Targeting and lysis of microthrombi associated with abnormal blood clotting

Abstract

The present invention provides means and methods for treating MVT and associated and / or similar conditions. Thus, the present invention provides a hybrid protein for the targeted delivery of a plasminogen activator to the platelet-VWF complex, or alternatively to the site where these complexes are located, in a fibrin-independent manner. The present invention further provides methods of treatment for conditions that can be prevented or treated by locally delivering / stimulating plasminogen activation to microvascular occlusion sites.

Description

Technical Field

[0001] This invention relates to the field of diseases or conditions associated with undesirable blood clotting, particularly microvascular thrombosis, such as thrombotic thrombocytopenic purpura. Specifically, this invention relates to a hybrid protein comprising a (urokinase) catalytic domain linked to an antibody-like molecule (particularly VHH) capable of promoting microthrombus dissolution. In a particular embodiment, the VHH binds to von Willebrand factor (VWF), such that the linked urokinase catalytic domain drives the conversion of plasminogen to plasmin, which then degrades fibrin and / or VWF into smaller fragments. Therefore, this invention provides means and methods for treating microvascular thrombosis and related conditions. Background Technology

[0002] Primary hemostasis is a procoagulant clot formation process associated with the initiation and formation of platelet thrombi, in which platelet binding to vWF is crucial. Secondary hemostasis is also a procoagulant clot formation process associated with fibrin formation via intrinsic and extrinsic coagulation cascades. In healthy individuals, a homeostatic balance exists between procoagulant, anticoagulant, and fibrinolytic forces. Many genetic, acquired, and environmental factors can tilt this balance towards coagulation, leading to the pathological formation of venous (e.g., deep vein thrombosis [DVT]), arterial (e.g., myocardial infarction, ischemic stroke), or intracardiac thrombi. Thrombi can obstruct blood flow at the site of formation or detach and embolize, blocking distal vessels (e.g., pulmonary embolism, embolic stroke). Here, we will focus on microvascular thrombosis.

[0003] Microvascular thrombosis (MVT) is characterized by the formation of microvascular platelet aggregates. These aggregates consist of at least platelets and vascular fibroblasts (VWF). This is particularly evident in thrombotic thrombocytopenic purpura (TTP), where platelet- and VWF-enriched but fibrin-poor microthrombi obstruct microvessels, leading to life-threatening consequences. Therefore, the fibrin seen in large vessel thrombosis is not essential for these microvascular obstructions. MVT is a common feature across multiple disease states, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, antiphospholipid antibody syndrome, and complement-mediated thrombotic microangiopathy (George et al., 2014, N Engl J Med. 371(7):654-66). In severe cases of MVT, multiple organ failure can occur, leading to fatal consequences. Even in less severe cases, organ damage can occur, reducing the patient's quality of life and life expectancy. Recent research suggests that microvascular disease is the underlying cause of cardiovascular events in a more prevalent patient population with cardiovascular disease who do not show obvious signs of large vessel blockage on radiographic examinations. This is believed to ultimately lead to heart failure, particularly in women.

[0004] Patients with TTP experience episodes of microvascular thrombosis when platelets form a complex with ultramacromers of von Willebrand factor (VWF). This is a consequence of severely reduced activity of the enzyme ADAMTS13 (a depolymerin and metalloproteinase with a type 1 platelet-reactive protein motif, member 13). ADAMTS13 normally regulates the thrombogenicity of VWF by enzymatically reducing the size of the VWF polymers. For this purpose, VWF needs to unfold from its globular form into an extended conformation, exposing its A2 domain for proteolysis. Most TTP patients have neutralizing autoantibodies against ADAMTS13. For a small subgroup, mutations in ADAMTS13 have been described as causing defects (Upshaw-Shulman syndrome).

[0005] Current TTP therapy involves extensive plasma exchange to simultaneously deplete inhibitory antibodies and restore ADAMTS13 activity. However, persistent autoantibodies hinder the clearance of microthrombi. This makes the therapy time-consuming and very expensive (Fijnheer et al., Ned Tijdsch Hematol 2016; 13(1): 18-24).

[0006] 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 found that systemic plasminogen activation (using streptokinase) had a therapeutic effect in a TTP mouse model, suggesting that plasmin could act as a functional substitute for ADAMTS13 (Tersteeg et al., 2014, Circulation, 129(12):1320-31). While plasmin(pro) can bind directly to extended (unfolded) VWF, 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 fibrin-poor, and it is uncertain whether fibrin is a prerequisite for other types of MVT. This renders molecules that are commonly used as thrombolytic agents in the treatment of large vessel diseases (i.e., tPA, uPA) ineffective in treating MVT. Summary of the Invention

[0007] This invention provides a hybrid protein for targeted delivery of plasminogen activators to platelet-VWF complexes, or alternatively to their sites, in a fibrin-independent manner. The invention further provides the use of this hybrid protein for treating MVT and associated and / or similar conditions, and methods for preventing or treating conditions by local delivery / stimulation of plasminogen activation to microvascular occlusion sites. Attached Figure Description

[0008] exist Figure 1 The results show the susceptibility of Microlyse variants to thrombin cleavage. Microlyse variant 1.1 contains glutamine at position 153, while Microlyse variant 1.0 contains arginine at position 153.

[0009] Figure 2 The EpiMatrix protein immunogenicity scale for ML1.0 and ML1.1 is shown.

[0010] Figure 3a This is the deconvolution MS spectrum of the intact ML1.0 protein (before PNGaseF treatment) in Example 4.

[0011] Figure 3b This is the deconvolutioned MS spectrum of ML1.0 after PNGaseF processing in Example 4.

[0012] Figure 4aThis is the reduced RP-HPLC chromatogram of ML1.1 in Example 4. The MS identification results of each peak are described in the legend. The X-axis represents the elution time (minutes), and the Y-axis represents the UV 214 nm signal.

[0013] Figure 4b It comes from Figure 4a Deconvolutional MS spectra of peaks 1, 5, and 7 with peak quality (Da).

[0014] Figure 5 The effect of alteplase infusion on bleeding time in the tail clip test of Example 6 is shown. The tail clip test was performed at 20 minutes (blue, mid-treatment infusion) or 40 minutes (red, end of treatment infusion). The dashed line at 600 seconds indicates the maximum allowable bleeding time before the test was stopped. ****P < 0.0001 or not significant (ns) relative to the solvent (0 mg / kg) group was determined using one-way ANOVA and Dunnet's multiple comparison test.

[0015] Figure 6 The effect of TGD001 and alteplase infusion on bleeding time is shown in the tail clip test of Example 6. TGD001 and alteplase (both 2.0 mg / kg) were administered as a 10% bolus followed by a 40-minute infusion (the remaining 90%). The tail clip test was performed at 20 minutes (during infusion) and 40 minutes (at the end of infusion). The dashed line at 600 seconds indicates the maximum allowable bleeding time before the test was stopped. ****P < 0.0001 or not significant (ns) relative to the solvent (0 mg / kg) group was determined using one-way ANOVA and Dunnet's multiple comparison test.

