertial cavitation and non linear stable cavitation result in emission of broad band ultrasound signals and ultrasound energy peaks at harmonic frequencies, thereby producing a unique acoustic signature that can be detected and isolated from background tissue signal. Exemplary reviews describing UCAs in ultrasound imaging applications can be found for example in Brown et al. (2019, Current Cardiology Reports 21(5) :30), Guvener et al. ( Methods 130:4-13) and Wang et al. (2017, Arterioscler Thromb Vase Biol. 37: 1029-1040).

[0227] Conventional MB agents are typically between 1 and 10 mm in diameter, have a core composed of air or high-molecular weight inert gas such as perfluorocarbons or sulfur hexafluoride, and a shell composed of lipids, polymers, surfactants or proteins, or mixtures thereof. Illustrative MBs are described for example by Luan et a/. (2012, Ultrasound Med Biol. 38(12) :2174-2185), Kooiman et al. (2008, J Control Release 133: 109- 118), Kothapalli et al. (2015, IEEE Trans Ultrason Ferroelectr Freq Control. 62(3) :451-462) and Emmer et al. (2013, IEEE Trans Ultrason Ferroelectr Freq Control. 60(l) :7-20).

[0228] NB agents are smaller than MBs, typically between 200-800 nm in diameter, have a core composed of air or high-molecular weight inert gas, and a shell composed of lipids, surfactants or polymers, or mixtures thereof. Non-limiting examples of NBs are described for example by Yin et al. (2012, Int J Nanomed. 7: 895-904), Rapoport et al. (2007, J Natl Cancer Inst. 99(14) : 1095-1106), Krupka et al. (2010, Mol Pharm. 7(1) :49- 59), Hwang et al. (2009, J Pharm Sci. 98(10) :3735-3747), Temme et al. (2015, circulation 131(16) : 1405-1414) and Peyman et al. (2016, Lab Chip. 16:679-687).

[0229] Attachment of the anti-GPIIb/IIIa antigen-binding molecule described herein to MBs or NBs may be accomplished through the use of a common coupling chemistry, such avidin-biotin, maleimide-thiol or carboxylic acid-amine (see, e.g., Klibanov et al., 2005. Bioconjugate Chem. 16:9-17; Wang et al., 2012. Circulation 125:3117-3126). Alternatively, attachment of a small molecule ligand to a MB or NB can be accomplished by coupling the ligand to the lipid prior to particle formation (see, e.g., Seo et al., 2008.

Bioconjugate Chemistry 19:2577-2584).

4.2 Half-life extending moieties

[0230] In some embodiments, the chimeric molecule comprises at least one heterologous moiety that is a "half-life extending moiety". Half-life extending moieties, can comprise, for example, (i) XTEN polypeptides; (ii) Fc; (iii) albumin, (iv) albumin binding polypeptide or fatty acid, (v) the C-terminal peptide (CTP) of the 13 subunit of human chorionic gonadotropin, (vi) PAS; (vii) HAP; (viii) transferrin; (ix) polyethylene glycol (PEG); (x) hydroxyethyl starch (HES), (xi) polysialic acids (PSAs); (xii) a clearance receptor or fragment thereof which blocks binding of the chimeric molecule to a clearance receptor; (xiii) low complexity peptides; (xiv) or any combinations thereof. In some embodiments, the half- life extending moiety comprises an Fc region. In other embodiments, the half-life extending moiety comprises two Fc regions fused by a linker. Exemplary heterologous moieties also include, e.g., FcRn binding moieties (e.g., complete Fc regions or portions thereof which bind to FcRn), single chain Fc regions (scFc regions, e.g., as described in U.S. Publ. No.

20080260738, WO 2008/012543 and WO 2008/1439545), or processable scFc regions. In some embodiments, a heterologous moiety can include an attachment site for a non polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these moieties.

[0231] In certain embodiments, a chimeric molecule of the disclosure comprises at least one (e.g., one, two, three, or four) half-like extending moiety which increases the in vivo half-life of the chimeric molecule compared with the in vivo half-life of the

corresponding chimeric molecule lacking such heterologous moiety. In vivo half-life of a chimeric molecule can be determined by any method known to those of skill in the art, e.g., activity assays (chromogenic assay or one stage clotting aPTT assay), ELISA, etc. In some embodiments, the presence of one or more half-life extending moieties results in the half-life of the chimeric molecule to be increased compared to the half-life of the corresponding chimeric molecule lacking such one or more half-life extending moieties. The half-life of the chimeric molecule comprising a half-life extending moiety is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the in vivo half-life of the corresponding chimeric molecule lacking such half-life extending moiety.

4.2.1 XTEN Polypeptides

[0232] "XTEN polypeptides" or "XTEN sequences" refer to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a chimeric molecule partner, XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a targeting moiety, or any other sequences or molecules on the chimeric molecule. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antigen-binding molecules such as single-chain antigen-binding molecules or Fc fragments of a light chain or a heavy chain.

[0233] In some embodiments, the XTEN sequence of the disclosure is a peptide or a polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or a polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues. [0234] In further embodiments, the XTEN polypeptide affects the physical or chemical property, e.g., pharmacokinetics, of the chimeric molecule of the present disclosure. The XTEN sequence used in the present disclosure can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. In a specific embodiment, the XTEN polypeptide linked to an anti-GPIIb/IIIa antigen-binding molecule of the disclosure increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the chimeric molecule described herein stays in vivo for an increased period of time compared to an anti-GPIIb/IIIa antigen-binding molecule lacking the XTEM polypeptide.

[0235] A variety of methods and assays can be employed to determine the physical/chemical properties of proteins comprising the XTEN polypeptide. Such methods include, but are not limited to analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Amau et al. (2006. Prot Expr and Purif 48: 1-13).

[0236] Non-limiting examples of XTEN polypeptides that can be used according to the present disclosure are disclosed in U.S. Pat. Nos. 7,855,279 and 7,846,445, US Patent Publication Nos. 2009/0092582 Al, 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, 2011/0172146 Al, 2013/0017997 Al, or 2012/0263701 Al, International Patent Publication Nos. WO 2010091122 Al, WO

2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2; or US 2012/0178691.

4.2.2 Fc and Single Chain Fc (scFc) Region

[0237] In certain embodiments, the chimeric molecule comprises at least one heterologous moiety comprising a Fc region. "Fc" or "Fc region" as used herein means a functional neonatal Fc receptor (FcRn) binding partner comprising an Fc domain, variant, or fragment thereof, unless otherwise specified. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Thus, the term Fc includes any variants of IgG Fc that are functional. Representative examples of Fc regions are described, e.g., in PCT Publication Nos. WO2011/069164, W02012/006623, W02012/006635, or WO 2012/006633.

4.2.3 Albumins

[0238] In certain embodiments, the chimeric molecule comprises a heterologous moiety comprising albumin or a functional fragment thereof. Human serum albumin (HSA, or HA), a protein of 609 amino acids in its full-length form, is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. The term "albumin" as used herein includes full-length albumin or a functional fragment or variant, thereof. Examples of albumin or the fragments or variants thereof are disclosed in US Pat. Publ. Nos. US2008/0194481, US2008/0004206,

US2008/0161243, US2008/0261877, or US2008/0153751 or PCT Appl. Publ. Nos.

W02008/033413, W02009/058322, or W02007/021494, which are incorporated herein by reference in their entireties. An exemplary mature human albumin amino acid sequence is provide below (NCBI Ref. Sequence NP_000468) :

4.2.4 Albumin Binding Polypeptides and Lipids

[0239] In certain embodiments, a heterologous moiety can comprise an albumin binding moiety, which comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin-binding antibody fragment, or any combinations thereof. For example, the albumin binding protein can be a bacterial albumin binding protein, an antibody or an antibody fragment including domain antibodies (see, e.g., U.S. Pat. No. 6,696,245). An albumin binding protein, for example, can be a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig and Skerra, 1998. J. Immunol. Methods 218:73-83). Other examples of albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaa₁-Xaa₂- Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp, Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gin, H is, lie, Leu, or Lys; Xaa₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thr as described in U.S. Pub. No.

US2003/0069395 or Dennis et ai. (2002. J. Biol. Chem. 277:35035-35043).

[0240] Domain 3 from streptococcal protein G, as disclosed by Kraulis et al. (1996. FEBS Lett. 378: 190-194) and Linhult et al. (2002. Protein Sci. 11 :206-213) is an example of a bacterial albumin-binding domain. Examples of albumin-binding peptides include a series of peptides having the core sequence DICLPRWGCLW [SEQ ID NO:36] such as: RLIEDICLPRWGCLWEDD [SEQ ID NO:37]; QRLMEDICLPRWGCLWEDDF [SEQ ID NO:38]; QGLIGDICLPRWGCLWGDSVK [SEQ ID NO:39]; and GEWWEDICLPRWGCLWEEED [SEQ ID NO:40].

4.2.5 C-terminai peptide

[0241] In certain embodiments, a chimeric molecule disclosed herein comprises at least one heterologous moiety comprising one b subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin or fragment or variant thereof. The insertion of one or more CTP peptides into a recombinant protein is known to increase the in vivo half-life of that protein. See, e.g., U.S. Pat. No. 5,712,122. Exemplary CTP peptides include

DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL [SEQ ID NO:41] or

SSSSKAPPPSLPSPSRLPGPSDTPILPQ [SEQ ID NO:42]. See, e.g., U.S. Patent Appl. Publ. No. US 2009/0087411. In some embodiments, the chimeric molecule comprises two

heterologous moieties that are CTP sequences. In some embodiments, three of the heterologous moieties are CTP sequences. In some embodiments, four of the heterologous moieties are CTP sequences. In some embodiments, five of the heterologous moieties are CTP sequences. In some embodiments, six or more of the heterologous moieties are CTP sequences. 4.2.6 PAS

[0242] In other embodiments, at least one heterologous moiety is a PAS sequence. A PAS sequence, as used herein, means an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions. Accordingly, the PAS sequence is a building block, an amino acid polymer, or a sequence cassette comprising, consisting essentially of, or consisting of alanine, serine, and proline which can be used as a part of the heterologous moiety in the chimeric molecule.

[0243] Non-limiting examples of the PAS sequences forming random coil conformation comprise an amino acid sequence selected from the group consisting of ASPAAPAPASPAAPAPSAPA [SEQ ID NO:43], AAPASPAPAAPSAPAPAAPS [SEQ ID NO:44], APSSPSPSAPSSPSPASPSS [SEQ ID NO:45], APSSPSPSAPSSPSPASPS [SEQ ID NO:46], SSPSAPSPSSPASPSPSSPA [SEQ ID NO:47], AASPAAPSAPPAAASPAAPSAPPA [SEQ ID NO:48], and ASAAAPAAASAAASAPSAAA [SEQ ID NO:49], or any combinations thereof. Additional examples of PAS sequences are known from, e.g., US Pat. Publ. No. 2010/0292130 and PCT Appl. Publ. No. WO2008/155134 Al.

