Targeted thrombolysis for the treatment of microvascular thrombosis

Abstract

To provide a fusion protein for targeted delivery of a plasminogen activator in a fibrin-independent manner to a platelet-VWF complex or a site where the platelet-VWF complex is located.SOLUTION: A pharmaceutical composition for preventing or treating microvascular thrombosis is provided, comprising a fusion protein, wherein the fusion protein comprises a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus comprising VWF and platelets, wherein the targeting agent comprises an antibody variable domain that specifically binds to VWF, and wherein the antibody variable domain comprises a CDR3 domain having the amino acid sequence of SAGLYVGDGPDDAMDDY.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of medicine and pharmacology, particularly to the field of biologics for use in the prevention or treatment of diseases or conditions associated with microvascular thrombosis, such as thrombotic thrombocytopenic purpura. More particularly, the present invention relates to fusion proteins comprising a targeting agent and a plasminogen activator, where the targeting agent targets the plasminogen activator to at least one of VWF, platelets, and activated vascular endothelium for the purpose of enzymatically degrading vascular obstructions in a fibrin-independent manner. The present invention further relates to gene therapy vectors encoding such fusion proteins. [Background technology]

[0002] Microvascular thrombosis (MVT) is characterized by the formation of platelet aggregates in small blood vessels. Platelet aggregates are composed, at a minimum, of platelets and VWF. This is evident in thrombotic thrombocytopenic purpura (TTP), in which platelet- and VWF-rich, but fibrin-poor, microthrombi occlude the microvasculature, resulting in life-threatening consequences. Thus, fibrin, as seen in macrovascular thrombosis, is not essential for such microvascular occlusion. MVT is a common feature of several conditions, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, antiphospholipid syndrome, and complement-mediated thrombotic microangiopathy (George et al., 2014, N Engl J Med. 371(7):654-66). Severe cases of MVT can result in multiple organ failure with potentially fatal outcomes. Even less severe cases can result in organ damage, potentially reducing both the patient's quality of life and life expectancy. Recent studies suggest that microvascular disease underlies cardiovascular disease / events in the more general population of patients with cardiovascular disease who do not have overt radiological signs of large-vessel obstruction. Microvascular disease is thought to ultimately lead to heart failure, particularly in women.

[0003] Patients with TTP experience episodes of microvascular thrombosis when platelets form complexes with very large multimers of von Willebrand factor (VWF). This episode results from a marked decrease in the activity of the enzyme ADAMTS13 (a disintegrin and thrombospondin type I motif-containing metalloproteinase member 13). ADAMTS13 normally regulates the thrombogenicity of VWF by enzymatically reducing the size of VWF multimers. To do so, VWF must unfold from its globular form to an unfolded conformation, exposing its A2 domain for proteolysis. The majority of TTP patients are affected by neutralizing autoantibodies against ADAMTS13. In a small subpopulation, mutations in ADAMTS13 resulting in deficiency have been described (Upshaw-Shulman syndrome).

[0004] Current therapies for TTP require massive plasma exchange to deplete inhibitory antibodies and simultaneously restore ADAMTS13 activity. However, persistent autoantibodies prevent clearance of microthrombi, making treatment time-consuming and prohibitively expensive (Fijnheer et al., Ned Tijdsch Hematol 2016, 13(1):18-24).

[0005] In addition to ADAMTS13, VWF can also be cleaved by the enzyme plasmin (Berkowitz et al., J Clin Invest 1987 Feb; 79(2):524-31). We previously identified the therapeutic potential of systemic plasminogen activation (by streptokinase) in a mouse model of TTP, suggesting that plasmin may act as a functional surrogate for ADAMTS13 (Tersteeg et al., 2014, Circulation 129(12):1320-31). Plasmin (gen) can directly bind to unfolded VWF, whereas natural plasminogen activators, such as tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), cannot. The natural targets of tPA and uPA are fibrin and the endothelial cell receptor uPAR, respectively. Furthermore, microthrombi in TTP are poor in fibrin, and it is unclear whether fibrin is essential for other types of MVT. This makes molecules commonly used as thrombolytic agents in the treatment of macrovascular disease (i.e., tPA, uPA) ineffective in treating MVT. Furthermore, for safety reasons (i.e., low platelet counts), it is desirable to avoid systemic plasminogen activation. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide means and methods for treating MVT and related conditions. Accordingly, the present invention provides fusion proteins for targeted delivery of plasminogen activators to platelet-VWF complexes or sites where platelet-VWF complexes are located in a fibrin-independent manner. The present invention further provides methods for treating conditions that can be prevented or treated by local delivery / stimulation of plasminogen activation to sites of microvascular obstruction. [Means for solving the problem]

[0007] In a first aspect, the present invention relates to 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. Preferably, the targeting agent in the fusion protein of the present invention specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium. Furthermore, it is preferred that the targeting agent in the fusion protein of the present invention is not a targeting agent that specifically binds only to the activated form of the GPIIb / IIIa receptor on platelets. More preferably, the targeting agent in the fusion protein of the present invention is selected from the group consisting of: a) a targeting agent that binds to at least unfolded VWF, preferably with a preference for unfolded VWF over globular VWF; b) a targeting agent that binds to the D3 domain of VWF; c) a targeting agent that binds to the GP1B receptor on platelets; d) a targeting agent that binds to the integrin αIIb / βIII on platelets; and e) a targeting agent that binds to a receptor preferentially expressed by activated endothelium, preferably the receptor being E-segment. and f) a targeting factor selected from the group consisting of lectin, 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 f) a targeting factor that binds to a membrane marker of activated or damaged endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine, and phosphatidylethanolamine. In one embodiment, the fusion protein of the present invention preferably comprises two or more targeting factors.

[0008] The fusion protein of the present invention is preferably a fusion protein in which 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 derived from a protein that naturally binds to VWF, platelets, and activated or damaged vascular endothelium, and that specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium. Thus, in the fusion protein of the present invention, the antibody variable domain is preferably a VHH, more preferably a humanized VHH. Alternatively, in the fusion protein of the present invention, the binding domain derived from a protein that naturally binds to VWF, platelets, and activated or damaged vascular endothelium preferably comprises a binding domain selected from the group consisting of: i) the platelet GP1B receptor-binding A1 domain of VWF; ii) a VWF-binding domain of one of ADAMTS13, factor XII, factor H (a complement regulator), plasminogen, and factor VIII; and iii) a membrane-binding domain selected from the vitamin K-dependent carboxylation / gamma-carboxyglutamic acid (GLA) domain, the C domain of factor V, and the C domain of factor VIII.

[0009] In the fusion proteins of the invention as defined above, the plasminogen activator preferably comprises the protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase, or staphylokinase. In the fusion proteins of the invention, the plasminogen activator preferably further comprises at least the cysteine-containing portion of the connecting peptide naturally occurring in plasminogen activators immediately upstream of the protease domain. Optionally, the fusion proteins of the invention comprise a linker amino acid sequence connecting the targeting agent and the plasminogen activator.

[0010] Thus, a fusion protein according to the invention preferably comprises, from N-terminus to C-terminus, a) one or more targeting factors as defined above, optionally linked by a linker amino acid sequence; b) optionally a linker amino acid sequence; and c) a plasminogen activator or plasminogen-derived protease domain as defined above.

[0011] In a second aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein according to the invention as defined above. Preferably, the nucleotide sequence encoding the fusion protein further comprises a nucleotide sequence encoding a signal peptide operably linked to the fusion protein. The nucleic acid molecule preferably further comprises regulatory elements operably linked to the nucleotide sequence that facilitate expression of the fusion protein.

[0012] In a third aspect, the present invention relates to a gene therapy vector comprising a nucleic acid molecule according to the invention.

[0013] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein according to the invention or a gene therapy vector according to the invention and a pharmaceutically acceptable excipient.

[0014] In a fifth aspect, the present invention relates to a fusion protein according to the present invention, a gene therapy vector according to the present invention, or a pharmaceutical composition according to the present invention for use in preventing or treating a disease or condition associated with thrombi comprising at least one of VWF and platelets, preferably a disease or condition associated with microvascular thrombosis. More preferably, the disease or condition associated with (micro)thrombi comprising at least one of VWF and platelets is selected from the group consisting of acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombus, occlusive thrombus formation, arterial thrombosis, acute coronary occlusion, peripheral arterial occlusive disease, coronary artery bypass graft, coronary valve replacement, and angioplasty, stenting, or restenosis and lesions due to coronary interventions such as atherectomy, thickening after angioplasty, atherectomy, or arterial stenting, occlusive syndromes of the vascular system or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction.

[0015] In a sixth aspect, the present invention relates to a method for treating or reducing the risk of a disease or condition associated with thrombi comprising at least one of VWF and platelets, preferably a disease or condition associated with microvascular thrombosis, comprising the step of administering to a subject in need thereof an effective amount of a fusion protein according to the present invention, a gene therapy vector according to the present invention, or a pharmaceutical composition according to the present invention. More preferably, the disease or condition associated with (micro)thrombi comprising at least one of VWF and platelets is selected from the group consisting of acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombus, occlusive thrombus formation, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and lesions resulting from coronary artery bypass grafting, coronary valve replacement, and coronary interventions such as angioplasty, stenting, or atherectomy, thickening after angioplasty, atherectomy, or arterial stenting, occlusive syndromes of the vascular system or lack of patency of affected arteries, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, and myocardial infarction. [Brief explanation of the drawings]