[0016] Figure 7 This illustrates the effect of TGD001 administration as a bolus injection on bleeding time in the tail clip test of Example 6. TGD001 was administered as a bolus injection. The tail clip test was performed immediately after the bolus injection. The dashed line at 600 seconds represents the maximum allowable bleeding time before the test was stopped. ****P < 0.0001 or not significant (ns) relative to the solvent (0 mg / kg) group was determined using one-way ANOVA and Dunnet's multiple comparison test.

[0017] Figure 8 The effect of prozymogen or activated TGD001 on bleeding time is shown in the tail clip model of Example 6. Mice received a bolus injection of TGD001. The dashed line at 600 seconds represents the maximum allowable bleeding time before the experiment was stopped. ****P < 0.0001 or not significant (ns) relative to the solvent (0 mg / kg) group, one-way ANOVA and Dunnet's multiple comparison test were used.

[0018] Figure 9The effect of TXA on the pharmacological activity of TGD001 in the tail clip model of Example 6 is shown. TGD001 (2 mg / kg) was administered by bolus injection, with or without TXA infusion. The tail clip experiment was performed immediately after the bolus injection. The dashed line at 600 seconds indicates the maximum allowable bleeding time before the experiment was stopped. ****P < 0.0001 or not significant (ns) relative to the solvent (0 mg / kg) group was determined using one-way ANOVA and Dunnet's multiple comparison test. Detailed Implementation

[0019] Thrombotic microangiopathy is a group of conditions characterized by catastrophic, diffuse thrombosis within microvessels. In thrombotic thrombocytopenic purpura (TTP), this is caused by a deficiency of functional ADAMTS13 (protein loss or functional protein loss, or both). This is most commonly a result of autoantibodies against ADAMTS13, which clear and neutralize the protease (acquired TTP [aTTP]). TTP is unpredictable in its episodic nature, and acute attacks are characterized by deep-seated, debilitating thrombocytopenia: microthrombi obstruct blood vessels, consuming platelets in the systemic circulation. This leads to ischemic damage in various tissues, with a significant risk of bleeding. Current standard of care for acute aTTP attacks includes plasma exchange to remove autoantibodies and restore ADAMTS13 activity. Long-term treatment strategies include immunomodulation to reduce autoantibody levels.

[0020] The interaction between platelet glycoprotein Ibα (GPIbα) and von Willebrand factor (VWF) drives microthrombus formation in transient ischemic attacks (aTTP). This does not require platelet activation, coagulation system activation, or fibrin formation. Recently, a bivalent single-chain antibody (VHH; nanobody) that blocks platelet binding to the VWF A1 domain has been developed for the treatment of aTTP. This nanobody (INN: caplacizumab) administered in combination with plasma exchange reduced the duration of acute TTP attacks from 5 days to 3-4 days. This was demonstrated by faster normalization of platelet counts and a reduction in the need for plasma exchange (39%), which has a significant impact on organ damage suffered during attacks. Essentially, shortening the duration of attacks is crucial for the management of TTP.

[0021] We previously found evidence of endogenous plasminogen activation during TTP episodes. We proposed that plasmin acts as an adjunct to VWF-cleaving enzymes in this context, and subsequently demonstrated that systemic plasminogen activation (using bacterial plasminogen activator) alleviated thrombocytopenia in a mouse TTP model. We then demonstrated that endogenous plasminogen activation modulates disease severity in an aTTP mouse model. These findings lead us to believe that utilizing the VWF-cleaving properties of plasmin has therapeutic value. While plasminogen can bind directly to VWF in a lysine-dependent manner, neither tissue plasminogen activator (tPA) nor urokinase plasminogen activator (uPA) can directly bind to VWF. According to the present invention, strategies to bring plasminogen activators to VWF are valuable for the treatment of TTP.

[0022] We have developed a novel thrombolytic agent for disrupting the platelet-VWF complex. In particular, the developed agent is a VWF-targeting plasminogen activator for plasmin-mediated disruption of the platelet-VWF complex, which is not strictly dependent on the presence of fibrin, thrombin, or platelet activation.

[0023] According to the present invention, the plasminogen activator, more specifically the urokinase catalytic domain of the plasminogen activator, has been found to have at least two factors that may negatively affect stability and thus therapeutic performance. First, there is a potential thrombin cleavage site leading to inactivation of the urokinase catalytic domain. This cleavage site has been found to be located at arginine position 153 of the urokinase catalytic domain. This cleavage site can be removed by altering the arginine at position 153. For this purpose, an amino acid that is not easily cleaved by thrombin should be selected. Preferably, this amino acid is structurally and / or chemically similar to arginine. The most similar amino acid is histidine, but histidine has a benzene ring, making it a less suitable candidate due to its structural effects. Therefore, considering other amino acids, glutamine is the most suitable candidate according to the present invention. The second observed effect that may negatively affect the stability of the urokinase catalytic domain is incorrect or incomplete glycosylation, which can severely affect the protein's half-life and may lead to immunogenicity problems. The urokinase catalytic domain has been found to contain an endogenous N-glycosylation site located at asparagine position 299. This potentially unwanted endogenous N-glycosylation site can be removed by altering the asparagine at position 299.

[0024] Therefore, the present invention provides a polypeptide comprising or composed of a urokinase catalytic domain, wherein the arginine residue at position 153 is substituted with a different amino acid residue, preferably a glutamine residue. In a further embodiment, the polypeptide comprises a substitution of an asparagine residue at position 299. Most preferably, the asparagine residue at position 299 is substituted with a glutamine residue. These mutants retain at least their structure and activity to a useful degree. Furthermore, according to our tests (computer simulations and others), the immunogenicity of these mutants is acceptable for therapeutic use. In another embodiment, the polypeptide according to the invention comprises a substitution of a lysine residue at position 297. Preferably, the lysine residue at position 297 is substituted with histidine. The term "polypeptide" refers to a molecule composed of a chain of amino acids, wherein the amino acids are linked by covalent (peptide) bonds. The term "urokinase catalytic domain" refers to a specific region or segment of a urokinase-type plasminogen activator (uPA) protein. The terms "urokinase-type plasminogen activator" and "urokinase" are used interchangeably to refer to an enzyme that functions in the breakdown of blood clots (a process known as fibrinolysis). Specifically, urokinase protein promotes the lysis of blood clots (i.e., microthrombi) by facilitating the conversion of plasminogen to plasmin, which then degrades fibrin (the protein network that forms the blood clot) into smaller fragments. Urokinase protein consists of several functional domains, each playing a unique role in its enzymatic activity. The “catalytic domain” is the part of the urokinase protein responsible for its enzymatic function, particularly its ability to activate plasminogen to plasmin. Specifically, urokinase is a serine protease capable of catalyzing plasmin activation via proteolytic cleavage of plasminogen. The term “residue” refers to a specific individual building block (i.e., amino acid) within a polypeptide chain. Each amino acid in the chain is called a residue. The term “position” as used herein refers to a specific location of a residue in the amino acid chain of a polypeptide. Residues are assigned starting from the first amino acid at the N-terminus of the polypeptide. The first N-terminal residue is assigned number 1, the second residue is assigned number 2, and so on. The residue positions referred to herein can be counted, for example, from the first amino acid in one of the following sequences: SEQ ID No. 1-3, preferably in SEQ ID No. 1. For example, position 153 refers to the 153rd amino acid counted from the N-terminus in the polypeptide chain, preferably in one of the following sequences: SEQ ID No. 1-3, more preferably SEQ ID No. 1. The term "substitution" refers to a specific type of mutation that occurs when an amino acid residue in the polypeptide chain is replaced by a different amino acid residue. For example, when an arginine residue is replaced by a glutamine residue.