4.2.7 HAP

[0244] In certain embodiments, at least one heterologous moiety is a glycine-rich homo-amino-acid polymer (HAP). The HAP sequence can comprise a repetitive sequence of glycine, which has at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids in length. In one embodiment, the HAP sequence is capable of extending half-life of a moiety fused to or linked to the HAP sequence. Non-limiting examples of the HAP sequence includes, but are not limited to (Gly)_n, (Gly₄Ser)_n, or Ser(Gly4Ser)_n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy et al. (2007. Protein Eng. Design Selection 20:273-284).

4.2.8 Transferrin

[0245] In certain embodiments, at least one heterologous moiety is transferrin or a peptide or fragment or variant thereof. Any transferrin can be used to make the chimeric molecules of the disclosure. As an example, wild-type human TF (TF) is a 679 amino acid protein, of approximately 75 KDa (not accounting for glycosylation), with two main domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to originate from a gene duplication. N domain comprises two subdomains, N1 domain and N2 domain, and C domain comprises two subdomains, C1 domain and C2 domain. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847 and S95936

(www.ncbi.nlm.nih.gov), all of which are herein incorporated by reference in their entirety. In one embodiment, the transferrin heterologous moiety includes a transferrin splice variant. In one example, a transferrin splice variant can be a splice variant of human transferrin, e.g., GenBank Accession AAA61140. In another embodiment, the transferrin portion of the chimeric molecule includes one or more domains of the transferrin sequence, e.g., N domain, C domain, N1 domain, N2 domain, C1 domain, C2 domain or any combinations thereof.

[0246] Transferrin transports iron through transferrin receptor (TfR)-mediated endocytosis. After the iron is released into an endosomal compartment and Tf-TfR complex is recycled to cell surface, the Tf is released back extracellular space for next cycle of iron transporting. Tf possesses a long half-life that is in excess of 14-17 days (Li et al., 2002. Trends Pharmacol. Sci. 23:206-209). Transferrin fusion proteins have been studied for half- life extension, targeted deliver for cancer therapies, oral delivery and sustained activation of proinsulin (Brandsma et al., 2011. Biotechnol. Adv, 29:230-238); Bai et al., 2005. Proc. Natl. Acad. Sci. USA 102:7292-7296); Kim et al., 2010. J. Pharmacol. Exp. Ther. 334:682-692); and Wang et al., 2011. J. Controlled Release 155:386-392).

4.2.9 PEG

[0247] In some embodiments, at least one heterologous moiety is a soluble polymer known in the art, including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol. In some embodiments, the chimeric molecule comprising a PEG heterologous moiety further comprises a heterologous moiety selected from an immunoglobulin constant region or portion thereof (e.g., an Fc region), a PAS sequence, HES, and albumin, fragment, or variant thereof.

[0248] The polymer can be of any molecular weight, and can be branched or unbranched. For polyethylene glycol, in one embodiment, the molecular weight is between about 1 kDa and about 100 kDa for ease in handling and manufacturing. Other sizes can be used, depending on the desired profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a protein or analog). For example, the polyethylene glycol can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,

20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0249] In some embodiments, the polyethylene glycol can have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No.

5,643,575; Morpurgo et al. (1996. Appl. Biochem. Biotechnol. 56: 59-72); Vorobjev et al.

(1999. Nucleosides Nucleotides 18:2745-2750) ; and Caliceti et al. (1999. Bioconjug. Chem. 10:638-646).

[0250] The number of polyethylene glycol moieties attached to each chimeric molecule of the disclosure (/.e., the degree of substitution) can also vary. For example, the PEGylated chimeric molecule can be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,

15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, Il 13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al. (1992. Crit. Rev. Thera. Drug Carrier Sys. 9:249-304).

4.2.10 HES

[0251] In certain embodiments, at least one heterologous moiety is a polymer, e.g., hydroxyethyl starch (HES) or a derivative thereof. Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight. HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics (Sommermeyer et al., 1987. Krankenhauspharmazie 8(8) :271-278; and Weidler et al. 1991. Arzneim.-Forschung/Drug Res. 41 :494-498).

[0252] Amylopectin contains glucose moieties, wherein in the main chain alpha- 1,4-glycosidic bonds are present and at the branching sites alpha-1, 6-glycosidic bonds are found. The physical-chemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the nicked alpha-1, 4-glycosidic bond, helical structures with about six glucose-monomers per turn are produced. The physico-chemical as well as the biochemical properties of the polymer can be modified via substitution. The introduction of a hydroxyethyl group can be achieved via alkaline hydroxyethylation. By adapting the reaction conditions it is possible to exploit the different reactivity of the respective hydroxy group in the unsubstituted glucose monomer with respect to a hydroxyethylation. Owing to this fact, the skilled person is able to influence the substitution pattern to a limited extent.

[0253] HES is mainly characterized by the molecular weight distribution and the degree of substitution. The degree of substitution, denoted as DS, relates to the molar substitution, is known to the skilled people. See Sommermeyer et ai, 1987, as cited above, in particular p. 273.

[0254] In some embodiments, hydroxyethyl starch has a mean molecular weight (weight mean) of from 1 to 300 kDa, from 2 to 200 kDa, from 3 to 100 kDa, or from 4 to 70 kDa. Hydroxyethyl starch can further exhibit a molar degree of substitution of from 0.1 to 3, preferably 0.1 to 2, more preferred, 0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6 substitution in the range of from 2 to 20 with respect to the hydroxyethyl groups. A non-limiting example of HES having a mean molecular weight of about 130 kDa is a HES with a degree of substitution of 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferably of 0.4 to 0.7 such as 0.4, 0.5, 0.6, or 0.7.

4.2.11 PSA

[0255] In certain embodiments, at least one heterologous moiety is a polymer, e.g., polysialic acids (PSAs) or a derivative thereof. Polysialic acids (PSAs) are naturally occurring unbranched polymers of sialic acid produced by certain bacterial strains and in mammals in certain cells (see, e.g., Roth J. et al. (1993) in Polysialic Acid: From Microbes to Man, eds. Roth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp 335-348. They can be produced in various degrees of polymerization from n=about 80 or more sialic acid residues down to n=2 by limited acid hydrolysis or by digestion with neuraminidases, or by fractionation of the natural, bacterially derived forms of the polymer. The composition of different polysialic acids also varies such that there are homopolymeric forms i.e., the alpha-2, 8-linked polysialic acid comprising the capsular polysaccharide of E. coli strain K1 and the group-B meningococci, which is also found on the embryonic form of the neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms also exist--such as the alternating alpha-2,8 alpha-2,9 polysialic acid of E. coli strain K92 and group C

polysaccharides of N. meningitidis. Various methods of attaching or conjugating polysialic acids to a polypeptide have been described (for example, see U.S. Pat. No. 5,846,951; WO0187922, and US 2007/0191597).

4.2.12 Clearance Receptors

[0256] In certain embodiments, the in vivo half-life of a chimeric molecule of the present disclosure can be extended where the chimeric molecule comprises at least one heterologous molecule comprising a clearance receptor, fragment or variant thereof. In specific embodiments wherein the chimeric molecule comprises Factor X, soluble forms of clearance receptors, such as the low density lipoprotein-related protein receptor LRP1, or fragments thereof, can block binding of Factor X to clearance receptors and thereby extend its in vivo half-life.

[0257] LRP1 is a 600 kDa integral membrane protein that is implicated in the receptor-mediate clearance of a variety of proteins, such as FVIII or X. See, e.g., Narita et al. (1998. Blood 91 : 555-560); Lenting et al. (2010. Haemophilia 16:6-16). The amino acid sequence of an exemplary human LRP1 protein is set out in NCBI Reference Sequence: CAA32112) :

[0258] Other suitable clearance receptors include for example LDLR (low-density lipoprotein receptor), VLDLR (very low-density lipoprotein receptor), and megalin (LRP-2), or fragments thereof. See, e.g., Bovenschen et al. (2005. Blood 106:906-912); Bovenschen (2010. Blood 116:5439-5440); and Martinelli et al. (2010. Blood 116: 5688-5697).

4.3 Therapeutic moieties

[0259] In some embodiments, at least one heterologous moiety is a therapeutic moiety. In certain embodiments, the therapeutic moiety is selected from an anti-cancer moiety (e.g., cytostatic/toxic, and/or anti-proliferative drugs), an immunotherapeutic moiety and an anti-inflammatory moiety. In some embodiments, the therapeutic agent is useful in the treatment of cancer. Useful classes of anti-cancer agents include chemotherapeutic agents, representative examples of which include antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

[0260] Specific examples of chemotherapeutic agents include erlotinib (TARCEVA, Genentech/OSI Pharm.), bortezomib (VELCADE, Millennium Pharm.), disulfiram,

epigallocatechin gaIlate , salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX, AstraZeneca), sunitib (SUTENT, Pfizer/Sugen), letrozole (FEMARA, Novartis), imatinib mesylate (GLEEVEC, Novartis), finasunate (VATALANIB, Novartis), oxaliplatin (ELOXATIN, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE, Wyeth), Lapatinib (TYKERB, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR, Bayer Labs), gefitinib (IRESSA, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and

methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin;

callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); ad renocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a-reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid,

mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW- 2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;

antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin yll and calicheamicin w1l (Angew Chem. Inti. Ed. Engl. 1994 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as

neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN (doxorubicin), morpholino-doxorubicin,

cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside;

aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;

sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;

vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol- Myers Squibb Oncology, Princeton, N J.), ABRAXANE (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and TAXOTERE (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR

(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;

vincristine; NAVELBINE (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0261] Other examples of chemotherapeutic agents include (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE (megestrol acetate), AROMASIN (exemestane; Pfizer), formestanie, fadrozole, RIVISOR (vorozole), FEMARA (letrozole; Novartis), and ARIMIDEX (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin;

buserelin, tripterelin, medroxyprogesterone acetate, diethylstiIbestrol, premarin,

fluoxymesterone, all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-a, Ralf and H- Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example,

ALLOVECTIN, LEUVECTIN, and VAXID; PROLEUKIN, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN; ABARELIX rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0262] Chemotherapeutic agents also includes "EGFR inhibitors," which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab;

ERBUTIX) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such as ABX- EGF or Panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al.

Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-a for EGFR binding

(EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J.

Biol. Chem. 279(29) :30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572,

6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: W098/14451, W098/50038,

W099/09016, and WO99/24037. Particular small molecule EGFR antagonists include OSI- 774 (CP-358774, erlotinib, TARCEVA Genentech/OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6- quin-azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA) 4-(3'-Chloro-4'- fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli- ne, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4- fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-- d] pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3- d]pyrimidin-6-yl]-phenol)- ; (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H- pyrrolo[2,3-d]pyrimi- dine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2- butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6- quinolinyl]-4-(- dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB,

GSK572016 or N-[3-chloro-4-[(3 fluorophenyl)methoxy]phenyl]- 6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-- furanyl]-4-quinazolinamine).