[0016] [Figure 1A] VHH-miniUPA (mUPA) construct. A) Schematic of the mUPA construct. B) Schematic of the mUPA construct containing VHH coding sequences (VHH-mUPA). [Figure 1B] VHH-miniUPA (mUPA) construct. A) Schematic of the mUPA construct. B) Schematic of the mUPA construct containing VHH coding sequences (VHH-mUPA). [Figure 2] Western blot of purified VHH-mUPA constructs. [Figure 3] Activity of VHH-mUPA constructs after activation with plasmin. [Figure 4] Plasminogen activation by VHH-mUPA constructs. [Figure 5A]Plasminogen activation by VHH-mUPA constructs in the presence of globular or unfolded VWF. A) Plasminogen activation by VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E) Plasminogen activation by VHH-A12. [Figure 5B] Plasminogen activation by VHH-mUPA constructs in the presence of globular or unfolded VWF. A) Plasminogen activation by VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E) Plasminogen activation by VHH-A12. [Figure 5C] Plasminogen activation by VHH-mUPA constructs in the presence of globular or unfolded VWF. A) Plasminogen activation by VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E) Plasminogen activation by VHH-A12. [Figure 5D] Plasminogen activation by VHH-mUPA constructs in the presence of globular or unfolded VWF. A) Plasminogen activation by VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E) Plasminogen activation by VHH-A12. [Figure 5E] Plasminogen activation by VHH-mUPA constructs in the presence of globular or unfolded VWF. A) Plasminogen activation by VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E) Plasminogen activation by VHH-A12. [Figure 6]Conversion of plasmin substrate after 5 min incubation at 37°C for constructs VHH-sVWF, VHH-D3, VHH-GP1B17, VHH-R2, and VHH-A12. [Figure 7A] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time. A) Microthrombolysis induced by VHH-sVWF, B) Microthrombolysis induced by VHH-D3, C) Microthrombolysis induced by VHH-R2, D) Microthrombolysis induced by VHH-GP1B17. [Figure 7B] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time. A) Microthrombolysis induced by VHH-sVWF, B) Microthrombolysis induced by VHH-D3, C) Microthrombolysis induced by VHH-R2, D) Microthrombolysis induced by VHH-GP1B17. [Figure 7C] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time. A) Microthrombolysis induced by VHH-sVWF, B) Microthrombolysis induced by VHH-D3, C) Microthrombolysis induced by VHH-R2, D) Microthrombolysis induced by VHH-GP1B17. [Figure 7D] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time. A) Microthrombolysis induced by VHH-sVWF, B) Microthrombolysis induced by VHH-D3, C) Microthrombolysis induced by VHH-R2, D) Microthrombolysis induced by VHH-GP1B17. [Figure 8A] A) Schematic example of the analytical method for determining when 50% microthrombus degradation has occurred. B) 50% microthrombus degradation by constructs VHH-sVWF, VHH-D3, VHH-GP1B17, VHH-R2, and VHH-A12. [Figure 8B]A) Schematic example of the analytical method for determining when 50% microthrombus degradation has occurred. B) 50% microthrombus degradation by constructs VHH-sVWF, VHH-D3, VHH-GP1B17, VHH-R2, and VHH-A12. [Figure 9] Microthrombolysis of VWF-platelet complexes adhered to human vascular endothelial cells analyzed in flow perfusion experiments. VWF-platelet complexes are visible as platelet strings, and the number of visible strings is counted as a function of time upon addition of the indicated fusion proteins. [Figure 10A] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time using a light transmission aggregometer. A) Microthrombolysis induced by caplacizumab (154.3 nM) or a VHH-D3 fusion protein containing UPA (154.3 nM) in the presence of plasminogen (100 μg / mL). B) Microthrombolysis induced by caplacizumab (154.3 nM) or a VHH-GP1B17 fusion protein containing UPA (154.3 nM) in the presence of plasminogen (100 μg / mL). [Figure 10B] Microthrombolysis (i.e., enzymatic degradation) of VWF-platelet aggregates. Dissolution of aggregates was monitored over time using a light transmission aggregometer. A) Microthrombolysis induced by caplacizumab (154.3 nM) or a VHH-D3 fusion protein containing UPA (154.3 nM) in the presence of plasminogen (100 μg / mL). B) Microthrombolysis induced by caplacizumab (154.3 nM) or a VHH-GP1B17 fusion protein containing UPA (154.3 nM) in the presence of plasminogen (100 μg / mL). DETAILED DESCRIPTION OF THE INVENTION

[0017] definition The terms "homology," "sequence identity," and the like are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing these sequences. In the art, "identity" also means the degree of sequence relatedness between such sequences, as determined by the match between strings of amino acid or nucleic acid sequences, as the case may be. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conservative amino acid substitutes of one polypeptide with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

[0018] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch), which optimally aligns sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Sequences can be said to be "substantially identical" or "essentially similar" when they share at least a certain minimum sequence identity percentage (as defined below) (when optimally aligned using default parameters, e.g., programs such as GAP or BESTFIT). GAP uses the Needleman and Wunsch global alignment algorithm, which aligns two sequences over their entire length (full length) to maximize the number of matches and minimize the number of gaps. Global alignment is preferably used to determine sequence identity when two sequences are of similar length. Generally, the GAP default parameters are used: gap creation penalty = 50 (nucleotides) / 8 (proteins), and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and sequence identity percentage scores can be determined using computer programs such as GCG Wisconsin Package version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open source software such as the EmbossWIN version 2.10.0 program "needle" (which uses the global Needleman-Wunsch algorithm) or "water" (which uses the local Smith-Waterman algorithm), using the same parameters or default settings as GAP described above. (For both "needle" and "water," and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5, and the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA.) When the overall lengths of the sequences are substantially different, local alignments such as those using the Smith-Waterman algorithm are preferred.

[0019] Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA and BLAST. Thus, the nucleic acid and protein sequences of the present invention can further be used as "query sequences" to search public databases to identify, for example, other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage (http: / / www.ncbi.nlm.nih.gov / ).

[0020] As will be apparent to those skilled in the art, when determining the degree of amino acid similarity, they may optionally take into account so-called "conservative" amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. Examples of classes of amino acid residues for conservative substitutions are shown in the table below. [Table 1] [Table 2] [Table 3]

[0021] Nucleotide sequences encoding the fusion proteins of the present invention can also be defined by their ability to hybridize under moderate, or preferably stringent, hybridization conditions with the nucleotide sequences of the encoding fusion proteins exemplified herein. Stringent hybridization conditions are defined herein as conditions under which a nucleic acid sequence of at least about 25, preferably about 50, 75, or 100, and most preferably about 200 or more nucleotides can hybridize in a solution containing about 1 M salt, preferably 6×SSC or any other solution with equivalent ionic strength, at a temperature of about 65°C, and washing in a solution containing about 0.1 M or less salt, preferably 0.2×SSC or any other solution with equivalent ionic strength, at 65°C. Hybridization is preferably carried out overnight, i.e., for at least 10 hours, and washing is preferably carried out for at least 1 hour with at least two changes of wash solution. These conditions typically allow specific hybridization of sequences with greater than about 90% sequence identity.

[0022] Moderate conditions are defined herein as conditions under which a nucleic acid sequence of at least 50 nucleotides, preferably about 200 or more nucleotides, can hybridize in a solution containing about 1 M salt, preferably 6×SSC or any other solution with equivalent ionic strength, at a temperature of about 45°C, and washing in a solution containing about 1 M salt, preferably 6×SSC or any other solution with equivalent ionic strength, at room temperature. Hybridization is preferably performed overnight, i.e., for at least 10 hours, and washing is preferably performed for at least 1 hour with at least two changes of wash solution. These conditions typically allow specific hybridization of sequences with up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions to specifically identify sequences with identities ranging from 50% to 90%.

[0023] The terms "nucleic acid construct" or "nucleic acid vector" are understood herein to mean an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, although a nucleic acid construct may contain (a portion of) a naturally occurring nucleic acid molecule, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules. The terms "expression vector" or "expression construct" refer to a nucleic acid molecule capable of effecting expression of a nucleotide sequence or gene in a host cell or host organism compatible with such expression vector or construct. These expression vectors typically contain regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed and effect expression of the nucleotide sequence. Such regulatory elements usually include at least suitable transcriptional regulatory sequences and, optionally, a 3' transcription termination signal. Additional elements necessary or useful for effecting expression, such as expression enhancer elements, may also be present. The expression vector is introduced into a suitable host cell and is capable of effecting expression of the coding sequence in an in vitro cell culture of the host cell. While the expression vector is suitable for replication in the host cell or host organism of the present invention, the expression construct is typically integrated into the genome of the host cell so that the host cell's genome is maintained. Techniques for introducing nucleic acids into cells are well established in the art, and any suitable technique can be used depending on the particular situation. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as adenovirus, AAV, lentivirus, or vaccinia. For microbial cells, such as bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduced nucleic acid may be carried on an extrachromosomal vector within the cell, or the nucleic acid may be integrated into the genome of the host cell. Integration may be facilitated by including sequences in the nucleic acid or vector that facilitate recombination with the genome, according to standard techniques. Expression of the nucleic acid may occur after introduction to produce the encoded fusion protein.In some embodiments, host cells may be cultured in vitro under conditions that allow expression of the nucleic acid (host cells are often the progeny of transformed cells, but may also include cells that were actually transformed), such that the encoded fusion protein polypeptide is produced; if an inducible promoter is used, expression may require activation of the inducible promoter.

[0024] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control transcription of one or more coding sequences, is located upstream of the transcription start site of the coding sequence relative to the direction of transcription, and is structurally specified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer.

[0025] The term "selectable marker" is a term familiar to those skilled in the art and is used herein to refer to any genetic entity that, when expressed, can be used to select for one or more cells containing the selectable marker. The term "reporter" is primarily used to refer to a visible marker such as green fluorescent protein (GFP), but is sometimes used interchangeably with marker. Selectable markers can be dominant, recessive, or bidirectional.

[0026] As used herein, the term "operably linked" refers to the association of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0027] The terms "protein" and "polypeptide" are used interchangeably and refer to molecules made up of chains of amino acids, without regard to a particular mode of action, size, three-dimensional structure, or origin.

[0028] The term "signal peptide" (sometimes referred to as signal sequence) refers to a short peptide (usually 16-30 amino acids long) present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. Signal peptides typically end with a stretch of amino acids that is recognized and cleaved by a signal peptidase, either during or after translocation (from the cytosol to the secretory pathway, i.e., the ER), to generate the free signal peptide and mature protein. Signal peptides are highly heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable, even across different species, although the efficiency of protein secretion can depend on the signal peptide. Suitable signal peptides are widely known in the art, e.g., from Kall et al. (2004, J. Mol. Biol. 338:1027-1036) and von Heijne (1985, J. Mol. Biol. 184(1):99-105).

[0029] The term "gene" refers to a DNA fragment comprising a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, operably linked to a suitable regulatory region (e.g., a promoter). A gene usually comprises several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) that contains a polyadenylation site. "Gene expression" refers to the process by which a DNA region operably linked to an appropriate regulatory region, particularly a promoter, is transcribed into RNA that is biologically active, i.e., can be translated into a biologically active protein or peptide.

[0030] The term "homologous," when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean essentially that the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably the same variety or strain. When homologous to a host cell, the nucleic acid sequence encoding the polypeptide is typically (but not necessarily) operably linked to a different (heterologous) promoter sequence from that in its natural environment, and, if applicable, to a different (heterologous) secretion signal sequence and / or terminator sequence. It is understood that regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relationship between two nucleic acid sequences, the term "homologous" means that one single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on several factors, including the amount of identity between the sequences and hybridization conditions, such as temperature and salt concentration, as discussed below.

[0031] The term "heterologous" when used with reference to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome, or DNA or RNA sequence in which the nucleic acid or protein is found, or that is found at one or more locations within a cell, genome, or DNA or RNA sequence different from that in which the nucleic acid or protein is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell, or is synthetically or recombinantly produced. Typically, although not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins are sometimes referred to as foreign nucleic acids or foreign proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which the nucleic acid or protein is expressed is encompassed herein by the term heterologous nucleic acid or heterologous protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, ie combinations in which at least two of the combined sequences are foreign to each other.

[0032] Unless otherwise specified, the terms "immunoglobulin" and "antibody", whether used herein to refer to a heavy chain antibody or a traditional four chain antibody, include a full-sized antibody, the individual chains of an antibody, as well as any part, domain, or fragment of an antibody (such as an antigen-binding domain or fragment, e.g., a VHH domain or VHH domain, respectively). H / V LThe term "sequence" is used as a general term that includes both the related amino acid sequence and the nucleic acid or nucleotide sequence that encodes the amino acid sequence (including, but not limited to, a sequence of a specific polypeptide, a polypeptide sequence ...