[0025] The present invention further provides a hybrid protein comprising the polypeptide, the polypeptide containing or being composed of a urokinase catalytic domain, wherein the polypeptide is linked to a specific binding molecule, such as an antibody-like molecule, preferably VHH. The term "hybrid protein" is defined as a complex of two or more polypeptide sequences or fragments thereof, which are generally unrelated but coupled together by fusing their coding genes (optionally via peptide linkers) or by chemically cross-linking components. The terms "VHH" and "VHH antibody" are used interchangeably and refer to a single-domain antibody or nanobody derived from camelids (camels, llamas, and alpacas) or cartilaginous fish (such as sharks and rays). Unlike conventional antibodies (IgG antibodies) with two heavy chains and two light chains found in humans and other mammals, VHH antibodies have a single polypeptide chain containing a single variable domain derived from the heavy chain. VHH antibodies have a frame region (FR) and a complementarity-determining region (CDR). The “framework region” typically consists of frame regions 1-4 (FR1-FR4) and forms the scaffold of the variable domain of the VHH antibody, providing structural support and maintaining the overall shape of the binding site. It contains relatively conserved amino acid sequences, stabilizing the antibody structure and facilitating its folding. The “complementarity-determining region (CDR),” also known in the art as the hypervariable region, is a short amino acid fragment within the variable domain of the antibody. CDRs are highly diverse and responsible for antigen recognition and binding specificity. There are typically three CDRs in the variable domain of a VHH antibody, generally named CDR1, CDR2, and CDR3. Complementarity-determining region 3 (CDR3) preferably contains any amino acid sequence listed in SEQ ID NO: 6-28, complementarity-determining region 1 (CDR1) preferably contains any amino acid sequence listed in SEQ ID NO: 29-51, and complementarity-determining region 2 (CDR2) preferably contains any amino acid sequence listed in SEQ ID NO: 52-74. Those skilled in the art will be able to determine the optimal combination of the disclosed CDRs. VHH antibodies preferably contain a variable domain that binds von Willebrand factor (vWF). Binding is defined as specific recognition with a higher affinity than background protein-protein binding. Preferably, the affinity is at least in the micromolar range, and more preferably in the nanomolar range. The basic human VWF monomer is a 2050-amino acid protein. Each monomer contains multiple specific domains with specific functions, including, for example, a D'D3 domain that binds factor VIII; an A1 domain that binds, in particular, the platelet GPIb receptor; an A2 domain (which must be partially unfolded to expose a cryptic cleavage site for the specific ADAMTS13 protease, which inactivates VWF by generating smaller multimers); an A3 domain that binds collagen; a C1 domain in which the RGD motif binds platelet integrin αllbβ3 upon activation; and a "cysteine ​​knot" domain (at the C-terminus of the protein).VWF polymers can be very large, typically greater than 20,000 kDa, and can consist of more than 80 subunits, each 250 kDa.

[0026] The VHH of the present invention can specifically bind to various forms, conformations, domains, and epitopes of VWF (depending on the amino acid composition of the CDR in the variable domain). For example, the VHH of the present invention can specifically bind to at least one of the extended (unfolded, activated) conformation and the globular (cycle-inactive) conformation of VWF. Preferably, the VHH of the present invention preferentially binds to extended VWF rather than globular VWF (i.e., it has a higher affinity for unfolded VWF than for globular VWF). Extended VWF exposes several important binding sites, such as the main binding domain of plasmin. Preferential binding to extended VWF helps improve the efficiency and potency of directing the urokinase catalytic domain to its desired site of action. More specifically, the VHH of the present invention binds to the extended (activated) conformation of VWF and does not bind to the cycle-inactive globular conformation of VWF. The VHH of the present invention can specifically bind to one or more domains of VWF, such as the D'D3 domain and / or the C-terminal cysteine ​​knot (CTCK) domain. Preferably, the VHH of the present invention specifically binds to the CTCK domain of the (extended) VWF.

[0027] Expressed in terms of the equilibrium dissociation constant (KD), the affinity of the VHH of the present invention for human VWF can be at least or stronger than 10 nM, at least or stronger than 5 nM, at least or stronger than 1 nM, at least or stronger than 500 pM, at least or stronger than 400 pM, or at least or stronger than 300 pM. Preferably, the affinity of the VHH of the present invention for extended human VWF is at least or stronger than 250 pM, at least or stronger than 200 pM, or at least or stronger than 150 pM. More preferably, the affinity of the VHH of the present invention for extended human VWF is at least or stronger than 100 pM. In a preferred embodiment, the affinity of the VHH of the present invention for extended human VWF is at least or stronger than 90 pM, for example, 70 pM.

[0028] The sequence encoding VHH is preferably codon-optimized and preferably contains humanized and / or deimmunized amino acid modifications for VHH. The terms "humanized" and / or "deimmunized" refer to modifications in which the amino acids of antibodies derived from non-human sources (e.g., camels, mice, or other species) are modified to make them more similar to human antibodies. The goal of humanization is to reduce the potential immunogenicity (the likelihood of triggering an immune response) of the antibody when used in humans for therapeutic or diagnostic purposes. When an antibody is humanized, one or more amino acid residues are replaced by one or more amino acid residues that predominate at the corresponding position in the VH domain of a conventional 4-chain antibody from humans. The hybrid protein described above can be a fusion protein or a chemically linked protein.

[0029] The hybrid protein of the present invention preferably further comprises a linker amino acid sequence located between VHH (on one hand) and the urokinase plasminogen activator (on the other hand). The preferred linker amino acid sequence for linking the target and the plasminogen activator has a sequence of (Gly-Gly-Gly-Gly-Ser)n, where n represents an integer (e.g., 1, 2, 3, etc.). The term "fusion protein," as understood by those skilled in the art, refers to a protein produced by conjugating two or more genes that originally encode individual proteins. The term "chemically linked protein," as understood by those skilled in the art, refers to a construct in which two or more proteins are covalently linked to each other using chemical methods, including the use of cross-linking agents, such as bifunctional reagents, click chemistry, or enzymatic conjugation. The hybrid protein preferably comprises (preferably in N-to-C-terminal order): an antibody or antigen-binding fragment, a linker sequence, and a portion capable of promoting microthrombus dissolution; most preferably, the hybrid protein comprises, in N-to-C-terminal order: a VHH antibody, a Gly / Ser linker sequence, and the urokinase plasminogen activator. Alternatively, the hybrid protein may contain more than one VHH, more than one adapter sequence, and / or more than one urokinase plasminogen activator. Preferably, the hybrid protein according to the invention comprises or is composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0030] Once administered, the hybrid protein of the present invention can be present in plasma in free form and / or in VWF-bound form. Preferably, the hybrid protein of the present invention, when in free form, can have an effective half-life in the range of 0.25 to 9 hours (preferably, more preferably 0.5 to 6 hours, e.g., 0.9 to 5.7 hours); and / or a terminal elimination half-life in the range of 8.5 to 15.5 hours, preferably 9.25 to 14.5 hours. Taking into account the VWF-binding species, the hybrid protein of the present invention can have a (total) effective half-life in the range of 6 to 8 hours, preferably 6.25 to 7 hours; and / or a (total) terminal elimination half-life in the range of 14 to 21.5 hours, preferably 14.5 to 20.5 hours. The half-life disclosed in this paragraph refers to the metabolism of the hybrid protein in healthy adults, without thrombus binding.