[0263] Additionally, chemotherapeutic agents include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both

HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo- SmithKIine), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC, available from Glaxo SmithKIine); multi -targeted tyrosine kinase inhibitors such as sunitinib (SUTENT, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib

(PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H- pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4- fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules {e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033

(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate (GLEEVEC); PKI 166 (Novartis); GW2016 (Glaxo SmithKIine); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer);

ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-lCll (Imclone), rapamycin (sirolimus, RAPAMUNE); or as described in any of the following patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016 (American Cyanamid); WO 1998/43960 (American

Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

[0264] Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0265] Further, chemotherapeutic agents include radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu);

miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18- OCH₃, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gal late, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as

chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone;

lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9- aminocamptothecin); podophyllotoxin; tegafur (UFTORAL); bexarotene (TARGRETIN) ; bisphosphonates such as clodronate (for example, BONEFOS or OSTAC), etidronate

(DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor {e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE) ; pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0266] In some embodiments, the therapeutic moiety is an immunomodulating agent, representative examples of which include tumor necrosis factor a (TNF-a) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra (Kineret), T-cell costimulation blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA); Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon a (IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; interleukins such as IL-2, IL-7, IL-12; cytokines such as granulocyte colony-stimulating factor (G-CSF), interferons; chemokines such as CXCL13, CCL26, CXCL7; antagonists of immune checkpoint blockades such as anti-CTLA-4, anti-PD1 or anti-PD-L1 (ligand of PD-1), anti-LAG3, anti-B7-H3; alemtuzumab (Campath), bevacizumab (AVASTIN, Genentech); cetuximab (ERBITUX, Imclone); panitumumab (VECTIBIX, Amgen), rituximab (RITUXAN, Genentech/Biogen Idee), pertuzumab (OMNITARG, 2C4, Genentech),

trastuzumab (HERCEPTIN, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the present disclosure include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab,

certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab,

motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, peefusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT- 874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgG₁l antibody genetically modified to recognize interleukin-12 p40 protein. [0267] In some embodiments, the therapeutic moiety is an anti-inflammatory agent. 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. Non-limiting examples of steroidal anti-inflammatory drugs include 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.

[0268] 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. In some embodiments, the non-steroidal anti- inflammatory agent is selected from immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC). [0269] In certain embodiments, the anti-inflammatory agent comprises CD39, which is an ecto-nucleoside triphosphate diphosphohydrolase that degrades adenosine 5'- diphosphate (ADP), a main platelet activating/recruiting agent. Targeting CD39 to activated platelets has been shown to enrich CD39 at developing clots, effectively preventing the ADP- induced autocrine and paracrine activation of platelets (Hohmann et al., 2013. Blood 121(16) : 3067-3075). Additionally, it has been shown that targeting CD39 to activated platelets can decrease systemic inflammation and mortality of sepsis (Granja et al., 2019.

Crit Care Med. 47(5) :e420-e427). In representative embodiments in which CD39 is employed in the chimeric molecule, the therapeutic moiety comprises a polypeptide corresponding to the extracellular domain of CD39, which suitably comprises, consists or consists essentially of the amino acid sequence:

. Suitably, the chimeric molecule 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 (antigen-binding sequences), a CD39 sequence downstream of the V_L and V_H sequences and an optional sequence that suitably comprises a flexible linker interposed between the antigen-binding sequences and the CD39 sequence.

[0270] 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;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker; · Lowercase text corresponds to variable light chain amino acid sequence of the anti-GPIIb/IIIa scFv SE; and • AAA is a flexible linker;

• Uppercase underlined text corresponds to the amino acid sequence of the extracellular domain of CD39.

[0271] In other representative examples, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

wherein :

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

• AAA is a flexible linker;

[0272] 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;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

• AAA is a flexible linker;

[0273] In other representative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

wherein :

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 11] is a flexible linker;

• AAA is a flexible linker;

[0274] In other representative 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 variable heavy chain amino acid

sequence of the anti-GPIIb/IIIa scFv SE;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 10] is a flexible linker;

• AAA is a flexible linker;

• Uppercase underlined text corresponds to the amino acid sequence of the extracellular domain of CD39;

. EQKLISEEDL [SEQ ID NO: 15] is a C-myc tag ; and

· His tags are underlined and italicized.

[0275] In still other representative 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 variable heavy chain amino acid sequence of the anti-GPIIb/IIIa scFv SE;

• GSASAPKLEEGEFSEARVS [SEQ ID NO: 10] is a flexible linker;

• AAA is a flexible linker;

. EQKLISEEDL [SEQ ID NO: 15] is a C-myc tag ; and

• His tags are underlined and italicized .

[0276] In some embodiments, the chimeric molecule comprises or consists essentially of the following amino acid sequence:

in which :

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

• X₁ is an optional linker that is suitably a flexible linker (e.g [.G, GGGS]_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

[0277] In representative examples of this type, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

in which :

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

• GGGGS is a flexible linker;

• Uppercase bold text corresponds to HSA amino acid sequence;

[0278] In other representative examples, the chimeric molecule comprises, consists or consists essentially of the following amino acid sequence:

in which :

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

• GGGGS is a flexible linker;

• Uppercase bold text corresponds to HSA amino acid sequence;

. EOKLISEEDL TSEO ID NO: 151 is a C-mvc taa : and

• His tag is italicized and underlined.

4.4 Peptide linkers

[0279] 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: 50]. 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: 54],

GGSGGSGGSGGSGGG [SEQ ID NO: 55], GGSGGSGGGGSGGGGS [SEQ ID NO: 56],

GGSGGSGGSGGSGGSGGS [SEQ ID NO: 57], or GGGGSGGGGSGGGGS [SEQ ID NO: 58]. In other embodiments, the linker is a poly-G sequence (GGGG)_n, where n can be an integer from 1-100.

[0280] 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.

[0281] 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).

4.5 Conjugation tags

[0282] In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein comprises one or more conjugation tags for conjugating to a payload (e.g., a detectable moiety, a half-life extending moiety or a therapeutic moiety). For example, recombinant engineering and incorporated selenocysteine (e.g., as described for example in U.S. Pat. No. 8,916,159) can be used to conjugate a payload to the anti- GPIIb/IIIa antigen-binding molecule or chimeric molecule (also referred to herein as

"payload conjugates"). Other methods of conjugation can include covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols, as described for example in Wu et al. (2005, Nat. Biotechnol, 23: 1137-1146).

[0283] Alternatively, the payload conjugates are obtained by means of site- specific sortase-enzyme mediated antibody conjugation. As disclosed in W02014/140317, sortases (also called sortase transpeptidases) form a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a specific peptide motif called "sortase recognition tag" or "sortase tag". Usually, a given sortase enzyme recognizes one or more sortase recognition tags. Sortase enzymes can be naturally occurring, or may have undergone genetic engineering (see, e.g., Dorr et al., 2014; Proc Natl Acad Sci. 111 : 13343- 13348).

[0284] In specific embodiments, the conjugate is obtained by means of site- specific sortase-enzyme mediated conjugation of (a) an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein carrying one or more sortase recognition tags, and (b) one or more payloads carrying a glycine or oligoglycine tag, Gly_(n). Suitably, the sortase recognition tag is fused or conjugated to the C-terminus of the anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule. Representative sortase recognition tags include LPXSG [SEQ ID NO: 59], LPXAG [SEQ ID NO:60], LPXTG [SEQ ID NO:61], LAXTG [SEQ ID NO:62], and NPQTG [SEQ ID NO:63], with X being any amino acid residue. In certain embodiments, the oligoglycine tag, Gly_n, has a length of 1 to 21 glycine residues, more typically a length of 3 to 5 amino acids, i.e., Gly₃, Gly₄, or Gly₅.

[0285] In other embodiments, the conjugating tag is a linker. The linker linking a payload to an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein ("anti-platelet-molecule") may be short, long, hydrophobic, hydrophilic, flexible or rigid, or may be composed of segments that each independently have one or more of the above- mentioned properties such that the linker may include segments having different properties. The linkers may be polyvalent such that they covalently link more than one agent to a single site on the antibody, or monovalent such that covalently they link a single agent to a single site on the anti-platelet-molecule.

[0286] As will be appreciated by skilled artisans, the linkers link a payload to the anti-platelet-molecule by forming a covalent linkage to the payload at one location and a covalent linkage to the anti-platelet-molecule at another. The covalent linkages are formed by reaction between functional groups on the linker and functional groups on the payload and anti-platelet-molecule. In the context of conjugation tags, the expression "linker" is intended to include (i) unconjugated forms of the linker that include a functional group capable of covalently linking the linker to a payload and a functional group capable of covalently linking the linker to an anti-platelet-molecule; (ii) partially conjugated forms of the linker that includes a functional group capable of covalently linking the linker to an anti- platelet-molecule and that is covalently linked to a payload, or vice versa; and (iii) fully conjugated forms of the linker that is covalently linked to both a payload and an anti- platelet-molecule.

[0287] The linkers are suitably, but need not be, chemically stable to conditions outside the cell, and may be designed to cleave, immolate and/or otherwise specifically degrade inside the cell. Alternatively, linkers that are not designed to specifically cleave or degrade inside the cell may be used. Choice of stable versus unstable linker may depend upon the toxicity of a payload (e.g., a therapeutic agent/moiety). For agents that are toxic to normal cells (e.g., chemotherapeutic agents), stable linkers are preferred. Agents that are selective or targeted and have lower toxicity to normal cells may utilize, chemical stability of the linker to the extracellular milieu is less important. A wide variety of linkers useful for linking drugs to anti-platelet-molecule in the context of antibody-drug conjugates (ADCs; also referred to as immuno-conjugates) are known in the art. Any of these linkers, as well as other linkers, may be used to link the payload to the anti-platelet-molecule of the disclosure.

[0288] Exemplary polyvalent linkers that may be used to link many cytotoxic and/or cytostatic agents to a single antibody molecule are described, for example, in WO 2009/073445; WO 2010/068795; WO 2010/138719; WO 2011/120053; WO 2011/171020; WO 2013/096901; WO 2014/008375; WO 2014/093379; WO 2014/093394; WO

2014/093640.

[0289] Additional examples of dendritic type linkers can be found in US

2006/116422; US 2005/271615; de Groot et ai. (2003. Angew. Chem. Int. Ed. 42:4490- 4494); Amir et ai (2003. Angew. Chem. Int. Ed. 42:4494-4499); Shamis et ai (2004. J.

Am. Chem. Soc. 126: 1726-1731); Sun et ai. (2002. Bioorganic & Medicinal Chemistry Letters 12:2213-2215); Sun et ai. (2003. Bioorganic & Medicinal Chemistry 11 : 1761-1768); King et al. (2002. Tetrahedron Letters 43: 1987-1990).

[0290] Exemplary monovalent linkers that may be used are described, for example, in Nolting (2013. Methods in Molecular Biology 1045:71-100); Kitson et al. (2013. Chemistry Today 31(4) :30-38); Ducry et al. (2010. Bioconjugate Chem. 21 : 5-13); Zhao et al. (2011. J. Med. Chem. 54:3606-3623); U.S. Pat. Nos. 7,223,837; 8,568,728; 8,535,678; and W02004010957.

[0291] In certain embodiments, the linker selected is cleavable in vivo. Cleavable linkers may include chemically or enzymatically unstable or degradable linkages. Cleavable linkers generally rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers generally incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker is noncleavable. In certain embodiments, a linker comprises a chemically labile group such as hydrazone and/or disulfide groups. Linkers comprising chemically labile groups exploit differential properties between the plasma and some cytoplasmic compartments. The intracellular conditions to facilitate drug release for hydrazone containing linkers are the acidic environment of endosomes and lysosomes, while the disulfide containing linkers are reduced in the cytosol, which contains high thiol concentrations, e.g., glutathione. In certain embodiments, the plasma stability of a linker comprising a chemically labile group may be increased by introducing steric hindrance using substituents near the chemically labile group.

