Antithrombotic agent

Abstract

The present invention addresses the problem of providing a substance which has an antithrombotic effect with a low risk of occurrence of a hemorrhagic complication that is commonly caused when an anticoagulant is used.　In this antithrombotic agent, a polypeptide or the like which contains a β2GPI domain V is used as an active ingredient, wherein the β2GPI domain V comprises an amino acid sequence which has a proline residue located at position-46 and a glutamic acid residue located at position-76 in the amino acid sequence represented by SEQ ID NO: 1 and has at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1.

Description

antithrombotic agents

[0001] The present invention relates to a polypeptide (hereinafter sometimes referred to as the "present D5-PE polypeptide") comprising a β2GPI domain V (hereinafter sometimes referred to as the "present β2GPI domain V") consisting of an amino acid sequence having a proline residue at position 46 and a glutamic acid residue at position 76 of the amino acid sequence shown in SEQ ID NO: 1 and having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; or a polynucleotide (hereinafter sometimes referred to as the "present D5-PE polynucleotide") comprising a polynucleotide encoding the present D5-PE polypeptide; as an active ingredient; a preventive or therapeutic agent for thromboembolic diseases, which contains the present D5-PE polypeptide or the present D5-PE polynucleotide as an active ingredient; a polypeptide comprising the present β2GPI domain V and the Fc region of immunoglobulin (hereinafter, sometimes referred to as the "present β2GPI domain V / Fc region polypeptide"); and a polynucleotide comprising a polynucleotide encoding the present β2GPI domain V / Fc region polypeptide (hereinafter, sometimes referred to as the "present β2GPI domain V / Fc region polynucleotide").

[0002] Thromboembolic disease, along with cancer, is one of the leading causes of death in adults. Thromboembolic disease occurs when a thrombus forms at the site of vascular injury, or when a formed thrombus breaks off and travels through the bloodstream to another blood vessel, where it occludes that vessel.

[0003] Antiphospholipid antibody syndrome (hereinafter sometimes referred to as "APS") is a systemic autoimmune disease that can cause thrombosis and pregnancy complications, and the number of APS patients in Japan is estimated to be approximately 20,000. APS is thought to develop when antiphospholipid antibodies (e.g., anti-β2GPI antibodies, anticardiolipin antibodies, lupus anticoagulants) whose primary antigen is β2-glycoprotein I (β2GPI) bound to negatively charged membrane phospholipids expressed on damaged cells activate vascular endothelium and platelets. The most common symptom of APS is cerebral infarction, followed by lower limb venous thrombosis and associated pulmonary embolism. Once APS develops, it often leads to irreversible multiple organ damage. Anticoagulant therapy and antiplatelet therapy using anticoagulants such as heparin and warfarin are known as palliative treatments. However, the recurrence rate of thrombosis after these therapies is estimated at 17% over a five-year period, making their effectiveness insufficient. Furthermore, bleeding complications due to nonspecific inhibition of blood coagulation reactions are also problematic. Direct oral anticoagulants (DOACs), which have fewer bleeding complications, were expected to be clinically applicable to APS. However, because their action site is downstream of the coagulation cascade, they have been ineffective, at least for thrombi in high-risk APS. Furthermore, self-injections of low-dose aspirin or heparin are used to treat pregnancy complications such as recurrent miscarriage due to APS. However, daily morning and evening self-injections of heparin pose a significant burden for pregnant women. APS is also known to be associated with a condition called catastrophic antiphospholipid syndrome (CAPS), which rapidly develops into thromboembolism in multiple organs. CAPS is an intractable condition with a high mortality rate and a high rate of residual organ damage, but there is currently no specific treatment. Furthermore, the mechanism of CAPS is thought to be thrombotic microangiopathy (TMA) following vascular endothelial damage.

[0004] On the other hand, it has been reported that components derived from barley seeds have antithrombotic effects (Patent Document 1). It has also been reported that chondroitin sulfate oligosaccharides have the effect of inhibiting platelet adhesion and platelet aggregation, and are useful for improving thrombosis (Patent Document 2). It has also been reported that suplatast tosilate has excellent antithrombotic effects (Patent Document 3). It has also been reported that human granulocyte colony-stimulating factor has antithrombotic effects (Patent Document 4). However, it has not been known until now that the present D5-PE polypeptide has antithrombotic effects.

[0005] Japanese Patent Application Laid-Open No. 2023-067102 Japanese Patent Application Laid-Open No. 2022-187126 International Publication No. 2011 / 065444 Pamphlet Japanese Patent Application Laid-Open No. 2-149527

[0006] An object of the present invention is to provide a substance having an antithrombotic effect with a low risk of bleeding complications that occur when anticoagulants are used.

[0007] Human (h)β2GPI loses its phospholipid-binding ability through plasmin-dependent limited proteolysis of its domain V. However, it is known that plasmin resistance (stable form) can be achieved by modifying two amino acid residues (K287 and K317) in domain V to K287P and K317E, respectively (see Shen L, et al. Best Pract Res Clin Rheumatol. 2018;32:572-590). Meanwhile, the epitope of antiphospholipid antibodies is thought to be primarily domain I of hβ2GPI (see de Laat B, et al. J Thromb Haemost. 2009;7:1767-1773).

[0008] Therefore, the present inventors, in the course of intensive research to solve the above-mentioned problems, have concluded that domain V of hβ2GPI (hereinafter sometimes referred to as "D5-PE polypeptide"; see Table 1) containing two amino acid substitution mutations (K287P and K317E) is resistant to degradation by plasmin and lacks domain I, and therefore may be effective in treating APS and, by extension, other thromboembolic diseases. Therefore, first, a recombinant polypeptide containing D5-PE polypeptide was produced and purified, and it was confirmed that the presence of the two amino acid substitution mutations in the D5-PE polypeptide improves polypeptide stability and specificity (binding ability) for phospholipids. Furthermore, it was found that administration of purified recombinant polypeptide containing D5-PE polypeptide to two types of thromboembolic disease model mice (SARS-CoV-2-infected NZW×BXSB F1 mice and economy class syndrome model mice) effectively suppressed thrombus formation. Furthermore, it has been confirmed that, unlike heparin and low molecular weight heparin (dalteparin), recombinant polypeptides containing D5-PE polypeptides do not prolong activated clotting time (hereinafter sometimes referred to as "ACT") or bleeding time, and do not adversely affect blood coagulation. The present invention has been completed based on these findings.