[0031] The present invention further provides a method for modulating, preferably reducing, the risk of microvascular thrombosis, wherein the method includes the step of administering an effective amount of the heterozygous protein disclosed above to a subject in need. The heterozygous protein is preferably administered at a dose of up to 1.5 mg, up to 1.0 mg, up to 0.9 mg, up to 0.8 mg, up to 0.7 mg, up to 0.6 mg, or up to 0.5 mg per kilogram of body weight. Preferably, the heterozygous protein is administered at a dose of at least 0.05 mg, at least 0.06 mg, at least 0.08 mg, at least 0.09 mg, or at least 0.1 mg per kilogram of body weight. For adult patients, the heterozygous protein may be administered at a dose of 0.5 to 100 mg, preferably 1.0 to 50 mg, more preferably 2.0 to 32 mg. The heterozygous protein is preferably administered via intravenous (IV) infusion or intravenous bolus. For example, the heterozygous protein may be administered as a controlled IV infusion over 0.5 to 60 minutes, preferably 0.5 to 5 minutes, for example, 1 minute. Alternatively, the hybrid protein can be administered via intravenous bolus. Preferably, the hybrid protein is administered via intravenous infusion or intravenous bolus at a dose of 0.02 to 1.5 mg per kilogram of body weight, for example, 0.04 to 1.0 mg. Alternatively, the hybrid protein is administered via intravenous infusion or intravenous bolus at a dose of 0.05 to 1.5 mg per kilogram of body weight. Preferably, the hybrid protein is administered to adult patients via intravenous infusion or intravenous bolus at a dose of 0.5 to 50 mg, more preferably 1 to 32 mg, 2 to 16 mg, for example, 8 mg. The hybrid protein of the present invention can be administered no more than 5 times within a week, preferably no more than 4 times within a week, no more than 5 times within two weeks, and most preferably no more than 4 times within two weeks.

[0032] Preferably, microvascular thrombosis is treated or its risk is reduced in the following diseases or conditions: acquired or hereditary thrombotic thrombocytopenic purpura (TTP); complement-mediated thrombotic microangiopathy; hemolytic uremic syndrome; antiphospholipid antibody syndrome; non-occlusive thrombosis; occlusive thrombosis; arterial thrombosis; acute coronary artery occlusion; peripheral artery occlusive disease; restenosis; and conditions resulting from coronary artery bypass grafting, coronary valve replacement, and coronary interventions (such as angioplasty, stenting, or atherosclerosis); hyperplasia after angioplasty, atherosclerosis, or arterial stenting; occlusive syndromes or obstruction of diseased arteries in the vascular system; transient ischemic attack; unstable or stable angina; cerebral infarction; HELLP syndrome; carotid endarterectomy; carotid stenosis; severe limb ischemia; cardioembolic disease; peripheral vascular disease; restenosis; sickle cell disease; and myocardial infarction. By providing a method for administering an effective amount of the hybrid protein, the inventors have provided means and methods for treating or reducing the risk of microvascular thrombosis.

[0033] A further method according to the invention for producing the heterozygous protein as described above includes the use of production cells, preferably eukaryotic cells. The term "production cell" refers to a cell capable of synthesizing and preferably secreting large quantities of a specific recombinant target protein. These cells are engineered to contain one or more genes encoding the desired protein, enabling them to produce and preferably secrete the recombinant protein into the surrounding environment. Production cells can be of various types, such as bacteria, yeast, insect cells, or mammalian cells, depending on the specific requirements of the protein and the intended application. Preferably, the production cell is a yeast cell, particularly Pichia pastoris cells or Saccharomyces cerevisiae cells, with Pichia pastoris cells being preferred. Alternatively, the production cell can be a mammalian cell, with Chinese hamster ovary cells being preferred. The heterozygous protein, or preferably a precursor of a heterozygous protein, may further comprise a secretion signal peptide, preferably an α-mating factor (preferably according to SEQ ID No. 4), or a secretion signal peptide such as mouse IgKVIII (preferably according to SEQ ID No. 5). The term "secretion signal peptide α-mating factor" describes a signal peptide that targets the secretion pathway in yeast cells (preferably Pichia pastoris). The term "secretion signal peptide mouse IgKVIII" is defined as a signal peptide commonly used in Chinese hamster ovary cells that guides the peptide to the cell's secretion pathway, ensuring proper processing and secretion. Methods for producing heterozygous proteins, as understood by those skilled in the art, include the following steps: the growth phase of the production cells; the induction phase of the production cells; harvesting; and one or more purification steps, such as: filtration, protein A-agarose purification; hydrophobic interaction chromatography (HIC); ion exchange chromatography (IEX).

[0034] The present invention further provides an isolated nucleic acid molecule according to the invention, which encodes a heterozygous protein as described above. Preferably, the isolated nucleic acid molecule is part of a circular nucleic acid molecule. The term "circular nucleic acid molecule" is defined as a nucleic acid (DNA or RNA) molecule that forms a closed circular structure, wherein the ends of the molecule are covalently linked together. The term "circular nucleic acid molecule" includes expression vectors or expression constructs known in the art, which are nucleic acid molecules capable of realizing the expression of nucleotide sequences or genes in host cells or host organisms compatible with such expression vectors or constructs. The circular nucleic acid molecule further preferably contains a promoter sequence operatively linked to the heterozygous protein. As used herein, the term "operatively linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operatively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter sequence affects the transcription of a coding sequence, then the promoter sequence is operatively linked to the coding sequence. The promoter sequence can be an inducible promoter sequence or a constitutive promoter sequence. Preferably, the inducible promoter sequence is an alcohol oxidase I (AOX1) promoter sequence, and the constitutive promoter sequence is a cytomegalovirus (CMV) promoter sequence.

[0035] The present invention further provides a pharmaceutical composition comprising the heterozygous protein disclosed above and a suitable excipient for administration. The pharmaceutical composition preferably comprises sodium acetate or sodium citrate, with sodium acetate being the most preferred. The pharmaceutical composition further preferably comprises one or more pharmaceutically acceptable excipients. The term "pharmaceutically acceptable" means a molecular entity or composition that does not produce an adverse, allergic, or other undesirable reaction when administered to a subject. Whether certain adverse reactions are acceptable is determined based on the severity of the disease. The pharmaceutical composition is preferably formulated as a liquid preparation or as a lyophilized preparation reconstituted with water for injection or a buffer solution.