[0292] Additional linkers which remain intact during systemic circulation and undergo hydrolysis and release the drug when the immuno-conjugate is internalized into acidic cellular compartments include carbonates. Such linkers can be useful in cases where the payload can be covalently attached through an oxygen.

[0293] Other acid-labile groups that may be included in linkers include cis- aconityl-containing linkers. c/s-Aconityl chemistry uses a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.

[0294] Cleavable linkers may also include a disulfide group. Disulfides are thermodynamically stable at physiological pH and are designed to release the drug upon internalization inside cells, wherein the cytosol provides a significantly more reducing environment compared to the extracellular environment. Scission of disulfide bonds generally requires the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that disulfide-containing linkers are reasonably stable in circulation, selectively releasing the drug in the cytosol. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds inside cells. GSH is reported to be present in cells in the concentration range of 0.5-10 mM compared with a significantly lower concentration of GSH or cysteine, the most abundant low-molecular weight thiol, in circulation at approximately 5 Tumor cells, where irregular blood flow leads to a hypoxic state, result in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations. In certain embodiments, the in vivo stability of a disulfide-containing linker may be enhanced by chemical modification of the linker, e.g., use of steric hindrance adjacent to the disulfide bond.

[0295] Another type of cleavable linker that may be used is a linker that is specifically cleaved by an 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.

[0296] In exemplary embodiments, the cleavable peptide is selected from tetrapeptides such as Gly-Phe-Leu-Gly [SEQ ID NO:64], Ala-Leu-Ala-Leu [SEQ ID NO:65] 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.

[0297] Enzymatically cleavable linkers may include a self-immolative spacer to spatially separate the drug from the site of enzymatic cleavage. The direct attachment of a drug to a peptide linker can result in proteolytic release of an amino acid adduct of the drug, thereby impairing its activity. The use of a self-immolative spacer allows for the elimination of the fully active, chemically unmodified drug upon amide bond hydrolysis.

[0298] Although cleavable linkers may provide certain advantages, the linkers contained in the immuno-conjugates of the disclosure need not be cleavable. For non- cleavable linkers, the release of drug does not depend on the differential properties between the plasma and some cytoplasmic compartments. The release of the drug is postulated to occur after internalization of the immuno-conjugate via antigen-mediated endocytosis and delivery to lysosomal compartment, where the immuno-conjugate is degraded to the level of amino acids through intracellular proteolytic degradation. This process releases a drug derivative, which is formed by the drug, the linker, and the amino acid residue to which the linker was covalently attached. The amino acid drug metabolites from conjugates with non- cleavable linkers are more hydrophilic and generally less membrane permeable, which leads to less bystander effects and less nonspecific toxicities compared to conjugates with a cleavable linker. In general, immuno-conjugates with non-cleavable linkers have greater stability in circulation than immuno-conjugates with cleavable linkers. Non-cleavable linkers may be alkylene chains, or maybe polymeric in natures, such as, for example, based upon polyalkylene glycol polymers, amide polymers, or may include segments of alkylene chains, polyalkylene glycols and/or amide polymers.

[0299] A variety of non-cleavable linkers used to link payloads such as drugs to antigen-binding molecules have been described. For example, reference may be made to Jeffrey et al. (2006, Bioconjug. Chem. 17:831-840; 2007, Bioorg. Med. Chem. Lett.

17:2278-2280); and Jiang et al. (2005, J. Am. Chem. Soc. 127: 11254-11255).

[0300] In specific embodiments, the antigen-binding molecules and chimeric molecules of the present disclosure are conjugated to payloads using methods disclosed in Polakis, P. (2016. Pharmacol Rev 68:3-19), Pietersz et al. (2017. Nanomedicine (Lond.) 12(15) : 1873-1889), Yap et al. (2017 Theranostics 7(10) :2565-2574), Yap et al. (2019. Theranostics 9(4) : 1154-1169) and Ziegler et al. (2019. Cardiovascular Research 2019 Mar 25. doi : 10.1093/cvr/cvz070),

5. Methods of Preparation

[0301] The present disclosure also provides a nucleic acid molecule or a set of nucleic acid molecules encoding (i) an anti-GPIIb/IIIa antigen-binding molecule disclosed herein, or (ii) a chimeric molecule disclosed herein.

[0302] 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.

[0303] The instant disclosure also provides methods for producing an anti-

GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein, such methods comprising culturing the host cell disclosed herein and recovering the antigen-binding molecule or chimeric molecule from the host cell or culture medium.

[0304] A variety of methods is available for recombinantly producing an anti- GPIIb/IIIa antigen-binding molecule or 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.

[0305] In some embodiments an expression vector or set of expression vectors from which a nucleic acid sequence encoding the amino acid sequence of an anti-GPIIb/IIIa antigen-binding molecule or 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 anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule. The anti-GPIIb/IIIa antigen-binding molecule or chimeric polypeptide may be recovered from the cell or culture medium.

[0306] 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). 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 an anti-GPIIb/IIIa antigen-binding molecule disclosed herein, or a chimeric molecule disclosed herein.

[0307] For recombinant production, a polynucleotide sequence encoding a polypeptide (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule 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.

[0308] 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 polypeptide (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule disclosed herein) can be recovered by lysing the cells.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] In other embodiments, 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.

[0313] 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.

[0314] Polynucleotides encoding the polypeptides of the present disclosure (e.g., an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule 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.

[0315] 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 ..,S2 or Sf9 cells).

[0316] 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.

[0317] Once expressed, the anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule 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. Ancillary therapeutic agents

[0318] The anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule of the present disclosure may be administered separately, sequentially or simultaneously with an ancillary therapeutic agent. In some embodiments, the anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule and the ancillary therapeutic agent are administered concurrently.

[0319] In some embodiments, the therapeutic agent is an anti-cancer agent including ones that exert antineoplastic, chemotherapeutic, anti-viral, anti-mitotic, anti- tumorigenic, and/or immunotherapeutic effects (e.g., prevent the development, maturation, or spread of neoplastic cells, directly on the tumor cell, e.g., by cytostatic or cytocidal effects, and not indirectly through mechanisms), as described for example in Section 4.3 supra.

[0320] In some embodiments, the therapeutic agent can be an anti- neurodegenerative agent in the treatment of neurodegenerative diseases such as, but not limited to, diseases and disorders in which the myelin which surrounds the neuron is either absent, incomplete, not formed properly, or is deteriorating. Such disease include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic

leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barre syndrome, Marie-Charcot-Tooth disease and Bell's palsy.

[0321] Examples of anti-neurodegenerative disease agents can include, but are not limited to L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators such as interferon beta-la and beta-lb (Avonex and Betaseron respectively), natalizumab (Copaxone) natalizumab (Tysabri), glatiramer acetate (Copaxone) or mitoxantrone.

[0322] In some embodiments, the therapeutic agent can be an anti- cardiovascular disease agent. Non-limiting examples of anti-cardiovascular disease agents include beta blockers, anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators, hormone antagonists, inotropes, diuretics, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2 antagonists and cytokine blockers/inhibitors, and HDAC inhibitors.

[0323] In some embodiments, the anti-cardiovascular disease agent can include an antihyperlipoproteinemic agent, an anti-arteriosclerotic agent, an anti- thrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an

antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof.

[0324] An antihyperlipoproteinemic agent can include, but is not limited to, aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequestrant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof, acifran, azacosterol, benfluorex, b- benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, y-oryzanol, pantethine, pentaerythritol tetraacetate, a-phenylbutyramide, pirozadil, probucol (LORELCO), b-sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.

[0325] The anti-cardiovascular disease agent can include an anti-arteriosclerotic agent such as pyridinol carbamate. An anti-cardiovascular disease agent can include an antithrombotic/fibrinolytic agent including, but not limited to anticoagulants (acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, Fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin); anticoagulant antagonists, antiplatelet agents (aspirin, a dextran, dipyridamole (PERSANTIN), heparin, sulfinpyrazone (ANTURANE) and ticlopidine (TICLID)); thrombolytic agents (tissue plasminogen activator (ACTIVASE), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (STREPTASE), reteplase (RETAVASE), tenecteplase (TNKase), anistreplase/APSAC (EMINASE));

thrombolytic agent antagonists or combinations thereof). Anti-cardiovascular disease agents may also include an blood coagulant including, but not limited to, thrombolytic agent antagonists (amiocaproic acid (AMICAR) and tranexamic acid (AMSTAT); antithrombotics (anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride tedelparin, ticlopidine and triflusal); and anti- coagulant antagonists (protamine and vitamine Kl).

[0326] An anti-cardiovascular disease agent can also include an anti-arrhythmic agent including, but not limited to, Class I anti-arrhythmic agents (sodium channel blockers), Class II anti-arrhythmic agents (beta-adrenergic blockers), Class II anti-arrhythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous anti-arrhythmic agents. Non-limiting examples of sodium channel blockers include Class IA (disppyramide (NORPACE), procainamide (PRONESTYL) and quinidine (QUINIDEX)); Class IB (lidocaine (XYLOCAINE), tocainide (TONOCARD) and mexiletine (MEXITIL)); and Class IC anti-arrhythmic agents, (encamide (ENKAID) and fiecamide (TAMBOCOR)).

[0327] Non-limiting examples of a beta blocker (also known as a b-adrenergic blocker, a b-adrenergic antagonist or a Class II anti-arrhythmic agent) include acebutolol (SECTRAL), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (BREVIBLOC), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (INDERAL), sotalol (BETAPACE), sulfmalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. In certain aspects, the beta Mocker comprises an aryloxypropanolamine derivative. Non-limiting examples of

aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol. Non-limiting examples of an agent that prolongs repolarization, also known as a Class III antiarrhythmic agent, include amiodarone (CORDARONE) and sotalol (BETAPACE).

[0328] Non-limiting examples of a calcium channel blocker, otherwise known as a Class IV anti-arrhythmic agent, include an arylalkylamine (e.g., bepridil, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazine derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a miscellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline. In certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine- type) calcium antagonist.

[0329] Non-limiting examples of miscellaneous anti-arrhythmic agents include adenosine (ADENOCARD), digoxin (LANOXIN), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide, ipatropium bromide, lidocaine, lorajmine, lorcamide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.

[0330] An anti-cardiovascular disease agent can also include an anti-hypertensive agent including, but not limited to, alpha/beta blockers (labetalol (NORMODYNE,

TRANDATE)), alpha blockers, anti-angiotensin II agents, sympatholytics, beta blockers, calcium channel blockers, vasodilators and miscellaneous anti-hypertensives.

[0331] Non-limiting examples of an alpha blocker, also known as an a-adrenergic blocker or an a-adrenergic antagonist, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. In certain embodiments, an alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin.