[0009] That is, the present invention is as follows: [1] An antithrombotic agent comprising a polypeptide comprising domain V of β2GPI, or a polynucleotide comprising a polynucleotide encoding the polypeptide, wherein domain V of β2GPI comprises a proline residue at position 46 and a glutamic acid residue at position 76 of the amino acid sequence shown in SEQ ID NO: 1, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. [2] The antithrombotic agent according to [1] above, wherein the polypeptide does not comprise domain I of β2GPI. [3] The antithrombotic agent according to [1] or [2] above, wherein the polypeptide comprises an Fc region of immunoglobulin. [4] The antithrombotic agent according to [3] above, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. [5] An agent for preventing or treating a thromboembolic disease, comprising the antithrombotic agent according to any of [1] to [4] above. [6] A polypeptide comprising domain V of β2GPI and an immunoglobulin Fc region, wherein domain V of β2GPI comprises a proline residue at position 46 and a glutamic acid residue at position 76 of the amino acid sequence shown in SEQ ID NO: 1, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. [7] The polypeptide according to [6] above, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. [8] A polynucleotide comprising a polynucleotide encoding the polypeptide according to [6] or [7] above.

[0010] Other embodiments of the present invention include: a method for inhibiting or preventing thrombus formation, which comprises administering the present D5-PE polypeptide or the present D5-PE polynucleotide (hereinafter, "the present D5-PE polypeptide" and "the present D5-PE polynucleotide" may be collectively referred to as "the present D5-PE polypeptide, etc.") to a subject in need of such inhibition or prevention; a method for preventing or treating a thromboembolic disease, which comprises administering the present D5-PE polypeptide, etc. to a subject in need of such prevention or treatment; the present D5-PE polypeptide, etc. for use in the inhibition or prevention of thrombus formation; the present D5-PE polypeptide, etc. for use in the prevention or treatment of thromboembolic disease; use of the present D5-PE polypeptide, etc. for the manufacture of an agent for inhibiting or preventing thrombus formation; and use of the present D5-PE polypeptide, etc. for the manufacture of an agent for the prevention or treatment of thromboembolic disease.

[0011] Since the present β2GPI domain V exhibits excellent antithrombotic effects without adversely affecting blood coagulation, the present D5-PE polypeptide, etc. (i.e., a polypeptide comprising the present β2GPI domain V, or a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the present β2GPI domain V) is useful as a preventive or therapeutic agent for thromboembolic diseases with a low risk of bleeding complications that occur when anticoagulants are used.

[0012] The left figure shows the results of immunoblotting using an anti-hβ2GPI antibody on a sample containing full-length hβ2GPI ("Fullβ2GPI" in the figure). The right figure shows the results of immunoblotting using an anti-human IgG antibody on samples containing four types of polypeptides (D5-PE / hIgG1-Fc polypeptide, D5-KK / hIgG1-Fc polypeptide, full-length hβ2GPI, or hIgG1-Fc polypeptide) ("D5-PE-IgG1," "D5-KK-IgG1," "Fullβ2GPI," and "Cont-hIgG1" in the figure, respectively). The arrowheads in the figure indicate bands of the expected size, and * indicates bands expected to be degradation products. The figures show the results of analyzing the binding ability of the above four polypeptides to cardiolipin by ELISA (enzyme-linked immunosorbent assay) using HRP-anti-human IgG antibody (Figure 2A) or HRP-anti-hβ2GPI antibody (which does not recognize domain V) (Figure 2B). Figures 3A to 3D show microscopic images of MSB (Martius-Scarlet-Blue)-stained kidney tissues from a SARS-CoV-2-uninfected NZW×BXSB F1 mouse (Figure 3A), a SARS-CoV-2-infected NZW×BXSB F1 mouse (Figure 3B), a SARS-CoV-2-infected NZW×BXSB F1 mouse administered with hIgG1-Fc polypeptide (Figure 3C), and a SARS-CoV-2-infected NZW×BXSB F1 mouse administered with D5-PE / hIgG1-Fc polypeptide (Figure 3D), respectively, on day 10 of infection. Arrows in the images indicate formed thrombi. Figure 3E shows microscopic images of MSB-stained kidney tissues from a SARS-CoV-2-infected NZW×BXSB F1 mouse administered with heparin on day 3 of infection. Figure 3F shows the results of analyzing the ratio of thrombi to renal glomeruli in SARS-CoV-2-infected NZW×BXSB F1 mice administered with hIgG1-Fc polypeptide ("Cont-hIgG1" in the figure) and SARS-CoV-2-infected NZW×BXSB F1 mice administered with D5-PE / hIgG1-Fc polypeptide ("D5-PE-IgG1" in the figure), based on the results of Figures 3C and 3D. "*" in the figure indicates a statistically significant difference (p<0.05).This figure shows the results of measuring the weight of thrombi formed in economy class syndrome model mice administered PBS ("Vehicle" in the figure) or 1 mg / mL D5-PE / hIgG1-Fc polypeptide ("D5-PE-IgG1" in the figure). "**" in the figure indicates a statistically significant difference (p<0.01). This figure shows the results of measuring the ACT when four types of test solutions (PBS ["Cont" in the figure], heparin-containing solution ["Heparin" in the figure], D5-PE / hIgG1-Fc polypeptide-containing solution ["D5-PE-IgG1" in the figure], and hIgG1-Fc polypeptide-containing solution ["Cont-hIgG1" in the figure]) were added to peripheral blood from healthy individuals. In the figure, "***" indicates a statistically significant difference (p<0.001), and "ns" indicates no statistically significant difference (p≧0.05). This figure shows the results of measuring bleeding time in mice administered with three test substances (hIgG1-Fc polypeptide ["Cont-hIgG1" in the figure], D5-PE / hIgG1-Fc polypeptide ["D5-PE-IgG1" in the figure], and dalteparin ["Dalteparin" in the figure]). In the figure, "*" and "**" indicate a statistically significant difference (p<0.05 and p<0.01), respectively, and "ns" indicates no statistically significant difference (p≧0.05).