[0036] The pharmaceutical compositions of the present invention may include a buffer system, such as those commonly used in pharmaceutical formulations for purposes such as maintaining the solubility, stability, bioavailability, and / or biological function of the active agent. Exemplary buffer systems suitable for the pharmaceutical compositions of the present invention may be phosphate-buffered saline (PBS), acetate (e.g., sodium acetate) buffer, citrate buffer, and Tris buffer. The selection of the type and composition of the buffer system takes into account various factors, such as the ionic strength of the composition, the excipients used, the route of administration and dosage requirements, manufacturing and storage conditions, etc. Preferably, the pharmaceutical compositions of the present invention comprise a buffer with a pH in the range of 5.0 to 7.5, such as a buffer with a pH of 5.5 or a buffer with a pH of 7.4.

[0037] The pharmaceutical compositions of the present invention may contain one or more excipients selected from solvents, tension modifiers, stabilizers, solubilizers, surfactants, preservatives and / or complexing agents.

[0038] The kit of parts according to the present invention comprises a heterozygous protein or pharmaceutical composition and one or more of the following: tranexamic acid (TXA), plasmin inhibitor (PI), and anti-urokinase antibody. Preferably, one or more of the following compounds are administered to the subject to achieve the modulating effect of the heterozygous protein: tranexamic acid (TXA), plasmin inhibitor (PI), and anti-urokinase antibody. In other words, the effect induced by administering the heterozygous protein to the subject is reduced or stopped. By providing a method for modulating the effect of the heterozygous protein, the inventors have established means and methods for improving the control of patients treated with heterozygous proteins.

[0039] Example Example 1 - Preparation of Microlyse variants 1.0 and 1.1 (ML1.0 and ML1.1) The cDNA sequences of human and mouse urokinase (PLAU) were obtained from the NCBI database (NM_002658.4 and NM_008873.3, respectively). The signal peptide, EGF-like domain, Kringle domain, and the first part of the linker peptide were removed. An N-terminal sequence encoding the Tobacco Etch Virus cleavage site was added to the remaining linker peptide and S1 peptidase domain (catalytic domain), followed by a GGGGS adapter. In the GGGGS adapter, [the following sequence was introduced without disrupting the amino acid sequence]. PstI and BamHI Digestion site. Added on the 5' side. EcoRI The digestion site, a NotI digestion site, was added on the 3' side after the STOP codon of PLAU. In the case of Microlyse variant 1.1 (ML1.1), the arginine residue at position 153 of the urokinase sequence was replaced with a glutamine residue before ordering two sequences (ML1.0 and ML1.1) from IDT (IntegratedDNA Technologies, Leuven, Belgium) as a custom gene construct.

[0040] The coding sequence of the nanobody (also known as VHH) remains the original llama sequence, or it is humanized via four different amino acid substitutions (A14P, E44G, K87R, P88A) within the nanobody framework. Subsequently, the sequence is codon-optimized via IDT for expression in human host cells. A sequence encoding the tobacco etch virus cleavage site is placed at the N-terminus of the VHH coding sequence, and a GGGGS adapter (encoding...) is placed at the C-terminus. PstI and BamHI (Digestion site). These DNA fragments were obtained from IDT as double-stranded DNA fragments (gBlocks).

[0041] Custom gene constructs were propagated in E. coli TOP10 and selected for resistance using ampicillin. The obtained plasmid DNA was then processed... EcoRI and NotI Digestion was performed. 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 AllergyClin Immunol Nov;30;138(5):1414-23). ​​pSM2 encodes an N-terminal mouse IgK secretion signal and a double STREP separation tag, which was subsequently ligated into a modified UPA construct.

[0042] According to the manufacturer's instructions (CloneJET PCR Cloning Kit; Thermo Fisher), gBlocks were ligated into the pJET1.2 cloning vector. The constructs were propagated in *E. coli* TOP10 and selected for ampicillin resistance. The obtained plasmid DNA was then processed... EcoRI and BamHI Digestion was performed. The resulting insert fragments were separated and isolated on an agarose gel and ligated into the pSM2 vector containing the miniUPA construct.

[0043] Using 239Fectin, the ML1.0 and ML1.1-pSM2 constructs were transfected into HEK293 FreeStyle™ cells in 6-well plates according to the manufacturer's (ThermoFisher) instructions. Samples were harvested 7 days after production and centrifuged at 2000×g for 10 min. The supernatant containing ML1.0 / ML1.1 was separated from the biomass and centrifuged again to remove final cell debris. Microlyse titers were determined by VWF-binding ELISA as previously disclosed (De Maat et al., 2022 Blood Jan;27;139(4):597-607). The titers of the Microlyse variants were adjusted to the same level by diluting with the supernatant of simulated transfected HEK293 FreeStyle™ cells.

[0044] Example 2 - Susceptibility of Microlyse variants ML1.0 and ML1.1 to thrombin cleavage 20 µL of the Microlyse variant supernatant obtained from Example 1 was incubated with 5 µL of vehicle solvent or thrombin (prediluted in 0.2% w / v BSA-HBS) at 37 °C for 30 min. The reaction was then terminated by adding sample reduction buffer (25 mM DTT). The sample was heated to 95 °C for 10 min and then separated on a 4–12% gradient Bis-Tris gel in MES buffer. The gel was transferred to an Immobilon-FL membrane at 125 volts for 1 h in transfer buffer. The membrane was blocked with 0.5 × Odyssey blocking buffer and then the construct was detected using a rabbit polyclonal anti-human UPA antibody conjugate IR800-labeled goat anti-rabbit antibody. Results were analyzed using a near-infrared Odyssey scanner (Licor) according to the manufacturer's instructions.

[0045] exist Figure 1 The results show the susceptibility of Microlyse variants ML1.0 and ML1.1 to thrombin cleavage. ML1.1 contains glutamine at position 153, while ML1.0 contains arginine at position 153. For both ML1.0 and ML1.1, a distinct band was observed at approximately 35 kDa, indicating that Microlyse is cleaved by thrombin.

[0046] Example 3 - PreDeFT Immunogenicity Analysis The immunogenic potential of the ML1.0 and ML1.1 sequences in humans was evaluated using computer simulations.

[0047] method The EpiMatrix system was used to screen the amino acid sequences of ML1.1 and ML1.0 for the presence of class II (HLA-DR) restricted HLA ligands or putative T-cell epitopes. The overall and regional immunogenic potential of the sequence subunits was assessed. To determine whether any putative T-cell epitopes identified by EpiMatrix were homologous to predicted epitopes found in the human proteome and thus unlikely to drive a therapeutically resistant immune response, all putative epitopes identified by EpiMatrix were screened against the human proteome using the JanusMatrix algorithm. Furthermore, to identify any predicted T-cell epitopes that may have been previously studied and evaluated, all putative T-cell epitopes identified by EpiMatrix were screened against a version of the Immunoepitaph Database (IEDB). The information was compiled to provide an EpiMatrix score as a measure of immunogenic potential.

[0048] result Regional analysis revealed 12 putative T-cell epitope clusters; five of these originated from the VhH domain and seven from the UPA domain. Of the 12 identified putative T-cell epitope clusters, six clusters (9-31, 33-48, 57-79, and 76-94 of the VhH domain; and 192-207 and 249-267 of the UPA domain) are expected to be tolerated by the human immune system and, under normal circumstances, may be actively tolerogenic in at least some human subjects. The remaining six T-cell epitope clusters scored high on the EpiMatrix and low on the JanusMatrix, and one cluster (44-58) overlapped with the antibody complementarity-determining region (CDR). Under normal circumstances, these peptides would likely be perceived as foreign by the human immune system and may contribute to inducing an anti-treatment immune response.