[0033] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH" or, in the case of heavy-chain antibodies such as camelid antibodies, "VHH" (which consist only of heavy chains). The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding site. The term "variable" refers to the fact that the sequence of certain segments of the variable domain varies widely among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed across the average 110-amino acid span of the variable domain. Rather, the V region consists of relatively invariant stretches of approximately 15-30 amino acids called framework regions (FRs), separated by short, highly variable regions called "hypervariable regions" (HVRs) or complementarity-determining regions (CDRs), each approximately 9-12 amino acids long. The variable domains of naturally occurring heavy and light chains each contain four FRs that largely adopt a β-sheet configuration, the β-sheets being connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are tightly held together by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0034] The terms "VHH," "VHH domain," and "nanobody" are used interchangeably herein and are used herein to refer to the variable domain of a heavy-chain antibody, i.e., an antibody that contains only a heavy chain and no light chain, as known, for example, from camelid antibodies. The amino acid sequence and structure of a VHH can be considered to have four framework regions, or "FRs," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, interposed between three complementarity-determining regions, or "CDRs," referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively, but are not limited to this configuration. The total number of amino acid residues in a VHH can be within the range of 110-120, preferably 112-115, and most preferably 113. However, it should be noted that parts, fragments or analogues of VHHs (described further hereinbelow) are not particularly limited with respect to their length and / or size, as long as such parts, fragments or analogues meet the further functional requirements outlined hereinbelow and are preferably suitable for the purposes described herein.

[0035] The amino acid residues of a VHH (or conventional variable domain) are the VHHs as shown by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH, Bethesda, Md., Paper No. 91). HThe domains are numbered according to the conventional numbering system of Riechmann and Muyldermans (1999, J. Immunol. Methods, 231:25-38; see e.g., Figure 2 of that reference) as applied to VHH domains of camelid antibodies. According to this numbering system, FR1 of VHH comprises amino acid residues 1-30, CDR1 of VHH comprises amino acid residues 31-36, FR2 of VHH comprises amino acid residues 36-49, CDR2 of VHH comprises amino acid residues 50-65, FR3 of VHH comprises amino acid residues 66-94, CDR3 of VHH comprises amino acid residues 95-102, and FR4 of VHH comprises amino acid residues 103-113. In this regard, as is well known in the art, VHH domains are numbered as they are applied to VHH domains of camelid antibodies. H It should be noted that for VHH domains and VHH domains, the total number of amino acid residues in each of the CDRs may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be used in the actual sequence, or the actual sequence may contain more amino acid residues than the number possible in the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. However, in general, according to the Kabat numbering, it can be said that position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4, regardless of the number of amino acid residues in the CDRs.

[0036] V HAn alternative method for numbering the amino acid residues of a domain, which can be applied in a similar manner to VHH domains of camelid antibodies, is the method described by Chothia et al. (1989, Nature 342, 877-883), the so-called "AbM definition" and the so-called "contact definition". However, in the present specification, claims and figures, the numbering system according to Kabat, as applied to VHH domains according to Riechmann and Muyldermans, will be followed unless otherwise stated.

[0037] For a general overview of heavy chain antibodies and variable VHH domains of heavy chain antibodies, see, inter alia, the following references which are cited as general background art: WO 94 / 04678, WO 95 / 04079, WO 96 / 34103, WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231, WO 02 / 48193, WO 97 / 49805 ...0310, WO 01 / 44301, EP 1 134 231, WO 02 / 48193, WO 97 / 49805 Reference is made to WO 01 / 21817, WO 03 / 035694, WO 03 / 054016, WO 03 / 055527, WO 03 / 050531, WO 01 / 90190, WO 03 / 025020, WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, and WO 04 / 062551, and to Hassanzadeh-Ghassabeh et al. (2013, Nanomedicine 8(6):1013-1026). For a more specific description of single domain VHH antibodies against von Willebrand factor or the platelet receptor GPIb, reference is made to WO 2004 / 062551 and WO 2006 / 122825.

[0038] In general, it should be noted that the term "VHH" (or nanobody), as used herein in the broadest sense, is not limited to a particular biological origin or a particular method of preparation. For example, VHHs used in the present invention can be prepared by (1) isolating the VHH domain of a naturally occurring heavy chain antibody, (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain, (3) "humanizing" a naturally occurring VHH domain (as described below) or expressing a nucleic acid encoding such a humanized VHH domain, or (4) synthesizing a naturally occurring VHH domain from any animal species, particularly a mammalian species, e.g., a human. H "Camelization" of a domain or any such camelization H (5) the use of synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences; (6) the use of nucleic acid synthesis techniques to prepare nucleic acids encoding VHHs followed by expression of the resulting nucleic acid; and / or (7) any combination of the foregoing. Suitable methods and techniques for accomplishing the foregoing are state of the art and therefore known to those skilled in the art.

[0039] One particularly preferred class of VHHs for use in the present invention corresponds to the amino acid sequence of a naturally occurring VHH domain, but is "humanized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence are changed to those of the VHH of a conventional four-chain antibody of human origin. HThe VHHs include VHHs having an amino acid sequence in which one or more of the amino acid residues occurring at the corresponding position(s) of the VHH domain have been substituted with one or more of the amino acid residues occurring at the corresponding position(s) of the VHH domain. Humanization can be carried out in essentially known manners that will be apparent to those skilled in the art, such as those described in, for example, Jones et al. (Nature 321:522-525, 1986), Riechmann et al. (Nature 332:323-329, 1988), Presta (Curr. Op. Struct. Biol. 2:593-596, 1992), Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol. 1:105-115, 1998), Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995), Hurle and Gross (Curr. Op. Biotech., 5:428-433, 1994), as well as specific prior art on VHH humanization, such as Vincke et al. (2009, J. Biol. Chem. 284:3273-3284). Again, it should be noted that such humanized VHHs of the present invention can be obtained in any suitable manner known per se, and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

[0040] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0041] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen or target). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen / target). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K d ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens / targets slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present invention. Certain exemplary embodiments are described below.

[0042] "K d " or "K dThe "value" can be measured using ELISA as described in the Examples herein, or by using a surface plasmon resonance assay with a BIAcore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at approximately 10-50 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min, achieving protein binding of approximately 10 response units (RU). After antigen injection, 1 M ethanolamine is injected to block unreacted groups. To measure kinetics, two-fold serial dilutions of antibody or Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min in PBS containing 0.05% Tween 20 (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2). d ) is k off / k on See, e.g., Chen, Y et al. (1999) J. Mol Biol 293:865-881. If the on-rate is 10 by the surface plasmon resonance assay described above, 6 M -1 S -1If the on-rate exceeds 100 kJ / s, the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, bandpass 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instrument) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred red cuvette.

[0043] The "on-rate" or "rate of association" or "association rate" or "k on " can also be determined using the same surface plasmon resonance as described above using a Biacore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) as described above.

[0044] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that, when properly administered to an animal, e.g., a human, do not produce adverse, allergic, or other untoward reactions, or produce an acceptable reaction. Whether a particular adverse effect is tolerable is determined based on the severity of the disease. The preparation of pharmaceutical compositions containing at least one chimeric polypeptide or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified in "Remington: The Science and Practice of Pharmacy" (Allen, LV, ed., 22nd ed., 2012, www.pharmpress.com), incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0045] As used herein, "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, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy, edited by Allen, LV, 22nd ed., 2012, www.pharmpress.com, incorporated herein by reference). Except insofar as a conventional carrier is incompatible with the active ingredient, use of any conventional carrier in the therapeutic or pharmaceutical compositions is contemplated.

[0046] Any reference herein to a nucleotide or amino acid sequence accessible in a public sequence database refers to the version of the sequence entry available on the filing date of this document.

[0047] Description of the embodiment The present inventors surprisingly found that targeting plasminogen activation to sites of MVT is a feasible approach for treating TTP. TTP patients experience attacks of MVT when platelets form complexes with extra-large VWF. We previously reported the therapeutic potential of systemic plasminogen activation (by streptokinase) in a mouse model of TTP, suggesting that plasmin may act as a functional substitute for ADAMTS13 (Tersteeg et al., 2014, supra). Plasmin (plasminogen) can directly bind to VWF, whereas natural plasminogen activators (tPA, uPA) cannot. Furthermore, microthrombi are poor in fibrin, and for safety reasons, it is desirable to avoid systemic plasminogen activation. Therefore, the present invention aims to stimulate plasmin cleavage of the thrombogenic multimeric protein VWF to increase therapeutic efficacy and safety. More specifically, the present invention relates to modified plasminogen activators that have acquired the ability to bind to either VWF, platelets, or activated / damaged (micro)vascular endothelial cells in a fibrin-independent manner to locally induce plasmin activity for clearance of MVT. The present invention further provides methods for treating conditions that can be prevented or treated by local delivery / stimulation of plasminogen activation to sites of MVT.

[0048] Thus, in a first aspect, the present invention relates to a fusion protein comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus containing at least one of VWF and platelets. The thrombus site to which the targeting agent targets the fusion protein of the present invention can, in principle, be any site where a thrombus exists or has developed, including sites of macrovascular thrombus and microvascular thrombus (MVT), as well as sites of (yet) non-occlusive macrovascular thrombus or microvascular thrombus. However, the fusion protein of the present invention is also intended to exclude sites of MVT, which are poor in fibrin but include sites where VWF, platelets, and vascular endothelium may be activated or damaged. Therefore, the targeting agent in the fusion protein of the present invention is preferably a targeting agent that specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium.

[0049] The targeting agent can be any ligand or binding molecule that specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium. However, it is preferred that the targeting agent is a proteinaceous targeting agent. More preferably, the proteinaceous targeting agent is part of a single amino acid chain of a fusion protein, which chain also includes a plasminogen activator.

[0050] A targeting agent that "binds" to a target of interest, e.g., VWF, platelets, or activated / damaged endothelium, is an agent that binds to the target with sufficient affinity so that the targeting agent is useful as a therapeutic agent when targeting structures, such as MVT, cells, or tissues, that express or expose the target, and that does not significantly cross-react with other proteins or molecules. In such embodiments, the extent of binding of the targeting agent to "non-target" molecules (e.g., proteins) will be less than about 10% of the binding of the targeting agent to the specific target molecule, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With respect to the binding of a targeting agent to a target molecule, the terms "specific binding," or "specifically binds" or "binds" or "is specific for" a particular target molecule or polypeptide, e.g., an epitope on a particular polypeptide target, refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that lacks binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding," or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target, refer to, for example, the K d (which may be determined as described above) is at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12M, or greater. In one embodiment, the term "specific binding" refers to binding by a targeting agent to a particular target molecule, polypeptide, or epitope on a particular polypeptide, and not to substantially bind to any other molecule, polypeptide, or epitope.

[0051] In one embodiment of the present invention, the targeting factor in the fusion protein specifically binds to von Willebrand factor (VWF), preferably human VWF. The basic human VWF monomer is a 2050-amino acid protein. Every monomer contains several specific domains with specific functions, including, for example, the D' / D3 domain that binds to factor VIII, the A1 domain that specifically binds to the platelet GPIb receptor, the A2 domain (which must partially unfold to expose a hidden cleavage site for the specific ADAMTS13 protease that inactivates VWF by producing much smaller multimers), the A3 domain that binds to collagen, the C1 domain (which binds the RGD motif to platelet integrin αIIbβ3 upon activation), and a "cysteine ​​knot" domain (at the C-terminus of the protein). VWF multimers can be quite large, exceeding 20,000 kDa, and can consist of more than 80 subunits of 250 kDa each. The main function of VWF is to bind to other proteins, particularly factor VIII, which is important for platelet adhesion to wound sites. VWF is not an enzyme and therefore does not have catalytic activity. VWF binds to several cells and molecules, including collagen (e.g., when exposed on endothelial cells due to vascular injury) and platelet GP1B receptors. The latter binding occurs under all conditions but is most efficient under high shear stress (i.e., rapid blood flow in narrow vessels). VWF binds to other platelet receptors when these are activated (i.e., when clotting is stimulated), for example, by thrombin.