[0332] Non-limiting examples of anti-angiotensin II agents include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. Non-limiting examples of angiotensin converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (VASOTEC), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril. Non-limiting examples of an angiotensin II receptor blocker, also known as an angiotensin II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan. Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherally acting sympatholytic. Non-limiting examples of a centrally acting sympatholytic, also known as a central nervous system (CNS) sympatholytic, include clonidine (CATAPRES), guanabenz (wytens guanfacine (TENEX) and methyldopa (ALDOMET). Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a b-adrenergic blocking agent or an al- adrenergic blocking agent. Non-limiting examples of a ganglion blocking agent include mecamylamine (INVERSINE) and trimethaphan (ARFONAD). Non-limiting of an adrenergic neuron blocking agent include guanethidine (ISMELIN) and reserpine (SERPASIL). Non- limiting examples of a b-adrenergic blocker include acenitolol (SECTRAL), atenolol

(TENORMIN), betaxolol (KERLONE), carteolol (CARTROL), labetalol (NORMODYNE,

TRANDATE), metoprolol (LOPRESSOR), nadanol (CORGARD), penbutolol (LEVATOL), pindolol (VISKEN), propranolol (INDERAL) and timolol (BLOCADREN). Non-limiting examples of alphal-adrenergic blocker include prazosin (MINIPRESS), doxazocin (CARDURA) and terazosin (HYTRIN).

[0333] In certain embodiments, an anti hypertensive agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator).

In particular embodiments, a vasodilator comprises a coronary vasodilator including, but not limited to, amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis(P-diethylaminoethyl ether), hexobendine, itramin tosylate, mannitol hexanitrane, rnedibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimethylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

[0334] A vasodilator may comprise a chronic therapy vasodilator or a

hypertensive emergency vasodilator. Non-limiting examples of a chronic therapy vasodilator include hydralazine (APRESOLINE) and minoxidil (LONITEN). Non-limiting examples of a hypertensive emergency vasodilator include nitroprusside (NIPRIDE), diazoxide (HYPERSTAT IV), hydralazine (APRESOLINE), minoxidil (LONITEN) and verapamil.

[0335] Non-limiting examples of miscellaneous antihypertensives include ajmaline, y-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyriclyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, pnmaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil. In certain aspects, an anti hypertensive may comprise an arylethanolamine derivative (amosulalol, bufuralol, dilevalol, labetalo, pronethalol, sotalol and sulfmalol); a benzothiadiazine derivative (althizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazide and

trichlormethiazide); a N-carboxyalkyl(peptide/lactam) derivative (alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril); a dihydropyridine derivative (amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine); a guanidine derivative (bethanidine, debrisoquine guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan); a hydrazines/phthalazine (budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine); an imidazole derivative (clonidine, lofexidine, phentolamine, tiamenidine and tolonidine); a quanternary ammonium compound (azamethonium bromide, chlorisondamine chloride, hexamethonium, penta.cynium bis(methylsulfate),

pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and

trimethidinium methosulfate); a reserpine derivative (bictaserpine, deserpidine,

rescinnamine, reserpine and syrosingopine); or a sulfonamide derivative (ambuside, clopamide, farosemide, indapamide, quinethazone, tripamide and xipamide).

[0336] Anti-cardiovascular disease agents can include a vasopressor.

Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure. Non-limiting examples of a vasopressor, also known as an anti hypotensive include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine. [0337] In some embodiments, an anti-cardiovascular disease agent can include treatment agents for congestive heart failure including, but not limited to, anti-angiotensin II agents, afterload-preload reduction treatment (hydralazine (APRESOLINE) and isosorbide dinitrate (ISORDIL, SORBITRATE)), diuretics, and inotropic agents.

[0338] Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, beizthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom, protheobromine, theobromine), steroids including aldosterone antagonists (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4'-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, triparnide, xipamide), a uracil (e.g., aminometradine, arnisornetradine), a potassium sparing antagonist (e.g., amiloride, triamterene) or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticrnafen and urea.

[0339] Non-limiting examples of a positive inotropic agent, also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, aminone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, strphanthin, sulmazole, theobromine and xamoterol.

[0340] In some embodiments, an inotropic agent is a cardiac glycoside, a beta- adrenergic agonist or a phosphodiesterase inhibitor. Non-limiting examples of a cardiac glycoside includes digoxin (LANOXIN) and digitoxin (CRYSTODIGIN). Non-limiting examples of a b-adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (DOBUTREX), dopamine (INTROPIN), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol,

hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include amrinone (INOCOR). 7. Pharmaceutical Compositions

[0341] 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) an anti-GPIIb/IIIa antigen-binding molecule disclosed herein; (ii) a chimeric molecule disclosed herein; (iii) a nucleic acid molecule or the set of nucleic acid molecules disclosed herein; or (iv) 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] Dosages and therapeutic regimens of the antigen-binding molecule or chimeric molecule can be determined by a skilled artisan. In certain embodiments, the antigen-binding molecule or 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 antigen-binding molecule or 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 an antigen-binding molecule or chimeric molecule of the present disclosure is 0.01-5 mg/kg, more suitably 0.03-2 mg/kg.

[0350] 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.

[0351] 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, 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 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.

[0352] 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.

8. Methods of Treatment

[0353] The agents of the disclosure (e.g., an anti-GPIIb/IIIa antigen-binding molecule disclosed herein, 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.

[0354] 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, ischemic 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.

[0355] 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.

[0356] 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.

9. Other Methods of Use

[0357] The instant disclosure also provides a method to target or deliver a therapeutic or prophylactic agent (e.g., an anticoagulant moiety) to the surface of platelets, wherein the method comprises fusing the agent to an anti-GPIIb/IIIa antigen-binding molecule disclosed herein.

[0358] The present disclosure also provides a method of measuring the level of activated platelets in a sample (e.g., a body fluid sample such as plasma, or a tissue sample) of a subject in need thereof comprising contacting the anti-GPIIb/IIIa antigen binding molecule or chimeric molecule disclosed herein with the sample from the subject and measuring the level of activated platelets in the sample. This method can further comprise fusing or conjugating the anti-GPIIb/IIIa antigen-binding molecule to a detectable moiety, for example, a fluorophore, metal-containing particle or radionuclide.

[0359] This disclosure also provides a method of isolating or separating activated platelets from other cells in a sample (e.g., a blood sample). The method comprises contacting the sample with an anti-GPIIb/IIIa antigen binding molecule or chimeric molecule disclosed herein and separating the cells that have bound to the anti-GPIIb/IIIa antigen binding molecule or chimeric molecule from the unbound fraction.

[0360] Furthermore, the disclosure includes methods of isolating or enriching activated platelets from a sample. This method involves contacting the sample with an anti- GPIIb/IIIa antigen binding molecule or chimeric molecule of the present disclosure and isolating the bound fraction of cells. The bound fraction predominantly contains the activated platelets.

[0361] Also, the disclosure encompasses the use of anti-GPIIb/IIIa antigen binding molecules or chimeric molecules of the instant disclosure as diagnostic tools for evaluating fibrinogen blocking. For example, the anti-GPIIb/IIIa antigen binding molecule or chimeric molecule can be used as a surrogate for fibrinogen, to block the ligand-binding site in assays. The anti-GPIIb/IIIa antigen binding molecule or chimeric molecule can also be used as probes (e.g., linked to a detectable moiety) to identify a sample that is capable of binding fibrinogen. In one embodiment, the disclosure provides a method involving, contacting a sample with a anti-GPIIb/IIIa antigen binding molecule linked to or conjugated with a detectable moiety and identifying cells to which the antigen binding molecule is bound as a sample that is capable of binding to fibrinogen when compared to those cells in the sample that are not bound by the antibody or antigen-binding fragment thereof.

[0362] The present disclosure further encompasses methods for detecting the presence of an activated platelet in a subject. These methods comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an activated platelet and the antigen-binding molecule to thereby detect the presence of the activated platelet in the subject.

[0363] Additionally, the disclosure includes methods for detecting presence of a thrombus in a subject. These methods comprise administering to the subject an anti- GPIIb/IIIa antigen-binding molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises a thrombus and the antigen-binding molecule to thereby detect the presence of the thrombus in the subject.

[0364] This disclosure also provides methods for detecting presence of an embolus. These methods comprise administering to the subject an anti-GPIIb/IIIa antigen binding molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an embolus and the antigen-binding molecule to thereby detect the presence of the embolus in the subject. [0365] Furthermore, the present disclosure provides methods for detecting presence of a tumor in a subject. These methods generally comprise administering to the subject an anti-GPIIb/IIIa antigen-binding molecule or chimeric molecule described herein that is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an activated platelet and the antigen-binding molecule or chimeric molecule (e.g., adjacent to the tumor such as in the microenvironment of the tumor) to thereby detect the presence of the tumor in the subject.

[0366] In a related aspect, the present disclosure provides methods for reducing or inhibiting proliferation, survival and viability of a tumor in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule described herein and an anti-cancer moiety (e.g., a chemotherapeutic agent).

[0367] In another related aspect, the present disclosure provides methods for treating or inhibiting the development of a cancer in a subject. These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule described herein and an anti-cancer moiety (e.g., a chemotherapeutic agent).

10. Kits

[0368] A further embodiment of the present disclosure is a kit for detecting activated platelets, thrombi or emboli, for detecting presence of a tumor, 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, for treating or inhibiting the development of a hematologic disorder, for reducing or inhibiting proliferation, survival or viability of a tumor, and/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.

[0369] 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

[0370] 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

[0371] 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

[0372] 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 CO₂ 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 3 mg/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 6 g/L. The flask was returned to the incubation and cultured at 37°C, with 5% CO₂, 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 1ml were collected and dialyzed against PBS.

Evaluation of the scFv proteins

[0373] 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.

[0374] 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

[0375] 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

[0376] 96-well plate light transmission aggregometry was performed using 100 mL of PRP. Platelet poor plasma (PPP) was obtained by centrifugation of blood at lOOOxg for lOmin at room temperature. PRP was mixed with 8 mM calcium chloride, 1 :50 thromboplastin (Siemens, USA), and 20pM 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 2pM 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

ScFv-CD39 GENERATION AND PRODUCTION

[0377] SE was PCR modified to contain Ncol and Notl flanking restriction sites with the sense strand primer: 5'- atgactCCATGGCGGAGG-TGCAGCTGGTGGAG-3' [SEQ ID NO:78] and antisense strand primer: 5'- tagcatGCGGCCGCA-GAAGAGGGCGGGAAC-3' 3'

[SEQ ID NO:79]. [0378] CD39 was modified via PCR using pcDNA3/CD39 (Crikis S, et al., 2010.

Am J Transplant. 10(12) :2586-2595; Dwyer KM, et al., 2004. J Clin Invest. 113(10) : 1440- 1446; Kaczmarek E, et al., 1996. J Biol Chem. 271(51) :33116-33122) as a template to obtain a truncated form, which was flanked by Ncol and Xhol restriction enzyme recognition sites (Figure 2A), cut by these restriction enzymes, and then ligated into a vector containing the scFvs (SE or Mut). After confirmation of successful cloning by control digests (Figure 2B) and PCR sequencing, the DNA was transfected into HEK293F cells and cultured for 7 or 8 days.