[0049] At the overall level, the Tregitope-adjusted EpiMatrix score of ML1.1 (-20.54) fell within the EpiMatrix protein immunogenicity scale ( Figure 2 The low range of ) indicates the least potential to drive anti-therapeutic immune responses. As expected, compared with ML-CTCK-hUPA (-14.83; Figure 2 Compared to the previous method, humanization of the VHH domain reduced the immunological potential of ML1.1. Studies of the ML1.1 domains revealed that the VHH domain (-25.16) and the linker (-25.58) fell in the low range, while the catalytic domain of UPA fell in the low-to-medium range (-13.97).

[0050] in conclusion ML1.1 falls in the low range of the EpiMatrix protein immunogenicity scale, indicating minimal potential to drive anti-therapeutic immune responses in humans.

[0051] Example 4 - Mass spectrometry analysis of ML1.0 and ML1.1 ML1.0 used in this embodiment (i.e., as described in Example 1) was transiently expressed in HEK cells. It was further purified via a streptavidin tag, as previously disclosed (De Maat et al., 2022 Blood Jan;27;139(4):597-607). The amino acid sequence of ML1.0 with an IgG secretion signal (cleaved during secretion) and an N-terminal purification tag is described in SEQ ID NO. 102.

[0052] The ML1.1 used in this embodiment was prepared as described in Example 1, except that it was recombinantly expressed in Pichia pastoris cells. The amino acid sequence of ML1.1 is described in SEQ ID NO. 1.

[0053] The obtained ML1.0 was analyzed using high-resolution mass spectrometry (MS). Figure 3a and 3b The images show the deconvolution MS spectra of ML1.0 before and after PNGase F treatment. In the PNGase-treated sample, the major mass measured was, as expected, the mass of ML1.0 with the N-terminal purification tag. The measured mass was 48768.3 Da, 341 Da higher than the expected mass from the amino acid sequence. This is likely due to the presence of biotin in the sample. This is not surprising, as the protein had already been purified with the N-terminal streptavidin tag. Therefore, we conclude that the correct sequence was found, and all N-linked glycosylations were removed. Figure 3a The spectrum of the undeglycosylated ML1.0 shows several mass variants (shifted towards higher mass), which can be attributed to the presence of N-glycans and sialylated core type 1 O-glycans. However, due to the presence of many mass variants (combinations of N-glycans with several O-glycans), MS deconvolution is complex. Nevertheless, it can be concluded that ML1.0 contains N-linked glycosylation.

[0054] For ML1.1, the host *Pichia pastoris* is capable of N-glycosylation. Since the amino acid sequence of ML1.1 does not contain N-glycosylation sites, N-glycosylation was not expected. The obtained ML1.1 was analyzed using reversed-phase high-performance liquid chromatography-mass spectrometry (RP-HPLC-MS). The results are shown in... Figure 4a As illustrated in the legend, the identity of each peak was confirmed by high-resolution mass spectrometry (HR-MS), such as... Figure 4b As shown. The peak marked (*) is most likely a TFA (trifluoroacetic acid) adduct.

[0055] Analysis confirmed that ML1.1 expressed in Pichia pastoris cells did not carry any N-glycosylation. The precise mass measured by HR-MS corresponded to the expected molecular mass. It was also shown that approximately 40% of ML1.1 carried one to three mannoses, and trace amounts carried up to five mannoses.

[0056] Example 5 - Repeated-dose toxicology study The ML1.1 used in this embodiment was prepared as described in Example 1, except that it was recombinantly expressed in Pichia pastoris cells and therefore did not carry any N-glycosylation. For this embodiment, ML1.1 was prepared into a 2 mg / mL solution in an aqueous buffer (pH 5.0) for parenteral application.

[0057] Repeat-dose toxicity studies were conducted in rodent (rat) species according to Good Laboratory Practice (GLP) principles. The aim of this study was to determine the toxicity of ML1.1 administered via IV bolus in Wistar Han IGS rats after 4 weeks. Furthermore, the toxicokinetics of ML1.1 were evaluated. Plasma pharmacokinetic (PK) assessments were included in the study. ADA was analyzed in rat plasma samples. Safety pharmacology was also assessed as part of the study.

[0058] The research conducted using ML1.1 is summarized in Table 1 below.

[0059] Table 1: Toxicological studies of ML1.1 Note: Tox = toxicology; PK = pharmacokinetics; IV = intravenous.

[0060] Male and female rats (n=10 per sex per group) were administered bolus injections at dose levels of 0, 1, 4, and 16 mg / kg (flow rate: 1 mL / min; volume: 8 mL / kg). Animals received ML1.1 three times during the first week, and once weekly thereafter (for a total of 7 administrations). Terminal sacrifice was performed on day 30 (one day after the last administration). Blood samples for TK assessment were collected from satellite group rats (n=6 per sex per group) on days 1 and 29, up to a maximum of 8 hours.

[0061] Toxicity assessment is based on mortality, clinical observation, body weight, weight gain, clinicopathological parameters (hematology, coagulation, clinical chemistry), organ weight, behavioral changes, and gross and microscopic examinations.

[0062] Systemic exposure to ML1.1 was confirmed on days 1 and 29. Overall, no adverse pathological findings were observed, and no ML1.1-related target organ effects were observed. In particular, there were no premature deaths, no ML1.1-related ophthalmological findings at the end of the 4-week dosing period, and no ML1.1-related changes in hematology, clinical chemistry, or coagulation parameters. Furthermore, no ML1.1-related clinical signs, gross findings, or organ weight changes were observed at any dose level.

[0063] In summary, ML1.1 administered via IV (bolus) at doses of 1, 4, and 16 mg / kg for 4 weeks (3 times in the first week, then once weekly for a total of 7 doses) was well tolerated in Wistar Han IGS rats. No target organ effects were observed. IV administration was well tolerated locally. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 16 mg / kg. At the NOAEL, the mean C0 and AUC in males were [not specified] after the first dose on day 1. tlast The values ​​were 263 µg / mL and 52.8 µg×h / mL for females and 170 µg / mL and 35 µg×h / mL for males.

[0064] Example 6 - Tail Clamp Bleeding Experiment Methodology The ML1.1 used in this embodiment was prepared as described in Example 1, except that it was recombinantly expressed in Pichia pastoris cells. For this embodiment, ML1.1 was prepared into a solution for parenteral application in an aqueous buffer (pH 5.0). The concentration of ML1.1 in the formulation was 2 mg / mL, or as otherwise specified in the experiments below. This formulation is referred to as "TGD001" in the corresponding figures.

[0065] Tail clamp hemorrhage experiments were conducted in adult wild-type mice (at least n=3 per group) at the animal facility of KU Leuven (Kortrijk, Belgium). Mice were anesthetized by an intraperitoneal injection of 10 mg / kg toluidine + 100 mg / kg ketamine. A 5 mm segment was surgically removed from the distal end of the tail. The tail was immediately immersed in 0.9% NaCl at 37°C. Hemorrhage cessation was monitored for up to 600 seconds.