[0052] The targeting agent in the fusion protein of the present invention can specifically bind to any and all forms, conformations, domains, and epitopes of VWF. Thus, the targeting agent can specifically bind to at least one of the unfolded (activated) conformation of VWF and the globular (circulating, non-activated) conformation of VWF (sVWF). In one embodiment, the targeting agent binds to at least unfolded VWF, preferably, the targeting agent preferentially binds to unfolded VWF over globular VWF (i.e., has a higher affinity for unfolded VWF than globular VWF), and more preferably, the targeting agent binds to the unfolded (activated) conformation of VWF and does not bind to the circulating, non-activated, globular form of VWF. More specifically, the targeting agent in the fusion protein of the present invention specifically binds to at least one of the VWF A1 domain, the A1 domain of activated VWF, the VWF A2 domain, the A2 domain of activated VWF, the VWF A3 domain, and the VWF D3 domain. A suitable example of a targeting agent that binds to VWF is the VHH camelid antibody fragment used in the Examples herein, as described in further detail below.

[0053] In another embodiment of the present invention, the targeting agent in the fusion protein specifically binds to platelets (also called thrombocytes), preferably human platelets. Together with VWF, platelets are the main component of MVT and are therefore also suitable targets for the targeting agent in the fusion protein of the present invention. The targeting agent in the fusion protein of the present invention can specifically bind to any and all forms of platelets. Thus, the targeting agent can specifically bind to at least one of activated platelets and non-activated platelets. Preferably, the targeting agent binds to (at least) non-activated platelets. Unlike large vessel thrombosis or conventional thrombosis, MVT necessarily involves activated platelets, and non-activated platelets in particular (together with VWF) are responsible for the problems in MVT, for example, in TTP. Therefore, it is preferred that the targeting agent in the fusion protein is not one that specifically binds only to activated platelets (and not to non-activated platelets). More specifically, the targeting agent is preferably not a targeting agent that specifically binds only to the activated form of the GPIIb / IIIa receptor on platelets, such as the single-chain antibody SCE5 described by Schwarz et al. (2004, FASEB J. 18:1704-1706). Thus, the targeting agent preferably specifically binds to the inactivated form of integrin αIIb / βIII. In a preferred embodiment, the targeting agent in the fusion protein of the present invention specifically binds to at least one of a) the platelet GP1B receptor and b) the platelet integrin αIIb / βIII. However, targeting agents that specifically bind to the platelet GP1B receptor are preferred. Suitable targeting agents that bind to platelets are known in the art, as exemplified by, for example, the anti-GPIbα antibody 6B4 described by Fontayne et al. (2006, Thromb Haemost. 96(5):671-84) or the anti-GP1B VHH antibody fragment used in the Examples herein.

[0054] In a further embodiment of the present invention, the targeting agent in the fusion protein specifically binds to activated, damaged, and / or exhausted endothelium (hereinafter collectively referred to as activated endothelium). The activated endothelium is preferably activated vascular endothelium, more preferably activated microvascular endothelium. Thus, the targeting agent can specifically bind to at least one of a receptor preferentially expressed by activated endothelium and a membrane marker of activated endothelium. Preferably, the receptor preferentially expressed by activated endothelium is selected from 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. Preferably, the membrane marker of activated endothelium is one or more of anionic phospholipids, phosphatidylserine, and phosphatidylethanolamine.

[0055] In one embodiment of the fusion protein of the invention, the targeting agent that specifically binds to one of the above-defined targets preferably comprises at least one of: a) an antibody variable domain that specifically binds to one of the targets; and b) a binding domain derived from a protein that naturally binds to one of the targets.

[0056] A preferred antibody variable domain present as a targeting agent in the fusion protein of the present invention is a VHH as defined herein above, more preferably the antibody variable domain is a humanized VHH.

[0057] In a preferred embodiment, the VHH present as a targeting agent in the fusion protein of the invention is a VHH that specifically binds to VWF. Suitable examples of VHHs that bind to VWF and are part of the fusion protein of the invention as a targeting agent are the VWF-binding VHHs that are part of the fusion proteins in the Examples herein or described in US Patent Application Publication No. 2013 / 0136736 A1, which is incorporated herein by reference.

[0058] A preferred VHH that binds to soluble globular VWF and is present as a targeting factor in the fusion protein of the present invention is a VHH-sVWF having the amino acid sequence of positions 55 to 178 of SEQ ID NO: 7, or an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with positions 55 to 178 of SEQ ID NO: 7, and exhibits K activity against VWF. d is less than 1, 0.5, 0.2, 0.1, 0.05 or 0.0306 nM.

[0059] A preferred VHH that binds to the D3 domain of VWF and is present as a targeting factor in the fusion protein of the present invention is VHH-D3, which has the amino acid sequence of positions 55 to 178 of SEQ ID NO: 8, or an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with positions 55 to 178 of SEQ ID NO: 8, and has a K domain for VWF. d is less than 1, 0.5, 0.4, 0.35, or 0.33 nM.

[0060] A preferred VHH that binds to the A1 domain of VWF and is present as a targeting factor in the fusion protein of the present invention is VHH-A12, which has the amino acid sequence of positions 55 to 179 of SEQ ID NO: 11, or an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with positions 55 to 179 of SEQ ID NO: 11, and has a K1 domain for VWF. d Another preferred VHH that binds to the A1 domain of VWF and is present as a targeting agent in the fusion protein of the invention is a VHH having the amino acid sequence of SEQ ID NO: 13 or a humanized version thereof having the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 or 14, and has a K d is less than 10, 5 or 2 nM.

[0061] A preferred VHH that binds to the platelet GP1B receptor and is present as a targeting factor in the fusion protein of the present invention is VHH-GP1B17, which has the amino acid sequence of positions 55 to 178 of SEQ ID NO: 12, or an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with positions 55 to 178 of SEQ ID NO: 12, and which binds to the platelet GP1B receptor. d Other preferred VHHs that bind to the platelet GP1B receptor and are present as targeting factors in the fusion proteins of the present invention include positions 55 to 175 of SEQ ID NO: 20, positions 55 to 178 of SEQ ID NO: 21, positions 55 to 172 of SEQ ID NO: 22, positions 55 to 171 of SEQ ID NO: 23, positions 55 to 174 of SEQ ID NO: 24, positions 55 to 176 of SEQ ID NO: 25, positions 55 to 178 of SEQ ID NO: 26, positions 55 to 178 of SEQ ID NO: 27, positions 55 to 178 of SEQ ID NO: 28, positions 55 to 166 of SEQ ID NO: 29, positions 55 to 180 of SEQ ID NO: 30, positions 55 to 182 of SEQ ID NO: 31, positions 55 to 184 of SEQ ID NO: 32, positions 55 to 186 of SEQ ID NO: 33, positions 55 to 188 of SEQ ID NO: 34, positions 55 to 189 of SEQ ID NO: 35, positions 55 to 190 of SEQ ID NO: 36, positions 55 to 192 of SEQ ID NO: 37, positions 55 to 194 of SEQ ID NO: 38, positions 55 to 196 of SEQ ID NO: 39, positions 60 to 610 of SEQ ID NO: 40, positions 60 to 612 of SEQ ID NO: 41, positions 60 to 614 of SEQ ID NO: 42, positions 60 to 616 of SEQ ID NO: 43, positions 60 to 618 of SEQ ID NO: 44, positions or having an amino acid sequence selected from the group consisting of amino acid sequences of positions 55 to 176 of SEQ ID NO: 31, positions 55 to 176 of SEQ ID NO: 32, positions 55 to 179 of SEQ ID NO: 33, positions 55 to 179 of SEQ ID NO: 34, positions 55 to 177 of SEQ ID NO: 35, positions 55 to 178 of SEQ ID NO: 36, and positions 55 to 181 of SEQ ID NO: 37, or having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence of this group, and having a K1 activity against the platelet GP1B receptor. d has an amino acid sequence with less than 20, 15, 10, 5, 2, 1, 0.5, 0.2 or 0.1 nM

[0062] Another preferred VHH that binds to the A1 domain of VWF comprises an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 46. Preferably, a VHH that binds to the A1 domain of VWF and comprises the amino acid sequence of SEQ ID NO: 46 is present as a targeting agent in a fusion protein of the invention, which is a VHH having the amino acid sequence of SEQ ID NO: 45 or a humanized version thereof having the amino acid sequence of SEQ ID NO: 44, or a VHH having an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 44 or 45 and having a Kd for VWF of less than 10, 5 or 2 nM.

[0063] In another embodiment of the fusion protein of the present invention, the targeting agent that specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium comprises a binding domain derived from a protein that naturally binds to at least one of VWF, platelets, and activated or damaged vascular endothelium. Preferably, the binding domain comprises: i) the A1 domain of VWF or at least a portion of the VWF A1 domain that binds to the platelet GP1B receptor; ii) a VWF-binding domain of one of ADAMTS13, factor XII, factor H (a complement regulator), plasminogen, and factor VIII, at least the portion of these domains that binds to VWF; and iii) a domain that binds to the membrane(s) of activated or damaged vascular endothelium, the domain being selected from the group consisting of a vitamin K-dependent carboxylation / gamma-carboxyglutamic acid (GLA) domain, the C domain of factor V, and the C domain of factor VIII.

[0064] In one embodiment of the present invention, the fusion protein comprises two or more targeting factors. Thus, the fusion protein may comprise, for example, two, three, four, five, six, or more targeting factors. When two or more targeting factors are present in the fusion protein, two or more copies of the same targeting factor may be present in the fusion protein. Alternatively, the fusion protein may comprise at least two different targeting factors. For example, the fusion protein may comprise at least two different targeting factors that bind (respectively) to at least two different domains of VWF or at least two different receptors on platelets. Alternatively, the fusion protein may comprise at least two different targeting factors, at least one of which binds to VWF and at least one other of which binds to platelets. The advantage of incorporating two or more targeting factors into the fusion protein is the multivalent binding avidity at the MVT site, i.e., the cumulative strength of the multiple affinities of the individual binding interactions of the individual targeting factors. When two or more targeting factors are present in the fusion protein, the individual targeting factors are preferably arranged in tandem, with suitable (flexible) spacer or linker amino acid sequences between the individual targeting factors.

[0065] Suitable flexible linker amino acid sequences are known in the art (e.g., from Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369). Flexible linkers are typically applied when the domains to be linked require some degree of movement or interaction. Flexible linkers are generally composed of small, non-polar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). The small size of these amino acids provides flexibility, allowing the mobility of the linked functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety. A preferred flexible linker has a sequence consisting primarily of a stretch of Gly and Ser residues (a "GS" linker). One example of a preferred (and widely used) flexible linker is (Gly-Gly-Gly-Gly-Ser). n The GS linker has the sequence: By adjusting the copy number "n", the length of this GS linker can be optimized to achieve appropriate separation of functional domains or to maintain necessary inter-domain interactions. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but may also contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility, as in the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 38) and EGKSSGSGSESKST (SEQ ID NO: 38) that have been applied to construct biologically active scFvs.