[0379] CD39 was PCR modified from pCDNA3-CD39 (Kaczmarek E, et al., 1996, supra ) in order to add restriction sites that allow for cloning into the pSectag2a vector

(Invitrogen, USA). For increased solubility, CD39 was additionally truncated to remove its two transmembrane domains. CD39 was then modified with the following primers to create SOICD39: Sense strand primer: 5'-tcagtaGCGGCCGCAACCCAGAACAAAGCATT-3' [SEQ ID NO:80] and antisense strand primer: 5'-atcgcaCTCGAGTGGTGGAGTGGGAGAGAG-3' [SEQ ID NO:81].

[0380] After amplification by PCR, the constructs were digested with their respective restriction enzymes and solCD39 was cloned into pSectag2A, which was prepared using the same restriction enzymes. The pSectag2A vector adds a 6x histidine tag (6xHis- tag) to the constructs, which allows for protein purification and for detection of the constructs in flow cytometric assays and by immunofluorescence microscopy. Ligation of the plasmids was performed with T4 ligase (NEB, USA) at 16°C overnight. The resulting plasmid constructs were transformed into Turbo Competent E. coli cells (NEB). Transformed bacteria were grown in LB media containing 100 mg/mL ampicillin at 37°C and the plasmids were purified using Plasmid Mini Kit (Qiagen, Australia). Obtained colonies were confirmed by DNA sequencing. This plasmid was then digested with Ncol and Notl in order to be ligated with either the SE scFv antibody or the Mut scFv to become SE-CD39 and Mut-CD39, respectively. Larger DNA preparations were performed by Plasmid Maxi Kit (Qiagen).

Preparation of the non-targeted scFv "Mut-"

[0381] A single-chain antibody Mut, which did not demonstrate specific binding, as described previously (Schwarz M, et al., 2004. FASEB J. 18(14) : 1704-1706) was PCR modified to also contain Ncol and Notl flanking restriction sites using the sense strand primer: 5'- atgactCCATGGCGGAA-GTGCAGCTGGTGCAGT-3' [SEQ ID NO:82] and antisense strand primer: 5'- tagcatGCGGCCGC-CGCGGACGGCGGGAAC-3' [SEQ ID NO:83].

Expression in mammalians cells and purification ofscFv constructs

[0382] Production of mammalian cells was performed using the human kidney cells (HEK293F) in suspension culture after transfection with polyethyleneimine (Polyscience Inc., Germany). The DNA plasmid for transfection was diluted to a ratio of 1 :4 with polyethyleneimine (PEI). 24 hours prior to transfection, HEK293F cells were diluted with Freestyle 293 expression medium (Invitrogen) 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 greater than 95%. The amount of Freestyle 293 expression medium to the PBS mixture of DNA and PEI was at a ratio of 9: 1. Appropriate amount of cell culture medium was transferred into a shaker flask and placed in an 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 to the concentration of 3 mg/mL, and vortexed shortly. The mixture was incubated for 15 min at RT. The DNA-PEI mixture was added to the pre-warmed medium while swirling gently. Glucose was added to a final concentration of 6 g/L. The flask was returned to the incubator and cultured at 37°C, with 5% C02, shaking at 110 - 140 rpm. The culture was supplemented with 5 g/L Lupin and 0.2 mM butyric acid after one day. At days 3, 5 and 7 after transfection, the culture was supplemented with 2 mM L-glutamine. At day 5, the culture was again 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 3000 g for 15 min at 4°C and supernatant was collected for protein purification. Proteins were purified with a nickel-based metal affinity chromatography column, Ni-NTA column (Invitrogen), according to the manufacturer's instructions. Fractions of 1 ml were collected and dialyzed against PBS.

EXAMPLE 3

BIOLUMINOMETRIC DETERMINATION OF CD39 FUNCTION

[0383] A malachite green phosphate assay kit from Gentaur was used to determine the enzymatic activity of SE-CD39, Mut-CD39, and commercially available recombinant human CD39 (R&D Systems) by measuring the release of phosphate during the conversion of ADP to adenosine 5'-monophosphate (AMP). For every molecule of ADP that is converted into AMP, 1 molecule of phosphate is released. Proteins were incubated at 37°C with a series of ADP concentrations from 0 to 100 mM. The reactions were stopped at several time points from 0 to 120 minutes. The samples were measured at a wavelength of 650 nm on a Victor 3V Multi-label counter (PerkinElmer). A standard series of phosphate

concentrations was used to convert raw data to the amount of AMP generated for each of the proteins. The amount of AMP generated vs time of incubation was then used to obtain the velocity of the reaction for each substrate (ADP) concentration. These velocity values were then graphed against the substrate (ADP) starting concentration to obtain Vmax and Km.

[0384] These procedures were performed according to a previously described method (Gorman et al., 2003. Luminescence 18(3) : 173-181). In PBS samples, serial concentrations of SE-CD39, Mut-CD39 and commercially available recombinant human CD39 from Abeam (UK) were incubated with 100 mM ADP for 10 min. Remaining ADP was then converted to ATP by the pyruvate kinase reaction: To 100 mL EDTA-PBS, 33 mL solution containing 40 U/mL pyruvate kinase, 4 pM phosphoenolpyruvate (PEP; Sigma-Aldrich, Australia), 10 mM KCI, and 40 mM MgSCU in 40 mM tricine buffer (pH 7.75) was added. After 5 min incubation, each sample was divided into two aliquots of 50 mL each. ATP

(representing the non-hydrolyzed remaining ADP) was determined in a bioluminescence assay using a microplate luminometer (Berthold MicroLumatPlus, Australia) by adding 50 mL luciferase reagent (ATP bioluminescence assay kit CLS II; Roche, Germany) to each sample. Hydrolyzed ADP levels were determined by subtracting the obtained ADP values from the

100 pM ADP starting concentration. Standard samples containing different concentrations of ADP in EDTA-PBS were also measured in order to establish a standard curve for the back- calculation of ADP levels.

[0385] The results presented in Table 4 show that the specific activity of the SE- CD39 construct in converting ADP to AMP is largely similar to that of soluble CD39 enzyme.

TABLE 4

EXAMPLE 4

IN VITRO PROOF OF ACTIVATED PLATELET TARGETING OF SE-CD39

[0386] Binding of SE-CD39 to activated human platelets was evaluated by flow cytometry: 0.1 mg/mL of SE-CD39, 0.2 mg/mL of Mut-CD39 (both activity matched), and 0.038 mg/mL of scFvSE (equimolar amount of scFvSE as in SE-CD39) were tested.

[0387] The results in presented in Figure 3 show that SE-CD39 binds selectively to activated platelets but not to non-activated platelets (n 5 3; **P , .01 ; Figure 3A,C). Flow cytometry also revealed significantly more binding of SE-CD39 to activated platelets compared with the Mut-CD39 control (n 5 3; ***P ,.001 ; Figure 3A), which demonstrated only background binding to activated platelets.

[0388] Neither SE-CD39 nor Mut-CD39 showed significant binding to resting nonactivated platelets (Figure 3A). Mut-CD39 also does not show binding to activated platelets (Figure 3A-B).

Methods

Preparation of platelet-rich, platelet-poor, and cell-free plasma

[0389] Citrated blood from volunteers was centrifuged at 180g for 10 minutes. The platelet-rich plasma (PRP) was collected and stored at 37°C. The remainder (infranatant) was centrifuged at 2500g for 10 minutes, and its supernatant was collected as platelet-poor plasma (PPP).

Binding of SE to activated platelets by flow cytometry

[0390] PRP was diluted 1 : 20 in phosphate-buffered saline (PBS; 100 mg/L calcium chloride, 100 mg/L magnesium chloride). To investigate the binding of scFv-CD39 constructs, the diluted PRP (45 mL) was either preincubated with a final concentration of 10 mM ADP or 5 mL of PBS for 15 minutes before addition of the constructs. The binding was then determined via a Penta-His Alexa Fluor-488-conjugated monoclonal antibody (Qiagen). To investigate the ecto-nucleoside triphosphate diphosphohydrolase efficiency, PRP was preincubated with scFv-CD39 constructs before administration of 20 mM ADP. Platelet activation status was measured by a phycoerythrin (PE)-labeled anti-P-selectin antibody (BD Bioscience). Samples were fixed using 13 Cellfix (BD Bioscience) and analyzed on a FACS Calibur (BD Bioscience).

EXAMPLE 5

IN VITRO ANTITHROMBOTIC EFFECTS OF SE-CD39

[0391] To analyze the antithrombotic efficacy of the SE-CD39 construct vs the Mut-CD39 construct, the inhibitory effects of these constructs were investigated on ADP- induced platelet activation. P-selectin expression assessed in flow cytometry was used as a measure of platelet activation: 0.1 mg/mL of SE-CD39 and 0.2 mg/mL of Mut-CD39, both activity matched, were tested.

[0392] SE-CD39 demonstrated strong inhibition of ADP-induced platelet activation compared with Mut-CD39 (n 5 3; ***P , .001; Figure 4), although non-targ-CD39 also showed a small inhibitory effect (n 5 3; *P , .05). Targ-CD39 is therefore shown to be significantly more potent at preventing platelet activation compared with non-targ-CD39. ScFvSCE5 (0.038 mg/mL of scFvSCE5, equimolar amount of scFvSCE5 as in targ-CD39) alone was not significantly different from the positive control.

EXAMPLE 6

IN VIVO ANTITHROMBOTIC EFFECTS OF SE-CD39

[0393] Male C57BL/6 mice of 20 to 25 g weight were obtained from Alfred Medical Research and Education Precinct (AMREP) Animal Services. All experiments involving animals were approved by the Alfred Medical Research and Education Precinct animal ethics committee.

[0394] Animals were anesthetized with ketamine (50 mg/kg; Parnell

Laboratories) and xylazine (10 mg/kg; Troy Laboratories). Body temperature was maintained at 37°C by placing mice on a heating mat to prevent hypothermia, which has been shown to activate platelets (Straub A, et al., 2011. Arterioscler Thromb Vase Biol. 31(7) : 1607-1616).

[0395] To demonstrate the advantage of using activated-platelet SE-CD39 as an antithrombotic treatment in vivo, a mouse thrombosis model was chosen based on a ferric chloride-induced injury of the carotid artery. SE-CD39 showed a significant prolongation of the vessel occlusion time compared with saline control, whereas the equimolar dose of Mut- CD39 showed no significant difference in vessel occlusion time compared with saline control (Figure 5A). To demonstrate that the improved effect of SE-CD39 is not caused by the SE part of the construct, but rather caused by the enrichment of CD39 at the growing thrombus, a SE-only control was also used. This control showed no significant prolongation compared with the saline control (Figure 5A). To achieve a similar occlusion time prolongation as SE, a high dose of Mut-CD39, equivalent to CD39 activity 10 times greater than SE-CD39, was required (Figure 5A). Overall, these data indicate that the benefits observed with the SE- CD39 are because of the targeting of the CD39 moiety to activated platelets.

[0396] In vivo mouse models of thrombosis and bleeding demonstrate SE-CD39's capability to protect against thrombosis without causing bleeding time prolongation when administered at a low dose (Figure 5B). Mut-CD39, however, was not sufficient in protecting against thrombosis at the same dose. The dose required for the non-targeted Mut-CD39 to prevent thrombosis caused significant bleeding time prolongation. Hence, targeting CD39 to activated platelets as SE-CD39 represents a novel promising strategy to break the link between efficient antithrombotic potency and associated bleeding risk.

