Targeted Thrombolysis for Treatment of Microvascular Thrombosis

Abstract

The present invention provides fusion proteins for targeted delivery of plasminogen activators to platelet-VWF complexes, or alternatively to the site where these are located, in a fibrin-independent manner. The fusion protein of the invention are for use in methods for the prevention or treatment of diseases or conditions associated with such platelet-VWF complexes, which may cause microvascular thrombosis in diseases such as e.g. thrombotic thrombocytopenic purpura. Preferred targeting agents for incorporation into the fusion proteins are e.g. nanobodies against VWF or platelets. Preferred plasminogen activators for use in the fusion proteins comprise the protease domains of uPA or tPA. The invention further pertains to nucleic acid molecule encoding the fusion proteins of the invention, e.g. a gene therapy vector, and to pharmaceutical compositions comprising the fusion proteins of the invention or such gene therapy vectors.

1 .- 9 . (canceled) 
     
     
         10 . A method for treating or reducing the risk of microvascular thrombosis, wherein the method comprises the step of administering to a subject in need thereof a fusion protein comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus comprising al least one of VWF and platelets, wherein the targeting agent is not a targeting agent that specifically binds to only the activated form of the GPIIb/IIIa receptor on platelets, for use in the prevention or treatment of microvascular thrombosis. 
     
     
         11 . The method of  claim 10 , wherein the microvascular thrombosis is treated or the risk of its occurrence is reduced in a disease or condition selected from the group consisting of: acquired or hereditary thrombotic thrombocytopenic purpura (TTP) complement-mediated thrombotic microangiopathy, haemolytic uremic syndrome, antiphospholipid antibody syndrome, non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease and myocardial infarct. 
     
     
         12 . A fusion protein comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus comprising at least one of VWF and platelets, wherein the targeting agent is one or more of:
 a) a targeting agent that at least binds unfolded VWF, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF;   b) a targeting agent that binds the D3 domain of VWF;   c) a targeting agent that binds integrin allb/(3III on platelets;   d) a targeting agent that binds to a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected form the group consisting of E-selectin, P-selectin, uPAR, c1q receptor, kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial protein C receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and,   e) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatidylethanolamine.   
     
     
         13 . A fusion protein according to  claim 12 , wherein the fusion protein comprises more than one targeting agent. 
     
     
         14 . A fusion protein according to  claim 12 , wherein the targeting agent comprises at least one of:
 a) an antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and,   b) a binding domain from a protein that naturally binds VWF, platelets and activated or injured vascular endothelium, which binding domain specifically binds to at least one of VWF, platelets, and activated or injured vascular endothelium.   
     
     
         15 . A fusion protein according to  claim 14 , wherein the antibody variable domain is a VHH, preferably a humanized VHH, or wherein the binding domain comprises a binding domain selected from the group consisting of:
 i) a VWF-binding domain from one of ADAMTS13, Factor XII, Factor H (complement regulator), plasminogen and Factor VIII; and,   ii) a membrane binding domain selected from the vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from factor V and the C-domain from factor VIII.   
     
     
         16 . A fusion protein according to  claim 12 , wherein the plasminogen activator comprises the protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase or staphylokinase,
 wherein preferably the plasminogen activator further comprises at least the cysteine-containing part of the connecting peptide that naturally occurs in the plasminogen activator immediately upstream of its protease domain,   and wherein the fusion protein optionally comprises a linker amino acid sequence linking the targeting agent and the plasminogen activator.   
     
     
         17 . A fusion protein according to  claim 16 , wherein the fusion protein comprises in a N- to C-terminal order:
 a) one or more targeting agents wherein the targeting agent is one or more of:
 a targeting agent that at least hinds unfolded VWP, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF; 
 ii) a targeting agent that binds the D3 domain of VWF; 
 ill) a targeting agent that binds integrin αIIb/βIII on platelets; 
 iv) a targeting agent that binds io a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected form the groan consisting of E-selection, P-selection, uPAR, c1q receptor, kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial protein C receptor thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and, 
 v) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatldylethanoiamine, whereby, optionally the targeting agents are linked by linker amino acid sequences; 
   b) optionally a linker amino acid sequence; and,   c) the plasminogen activator or plasminogen-derived protease domain.   
     
     
         18 . A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as defined  claim 12 , wherein the nucleotide sequence encoding the fusion protein further preferably comprises a nucleotide sequence encoding a signal peptide operably linked to the fusion protein, and wherein nucleic acid molecule further preferably comprises regulatory elements conducive to the expression of the fusion protein, which regulatory elements are operably linked to the nucleotide sequence. 
     
     
         19 .- 20 . (canceled) 
     
     
         21 . The method of  claim 10 , wherein the targeting agent specifically binds to at least one of VWF, platelets and activated or injured vascular endothelium. 
     
     
         22 . The method of  claim 10 , wherein the targeting agent is one or more of;
 a) a targeting, agent that at least binds unfolded VWF, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF.   b) a targeting agent that binds the D3 domain of VWF;   c) a targeting agent that binds the GP1B receptor on platelets,   d) a targeting agent that binds integrin αIIb/βIII on platelets;   e) a targeting agent that binds to a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected from the group consisting of E-setecan, P-selectin, uPAR, c1q receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and,   f) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatidylethanolamine.   
     
     
         23 . The method of  claim 10 , wherein the fusion protein comprises more than one targeting agent. 
     
     
         24 . The method of  claim 10 , wherein the targeting agent comprises at least one of:
 a) ao antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and,   b) a binding domain from a protein that naturally binds VWF, platelets and activated or injured vascular endothelium, which binding domain specifically binds to at least one of VWF, platelets, and activated or injured vascular endothelium.   
     
     
         25 . The method of  claim 24 , wherein the antibody variable domain is a VHH, preferably a humanized VHH, or wherein the binding domain comprises a binding domain selected front the group consisting of:
 i) the platelet GP1B receptor-binding A1 domain from VWF;   ii) a VWF-binding domain from one of ADAMTS13, Factor XII, Factor H (complement regulator), plasminogen and Factor VIII, and,   iii) a membrane binding domain selected from the vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from factor V and the C-domain from factor VIII.   
     
     
         26 . The method of  claim 10 , wherein the plasminogen activator comprises the protease domain of tissue plasminogen activator ((PAT urokinase plasminogen activator (nPA), plasminogen, streptokinase or staphylokinase,
 wherein preferably the plasminogen activator further comprises at least the cysteine-containing part of the connecting peptide that naturally occurs in the plasminogen activator immediately upstream of its protease domain,   and wherein the fusion protein optionally comprises a linker amino acid sequence linking the targeting agent and the plasminogen activator   
     
     
         27 . The method of  claim 26 . wherein the fusion protein comprises in a N- to C-terminal order;
 a) the one or more targeting agents, whereby, optionally the targeting agents axe linked by linker amino acid sequences;   b) optionally a linker amino acid sequence; and,   c) the plasminogen activator or plasminogen-derived protease domain.