[0066] In addition to one or more targeting factors described above for targeting MVT, the fusion proteins of the present invention further comprise at least a plasminogen activator. Plasminogen activators are serine proteases that catalyze the activation of plasmin through the proteolytic cleavage of zymogen plasminogen. Although plasmin is an important factor in fibrinolysis, the scope of the present invention relies on plasminogen activation for fibrin-independent thrombolytic activity against MVT. Therefore, the fusion proteins of the present invention comprise a plasminogen activator, preferably comprising the (catalytic) protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase, or staphylokinase. The catalytic protease domains of tPA, uPA, plasminogen, streptokinase, and staphylokinase are well known in the art. A preferred plasminogen-activating catalytic protease domain for incorporation into the fusion protein of the present invention is the catalytic protease domain of uPA, preferably human uPA, comprising the amino acid sequence of positions 16 to 268 of SEQ ID NO: 1, or the catalytic protease domain of tPA, preferably human tPA, comprising the amino acid sequence of positions 15 to 266 of SEQ ID NO: 19.

[0067] In one embodiment, where the catalytic domain protease domain for incorporation into the fusion protein of the invention is the catalytic protease domain of uPA, preferably human uPA, the (human) uPA domain comprises a mutation in its sequence that stabilizes (human) uPA. Stabilizing mutations are described in U.S. Pat. No. 5,472,692 and by Sun et al. (J. Biol. Chem. 19 Sep. 1997; 272(38):23818-23823), which are incorporated herein in their entireties. In a preferred embodiment, the catalytic domain protease comprises and / or consists of SEQ ID NO: 1, in which the lysine (K) at position 157 of SEQ ID NO: 1 has been mutated to histidine (H). In a preferred embodiment, the catalytic domain protease comprises and / or consists of SEQ ID NO: 2, in which the lysine (K) at position 158 of SEQ ID NO: 2 has been mutated to histidine (H).

[0068] In one embodiment, the plasminogen activator incorporated into the fusion protein of the invention comprises a variant of the catalytic protease domain that has reduced sensitivity to its natural inhibitor, such as plasminogen activator inhibitor 1 (PAI-1). Such variants include, for example, variants of the protease domain of uPA or tPA with one or more modifications in the exosite loops involved in the interaction with PAI-1, such as the 37 and 147 loops in the protease domain of uPA, or analogous loops in the protease domain of tPA, such as the 37, 60, 97, 147, and 217 loops (see Lin et al., J. Biol. Chem. 2011, Mar. 4; 286(9):7027-7032). Examples of such modifications are the deletion of an exosite specific for interaction with PAI-1 (tPA del 296-302) or the substitution of specific amino acids that specifically mediate interaction with PAI-1 (tPA Arg→Glu 304 or Arg→Ser 304) (Madison et al., Nature. 1989 Jun 29;339(6227):721-4).

[0069] In a preferred embodiment, the plasminogen activator incorporated into the fusion protein of the invention further comprises at least a cysteine-containing portion of a connecting peptide naturally occurring in plasminogen activators immediately upstream (N-terminal) of the protease domain, where the presence of the cysteine-containing connecting peptide prevents the corresponding cysteine ​​in the protease domain from forming unwanted disulfide bridges.

[0070] Preferred connecting peptides to be included in the plasminogen activator incorporated into the fusion protein of the present invention are: a) a connecting peptide of uPA comprising at least amino acids 17 to 27 of SEQ ID NO: 16, more preferably a connecting peptide comprising at least amino acids 13 to 27 of SEQ ID NO: 16; b) a connecting peptide of tPA comprising at least amino acids 3 to 14 of SEQ ID NO: 17, more preferably a connecting peptide comprising at least amino acids 1 to 14 of SEQ ID NO: 17; or c) a connecting peptide of plasminogen comprising at least amino acids 5 to 18 of SEQ ID NO: 18, more preferably a connecting peptide comprising at least amino acids 1 to 18 of SEQ ID NO: 18.

[0071] In another preferred embodiment, the fusion protein of the present invention preferably comprises a linker amino acid sequence located between one or more targeting factors on the one hand and a plasminogen activator on the other hand. Thus, when the plasminogen activator comprises, for example, a connecting peptide as described above, it is understood that the linker amino acid sequence is located upstream of the connecting peptide of the plasminogen activator. The linker amino acid sequence linking the targeting factor and the plasminogen activator is preferably a flexible linker amino acid sequence as described above. A preferred linker amino acid sequence linking the targeting factor and the plasminogen activator is (Gly-Gly-Gly-Gly-Ser) n wherein n is 1 to 4, more preferably 2 or 3, and most preferably 2. A particularly preferred linker amino acid sequence has the amino acid sequence of SEQ ID NO:6.

[0072] Thus, a preferred fusion protein of the present invention is a fusion protein comprising, from N-terminus to C-terminus, a) one or more targeting factors as defined above, optionally linked by a linker amino acid sequence as defined above; b) optionally a linker amino acid sequence as defined above; and c) a plasminogen activator as defined above. The fusion protein may further comprise additional functional elements, such as an isolation tag, such as a His tag or a STREP isolation tag, or a cleavage site recognized only by a specific protease, such as a tobacco etch virus cleavage site, so that undesired amino acid sequences (e.g., the isolation tag) can be cleaved from the fusion protein. An example of a preferred fusion protein configuration of the present invention is shown in Figures 1A and 1B, where the nucleotide sequence encoding the fusion protein also encodes an (Igκ) signal sequence upstream of the fusion protein, directing secretion of the protein from the cells in which it is produced.

[0073] In a second aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the present invention as defined above. The nucleotide sequence encoding the fusion protein preferably further comprises a nucleotide sequence encoding a signal peptide operably linked to the fusion protein. A preferred signal peptide for directing the secretion of the fusion protein of the present invention is the Igκ signal peptide having the amino acid sequence of SEQ ID NO: 3. The nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the present invention preferably further comprises a regulatory element operably linked to the nucleotide sequence, which regulatory element facilitates the expression of the fusion protein in a suitable host cell.

[0074] In a third aspect, the present invention relates to a vector comprising a nucleic acid molecule according to the invention. Optionally, the vector according to the invention is a gene therapy vector.

[0075] Preferably, the gene therapy vector is a viral gene therapy vector, such as a viral gene therapy vector selected from adenovirus, adeno-associated virus (AAV), herpes virus, pox virus, oncolytic virus vector, and retrovirus-based gene therapy vector. Preferred viral gene therapy vectors are AAV vectors or lentiviral vectors.

[0076] In a fourth aspect, the present invention relates to a host cell comprising a vector according to the invention, which host cell expresses a fusion protein according to the invention.

[0077] The cells are preferably isolated or cultured. Host cells that can be used include prokaryotes, yeast cells, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell line (Gluzman et al., 1981, Cell 23:175), L cells, HEK293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines, and the CVI / EBNA cell line, derived from the African green monkey kidney cell line CVI, described in McMahan et al., 1991, EMBO J. 10:2821. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian host cells are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0078] The transformed cells can be cultured under conditions that promote the host cell to express the fusion protein of the invention. Thus, in one aspect, the invention relates to a method for producing a fusion protein of the invention, comprising culturing a cell comprising at least one expression vector as defined herein under conditions that favor the expression of the fusion protein of the invention, and optionally recovering the fusion protein of the invention.

[0079] Fusion proteins of the invention can be recovered by conventional protein purification means including, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography using, for example, streptavidin / biotin (see, e.g., Low et al., 2007, J. Chromatography B, 848:48-63; Shukla et al., 2007, J. Chromatography B, 848:28-39).

[0080] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising and / or consisting of a fusion protein according to the invention, a nucleic acid according to the invention, a vector or gene therapy vector according to the invention, or a host cell according to the invention, and a pharmaceutically acceptable excipient.

[0081] The pharmaceutical composition preferably further comprises at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier, such as an adjuvant or vehicle, is for administering the antibody or antibody fragment to a subject. The pharmaceutical composition can be used in the treatment methods described herein below by administering an effective amount of the composition to a subject in need thereof. The term "subject" as used herein refers to all animals classified as mammals, including, but not limited to, primates and humans. The subject is preferably a human, male or female, of any age or race.

[0082] The term "pharmaceutically acceptable carrier," as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration (see, e.g., "Handbook of Pharmaceutical Excipients," Rowe et al., 7th ed., 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn 2+- protein complexes), and / or non-ionic surfactants such as Tween™, PLURONICS™, or polyethylene glycol (PEG).

[0083] Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the present invention. Thus, in certain embodiments, the pharmaceutical compositions of the present invention may contain two or more active compounds, preferably those with complementary activities that do not adversely affect each other, as needed for the particular indication being treated. For example, it may be desirable to additionally provide a chemotherapeutic agent, cytokine, analgesic, thrombolytic agent, or immunomodulatory agent, such as an immunosuppressant or immunostimulator. The effective amount of such other active agent will depend, among other factors, on the amount of the antibody of the present invention present in the pharmaceutical composition, the type of disease or disorder, or treatment.

[0084] In some embodiments, the fusion proteins of the present invention are formulated with carriers that protect the compound from rapid elimination from the body, such as sustained-release formulations, including implants and microencapsulated delivery systems, e.g., liposomes. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may also be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposomal suspensions, including targeted liposomes, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 or WO 2010 / 095940.

[0085] The route of administration of the fusion protein of the present invention can be parenteral. As used herein, the term "parenteral" includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. Intravenous forms of parenteral administration are preferred. "Systemic administration" refers to oral, intravenous, intraperitoneal, and intramuscular administration. The amount of fusion protein required for therapeutic or prophylactic effect will, of course, vary depending on the fusion protein selected, the nature and severity of the condition being treated, and the patient. Furthermore, the fusion protein may be suitably administered by pulse infusion, e.g., with declining doses of the fusion protein. Preferably, dosing is administered by injection, most preferably intravenous or subcutaneous, depending in part on whether the administration is brief or chronic.

[0086] Thus, in certain embodiments, the pharmaceutical compositions of the present invention may be in a form suitable for parenteral administration, such as, for example, a sterile solution, suspension, or lyophilized product in an appropriate unit dosage form. 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. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must 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, a pharmaceutically acceptable polyol, such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0087] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0088] Sterile injectable solutions can be prepared by incorporating the required amount of active compound (e.g., fusion protein) into a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces powders from a previously sterile-filtered solution of the active ingredient and any additional desired ingredients.

[0089] In certain embodiments, the pharmaceutical compositions are administered intravenously (IV) or subcutaneously (SC). Suitable excipients, such as bulking agents, buffers, or surfactants, can be used. The formulations listed herein are prepared using standard methods for preparing parenterally administrable compositions, as are well known in the art and described in more detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (Allen, LV, ed., 22nd ed., 2012, www.pharmpress.com).

[0090] It is particularly advantageous to formulate pharmaceutical compositions, i.e., parenteral compositions, in unit dosage forms for easy administration and uniform dosage. As used herein, unit dosage form refers to physically discrete units suitable as dosage units for the subject to be treated, each containing a predetermined amount of active compound (antibody of the present invention) calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined and directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the field of formulating such active compounds to treat individuals.

[0091] Generally, for the prevention and / or treatment of the diseases and disorders mentioned herein, depending on the particular disease or condition to be treated and its severity, the potency of the particular fusion protein of the invention used, the particular route of administration, and the particular pharmaceutical formulation or composition used, the fusion protein of the invention will generally be administered continuously (e.g., by infusion) in the range of 0.001 to 1,000 mg / kg body weight / day, preferably about 0.01 to about 100 mg / kg body weight / day, and most preferably about 0.05 to 10 mg / kg body weight / day, e.g., about 1, 10, 100, or 1,000 micrograms / kg body weight / day, either as a single daily dose or as multiple doses divided throughout the day. A clinician will generally be able to determine a suitable daily dose depending on the factors mentioned herein. It will be apparent, however, that in certain cases, a clinician may deviate from these amounts, based, for example, on the factors mentioned above and the clinician's own professional judgment.