[0013] The antithrombotic agent of the present invention is a formulation containing the present D5-PE polypeptide and / or the present D5-PE polynucleotide, specified for use as an "antithrombotic agent" (hereinafter, sometimes referred to as the "present antithrombotic agent"). Furthermore, the preventive or therapeutic agent for thromboembolic disease of the present invention is a formulation containing the present D5-PE polypeptide and / or the present D5-PE polynucleotide, specified for use as an "antithrombotic agent" and for use as "for preventing or treating thromboembolic disease" (hereinafter, sometimes referred to as the "present preventive / therapeutic agent"). Hereinafter, the "present antithrombotic agent" and the "present preventive / therapeutic agent" will be collectively referred to as the "present agent"). The present agent may be used alone as a pharmaceutical (preparation), or may be used in the form of a composition (pharmaceutical composition) by further mixing with additives. Examples of such additives include, for example, pharmaceutically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, isotonic agents, coating agents, solubilizers, dissolution aids, and other compounding components.

[0014] As used herein, "antithrombotic" refers to inhibiting or preventing the formation of thrombi (blood clots) in mammalian subjects. When a blood vessel is clogged with a thrombus, blood flow beyond that point is significantly impaired, causing thromboembolic diseases. For example, when a thrombus clogs a blood vessel in the heart, brain, or lungs, it can cause myocardial infarction, cerebral infarction, pulmonary embolism, etc., which can be life-threatening.

[0015] As used herein, "thromboembolic disease" refers to thrombosis or its complication, thromboembolism. Here, "thrombosis" refers to a disease in which a thrombus formed due to some cause (e.g., antiphospholipid syndrome, cancer, or other disease; obesity; surgery; trauma; etc.) blocks a blood vessel, causing organ damage due to peripheral circulatory failure. Furthermore, "thromboembolism" refers to a disease in which a formed thrombus is carried away by the bloodstream and blocks a blood vessel at a site other than the site of thrombus formation, causing organ damage.

[0016] Examples of the thromboembolic diseases include thrombotic microangiopathy (TMA), arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, arterial cerebrovascular thromboembolic disorders, venous cerebrovascular thromboembolic disorders, and thromboembolic disorders in the cardiac cavity or peripheral circulation.

[0017] As used herein, "thrombotic microangiopathy (TMA)" refers to a collective term for a disease characterized by the following three characteristics: 1) the formation of platelet clumps within small blood vessels (intracapillary platelet thrombus), 2) the destruction and reduction of platelets (destructive thrombocytopenia), and 3) the subsequent destruction of red blood cells, resulting in anemia (microangiopathic hemolytic anemia). TMA is the pathological condition of thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), but is also known to develop following various diseases, such as antiphospholipid syndrome, systemic lupus erythematosus (SLE), and complement deficiency.

[0018] Examples of the arterial cardiovascular thromboembolic disorder include coronary artery disease, ischemic cardiomyopathy, acute coronary syndrome, coronary artery thrombosis, ischemic complications of unstable angina or non-Q wave myocardial infarction, angina pectoris (variant angina pectoris, unstable angina pectoris, etc.), myocardial infarction (primary myocardial infarction, recurrent myocardial infarction, acute myocardial infarction, etc.), vascular reocclusion or stenosis (e.g., vascular reocclusion or stenosis after surgery such as coronary artery bypass surgery or percutaneous transluminal angioplasty), sudden ischemic death, etc.

[0019] Examples of the above-mentioned venous cardiovascular thromboembolic diseases include deep vein thrombosis or pulmonary embolism in surgeries such as abdominal surgery, total hip replacement surgery, knee joint replacement surgery, and hip fracture surgery, as well as in multiple fractures, multiple trauma, trauma, spinal cord injury, and burns.

[0020] Examples of the arterial cerebrovascular thromboembolic disorder include stroke (stroke in patients with non-valvular atrial fibrillation or valvular atrial fibrillation, ischemic stroke, etc.), cerebral artery thrombosis, cerebral infarction (atherothrombotic cerebral infarction, asymptomatic cerebral infarction, etc.), transient ischemic attack (TIA), lacunar infarction, cerebral artery embolism, cerebral thrombosis, cerebrovascular disorder, cerebrovascular dementia, etc.

[0021] Examples of the venous cerebrovascular thromboembolic disorders include intracranial venous thrombosis, cerebral embolism, cerebral thrombosis, cerebral venous sinus thrombosis, intracranial venous sinus thrombosis, and cavernous sinus thrombosis.

[0022] Examples of the thromboembolic diseases in the cardiac chamber or peripheral circulation include venous thrombosis (lower limb venous thrombosis, thrombophlebitis, retinal vein thrombosis, thrombotic pulmonary hypertension, deep vein thrombosis, postoperative deep vein thrombosis, pulmonary embolism, economy class syndrome, etc.), systemic venous thromboembolism, recurrent venous thromboembolism, thrombophlebitis, non-valvular or valvular atrial fibrillation, cardiogenic embolism, disseminated intravascular coagulation (DIC), superior mesenteric artery occlusion, portal vein thrombosis, retinal vein occlusion, atherosclerosis, atherothrombosis, peripheral arterial occlusive disease (PAOD), peripheral arterial disease, and arterial embolism.