[0066] The effects of several variables were assessed in five experiments; see Table 1 for an overview of the experimental setup. In Experiment 1, a tail clip model was validated using recombinant human tissue plasminogen activator (rh-tPA; alteplase). Mice received an infusion of 1 or 2 mg / kg alteplase (10% bolus followed by 90% infusion over 40 minutes). Tail clips were applied 20 or 40 minutes after the start of treatment. In Experiment 2, ML 1.1 (2.0 mg / kg) was administered as a 10% bolus followed by an infusion over 40 minutes (the remaining 90%). Tail clip experiments were performed 20 minutes (during infusion) and 40 minutes (after infusion) after the start of treatment. Experiment 3 assessed the bolus administration of ML 1.1 dose ranges. Mice received bolus injections of ML 1.1 (0.25, 1, and 2 mg / kg), with tail clips applied immediately after treatment. Experiment 4 investigated the effect of the spontaneous activity of ML 1.1 on bleeding time in the tail clip model. Mice received bolus injections (2 mg / kg) of either ML 1.1 samples with high levels of spontaneous intrinsic activity resulting from downstream processing alterations (freeze-thaw in PBS) or standard ML 1.1 (batch) with <10% spontaneous activity. Experiment 5 investigated the effect of tranexamic acid (TXA) (a commercially available antifibrinolytic drug) on ​​the thrombolytic activity of ML1.1. Mice received bolus injections of ML 1.1 (2 mg / kg), with TXA infusion initiated immediately after ML 1.1 administration.

[0067] Table 2: Experimental setup for each experiment Note: rh-tPA = recombinant human tissue plasminogen activator; TXA = tranexamic acid; *: TXA infusion begins 15 minutes before the ML1.1 bolus / tail clip. Infusion continues during the bleeding period, up to 10 minutes, after which the mice are euthanized.

[0068] Results and Discussion Experiment 1: Alteplase Infusion To characterize the tail clip model, the efficacy of alteplase (recombinant human tPA; rh-tPA) treatment was first tested. Alteplase has a very short half-life (6 minutes in humans), thus requiring a 10% bolus and a 90% infusion to generate sufficient exposure to initiate thrombolysis. Therefore, mice were infused with 1 or 2 mg / kg alteplase and compared to the solvent (0 mg / kg). For the solvent group, mice showed normal bleeding times, with a mean of 77.2 (standard deviation: 26.8) seconds, but when treated with alteplase, mice showed a maximum bleeding time of 600 seconds at 20 minutes after the start of infusion (i.e., mid-infusion). Interestingly, when the experiment was repeated but tail clipping was initiated at the end of the infusion (40 minutes), 1 mg / kg alteplase showed a reduced increase in bleeding time (no significant difference from the solvent group), while 2 mg / kg still showed a maximum bleeding time of 600 seconds. Figure 5 These data confirm that the tail clip test is sensitive to the thrombolytic activity of plasminogen activator. Recombinant human tissue plasminogen activator has previously been shown to prolong bleeding time in the tail clip test (Danielyan et al., 2008; Jankun et al., 2010). It should be noted that alteplase was used at a dose of 10 mg / kg in our mouse efficacy model and did not induce a tendency for spontaneous bleeding. Therefore, the tail clip model does not necessarily predict the risk of spontaneous bleeding in vivo.

[0069] Experiment 2: ML 1.1 Infusion In subsequent experiments, following the same infusion strategy as alteplase, the effect of ML 1.1 was tested at 2 mg / kg in a tail clip model for comparison. Similar to alteplase, at the 20-minute timepoint (mid-infusion; P < 0.0001), Figure 6 An increase in bleeding time was observed with ML 1.1. Interestingly, compared to tPA, ML 1.1 did not increase bleeding time to the same extent as alteplase. Figure 6 Furthermore, as observed with alteplase, when the tail clamp was applied at the end of the infusion (40 minutes), the bleeding time was shorter and there was no significant difference compared to the solvent group. Figure 6 This indicates that the thrombolytic activity of ML 1.1 is rapidly initiated after the start of infusion and decreases within 40 minutes.

[0070] Experiment 3: Injection of ML 1.1 The effect of bolus application on the tail clip model was also tested, with tail clipping initiated immediately after bolus injection.

[0071] In mice treated with up to 1 mg / kg, no increase in bleeding time was observed; however, at 2 mg / kg, bleeding time increased to a maximum of 600 seconds. Figure 7This indicates that at the dose at which ML 1.1 demonstrates efficacy, the bleeding potential in response to major trauma is not affected.

[0072] It is believed that the prolonged bleeding time in the tail clip test does not indicate spontaneous bleeding events in the body, but rather that the expected thrombolytic activity disrupts the thrombus formation required for hemostasis.

[0073] Experiment 4: Spontaneous activity of ML 1.1 ML 1.1 is produced in proenzyme form, exhibiting very limited enzymatic activity. This limited activity is only sufficient to induce initial plasmin formation when ML 1.1 binds to its target (VWF) and plasminogen. Plasmin can then activate ML 1.1, thereby achieving maximal local plasmin formation. Therefore, a high proenzyme concentration of ML 1.1 is crucial for limiting progression to systemic activation, as is typically seen with fully activated urokinase.

[0074] To investigate the differences between proenzyme-1 or activated ML 1.1, a batch of ML 1.1 (prepared in PBS) was frozen and thawed to initiate self-activation and compared in a tail clip model with a standard batch of ML 1.1 having high levels of proenzyme-1.1.

[0075] A bolus injection of ML 1.1 with high levels of spontaneous or intrinsic activity at 1 mg / kg caused prolonged bleeding time. Figure 8 Standard ML 1.1 (with less than 10% spontaneous activity (proenzyme state)) prolonged bleeding time at 2 mg / kg instead of 1 mg / kg. These data suggest that activated ML1.1 has higher thrombolytic activity compared to its proenzyme form. Therefore, it may be more likely to cause potential bleeding problems.

[0076] Experiment 5: ML 1.1 and TXA While thrombolytic agents such as rh-tPA have shown clinical value, their potential to induce bleeding is a concern for clinicians. Although no bleeding tendency was observed in all of our preclinical models (including a GLP toxicology study in rats at doses up to 16 mg / kg ML 1.1), intervention strategies may be valuable.

[0077] Patients with hyperfibrinolysis exhibit a bleeding tendency. Here, the plasmin generated overrides its natural inhibitors, allowing persistent and systemic plasmin activity. To limit plasminogen / plasmin activity in these patients, treatment with lysine analogs such as tranexamic acid (TXA) is employed. Both plasminogen and plasmin bind to their targets, such as fibrin and VWF, in a lysine-dependent manner. Lysine analogs competitively bind to the natural targets of plasmin(pro), thereby preventing plasminogen from becoming activated or plasmin from interacting with its targets. Since ML 1.1 functions via plasmin formation, TXA can also be considered a potential reversal agent.

[0078] To test the efficacy of TXA, mice were immediately treated with TXA infusion following a 2 mg / kg ML 1.1 bolus injection. In the absence of TXA, mice exhibited bleeding times exceeding 600 seconds, while in the presence of TXA, the bleeding time returned to normal. Figure 9 This indicates that TXA effectively limits the activity of ML 1.1 by interfering with the formation of plasmin.