[0092] In addition to administering the fusion proteins of the present invention to a patient, the present application contemplates administering the fusion proteins by gene therapy.

[0093] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0094] The fusion proteins and pharmaceutical compositions of the present invention may be used with other drugs to provide combination therapy, which may form part of the same composition or may be provided as separate compositions administered at the same or different times.

[0095] In a sixth aspect, the present invention relates to at least one of a fusion protein, a gene therapy vector, and a pharmaceutical composition (each as defined above) for use in treating or preventing a disease or condition associated with thrombosis. The disease or condition to be prevented or treated may be any disease or condition associated with or involving thrombi comprising at least one of VWF and platelets. Such a disease or condition associated with thrombi comprising at least one of VWF and platelets may be any disease or condition involving macrovascular thrombosis and / or microvascular thrombosis (MVT), and / or any disease or condition involving sites of (yet) non-occlusive macrovascular thrombosis or microvascular thrombosis. However, it is preferred that the disease or condition to be prevented or treated is at least a disease or condition associated with microvascular thrombosis (MVT). In this specification, the term "preventing a disease or condition" should be understood to include and / or be equivalent to reducing the risk of the disease or condition occurring or developing.

[0096] In a preferred embodiment, the thrombus-related disease or condition involving at least one of VWF and platelets is acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy (George et al., 2014, N Engl J 371(7):654-66), hemolytic uremic syndrome, antiphospholipid syndrome, formation of occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and lesions due to coronary artery bypass graft, coronary artery valve replacement, and coronary interventions such as angioplasty, stenting, or atherectomy, thickening after angioplasty, atherectomy, or arterial stenting, occlusive syndrome of the vascular system or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome (HELLP is hemolysis, elevated liver enzymes, and decreased platelet count), carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction.

[0097] Additional diseases or conditions associated with thrombi containing at least one of VWF and platelets that may be treated or prevented by the methods of the present invention are selected from the group consisting of unstable angina, stable angina, angina, embolism, deep vein thrombosis, hemolytic anemia, acute renal failure, thrombolytic complications, disseminated intravascular coagulation (DIC), thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and atrial thrombosis in atrial fibrillation, chronic unstable angina, transient ischemic attack and stroke, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and / or thrombosis after angioplasty, anastomosis of vascular grafts, and chronic exposure to cardiovascular devices. Such conditions may result from thromboembolism and reocclusion during and after thrombolytic therapy, after angioplasty, and after coronary artery bypass surgery.

[0098] In further embodiments, the fusion protein, gene therapy vector, and pharmaceutical composition (each as defined above) are for use in treating or preventing plaque or thrombus in an individual. The formation of such plaque or thrombus can occur under conditions of high shear. In both thrombosis and reocclusion, reversible adhesion or tethering of platelets at high shear rates is followed by strong adhesion via collagen receptors, leading to platelet activation. Tethering of platelets by VWF to collagen exposed in damaged vascular walls is particularly important under high shear conditions. The fusion proteins of the present invention function well under high shear conditions.

[0099] In a seventh aspect, the present invention relates to a method for treating or reducing the risk of a disease or condition associated with thrombi comprising at least one of VWF and platelets, preferably a disease or condition associated with microvascular thrombosis, comprising the step of administering an effective amount of the fusion protein as defined above, the gene therapy vector as defined above, or the pharmaceutical composition as defined above to a subject in need thereof. Preferably, in this method, the disease or condition associated with thrombi comprising at least one of VWF and platelets is a disease or condition associated with microvascular thrombosis, such as acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombus, occlusive thrombus formation, arterial thrombosis, acute coronary occlusion, peripheral arterial occlusive disease, coronary artery bypass graft, coronary valve replacement, and angioplasty, stenting, or atherectomy. restenosis and lesions due to coronary intervention, thickening after angioplasty, atherectomy or arterial stenting, occlusive syndromes of the vascular system or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction, or a further disease or condition as defined above.

[0100] In an eighth aspect, the present invention relates to a method for treating or reducing the risk of at least one of microvascular thrombi, fibrin-independent thrombi, and thrombi comprising at least one of VWF and platelets. Preferably, the method includes treating acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombi, occlusive thrombus formation, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, coronary artery bypass graft, coronary valve replacement, and restenosis and disorders resulting from coronary interventions such as angioplasty, stenting, or atherectomy, angioplasty, atherectomy, or stenting. At least one of these thrombi is treated or the risk of occurrence is reduced in a disease or condition selected from the group consisting of thickening after thrombus surgery, vascular occlusive syndrome or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction, or a further disease or condition as defined above.

[0101] In this document and in the claims, the verb "comprise" and its conjugations are used in an open-ended sense to mean that items following the word are included, but items not specifically listed are not excluded. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the presence of more than one of that element, unless the context clearly requires that only one of that element be present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0102] The word "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably means that the value may be 0.1% more or less than the given value (10).

[0103] All patents and references cited herein are incorporated by reference in their entirety.

[0104] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0105] Example 1 Methods and Materials Construction of nanobody-mUPA The cDNA sequences of both human and mouse urokinase (PLAU) were obtained from the NCBI database (NM_002658.4 and NM_008873.3, respectively). The signal peptide, EGF-like, and Kringle domain sequences were removed, along with the initial portion of the connecting peptide. An N-terminal sequence encoding a tobacco etch virus cleavage site followed by a GGGGS linker was added to the remaining connecting peptide and the S1 peptidase domain (catalytic domain). A PstI-BamHI digestion site was incorporated into the GGGGS linker without disrupting the amino acid sequence. An EcoRI digestion site was added to the 5' end, and a NotI digestion site was added to the 3' end after the PLAU stop codon. This construct was obtained as a custom gene construct from IDT (Integrated DNA Technologies, Leuven, Belgium).

[0106] Nanobody (also known as VHH) coding sequences were codon-optimized for expression in human host cells via IDT. A tobacco etch virus (TEV) cleavage site was inserted at the N-terminus of the VHH coding sequence, and a GGGGS linker (encoding a PstI-BamHI digestion site) was inserted at the C-terminus. These DNA segments were obtained from IDT as double-stranded DNA fragments (gBLocks).

[0107] The custom gene construct was propagated in E. coli TOP10 and selected for ampicillin resistance. The resulting plasmid DNA was digested with EcoRI and NotI. The resulting insert (886) was separated and isolated on an agarose gel and ligated into a modified pcDNA6 expression vector (pSM2) (De Maat et al., 2016, J Allergy Clin Immunol, November 30;138(5):1414-23). ​​pSM2 encodes an N-terminal mouse IgK secretion signal and a double STREP isolation tag, after which the modified UPA construct is ligated.

[0108] The gBlocks were ligated into the pJET1.2 cloning vector (CloneJET PCR Cloning Kit, Thermo Fisher Scientific) according to the manufacturer's instructions. These constructs were propagated in E. coli TOP10 and selected for ampicillin resistance. The resulting plasmid DNA was digested with EcoRI and BamHI. The resulting insert was separated and isolated on an agarose gel and ligated into the pSM2 vector containing the miniUPA construct. These gene constructs are shown schematically in Figure 1A and Figure 1B.

[0109] Table 1 lists the nanobody-mUPA fusion protein constructs that were prepared, including their nanobody targets and their names. [Table 4]

[0110] Generation of nanobody-mUPA The nanobody / mUPA-pSM2 construct was transfected into HEK293 FreeStyle™ cells using 239fectin as described by the manufacturer (ThermoFisher). After one day, the cells were expanded to 20 mL. Two days later, the cells were placed under selection with blasticidin (5 μg / mL). Transfected cells were further cultured according to the manufacturer's instructions until the construct was stably integrated into the HEK genome. Cells were expanded (1.1 × 10 6 After 7 days of protein production, the cells were spun down at 2000 × g for 5 minutes, the supernatant was then collected, and benzamine (0.174 mg / mL) was added, after which the supernatant was stored at −20°C until further use.

[0111] Purification of nanobody-mUPA The collected supernatant (400 mL) was concentrated to 150 mL in a Quixstand using a 10 kDA cutoff membrane (GE Healthcare). The concentrate was then dialyzed against 2 L of 1x STREP buffer containing benzamidine (100 mM Tris, 150 mM NaCl, 0.174 mg / mL benzamidine, pH 8.0). The concentrate was loaded onto a column containing 8 mL of Strep-Tactin Superflow beads (IBA). After washing the column with 20 mL of 1x STREP buffer, the protein was eluted with 1x STREP buffer containing d-desthiobiotin (2.5 mM, Sigma-Aldrich). The purified protein was dialyzed against 2 L of sodium acetate (4 mM sodium acetate, 150 mM NaCl, pH 5.4) and stored at -80°C.

[0112] result Western blot of purified nanobody-mUPA constructs The nanobody-mUPA construct was diluted to a concentration of 100 μg / mL in sample buffer (25 mM DTT). 10 μL of sample was loaded onto a 4-12% gradient Bis-Tris gel containing MES buffer. Sample separation was performed at 165 volts for 50 minutes. The gel was transferred onto an Immobilon-FL membrane in blotting buffer at 125 volts for 1 hour. The membrane was blocked with 0.5x Odyssey blocking buffer, and then the construct was detected using a rabbit polyclonal anti-human UPA antibody in combination with an IR800-conjugated goat anti-rabbit antibody. Results were analyzed using a near-infrared Odyssey scanner (Licor) according to the manufacturer's instructions. Figure 2 shows that all fusion proteins, except A11-mUPA, were expressed at approximately equal levels.

[0113] Urokinase activity of nanobody-mUPA constructs The nanobody-mUPA construct should exhibit non-spontaneous (i.e., non-inducible) activity but be activatable by molecular cleavage by plasmin.

[0114] To demonstrate that none of the nanobody-mUPA constructs exhibited spontaneous activity, the constructs (1 μg / mL) were incubated in the presence of 0.5 mM urokinase substrate (I1140; Bachem) in 0.2% BSA-HBS. Substrate conversion was measured at 37°C according to the manufacturer's instructions. None of the fusion proteins exhibited detectable spontaneous activity toward the urokinase substrate (data not shown).

[0115] To test whether the nanobody-mUPA construct could be activated by plasmin, plasminogen was preactivated with streptokinase (hereafter referred to as plasmin) for 15 min at 37 °C. The nanobody-mUPA fusion construct (final concentration 1 μg / mL) was diluted in 0.2% BSA-HBS and incubated with plasmin (final concentration 1 μg / mL) for 12 min. Urokinase substrate (0.5 mM I1140, Bachem) was then added, and its conversion was measured at 37 °C according to the manufacturer's instructions. Figure 3 shows that the fusion protein can be successfully activated.

[0116] Plasminogen activation The principle of plasminogen activation by urokinase relies on the reciprocal cleavage between urokinase and plasminogen. To test plasminogen activation by the nanobody-mUPA constructs, the constructs (1 μg / mL) were incubated in 0.2% BSA-HBS in the presence of 100 μg / mL plasminogen and 0.2 mM plasmin substrate (I1390; Bachem). Substrate conversion was measured at 37°C according to the manufacturer's instructions. Figure 4 shows that all constructs showed comparable enzymatic activity generation.