[0023] As used herein, "having a proline residue at position 46 in the amino acid sequence shown in SEQ ID NO: 1" means having an amino acid residue corresponding to a mutation (K287P) in which the lysine residue (K) at position 287 in full-length hβ2GPI (the amino acid sequence shown in SEQ ID NO: 2) is replaced with a proline residue (P). Furthermore, as used herein, "having a glutamic acid residue at position 76 in the amino acid sequence shown in SEQ ID NO: 1" means having an amino acid residue corresponding to a mutation (K317E) in which the lysine residue (K) at position 317 in full-length hβ2GPI (the amino acid sequence shown in SEQ ID NO: 2) is replaced with a glutamic acid residue (E). Having an amino acid residue corresponding to K287P and an amino acid residue corresponding to K317E in β2GPI domain V improves the stability and specificity for phospholipids of β2GPI domain V, as demonstrated in the Examples described below. Whether or not a protein has an amino acid residue corresponding to K287P or K317E can be determined by performing alignment analysis with the amino acid sequence of SEQ ID NO: 2 and / or the amino acid sequence of SEQ ID NO: 1.

[0024] The present β2GPI domain V is not particularly limited as long as it is a polypeptide having an amino acid sequence that contains amino acid residues corresponding to K287P and K317E and that has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. The present β2GPI domain V includes human β2GPI domain V having two amino acid substitution mutations (K287P and K317E) (a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 having K287P and K317E), or a variant, homolog, or ortholog thereof that has a biological function (specifically, phospholipid-binding ability). Such variants, homologs, or orthologs may be derived from humans or non-human mammals.

[0025] The D5-PE polypeptide of the present invention is not particularly limited as long as it contains the β2GPI domain V, and the β2GPI domain V / Fc region polypeptide is not particularly limited as long as it contains the β2GPI domain V and an Fc region of an immunoglobulin. When administered to patients with APS, these polypeptides preferably do not contain β2GPI domain I, and more preferably do not contain β2GPI domain I and one or more domains selected from β2GPI domains II, III, and IV (preferably all of β2GPI domains II to IV). Specific examples of β2GPI domain I include polypeptides consisting of an amino acid sequence that shares at least 80% sequence identity with amino acid residues 1 to 64 of the amino acid sequence of SEQ ID NO: 2. Specific examples of β2GPI domains II to IV include polypeptides consisting of an amino acid sequence that shares at least 80% sequence identity with amino acid residues 65 to 241 of the amino acid sequence of SEQ ID NO: 2.

[0026] From the viewpoints of convenient polypeptide purification and extending the blood half-life of the polypeptide, the present D5-PE polypeptide preferably contains a polypeptide tag (e.g., an immunoglobulin Fc region, GST [glutathione-S-transferase], a His tag, a FLAG tag, etc.). Since the effectiveness of this tag has been demonstrated in the Examples described below, a suitable example is one containing an immunoglobulin Fc region (i.e., the present β2GPI domain V / Fc region polypeptide). Furthermore, from the viewpoint of convenient polypeptide purification, the present D5-PE polypeptide may contain a signal peptide (e.g., within the range of 10 to 50 amino acid residues). Examples of such signal peptides include a CD8α-derived signal peptide and an IL2-derived signal peptide. The present β2GPI domain V and the polypeptide tag, or the present β2GPI domain V and the signal peptide, may be linked via a linker peptide (e.g., within the range of 3 to 10 amino acid residues).

[0027] As used herein, the term "immunoglobulin Fc region" refers to an Fc region (native Fc region) that is one of the two fragments (Fab and Fc regions) obtainable by treating an immunoglobulin with papain (a protease), or a variant of a native Fc region that maintains its function. The immunoglobulin Fc region may be an Fc region that has disulfide bonds (S-S bonds), or an Fc region that has been modified so as not to have S-S bonds. An example of an Fc region that has S-S bonds is a polypeptide that comprises an amino acid sequence that has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 6 and that has cysteine ​​(C) at position 4, C at position 10, and C at position 13 of SEQ ID NO: 6. Furthermore, examples of Fc regions modified to have no S-S bonds include polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, in which the 4th, 10th, and 13th amino acid residues of SEQ ID NO: 8 are amino acid residues other than C, where "amino acid residues other than C" are preferably S, since serine (S) does not form bonds such as S-S bonds and its amino acid properties and chemical properties are similar to those of C. Furthermore, from the viewpoint of improving the secondary structure and overall stability of the protein, preferred Fc regions modified to have no S-S bonds are polypeptides having S at position 22 of SEQ ID NO: 8.

[0028] Examples of the immunoglobulin include IgG (IgG1, IgG2), IgM, IgA, IgD, and IgE, and IgG (particularly IgG1) is a preferred example because its effects have been demonstrated in the Examples described below. The biological species from which the immunoglobulin is derived is not limited as long as it is a mammal, but humans are preferred.

[0029] Examples of the D5-PE polypeptide of the present invention that comprises an immunoglobulin Fc region (i.e., the β2GPI domain V / Fc region polypeptide) include, for example, a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 (preferably, a polypeptide having C at position 120, C at position 126, C at position 129, and P at position 138 of SEQ ID NO: 7), and a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 (preferably, a polypeptide having an amino acid residue other than C at position 120, 126, and 129 of SEQ ID NO: 9, more preferably, a polypeptide having S at position 120, S at position 126, S at position 129, and S at position 138 of SEQ ID NO: 9).

[0030] The D5-PE polypeptide of the present invention can be synthesized using chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method), or can be synthesized by conventional methods using various commercially available peptide synthesizers. Alternatively, the D5-PE polypeptide of the present invention can be obtained by introducing the D5-PE polynucleotide of the present invention in the form of a vector described below into a host cell line (e.g., a human-derived cell line) to obtain a transformed cell line, and then producing the D5-PE polypeptide in the culture supernatant of the transformed cell line.