[0079] in conclusion In the tail clip experiment, thrombolytic activity sufficient to disrupt thrombus formation was evident in a concentration-dependent manner after treatment with alteplase (infusion) and ML 1.1 (both bolus and infusion). For ML 1.1, thrombolytic activity affected bleeding time within minutes after bolus administration and decreased within 40 minutes after infusion. Higher levels of spontaneous intrinsic activity increased the thrombolytic activity of ML 1.1, while TXA infusion silenced the thrombolytic effect of ML 1.1.

[0080] Interestingly, infusion data showed that the bleeding time at 2 mg / kg ML 1.1 was shorter than that at 1 mg / kg alteplase. It is believed that the prolonged bleeding time observed in the tail clip experiment is not equivalent to spontaneous bleeding events in vivo, but rather that the anticipated thrombolytic activity disrupted the thrombus formation required for hemostasis.

Claims

1. A polypeptide comprising a urokinase catalytic domain, wherein an arginine residue corresponding to position 153 of SEQ ID No. 1 is replaced by a glutamine residue.

2. The polypeptide according to claim 1, wherein the urokinase catalytic domain further comprises a substitution of the asparagine residue corresponding to position 299 in SEQ ID No.

1.

3. The polypeptide according to claim 2, wherein the asparagine residue corresponding to position 299 in SEQ ID No. 1 is replaced by a glutamine residue.

4. The polypeptide according to any one of claims 1-3, wherein the urokinase catalytic domain further comprises a substitution of the lysine residue corresponding to position 297 in SEQ ID No.

1.

5. The polypeptide according to claim 4, wherein the lysine residue corresponding to position 297 in SEQ ID No. 1 is replaced by histidine.

6. The polypeptide according to any one of claims 1-5, comprising the following amino acid sequence: , where X represents K or H, and Z represents N or Q.

7. The urokinase catalytic domain according to any one of claims 1-6, which can promote the dissolution of microthrombi.

8. A hybrid protein comprising a polypeptide comprising a urokinase catalytic domain according to any one of claims 1-7, and further comprising VHH.

9. The hybrid protein of claim 8, wherein the variable domain of the VHH comprises complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3.

10. The hybrid protein of claim 9, wherein the complementarity-determining region 3 (CDR3) comprises at least one of the amino acid sequences SEQ ID NO: 6-28.

11. The hybrid protein of claim 9, wherein complementarity-determining region 1 (CDR1) comprises at least one of the amino acid sequences SEQ ID NO: 29-51.

12. The hybrid protein of claim 9, wherein the complementarity-determining region 2 (CDR2) comprises at least one of the amino acid sequences SEQ ID NO: 52-74.

13. The hybrid protein of claim 9, wherein the CDR1, CDR2, and CDR3 of the VHH variable domain comprise sequences selected from the following: SEQ ID NO: 6, 29, 52; SEQ ID NO: 7, 30, 53; SEQ ID NO: 8, 31, 54; SEQ ID NO: 9, 32, 55; SEQ ID NO: 10, 33, 56; SEQ ID NO: 11, 34, 57; SEQ ID NO: 12, 35, 58; SEQ ID NO: 13, 36, 59; SEQ ID NO: 14, 37, 60; SEQ ID NO: 15, 38, 61; SEQ ID NO: 16, 39, 62; SEQ ID NO: 17, 40, 63; SEQ ID NO: 18, 41, 64; SEQ ID NO: 19, 42, 65; SEQ ID NO: 20, 43, 66; SEQ ID NO: SEQ ID NO: 22, 45, 68; SEQ ID NO: 23, 46, 69; SEQ ID NO: 24, 47, 70; SEQ ID NO: 26, 48, 71; SEQ ID NO: 27, 49, 72; SEQ ID NO: 27, 50, 73; SEQ ID NO: 28, 51, 74.

14. The hybrid protein of claim 9, wherein the variable domain comprises or is composed of sequences selected from or consisting of SEQ ID NO: 75-SEQ ID NO:

101.

15. The hybrid protein according to any one of claims 8-14, wherein the variable domain has binding specificity for von Willebrand factor.

16. The hybrid protein according to any one of claims 8-14, wherein at least a portion of the coding sequence is codon-optimized.

17. The hybrid protein of claim 16, wherein the codon optimization comprises humanization and / or deimmunization.

18. The heterozygous protein according to claims 16-17, wherein the humanization and / or deimmunization results in one or more of the following mutations at the position corresponding to SEQ ID No. 1: A14P, E44G, A45P, E47G, K75R, K87R, P88A.

19. The hybrid protein according to any one of claims 8-18, wherein it is a chemically linked protein.

20. The hybrid protein according to any one of claims 8-18, wherein it is a fusion protein.

21. The hybrid protein according to any one of claims 8-20, further comprising a linker sequence connecting the polypeptide comprising the urokinase catalytic domain to the VHH.

22. The hybrid protein of claim 21, wherein the adapter sequence is a Gly / Ser-adaptor, and preferably the adapter sequence comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n.

23. The hybrid protein according to any one of claims 8-22, wherein the hybrid protein comprises, in order from N- to C-terminus: a. VHH as defined in claims 8-18; b. Optionally, the connector sequence as defined in claim 21; and c. A polypeptide comprising a urokinase catalytic domain as defined in any one of claims 1-7.

24. The hybrid protein according to any one of claims 8-23, wherein the amino acid sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

25. The hybrid protein according to any one of claims 8-24, for the prevention or treatment of microvascular thrombosis.

26. The hybrid protein for the use described in claim 25, which has binding specificity to von Willebrand factor.

27. A method for treating microvascular thrombosis or reducing the risk of microvascular thrombosis, wherein the method comprises administering an effective amount of the heterozygous protein according to any one of claims 8-24 to a subject in need of such treatment.

28. The method of claim 27, wherein the heterozygous protein is administered at a dose of 1 to 32 mg.

29. An isolated nucleic acid molecule encoding a heterozygous protein as defined in any one of claims 8-24.

30. The nucleic acid molecule according to claim 29 further comprises a promoter sequence.

31. The nucleic acid molecule according to claim 31, wherein the promoter sequence is an inducible promoter sequence.

32. The nucleic acid molecule according to claim 32, wherein the inducible promoter sequence is an alcohol oxidase I (AOX1) promoter sequence.

33. A pharmaceutical composition comprising a heterozygous protein according to any one of claims 8-24, and further comprising one or more pharmaceutically acceptable excipients.

34. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition is formulated as a liquid formulation or a lyophilized formulation.

35. A kit or aliquot kit comprising a heterozygous protein as defined in any one of claims 8-26 or a pharmaceutical composition according to claim 33 or 34, further comprising one or more of the following: a) Tranexamic acid (TXA); b) Plasmin inhibitors (PIs); c) Anti-urokinase antibody.

36. The heterozygous protein as defined in any one of claims 8-26 or the pharmaceutical composition according to claim 33 or 34, for use in combination with one or more of the following to prevent or treat microvascular thrombosis: a) Tranexamic acid (TXA); b) Plasmin inhibitors (PIs); c) Anti-urokinase antibody.

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