[0117] Binding of anti-VWF constructs to VWF determined by ELISA Nunc Maxisorp plates (Thermo) were coated overnight with 1 μg / mL VWF in PBS. The next day, the plates were blocked with 1% BSA-PBS. Anti-VWF nanobody-mUPA fusion constructs were diluted to various concentrations in 1% BSA-PBS, and 50 μL was added to each well and incubated for 1 hour. The plates were then washed with PBS-Tween 20 (PBST, 0.05% v / v). Bound constructs were detected with a rabbit anti-UPA polyclonal antibody combined with a goat anti-rabbit HRP secondary antibody (Abcam). The wells were rinsed with PBST, and then 100 μl of TMB was added at room temperature. The substrate was allowed to develop for 5 minutes, after which 50 μl of H2SO4 (0.3 M) was added. Results were analyzed by absorbance at 450 nm. Results were analyzed using Graphpad Prism 7.02, and K was calculated by nonlinear regression curve fitting. d The binding affinities (in nM) were determined. Table 2 lists the binding affinities. [Table 5]

[0118] Plasminogen activation in the presence of globular or unfolded VWF VWF is a scaffold for microthrombus formation. It also has plasmin(ogen)-binding properties that depend on the protein's conformation (Tersteeg et al., 2014, supra). VWF unfolds under shear stress (and also during immobilization in microtiter plates (see previous experiments)). This can be mimicked by incubation with the small molecule ristocetin. We investigated whether plasminogen activation by the targeted fusion proteins of the present invention is affected by VWF conformation. Nanobody fusion constructs (0.25 μg / mL) and VWF (5 μg / mL) were diluted in 0.2% BSA-HBS. Ristocetin (0.6 mg / mL) or buffer was then added to unfold or maintain VWF in a globular form, respectively, while incubating at 37°C for 5 minutes. Plasminogen (100 μg / mL) was then added, followed by the plasmin substrate (I1390 Bachem; 0.2 mM). Substrate conversion was measured at 37°C according to the manufacturer's instructions. For comparison, the substrate conversion after 5 minutes for the different constructs is shown in the bar graph below. Data were processed with Graphpad Prism 7.02 and analyzed by one-way ANOVA. *P<0.05. Figures 5A-5E and 6 show that the time to the onset of plasmin activity is significantly reduced for open VWF (in the presence of ristocetin) compared to closed VWF.

[0119] Microthrombolysis of VWF-platelet aggregates.

[0120] Platelets were isolated from citrated whole blood as previously described (Tersteeg et al., 2014, supra). Isolated platelets (200,000 / mL) were incubated with VWF (5 μg / mL), plasminogen (100 μg / mL), and the aggregation inhibitors RGDW (200 μM) and iloprost (0.4 μg / mL) in a light transmission aggregometer for 15 min at 37°C. Aggregation was induced by the addition of ristocetin (0.6 mg / mL). Six min later, the nanobody-mUPA construct (1 μg / mL) was added, and aggregate dissolution was monitored over time (Figures 7A–7D). The time point at which 50% microthrombus dissolution occurred was determined for all samples (a schematic example of the analytical method is shown in Figure 8A). The results were processed in Graphpad Prism 7.02 and analyzed by one-way ANOVA (*P<0.05). The results are shown in Figure 8B. Clearly, targeting plasminogen activation by at least the fusion proteins sVWF-mUPA, D3-mUPA, and GP1B17-mUPA accelerates microthrombolysis.

[0121] Example 2: VWF-platelet microthrombolysis of endothelial cells in flow perfusion Materials and Methods Cultivation of human vascular endothelial cells (Huvecs) on glass coverslips Huvecs (passage 0) stored in liquid nitrogen were thawed at 37°C and added to medium (EBM-2, Lonza, or Promocel supplemented with the Huvec growth factor EGM2) at a 1:10 ratio and spun down at 100g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 5 mL of medium and cultured in a T25 flask at 37°C and 5% CO2. The next day, the cells were passaged into three T75 flasks. On day 6, the cells were passaged 1:6 onto glass coverslips pretreated with HT buffer (HEPES Tyrode's buffer: 10 mM HEPES, 0.5 mM NaHPO4, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4) at pH 7.4 containing 1.25% glutaraldehyde.

[0122] To coat the coverslips with glutaraldehyde, they were rinsed with demineralized water and ethanol. They were then incubated in 37% HCl:methanol (1:1) for 30 minutes and then rinsed with demineralized water for 5 minutes. Next, they were incubated in aminopropyltriethoxysilane:ethanol (1:100) for 30 seconds and then rinsed with demineralized water and ethanol. The coverslips were then dried and incubated with 20% glutaraldehyde:HT buffer pH 7.4 (1:20) for 1 hour. They were then rinsed with demineralized water and stored in ethanol until use.

[0123] Huvecs were cultured on glass coverslips for approximately 10 to 15 days before use.

[0124] Preparation of heat-inactivated plasma Heat-inactivated plasma was prepared by mixing two bags of plasma (Octapharma Ominplasma, both blood type AB, lot number: C442A9521, bags: X000214223782 and X000214223577). 200 mL of this mix was distributed into 10 Falcon tubes (50 mL, 20 mL each) and incubated for 30 minutes in a water bath set at exactly 56°C (20 mL was completely submerged), with the Falcon tubes mixed midway (15 minutes). After the 30-minute incubation, the Falcon tubes were covered with ice and kept on ice until centrifugation (note: the tubes were still somewhat warm when removed from the ice for centrifugation). The tubes were centrifuged at 15,000 g for 5 minutes (without cooling). The supernatants were combined and kept on ice until divided into 1 mL aliquots.

[0125] Flow chamber setup The laminar flow perfusion chamber was filled with pre-warmed medium, and all air was removed from the tubing before placing the coverslip. The inlet tubing was cut to a length corresponding to a volume of 90.6 μL, so that the fluid entering the inlet during perfusion would reach the perfusion chamber exactly 1 minute later. The syringe diameter used was 16 mm (only for Braun 12 ml syringes), and the syringe pump was set to 90.6 μL / min. This allowed for a flow rate of 300 s (when using 3 mm tubing). -1 The HUVEC coverslip was placed in the medium and the perfusion chamber was placed under an inverted microscope (Zeiss observer Z.1, Carl Zeiss) equipped with a vacuum generator set at 10 bar and a heating module that maintained the perfusion chamber at 37 °C. Any remaining air bubbles were removed by perfusing the medium.

[0126] Washed platelets in heat-inactivated plasma Blood from consenting healthy volunteers was collected in 0.1 volume of 3.2% (10.9 mM) trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation (160 g for 15 minutes at room temperature). PRP supplemented with 10% (v / v) acid-citrate dextrose, 85 mM trisodium citrate, 71 mM citric acid, and 111 mM D-glucose was centrifuged (400 g for 15 minutes at room temperature), and platelets were resuspended in HEPES Tyrode's buffer, pH 6.5, containing 0.145 M NaCl, 5 mM KCl, 0.5 mM NaHPO, 1 mM MgSO, 10 mM HEPES, and 5.5 mM D-glucose. After adding 10 μg / mL of PGI2 to the platelet suspension, another centrifugation step (400 g for 15 min at room temperature) was performed, and the platelets were resuspended in heat-inactivated plasma and the platelet count was adjusted to a final count of 200 G / L.

[0127] Setting up the perfusion experiment Heat-inactivated plasma containing 200 g / L platelets (pre-warmed in a 37°C water bath) was dispensed into 2 mL Eppendorf cups (e.g., 2 × 2 mL required for a 40-minute experiment). Prior to the start, all Eppendorf cups were supplemented with iloprost (8 μL of 0.1 mg / mL stock (250-fold dilution), final concentration: 0.4 μg / mL, Bayer Schering Pharma AG), followed by histamine (4 μL of 500 μM stock in medium (500-fold dilution), final concentration: 100 μM). Immediately after addition of iloprost and histamine, perfusion was initiated by transferring the inlet tubing from the 2 mL Eppendorf cup containing medium to the Eppendorf cup containing heat-inactivated plasma with platelets by squeezing the tubing to ensure no air was introduced. Approximately 1 minute later, the experiment began in frame 1, when the first platelets entered the flow chamber (visible change). After 7 min, add the construct to the remaining Eppendorf cup (7 × 90.6 = 634 µL used at this point, so 2000 - 634.2 = 1365.8 µL) by pipetting the required volume directly into the Eppendorf cup and mixing with a plastic pipette to a final concentration of 10 µg / mL. At this point, add the construct to an additional Eppendorf cup to ensure equal preincubation times with plasma. During perfusion, the 2 mL Eppendorf cup must be refilled by carefully adding plasma drop by drop with a plastic pipette. This refilling occurs at frames 150, 250, etc., so that any disturbances that may result from refilling can be matched to the corresponding frame / time point. DIC images are taken every 5 s during the experiment. At the end of the experiment, five screenshots of different areas within the perfusion chamber are taken and the number of platelet-VWF complexes visible in the form of "platelet strings" are counted.

[0128] result The results are shown in Figure 9. Figure 9 shows that targeted plasminogen activation by at least the fusion proteins GP1B17-mUPA, sVWF-mUPA, and D3-mUPA accelerates thrombolysis of platelet-VWF complexes bound to Huvecs compared with the control fusion protein R2-mUPA. In particular, the fusion protein GP1B17-mUPA rapidly eliminated platelet-VWF complexes, indicating that targeting platelets in particular is efficient for eliminating platelet-VWF complexes in microthrombi.

[0129] Example 3: Methods and Materials Caplacizumab production Cloning Caplacizumab (a bivalent variant of the Kabribi VHH) was produced in E. coli and purified by HIS-tag affinity chromatography. The caplacizumab protein sequence was obtained from the EMA review report (EMA / 490172 / 2018, procedure number EMEA / H / C / 004426 / 0000) and codon-optimized for expression in E. coli using the codon optimization tool from IDT (integrated DNA technologies). An N-terminal BamHI digestion site and a C-terminal NotI digestion site were added to the construct, which was then ordered from IDT as a double-stranded DNA fragment (SEQ ID NO: 40).

[0130] The DNA fragment was dissolved in 5 mM Tris buffer (pH 8.5) and heated to 50°C for 20 minutes. The DNA fragment was then ligated into the pJET1.2 vector (CloneJET PCR Cloning Kit, Thermo Fisher Scientific) according to the manufacturer's instructions. The ligated product was transformed into chemically competent E. coli TOP10 (Thermo Fisher Scientific) by heat shock according to the manufacturer's instructions. The transformed bacteria were cultured in 10 mL of 2xYT medium (containing 100 μg / mL ampicillin) and grown overnight at 37°C. Plasmid DNA was isolated using a plasmid isolation kit (M&N) according to the manufacturer's instructions. The insert was digested with Cutsmart buffer containing BamHI-HF and NotI-HF (NEB) and separated on a 0.7% (w / v) agarose gel (1x TBE buffer, 1:10.00 Gel Red) at 130 V for 1 hour. The insert was excised from the gel and purified with a PCR & Gel Cleanup Kit (M&N) according to the manufacturer's instructions.