[0031] The D5-PE polypeptide of the present invention produced in the culture supernatant of the above-mentioned transformed cell line can be isolated and purified by affinity purification using a protein that binds to the Fc region (e.g., protein A, protein G, etc.) if the D5-PE polypeptide contains an immunoglobulin Fc region (i.e., the β2GPI domain V / Fc region polypeptide); it can be isolated and purified by affinity purification using glutathione if the D5-PE polypeptide contains GST; it can be isolated and purified by affinity purification using a metal that binds to the His tag, such as cobalt or nickel, if the D5-PE polypeptide contains a His tag; and it can be isolated and purified by affinity purification using an anti-FLAG antibody if the D5-PE polypeptide contains a FLAG tag. The D5-PE polypeptide produced by the transformed cell line may be post-translationally modified within the cell, and post-translationally modified polypeptides are also included in the D5-PE polypeptide as long as they have antithrombotic activity. Examples of such post-translational modifications include N-terminal methionine removal, N-terminal acetylation, glycosylation, limited degradation by intracellular proteases, myristoylation, isoprenylation, and phosphorylation.

[0032] In this specification, examples of "mammals" include humans as well as non-human mammals (e.g., rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees), with humans being a preferred example.

[0033] The D5-PE polynucleotide is not particularly limited as long as it comprises a polynucleotide encoding a polypeptide comprising the β2GPI domain V of the present invention. Furthermore, the β2GPI domain V / Fc region polynucleotide is not particularly limited as long as it comprises a polynucleotide encoding a polypeptide comprising the β2GPI domain V and an immunoglobulin Fc region (i.e., a polynucleotide encompassed by the D5-PE polynucleotide of the present invention). Suitable examples of these polynucleotides include polynucleotides (vectors) in which a promoter is operably linked upstream of a polynucleotide encoding a polypeptide comprising the β2GPI domain V of the present invention or upstream of a polynucleotide encoding a polypeptide comprising the β2GPI domain V and an immunoglobulin Fc region. Such vectors are not particularly limited as long as they are capable of transcribing mRNA and can be selected appropriately depending on the purpose. Examples include non-viral vectors (e.g., episomal vectors, artificial chromosome vectors, and plasmid vectors) and viral vectors. Furthermore, the vectors may be circular or linear.

[0034] The promoter used in the above vector is not particularly limited, as long as it is a region to which RNA polymerase (preferably RNA polymerase and general transcription factor) binds and initiates transcription of a downstream polynucleotide encoding the present D5-PE polypeptide. Examples include the SRα promoter, SV40 early promoter, retroviral LTR, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoter, and heat shock promoter. Furthermore, an enhancer of the IE gene of human CMV may be used together with the promoter. For example, the CAG promoter (containing the cytomegalovirus enhancer, chicken β-actin promoter, and β-globin gene poly(A) signal region) can be used.

[0035] The episomal vector is a vector capable of autonomous replication outside of a chromosome. Specific means for using an episomal vector are disclosed in Yu et al., Science, 324, 797-801 (2009). In a particularly preferred embodiment of the present invention, an episomal vector can be used in which loxP sequences are arranged in the same orientation on the 5' and 3' sides of vector elements necessary for replication of the episomal vector. Because episomal vectors are capable of autonomous replication outside of a chromosome, they can provide stable expression in host cells even without being integrated into the genome.

[0036] Examples of the episomal vector include vectors containing, as vector elements, sequences necessary for autonomous replication derived from EBV, SV40, etc. Specific examples of the vector elements necessary for autonomous replication include an origin of replication and a gene encoding a protein that binds to the origin of replication and controls replication, such as the replication origin oriP and EBNA-1 gene for EBV, and the replication origin ori and SV40LT gene for SV40.

[0037] Examples of the artificial chromosome vector include a YAC (Yeast artificial chromosome) vector, a BAC (Bacterial artificial chromosome) vector, and a PAC (P1-derived artificial chromosome) vector.

[0038] Examples of the above-mentioned plasmid vector include pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0039] The viral vector refers to a gene vector that utilizes the infectivity and replication ability of a virus, and more specifically refers to a viral particle (also referred to as a "recombinant virus") that contains a viral vector plasmid (which may be DNA or RNA) into which a pathogenic gene has been removed from the viral genome and a foreign gene (in this case, a mammalian YAP polynucleotide) has been incorporated. Examples of the viral vector include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0040] The AAV vector, which is one embodiment of the present D5-PE polynucleotide, can be produced in the nucleus by transfecting, for example, HEK293 cells containing genes encoding AAV proteins required for AAV replication and AAV particle formation (rep genes, cap genes) and genes encoding proteins required for AAV proliferation (E2A genes, E4 genes, VARNA genes) with an AAV vector containing a polynucleotide encoding a polypeptide comprising the present β2GPI domain V, or an AAV vector containing a polynucleotide encoding a polypeptide comprising the present β2GPI domain V and an immunoglobulin Fc region. After freeze-thawing, the AAV vector can be recovered, separated, and purified from the nucleus by density gradient centrifugation using cesium chloride, a column method, or the like (see Li, XG et al. Mol Ther 13, 160-166 (2006); Matsushita, T. et al. Gene Ther 5: 938-945 (1998); Urabe, M. et al. Mol Ther 13: 823-828(2006)”).

[0041] In addition to the promoter, the vector may optionally contain an enhancer, a poly(A) addition signal, a marker gene, a replication origin, a gene encoding a polypeptide that binds to the replication origin and controls replication, and the like. The marker gene refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Specific examples of the marker gene include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination. Specific examples of the drug resistance gene include neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, and hygromycin resistance genes. Specific examples of the fluorescent protein gene include green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes. Specific examples of the luciferase gene include luciferase genes. Specific examples of the chromogenic enzyme gene include β-galactosidase genes, β-glucuronidase genes, and alkaline phosphatase genes.