EXAMPLE 7

IN VIVO ACTION OF SE-CD39 IN MOUSE MODEL OF ISCHEMIA/REPERFUSION

[0397] Whether the low systemic concentration of CD39 targeted to activated platelets (SE-CD39) has potential to harness the benefits of CD39 at the site of a complex ischemic event such as myocardial infarction (MI), but at the same time avoiding bleeding complications was assessed using a mouse model of myocardial ischemia/reperfusion (I/R). Mice were treated with SE-CD39, Mut-CD39 (a mutated, non-binding version of SE-CD39) or PBS (Figure 6). Treatment with SE-CD39 resulted in continuous improvement of

echocardiographically determined ejection fraction (EF, Figure 7A and B) and fractional shortening (FS, Figure 8) 1, 2, 3, and 4 weeks post-I/R. A significant increase in EF and FS was observed at week 3 and 4 for the SE-CD39 mice, compared with PBS and Mut-CD39 mice.

[0398] The present inventors observed that pathological changes in strain were prevented by treatment with SE-CD39, compared with Mut-CD39 and PBS treated mice. This result is consistent with a clinical study observing less myocardial damage and deformation in patients who had early reperfusion, as compared to those with delayed reperfusion (Bertini M, et al., 2009. Am J Cardiol 104:480-485). Radial and longitudinal strain analysis indicated that this cardiac performance was retained 4 weeks post-I/R for the SE-CD39 treated animals, but reduced for PBS and Mut-CD39 treated animals (Figure 7C and D, Figure 9).

Methods

Myocardial ischemia-reperfusion injury in mice

[0399] The ligation of the left coronary artery was performed as described previously (Ziegler M, et al., 2012. Circulation 125:685-696). Briefly, 20 - 25g male, C57BL/6 mice were anesthetized 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, SE-CD39 (0.4 mg/g body wt), or Mut-CD39 (0.4 mg/g body wt).

[0400] Following surgery, mice were culled at three different time points. The short-term group (n=8) was culled after 2 hours for the analysis of cardiac biomarkers, bleeding time, blood gases and microthrombosis. A second group (n=5) was culled 3 days post-surgery for the determination of cytokines within the infarcted myocardium. The last group (long-term, n=6 - 7) underwent echocardiography prior to surgery and weekly post surgery. After 28 days mice were culled for histological analysis. We registered two deaths out of 60 mice throughout the entire study; 1 PBS treated mouse and 1 mouse treated with Mut-CD39. Therefore, no correlation of mortality with the treatment was observed. Also no change in infection rate within the different treatment groups was seen.

Ultrasound and echocardiographic analysis

[0401] 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).

[0402] 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.

[0403] 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.

[0404] 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). For an accurate strain analysis to be performed, the image quality of the ventricular apex is imperative. For this reason, we excluded animals with sub-optimal images from strain analysis. Exclusion criteria for this analysis includes 1) shortened image, 2) images with shadowing of ribs (a common problem in murine ultrasound imaging) or 3) distorted image quality of the ventricular apex. 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.

Measurement of the cardiac biomarkers H-FABP and troponin I

[0405] Blood was collected 2 hours post-I/R. Blood was diluted 1 : 25 in PBS and troponin I (Tnl) was measured using the Abbott Architect i-STAT cTnl cartridges (Abbott Diagnostics) according to the manufacturer's protocol. Due to the high conservation between rodent and human Tnl, this system is reliable in measuring rodent Tnl levels. 5 PBS treated mice, 7 SE-CD39 treated mice and 6 Mut-CD39 treated mice were successfully measured, the other 6 blood samples caused errors in the cartridge reading process. The remai ning blood samples were then spun (300 g, 10 min) and plasma samples were collected. Plasma samples were diluted 1 : 100 in diluent to measure Heart-type fatty acid binding protein (H- FABP) and 1 : 25 to measure Tnl. H-FABP and Tnl plasma levels were measured by a mouse- specific ELISA (Life Diagnostics, USA) according to the manufacturer's protocol.

Tail bleeding time

[0406] Tail bleeding times were performed as previously described (Stoll P, et ai , 2007. Arter Thromb Vase Biol 27: 1206-1212). Two hours after I/R injury and injection of constructs, the tail was transected 5 mm from the tip and immediately submersed in 37°C saline. Time until blood flow ceased for 1 minute was reported.

Blood gases

[0407] Mice were anesthetized using a combination of ketamine and xylazine. Blood was collected by cardiac puncture 2 hours post-I/R. Blood gases were immediately measured via the use of the Abbott Architect i-STAT G3⁺ blood gas cartridges (Abbott Diagnostics) according to the manufacturer's protocol.

Evans Blue/TTC staining

[0408] Mice were anesthetized 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.

Immunohistology

[0409] Hearts were harvested after 3 or 28 days, fixed in 4% paraformaldehyde (PFA), embedded in paraffin, followed by cutting of 6 mm thick sections. Three random cardiac sections for each mouse were immunostained using rat anti-mouse CD31 monoclonal antibody (0.3 ug/ml, BD Pharmingen, USA), and a corresponding isotype control antibody. The appropriate biotinylated secondary antibody (Vector Laboratories, USA) was applied for 30 min at RT. Immunostaining of cardiac sections was performed with a streptavidin-biotin- immunoperoxidase method (Vectostatin ABC-Peroxidase and diaminobenzidine; Vector Laboratories) according to the manufacturer's protocol. Samples were then imaged on an Olympus BX50 microscope using a 200x magnification.

[0410] Hearts were harvested after 2 hours, sliced and embedded in OCT Tissue Tek compound. Heart sections were immunostained using rat anti-mouse GPIIb (CD41) monoclonal antibody (1 :200, Clone MWReg30, GeneTex). Samples were imaged on an Olympus BX50 microscope using a 400x magnification and six random images of each section were analyzed using ImageJ 1.47 software and computerized batch processing procedure. Results are expressed as % platelet surface coverage/High Power Field (HPF).

Ribonucleic acid (RNA) isolation and cytokines profiles

[0411] Two hours or 3 days post-I/R, hearts were flushed and the part downstream of the LAD ligation site was used for RNA isolation. RNA was isolated from the heart using the TRIzol (Life Technologies, USA) and RNA cleanup kit (Qiagen, Germany) according to the manufacturer's instruction. The RNA was converted to complementary DNA (cDNA) using the QuantiTect Rev. Transcription Kit (Qiagen, Germany), as according to manufacturer's instruction. Custom primers were used for TGFb1, IL4 and CD41. These qPCR were performed with SYBR Green (Applied Biosystems, USA). Cytokine profiles were analyzed using the 384 well RT² Profiler™ PCR Array Mouse Inflammatory Cytokines 8i Receptors (Qiagen). The array was performed using the QuantStudio 6k Flex (Applied Biosystems, USA). The data of this array was analyzed using the GeneGlobe Data Analysis Centre (Qiagen).

Statistical analysis

[0412] 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. Multiple comparisons show adjusted P-values. Statistical analyses were performed using GraphPad Prism Software, with p<0.05 considered statistically significant.

Preparation of SE-HSA-CD39

[0413] HSA-CD39 was PCR modified to contain EcoRV and PspOMI flanking restriction sites with the sense strand primer: 5'- atatGATATCaATGGATGCACACAAGAGTG-3' [SEQ ID NO: 84] and antisense strand primer: 5'- ttcGGGCCCTCCTCGAGTGGTGGAGTGGG-3'

[SEQ ID NO: 85].

[0414] HSA-CD39 was modified via PCR using pSectag2A/HSA-CD39 as a template to obtain a truncated form, which was flanked by EcoRV and PspOMI restriction enzyme recognition sites, cut by these restriction enzymes, and then ligated into a vector containing the scFvs (SE or Mut). After confirmation of successful cloning by control digests and PCR sequencing, the DNA was transfected into HEK293F cells and cultured for 7 or 8 days. Supernatant was purified using a nickel agarose column with strong affinity toward the 6xHis-tag, included on the C terminus of the scFv-CD39 constructs.

EXAMPLE 8

SE-HUMAN SERUM ALBUMIN-CD39 PREPARATION

[0415] A SE-HSA-CD39 construct was prepared with the following sequence:

[0416]

in which :

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

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

• Uppercase bold text corresponds to HSA amino acid sequence; • Uppercase underlined text corresponds to the amino acid sequence of the extracellular domain of CD39;

. EQKLISEEDL [SEQ ID NO: 15] is a C-myc tag; and

• a His tag is underlined.

EXAMPLE 9

SE-HSA-CD39 BINDING TO ACTIVATED PLATELETS

[0417] Binding of SE-HSA-CD39 to activated human platelets was evaluated by flow cytometry according to the protocols described in Example 4.

[0418] The results in presented in Figure 11 show that SE-HSA-CD39 binds selectively to activated platelets (Figure 11B) but not to non-activated platelets (Figure 11A).

EXAMPLE 10

IN VITRO ANTITHROMBOTIC EFFECTS OF SE-HSA-CD39

[0419] The antithrombotic efficacy of the SE-HSA-CD39 construct was investigated on ADP-induced platelet activation, following the protocols described in Example 5.

[0420] The results presented in Figure 12 demonstrate that SE-HSA-CD39 effectively hydrolyzes ADP to AMP, thereby preventing platelet activation.

EXAMPLE 11

PRODUCTION OF MECOSAR-NHS-ESTER AND SE-MECOSAR

[0421] MeCOSar-NHS-ester (where MeCOSar = l-methyl-8-NHC0(CH2)3C02H- sarcophagine; sarcophagine = 3, 6, 10, 13, 16, 19-hexaazabicyclo[6.6.6]-icosane = sar) as well as the conjugation of the MeCOSar to the scFv were prepared as described previously (Alt, K. et al., 2014. Mol Pharm 11 : 2855-2863; Paterson, B. M. et al., 2014. Dalton Trans 43: 1386-1396).

[0422] Briefly, the activated ester, (t-Boc)4-5MeCOSar-NHS-ester, was obtained by reacting (t-Boc)4-5Me-COSar with l-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in dimethylformamide (DMF), followed by purification by silica gel chromatography. The t-Boc groups were removed with trifluoroacetic acid, and MeCOSar-NHS-ester was isolated as the tris-trifluoroacetic acid tris-hydrate salt.

[0423] Conjugation of the MeCOSar to the scFv was performed by incubating the scFv with MeCOSar-NHS-ester in PBS for 3 h at RT with shaking.

Materials

Reagents

[0424] · MillQ water:

[0425] · PBS (pH 7.4) - made fresh in house from: 137 mM NaCI (Merck, Cat#

106404); 1.7 mM KCI (ChemSupply, AR grade, Cat# PA054); 10 mM NaH2P04 (ChemSupply, AR grade, Cat# SA061) ; 1.8 mM K2HP04 (ChemSupply, AR grade, Cat# PA020); and NaOH (ChemSupply, AR grade, Cat# SA178).

[0426] · MeCOSar-NHS (29 mg/mL in DMSO) .