[0131] The purified insert was ligated into the pTH4.0 vector, a modified pET32a(+) vector encoding an N-terminal PelB signal peptide, a His6 tag for purification purposes, and a sequence encoding a tobacco etch virus (TEV) protease cleavage site followed by a BamHI digestion site. A C-terminal NotI digestion was followed by a myc tag for detection purposes, followed by a stop codon (Table 1). The pTH4.0 vector was digested with BamHI-HF and NotI-HF, and then the fragment was purified as described. The fragment was ligated into the digested pTH4.0 vector at a 3:1 ratio using 1x T4 ligation buffer containing T4 ligase, according to the manufacturer's instructions. The ligation mixture was transformed into TOP10 bacteria as described above, and the transformed bacteria were grown overnight at 37°C on YT agar plates (100 μg / mL ampicillin, 2% (w / v) glucose). Colonies were picked and grown in 10 mL of 2xYT medium (containing 100 μg / mL ampicillin and 2% (w / v) glucose). Plasmid DNA was isolated as described above. The DNA sequence was confirmed by Sanger sequencing by Macrogen (SEQ ID NO: 41).

[0132] production Caplacizumab in pTH4.0 plasmid DNA was transformed into chemically competent BL21 pLysS E. coli (Thermo) according to the manufacturer's instructions. Transformed bacteria were grown overnight at 37°C in 10 mL of 2xYT medium (containing 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, and 2% (w / v) glucose). The overnight culture was diluted 1:10 into 2xYT medium (containing 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, and 2% (w / v) glucose) and grown for 3 hours at 37°C. The culture was then diluted 1:100 into 2xYT medium (containing 100 μg / mL ampicillin, 34 μg / mL chloramphenicol) and grown for approximately 3 hours at 37°C. When the bacteria reached an OD of 0.6, protein production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (final concentration of 0.1 mM). Protein production was carried out overnight at 24°C. Bacteria were pelleted at 5000 × g for 15 minutes, and the supernatant was discarded. The bacterial pellet from a 400 ml culture was resuspended in 25 mL of Dulbecco's phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.2 mM Na2HPO4, pH = 7.4) and frozen at -20°C.

[0133] purification Frozen bacteria were thawed at 37°C and pelleted by centrifugation at 10,000 x g for 15 minutes. The supernatant was transferred to a new tube. 5 mL of cobalt Sepharose beads (TALON Superflow, G&E Heathcare, 50% solution) were washed with PBS according to the manufacturer's instructions, added to the supernatant, and incubated for 2 hours at room temperature on a roller bench. TALON was pelleted by centrifugation at 1,000 x g for 5 minutes. The supernatant was discarded, and the pellet was dissolved in 20 mL of PBS. TALON washing was repeated three times in total. After the last step, TALON was dissolved in 8 mL of PBS and loaded onto a PD-10 column. The column was rinsed with excess PBS. The column was eluted with imidazole (150 mM in PBS), and 0.5 mL fractions were collected. Protein-containing fractions were pooled and dialyzed overnight against HEPES-buffered saline (HBS: 10 mM HEPES, 150 mM NaCl, pH 7.4) using a 3.500 MWCO dialysis membrane (3 RC tubing; Spectra / Por). Protein concentration was determined by absorbance at 280 nm in a DeNovix spectrophotometer (DS-11), after which the concentration was corrected for the extinction coefficient (calculated by ProtParam). Purity was assessed by SDS-PAGE with Coomassie Page Blue staining.

[0134] Microthrombolysis of VWF-platelet aggregates.

[0135] Platelets were isolated from citrated whole blood as previously described (Tersteeg et al., 2014, supra). Isolated platelets (200,000 / μL) were incubated with VWF (5 μg / mL), plasma-purified plasminogen (100 μg / mL), and the aggregation inhibitors RGDW (200 μM) and iloprost (0.4 μg / mL) in a light transmission aggregometer for 15 min at 37°C. Aggregation was induced by the addition of ristocetin (0.6 mg / mL). Six min later, nanobody-mUPA constructs or caplacizumab were added, and aggregate dissolution was monitored over time. The time point at which 50% of microthrombi had disintegrated was determined for all samples (a schematic example of the analytical method is shown in Figure 8A). The results were processed using Graphpad Prism 7.02 and analyzed by one-way ANOVA (*P<0.05). The results are shown in Figures 10A and 10B.

[0136] result The results are shown in Figures 10A and 10B. It can be clearly seen that targeting plasminogen activation by at least the fusion proteins D3-mUPA and GP1B17-mUPA accelerates microthrombolysis compared to that induced by caplacizumab. [Table 6] TIFF2026031952000007.tif209149 TIFF2026031952000008.tif216149 TIFF2026031952000009.tif189149 TIFF2026031952000010.tif193149 TIFF2026031952000011.tif197149 TIFF2026031952000012.tif209149 TIFF2026031952000013.tif187149 TIFF2026031952000014.tif51149

Claims

1. A fusion protein for use in preventing or treating microvascular thrombosis, comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to the site of a thrombus comprising at least one of VWF and platelets, wherein the targeting agent is not a targeting agent that specifically binds only to the activated form of the GPIIb / IIIa receptor on platelets.

2. 2. The fusion protein for use according to claim 1, wherein the targeting agent specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium.

3. the targeting agent is a) a targeting agent that binds to at least unfolded VWF, preferably with preferential binding to unfolded VWF over globular VWF; b) a targeting agent that binds to the D3 domain of VWF; c) a targeting agent that binds to the GP1B receptor of platelets; d) a targeting agent that binds to platelet integrin αIIb / βIII; e) a targeting agent that binds to a receptor preferentially expressed by activated endothelium, preferably said receptor being selected from 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 f) a targeting agent that binds to a membrane marker of activated or damaged endothelium, said membrane marker being one or more of anionic phospholipids, phosphatidylserine, and phosphatidylethanolamine; 3. The fusion protein for use according to claim 1 or 2, which is one or more of:

4. The fusion protein for use according to any one of claims 1 to 3, wherein the fusion protein comprises two or more targeting factors.

5. the targeting agent is a) an antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and b) a binding domain derived from a protein that naturally binds to VWF, platelets, and activated or damaged vascular endothelium, wherein the binding domain specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium; The fusion protein for use according to any one of claims 1 to 4, comprising at least one of:

6. The antibody variable domain is a VHH, preferably a humanized VHH, or the binding domain is i) the platelet GP1B receptor-binding A1 domain of VWF; ii) the VWF-binding domain of one of ADAMTS13, factor XII, factor H (a complement regulator), plasminogen, and factor VIII; and iii) a membrane-binding domain selected from a vitamin K-dependent carboxylation / gamma-carboxyglutamic acid (GLA) domain, a factor V C domain, and a factor VIII C domain; 6. A fusion protein for use according to claim 5, comprising a binding domain selected from the group consisting of:

7. the plasminogen activator comprises the protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase, or staphylokinase; Preferably, said plasminogen activator further comprises at least a cysteine-containing portion of a connecting peptide naturally occurring in said plasminogen activator immediately upstream of its protease domain; 7. The fusion protein for use according to any one of claims 1 to 6, wherein the fusion protein optionally comprises a linker amino acid sequence connecting the targeting agent and the plasminogen activator.

8. The fusion protein comprises, in order from the N-terminus to the C-terminus: a) one or more targeting agents according to any one of claims 1 to 6, optionally linked by a linker amino acid sequence; b) optionally a linker amino acid sequence; and c) the plasminogen activator or plasminogen-derived protease domain according to claim 7; 8. The fusion protein for use according to claim 7, comprising:

9. Restenosis and disorders resulting from acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombus, occlusive thrombus formation, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, coronary artery bypass graft, coronary valve replacement, and coronary interventions such as angioplasty, stenting, or atherectomy, angioplasty, atherectomy, or arterial stenting 9. The fusion protein for use according to any one of claims 1 to 8, wherein said microvascular thrombosis is prevented or treated in a disease or condition selected from the group consisting of post-operative thickening, occlusive syndrome of the vascular system or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction.

10. 10. A method for treating or reducing the risk of microvascular thrombosis, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1 to 8.

11. Acquired or hereditary thrombotic thrombocytopenic purpura (TTP), complement-mediated thrombotic microangiopathy, hemolytic uremic syndrome, antiphospholipid syndrome, non-occlusive thrombus, occlusive thrombus formation, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, coronary artery bypass graft, coronary valve replacement, and restenosis and lesions resulting from coronary interventions such as angioplasty, stenting, or atherectomy, angioplasty, atherectomy, or arterial stenosis 11. The method of claim 10, wherein the microvascular thrombosis is treated or the risk of occurrence is reduced in a disease or condition selected from the group consisting of post-tent thickening, vascular occlusive syndrome or lack of patency of the affected artery, transient ischemic attack, unstable or stable angina, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease, and myocardial infarction.

12. 1. 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 comprises: a) a targeting agent that binds to at least unfolded VWF, preferably with preferential binding to unfolded VWF over globular VWF; b) a targeting agent that binds to the D3 domain of VWF; c) a targeting agent that binds to platelet integrin αIIb / βIII; d) a targeting agent that binds to a receptor preferentially expressed by activated endothelium, preferably said receptor being selected from 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 of activated or damaged endothelium, said membrane marker being one or more of anionic phospholipids, phosphatidylserine, and phosphatidylethanolamine. A fusion protein, wherein the fusion protein is one or more of:

13. The fusion protein of claim 12 , wherein the fusion protein comprises two or more targeting factors.

14. the targeting agent is a) an antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and b) a binding domain derived from a protein that naturally binds to VWF, platelets, and activated or damaged vascular endothelium, wherein the binding domain specifically binds to at least one of VWF, platelets, and activated or damaged vascular endothelium; 14. The fusion protein of claim 12 or 13, comprising at least one of:

15. The antibody variable domain is a VHH, preferably a humanized VHH, or the binding domain is i) a VWF-binding domain of one of ADAMTS13, factor XII, factor H (a complement regulator), plasminogen, and factor VIII; and ii) a membrane-binding domain selected from a vitamin K-dependent carboxylation / gamma-carboxyglutamic acid (GLA) domain, a factor V C domain, and a factor VIII C domain.

15. The fusion protein of claim 14, comprising a binding domain selected from the group consisting of:

16. the plasminogen activator comprises the protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase, or staphylokinase; Preferably, said plasminogen activator further comprises at least a cysteine-containing portion of a connecting peptide naturally occurring in said plasminogen activator immediately upstream of its protease domain; The fusion protein of any one of claims 12 to 15, wherein the fusion protein optionally comprises a linker amino acid sequence connecting the targeting agent and the plasminogen activator.

17. The fusion protein comprises, in order from the N-terminus to the C-terminus: a) one or more targeting agents according to any one of claims 12 to 14, optionally linked by a linker amino acid sequence; b) optionally a linker amino acid sequence; and c) a plasminogen activator or plasminogen-derived protease domain according to claim 16; 17. The fusion protein of claim 16, comprising:

18. 18. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein according to any one of claims 12 to 17, wherein said nucleotide sequence encoding said fusion protein preferably further comprises a nucleotide sequence encoding a signal peptide operably linked to said fusion protein, and wherein the nucleic acid molecule preferably further comprises regulatory elements which assist in the expression of said fusion protein, said regulatory elements being operably linked to said nucleotide sequence.

19. A gene therapy vector comprising the nucleic acid molecule of claim 18.

20. A pharmaceutical composition comprising a fusion protein according to any one of claims 12 to 18 or a gene therapy vector according to claim 19, and a pharmaceutically acceptable excipient.

Images