[0042] The D5-PE polynucleotide may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA from the viewpoint of stability. Furthermore, the polynucleotide may be partially or entirely substituted with artificial nucleotides such as PNA (polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, bridged nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acid), or TNA (threose nucleic acid). The D5-PE polynucleotide may comprise a single-stranded (sense strand) polynucleotide encoding the D5-PE polypeptide, or a double-stranded polynucleotide consisting of the sense strand and its complementary antisense strand; however, a double-stranded polynucleotide is preferred.

[0043] The D5-PE polynucleotides can be readily prepared by conventional methods based on the amino acid sequence of the D5-PE polypeptide. A nucleotide sequence encoding the amino acid sequence can be obtained based on the amino acid sequence listed in the Sequence Listing, and the D5-PE polynucleotides can be prepared using standard molecular biological and / or chemical procedures. For example, polynucleotides can be synthesized based on the nucleotide sequence, and the D5-PE polynucleotides can be prepared by combining DNA fragments obtained from a cDNA library using the polymerase chain reaction (PCR).

[0044] As used herein, "at least 80% sequence identity with an amino acid sequence" means that the percentage of amino acids identical to the amino acid sequence being compared is 80% or more, preferably 85% or more, more preferably 87% or more, even more preferably 88% or more, even more preferably 89% or more, particularly preferably 90% or more, especially more preferably 93% or more, especially more preferably 96% or more, especially even more preferably 98% or more, and most preferably 99% or more sequence identity (e.g., 100% sequence identity). Amino acid sequence identity can be determined using known programs such as ClustalW, GENETYX, and BLAST.

[0045] As used herein, "at least 80% sequence identity to an amino acid sequence" means, in other words, an amino acid sequence in which zero, one, or several amino acid residues have been deleted, substituted, inserted, and / or added in the amino acid sequence being compared, and which has the same function as a polypeptide consisting of the amino acid sequence being compared. Here, "an amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted, and / or added" refers to an amino acid sequence in which, for example, 1 to 30 amino acid residues have been deleted, substituted, inserted, and / or added, preferably 1 to 20 amino acid residues, more preferably 1 to 15 amino acid residues, even more preferably 1 to 10 amino acid residues, even more preferably 1 to 5 amino acid residues, even more preferably 1 to 3 amino acid residues, and even more preferably 1 to 2 amino acid residues have been deleted, substituted, inserted, and / or added. Mutation of these amino acid residues can be carried out by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.

[0046] Of the agents, the antithrombotic agent is administered to subjects in need of inhibition or prevention of thrombus formation, and the prophylactic / therapeutic agent is administered to subjects in need of inhibition or prevention of thrombus formation and in need of prevention or treatment of thromboembolic disease. The agent may be administered to a subject by any method that allows the administered D5-PE polypeptide or the D5-PE polypeptide expressed from the administered D5-PE polynucleotide to reach the blood vessels, and examples of such methods include intravenous administration, intraperitoneal administration, and administration to a tissue or organ.

[0047] The dosage and administration period of the D5-PE polypeptide etc. contained in the agent of the present invention are determined appropriately depending on the age, body weight, sex, symptoms, drug sensitivity, etc. The dosage is, for example, in the range of 1 μg to 1000 mg / day, and the administration period is, for example, at least 1 to 10 days. The agent of the present invention may be administered once a day, or multiple times (for example, 2 to 4 times) as necessary.

[0048] The agent may contain a substance with antithrombotic activity in addition to the D5-PE polypeptide of the present invention, but since the D5-PE polypeptide of the present invention alone exhibits excellent antithrombotic activity, it is preferable that the agent does not contain any substance with antithrombotic activity (e.g., a compound, a polypeptide [e.g., an antibody], DNA, or RNA) in addition to the D5-PE polypeptide of the present invention.

[0049] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0050] [Isolation and Purification of D5-PE / hIgG1-Fc Polypeptide] First, the D5-PE polypeptide was expressed as a recombinant polypeptide and isolated and purified. Furthermore, to facilitate polypeptide purification and extend the serum half-life of the polypeptide, a D5-PE polypeptide was prepared to which hIgG1-Fc was added as a polypeptide tag. To prepare DNA encoding the D5-PE polypeptide, site-directed mutagenesis was used to introduce mutations (K287P and K317E) into a plasmid containing DNA encoding domain V of hβ2GPI (a polypeptide consisting of amino acid residues 242 to 326 of full-length hβ2GPI [see Table 2]) by substituting K287 (corresponding to the 287th lysine residue of full-length hβ2GPI) and K317 (corresponding to the 317th lysine residue of full-length hβ2GPI) with proline and glutamic acid, respectively.