[0427] »SE (9.6 mg/mL) in PBS buffer, diluted to a concentration of 1.8 mg/mL in PBS.

Equipment

[0428] · Centrifuge tubes, 1.5 mL - Eppendorf 3810X

[0429] · 10 kDa Spin filters - Amicon Ultra - 0.5 mL 10 kDa, Cat#: UFC501096

(Merck-Millipore)

[0430] Centrifuge - Eppendorf Centrifuge 5418

[0431] · NanoDrop (ThermoFisher Scientific)

[0432] · Agilent 6200 LC/MS QTOF (see Equipment setup)

Equipment Setup

[0433] Parameters used in this protocol :

[0434] All data were acquired, and reference mass corrected via a dual-spray electrospray ionization (ESI) source. Acquisition was performed using the Agilent Mass Hunter Acquisition software version B.02.01 (B2116.30). Ionization mode: Electrospray Ionization; Drying gas flow: 7 L/min; Nebulizer: 35 psi; Drying gas temperature: 325°C; Capillary Voltage (Vcap) : 4000 V; Fragmentor: 300 V; Skimmer: 65 V; OCT RFV: 250 V; Scan range acquired: 300-3200 m/z internal reference ions: positive ion mode = m/z = 121.050873 & 922.009798.

[0435] Column: Phenomenex Jupiter C5 5 5 mm 300 A 2.1 x 50 mm.

[0436] 1. Transfer 70 mg of antibody in buffer (40 mL) to a 1.5 mL Eppendorf tube.

[0437] 2. Add MeCOSar-NHS (16 pg, 0.5 mL of 29 mg/mL mixture in DMSO, 10 equivalents,) and incubate at room temperature with gentle shaking for 30 min. [0438] 3. Dilute the reaction mixture to a total volume of 450 mL with PBS and transfer the contents of the Eppendorf tube to a centrifugal filter unit (10,000 Da cut off) and centrifuge at 14,000 ref for 7 min.

[0439] 4. Add 450 mL of PBS (pH 7.4) and centrifuge at 14,000 ref for 7 min and repeat twice.

[0440] 5. Invert the centrifugal filter unit into a fresh tube and centrifuge at 1,00 ref for 2 min.

[0441] 6. Determine concentration using NanoDrop utilizing the A280.

[0442] 7. Take a 10 pg mass equivalent aliquot of the solution and dilute to 60- 100 mL with PBS (pH 7.4) and inject into the LC/MS using Method 1.

[0443] 8. Analyze using Mass Hunter Qualitative analysis using the following parameters a. Deconvolution algorithm : Maximum entropy

[0444] b. Mass range : 35000-55000 Daltons

[0445] c. Mass step: 1 Dalton

[0446] d. Baseline

[0447] i. Subtract baseline: Checked

[0448] ii. Baseline Factor: 7.00

[0449] e. Adduct: Proton

[0450] f. Isotope width : Automatic

Result

[0451] Protein isolated from the MeCOSar conjugation was analyzed by liquid chromatography/mass spectrometry. Deconvolution of the data presented in Figure 13 revealed a mixture of conjugated species SE-MeCOSar_n species with n = 1, 2, 3, 4 corresponding to mass increments of about 461 Daltons.

EXAMPLE 12

RADIOLABELING OF SE-MECOSAR

[0452] SE-MeCOSar (100 mg) is incubated with 25-35 MBq of 64Cu in PBS for 30 min at room temperature. A solution of 10 mM ethylenediaminetetraacetic acid (EDTA, 10 mL) is added and the reaction incubated for 5 min. All samples are washed twice with PBS using spin columns (Millipore, cut off 10,000 MWCO). Analysis/quality control is performed using thin layer chromatography (silica gel 60, F254; Merck) and 0.1 M citrate buffer (pH 5) as the mobile phase. Radiolabeled immunoconjugate (1.5 mL) is spotted at the origin, the strip is allowed to air-dry and is developed. The strip is cut into three pieces and the radioactivity in each section is counted using a gamma counter (Wizard single detector gamma-counter, Perkin Elmer). EXAMPLE 13

PET STUDIES AND POST-MORTEM BlODISTRIBUTION.

[0453] The animals are injected with SE-64CuMeCOSar (20 mg, 3-5 MBq), via lateral tail-vein injection. After 30 min incubation, a PET scan was performed using

NanoPET/CT In Vivo Preclinical Imager (Mediso, Hungary) with a 30 min PET acquisition time, and coincidence relation of 1-3. Image reconstruction is performed with the following parameters OSEM with SSRB 2D LOR, energy window, 400-600 keV; filter Ramlak cut off 1, number of iteration/subsets, 8/6. For the CT scans, an X-ray voltage of 45 kVp, an exposure time of 900 ms and a pitch of 1 are used. A total projection of 240 projects over 360° of rotation was acquired. Projection data are rebinned by 1 :4 and reconstructed using a RamLak filter. Image files of PET and CT scan were fused using InVivoScope version 2.00. For quantitative tracer uptake analysis after the CT scan, the animals are perfused with PBS, the heart (divided into an ischemic and a non-ischemic part) and the quadriceps femoris (muscle control tissue) is removed and radioactivity is measured in the gamma counter (Perkin Elmer) using an energy window between 450 and 650 keV. Results are expressed as % injected dose per g (% ID/g) of tissue.

[0454] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0455] 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.

[0456] Throughout the specification the aim has been to describe the preferred embodiments of the present disclosure without limiting the invention 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 disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An antigen-binding molecule that binds to activated glycoprotein Ilb/IIIa (GPIIb/IIIa), comprising :

(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. The antigen-binding molecule of claim 1, which is a monovalent antigen-binding molecule.

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

4. The antigen-binding molecule of claim 1, which is a multivalent antigen-binding molecule.

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

6. The antigen-binding 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. A chimeric molecule comprising a GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, and a heterologous moiety.

8. The chimeric molecule of claim 7, wherein the heterologous moiety comprises a payload.

9. The chimeric molecule of claim 7 or claim 8, wherein the heterologous moiety is a detectable moiety, a half-life extending moiety, or a therapeutic moiety (e.g., an anti- inflammatory moiety, an immunomodulating moiety, or an anti-cancer moiety).

10. The chimeric molecule of any one of claims 7 to 9, wherein the heterologous moiety is a proteinaceous molecule and the chimeric molecule is in the form of a single chain chimeric polypeptide.

11. The antigen-binding molecule of any one of claims 1 to 6, or the chimeric molecule of any one of claims 7 to 10, which is contained in a delivery vehicle.

12. The chimeric molecule of claim 11, wherein the delivery vehicle is a liposome, a nanoparticle, a microparticle, a dendrimer or a cyclodextrin.

13. An isolated polynucleotide comprising a nucleic acid sequence encoding the anti- GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, or the chimeric molecule of any one of claims 7 to 10.

14. A construct comprising a nucleic acid sequence encoding the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, or the chimeric molecule of any one of claims 7 to 10 in operable connection with one or more control sequences.

15. A host cell that contains the construct of claim 14.

16. A pharmaceutical composition comprising the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, and a pharmaceutically acceptable carrier.

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

conformation, the method comprising contacting the GPIIb/IIIa with the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, to thereby inhibit binding of the ligand to the

GPIIb/IIIa.

18. A method for inhibiting binding of a ligand to an activated platelet, the method comprising contacting the activated platelet with the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, to thereby inhibit binding of the ligand to the activated platelet.

19. The method of claim 17 or claim 18, wherein the ligand is selected from fibrinogen, von Willebrand factor, vitronectin, thrombospondin and CD40 ligand.

20. The method of claim 17 or claim 18, wherein the ligand is fibrinogen.

21. A method for inhibiting platelet aggregation in a subject, the method comprising administering to the subject an effective amount of the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16, to thereby inhibit platelet aggregation in the subject.

22. A method for inhibiting thrombus formation in a subject, the method comprising administering to the subject an effective amount of the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16, to thereby inhibit thrombus formation in the subject.

23. A method for inhibiting embolus formation in a subject, the method comprising administering to the subject an effective amount of the anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16, to thereby inhibit embolus formation in the subject.

24. 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 anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16.

25. The method of claim 24, 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.

26. 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 anti-GPIIb/IIIa antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16.

27. The method of claim 26, 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.

28. The method of claim 26, 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 extracorporeal circulation (ECMO, cardiopulmonary bypass) procedure in which blood is exposed to an artificial surface that promotes thrombosis.

29. The method of claim 28, 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.

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

cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis.

31. The method of claim 26, wherein the thromboembolism-associated conditions is acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism.

32. 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 GPIIb/IIIa-specific antigen-binding molecule according to any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16.

33. The method of claim 32, wherein the hematologic disorder is sickle cell disease or thrombophilia.

34. A method for reducing or inhibiting proliferation, survival and viability of a tumor in a subject, the method comprising administering to the subject an effective amount of a chimeric molecule comprising the GPIIb/IIIa-specific antigen-binding molecule according to any one of claims 1 to 6, 11 and 12 and an anti-cancer moiety (e.g., a chemotherapeutic agent or immunomodulating agent).

35. A method for treating or inhibiting the development of a cancer in a subject, the method comprising administering to the subject an effective amount of a chimeric molecule comprising the GPIIb/IIIa-specific antigen-binding molecule according to any one of claims 1 to 6, 11 and 12 and an anti-cancer moiety (e.g., a chemotherapeutic agent or

immunomodulating agent).

36. A method for detecting the presence of an activated platelet, the method comprising contacting the activated platelet with an anti-GPIIb/IIIa antigen-binding molecule as defined in any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, which is operably connected to a detectable moiety, to form a complex that comprises the activated platelet and the antigen-binding molecule or chimeric molecule, and detecting the complex to thereby detect the presence of the activated platelet.

37. A method for detecting the presence of an activated platelet in a subject, the method comprising administering to the subject a an anti-GPIIb/IIIa antigen-binding molecule as defined in any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, which is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an activated platelet and the antigen- binding molecule or chimeric molecule to thereby detect the presence of the activated platelet in the subject.

38. A method for detecting presence of a thrombus in a subject, the method comprising administering to the subject an anti-GPIIb/IIIa antigen-binding molecule as defined in any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, which is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises a thrombus and the antigen-binding molecule or chimeric molecule to thereby detect the presence of the thrombus in the subject.

39. A method for detecting presence of an embolus in a subject, the method comprising administering to the subject an anti-GPIIb/IIIa antigen-binding molecule as defined in any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, which is operably connected to a detectable moiety, and detecting the presence in the subject of a complex that comprises an embolus and the antigen-binding molecule or chimeric molecule to thereby detect the presence of the embolus in the subject.

40. The method of any one of claims 21 to 39, wherein subject has or is suspected of having a condition associated with the presence of activated platelets.

41. The method of claim 40, 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.

42. A kit for detecting an activated platelet, thrombus and/or embolus, for detecting presence of a tumor, 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, for treating or inhibiting the development of a hematologic disorder, for reducing or inhibiting proliferation, survival or viability of a tumor, and/or for treating or inhibiting the development of a cancer, the kit comprising the anti-GPIIb/IIIa antigen-binding molecule of any one of claims 1 to 6, 11 and 12, or the chimeric molecule of any one of claims 7 to 12, or the composition of claim 16.