[0051] To prepare a vector expressing a fusion protein of D5-PE polypeptide and hIgG1-Fc (sometimes referred to herein as "D5-PE / hIgG1-Fc polypeptide"), DNA encoding the prepared D5-PE polypeptide, DNA encoding the IL-2 signal peptide (see Table 3), DNA encoding linker 1 (see Table 3), DNA encoding linker 2 (see Table 3), and DNA encoding hIgG1-Fc (see Table 4) were cloned into the EcoRI and NheI restriction enzyme sites of the pFUSE-hIgG2-Fc2(IL2ss) vector (pfuse-hfc2, Invivogen) using an In-Fusion HD Cloning Kit (Clontech). As comparative controls, a vector expressing a fusion protein of hIgG1-Fc with domain V of hβ2GPI (sometimes referred to herein as "D5-KK polypeptide") without the above mutation (sometimes referred to herein as "D5-KK / hIgG1-Fc polypeptide"), a vector expressing full-length hβ2GPI, and a vector expressing linker 2 and hIgG1-Fc (sometimes referred to herein as "hIgG1-Fc polypeptide") were also prepared in the same manner. These constructed vectors were introduced into Expi293F cells (high-expressing Human Embryonic Kidney 293 cell line) using the Expi293 Expression System (Thermo Fisher Scientific), and four types of polypeptides (D5-PE / hIgG1-Fc polypeptide, D5-KK / hIgG1-Fc polypeptide, full-length hβ2GPI, and hIgG1-Fc polypeptide) were expressed in the culture supernatant. The culture supernatant was then collected, and the four polypeptides were isolated and purified by affinity purification using Protein A. The D5-PE / hIgG1-Fc polypeptide is a polypeptide consisting of, in order from the amino (N) terminus, an IL-2 signal peptide, linker 1, D5-PE polypeptide, linker 2, and hIgG1-Fc (see Table 5).The D5-KK / hIgG1-Fc polypeptide is a polypeptide consisting of, in order from the N-terminus, an IL-2 signal peptide, linker 1, D5-KK polypeptide, linker 2, and hIgG1-Fc.

[0052] Furthermore, the D5-PE / hIgG1-Fc (dissipation-free) polypeptide was isolated and purified by a similar method using a DNA encoding hIgG1-Fc (dissipation-free bond) (see Table 6) instead of the DNA encoding hIgG1-Fc. The D5-PE / hIgG1-Fc (dissipation-free bond) polypeptide is a polypeptide consisting of, in order from the N-terminus, an IL-2 signal peptide, linker 1, the D5-PE polypeptide, linker 2, and hIgG1-Fc (dissipation-free bond) (see Table 7).

[0053] In the table, "P １ " indicates K287P, and "E ２ " indicates K317E.

[0054] In the table, the single underlined part indicates domain I, the double underlined part indicates domain V, and the remaining region indicates domains II to IV. １ " indicates K287, and "K ２ " indicates K317.

[0055]

[0056]

[0057]

[0058] The amino acid residues boxed in the table indicate amino acid residues that have been modified in hIgG1-Fc so that no disulfide bonds are formed.

[0059] The amino acid residues boxed in the table indicate amino acid residues that have been modified in hIgG1-Fc so that no disulfide bonds are formed.

[0060] [Detection of D5-PE / hIgG1-Fc Polypeptide] To confirm whether the above four types of polypeptides (D5-PE / hIgG1-Fc polypeptide, D5-KK / hIgG1-Fc polypeptide, full-length hβ2GPI, or hIgG1-Fc polypeptide) were purified in an intact state, 2-mercaptoethanol (2ME) was added to a sample containing the above four types of purified polypeptides to cleave the S—S bonds in the polypeptides, followed by polyacrylamide gel electrophoresis (SDS-PAGE). The polypeptides contained in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane, and then the PVDF was probed with an HRP (Horse Radish Peroxidase)-conjugated anti-hβ2GPI antibody (HRP-anti-hβ2GPI antibody) (Aviscera) to detect full-length hβ2GPI. To detect other polypeptides containing hIgG1-Fc, immunoblotting was performed using an HRP-anti-human IgG antibody. A TMB (3,3',5,5'-tetramethylbenzidine) solution was used to detect the HRP-anti-human IgG antibody.

[0061] As a result, for the D5-KK / hIgG1-Fc polypeptide, only a portion of the bands were detected at the expected size, and most of the bands were detected at a position with a molecular weight smaller than the expected size, whereas for the D5-PE / hIgG1-Fc polypeptide, most of the bands were detected at the expected size (see Figure 1).

[0062] This result indicates that the introduction of two types of mutations (K287P and K317E) into domain V of hβ2GPI makes it less susceptible to degradation, enabling it to be purified in an intact state.

[0063] [Investigation of the specificity of D5-PE / hIgG1-Fc polypeptide for phospholipids] Next, to investigate the specificity of the purified D5-PE / hIgG1-Fc polypeptide, the binding ability to the negatively charged phospholipid (cardiolipin), with which domain V of hβ2GPI forms a complex, was analyzed using ELISA. Specifically, bovine heart-derived cardiolipin (Sigma) was coated onto an ELISA plate, and blocking was performed using PBS (phosphate-buffered saline) containing 1% BSA at room temperature for 1 hour. Four types of purified polypeptides (D5-PE / hIgG1-Fc polypeptide, D5-KK / hIgG1-Fc polypeptide, full-length hβ2GPI, or hIgG1-Fc polypeptide) were then added at a concentration of 20 μg / mL, and the plate was incubated at room temperature for 1 hour. After washing with PBS, HRP-anti-human IgG antibody (Figure 2A) or HRP-anti-hβ2GPI antibody (Figure 2B) was added, and the plate was washed with PBS, treated with TMB solution, and color development was measured.

[0064] As a result, although all three types of polypeptides other than the hIgG1-Fc polypeptide (D5-PE / hIgG1-Fc polypeptide, D5-KK / hIgG1-Fc polypeptide, and full-length hβ2GPI) were found to have the ability to bind to cardiolipin (see Figures 2A and 2B), the D5-PE / hIgG1-Fc polypeptide was shown to have a higher binding ability to cardiolipin than the D5-KK / hIgG1-Fc polypeptide (see Figure 2A).

[0065] These results indicate that the specificity of the D5-PE polypeptide for phospholipids in the D5-PE / hIgG1-Fc polypeptide is improved by introducing two types of amino acid substitution mutations (K287P and K317E) into domain V of hβ2GPI. Note that, in Figure 2B, the reason that binding between the D5-PE / hIgG1-Fc polypeptide or the D5-KK / hIgG1-Fc polypeptide and cardiolipin was not detected is thought to be because the HRP-anti-hβ2GPI antibody used here is an antibody that binds to a region of hβ2GPI other than domain V.

[0066] [Confirmation that D5-PE / hIgG1-Fc Polypeptide Has Antithrombotic Effect 1] Next, we analyzed whether the purified D5-PE / hIgG1-Fc polypeptide has an antithrombotic effect. Previous analyses by the inventors have shown that intranasal infection with SARS-CoV-2 in NZW×BXSB F1 mice, a model animal for spontaneous APS, leads to the development of acute thrombosis. Therefore, 19-week-old NZW×BXSB F1 mice (male, obtained from Japan SLC) were intraperitoneally administered a triple anesthetic solution (containing medetomidine hydrochloride, midazolam, and butorphanol tartrate) at a dose of 0.1 mL / 10 g (body weight) and then intranasally infected with 2 LD50 equivalents of SARS-CoV-2 (QHmusX). QHmusX is a virus strain that originated from a European strain and acquired infectivity in mice through repeated passage in BALB / c mice (see "Iwata-Yoshikawa N, et al. A lethal mouse model for evaluating vaccine-associated enhanced respiratory disease during SARS-CoV-2 infection. Sci Adv. 2022 Jan 7;8(1):eabh3827"). The lethal dose determined by infection in BALB / c mice is 1 LD50.

[0067] Immediately after nasal infection with SARS-CoV-2, two purified polypeptides (1 mg / mL D5-PE / hIgG1-Fc polypeptide and 0.74 mg / mL hIgG1-Fc polypeptide) were intraperitoneally administered at a dose of 200 μL per mouse. The amounts of D5-PE / hIgG1-Fc polypeptide and hIgG1-Fc polypeptide per administration were equivalent to 5.6 nmol. Thereafter, intraperitoneal administration of the polypeptides was continued every 24 hours until day 4 post-infection. On day 10 post-infection, the mice were intraperitoneally administered the above-mentioned three-component anesthetic solution, dissected, and both kidneys were collected. The kidneys were fixed in formalin, embedded in paraffin, thin sections were prepared, and MSB staining was performed. Using MSB, thrombi in renal glomeruli were identified, and 30 glomerular fields per mouse were photographed under a 400x optical microscope. Using WinROOF Education (MITAN), the total area of ​​all glomeruli included in the 30 fields (a) and the total area of ​​thrombi in the glomeruli (b) were calculated, and the ratio of thrombi to glomeruli (● in Figure 3F) per mouse (= (b) × 1000 / (a)) was calculated. To confirm that existing anticoagulant therapy suppresses renal thrombus formation, PBS (200 μL) and low-molecular-weight heparin (dalteparin, 6 IU / 200 μL) were administered intraperitoneally immediately after infection and every 24 hours until day 3 postinfection. On day 3 postinfection, the mice were dissected and both kidneys were similarly stained with MSB.

[0068] First, in a preliminary experiment, renal thrombi, which were not observed in SARS-CoV-2-uninfected NZW×BXSB F1 mice, were observed when NZW×BXSB F1 mice were infected with SARS-CoV-2 (see Figures 3A and 3B). It was confirmed that such renal thrombus formation was suppressed by administering low-molecular-weight heparin, an anticoagulant (see Figure 3E).

[0069] Next, administration of the hIgG1-Fc polypeptide to SARS-CoV-2-infected NZW×BXSB F1 mice did not suppress renal thrombus formation, whereas administration of the D5-PE / hIgG1-Fc polypeptide to SARS-CoV-2-infected NZW×BXSB F1 mice significantly suppressed renal thrombus formation (see Figures 3C, 3D, and 3F). These results indicate that the D5-PE / hIgG1-Fc polypeptide suppressed renal thrombus formation that occurred during SARS-CoV-2 infection in APS model mice.

[0070] [Confirmation that D5-PE / hIgG1-Fc Polypeptide Has Antithrombotic Effect 2] Mice in which thrombosis can be induced by ligation of the inferior vena cava are used as a mouse model of economy class syndrome (see Payne H, et al. Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis. J Vis Exp. 2017 Dec 22;(130):56697.). Therefore, the antithrombotic effect of purified D5-PE / hIgG1-Fc polypeptide was analyzed using the above-mentioned mouse model of economy class syndrome. Specifically, the above-mentioned triple anesthetic solution was intraperitoneally administered to elderly BALB / c mice (retired mice), and then the bilateral renal veins, inferior vena cava (IVC), and abdominal aorta were exposed. The inferior vena cava was separated from the aorta and retroperitoneum distal to the origin of both renal veins using forceps (curved tip), and the separated inferior vena cava was ligated with 4-0 suture. After ligation, the peritoneum and abdominal wall were sutured, and after wound closure, 75 μg / mL of an anesthetic reversal agent (atipamezole hydrochloride) was administered intraperitoneally at a dose of 0.1 mL / 10 g (body weight), followed by intraperitoneal administration of vehicle (PBS) or 1 mg / mL of D5-PE / hIgG1-Fc polypeptide at a dose of 200 μL / animal. Eighteen hours after wound closure and administration of the anesthetic reversal agent, surviving animals were intraperitoneally administered the above-mentioned triple anesthetic solution, followed by dissection, the aorta was cut, and the inferior vena cava was collected after blood collapse. The vessel wall of the inferior vena cava was incised, and the thrombus formed distal to the ligation site was collected and weighed.

[0071] As a result, it was shown that administration of the D5-PE / hIgG1-Fc polypeptide to economy class syndrome model mice significantly reduced the thrombus weight in economy class syndrome model mice that had not been administered the polypeptide (PBS administration) (see Figure 4).

[0072] This result indicates that the D5-PE / hIgG1-Fc polypeptide inhibited thrombus formation in wild-type mice.

[0073] [Confirmation that D5-PE / hIgG1-Fc polypeptide does not prolong ACT] To analyze whether the antithrombotic effect of D5-PE / hIgG1-Fc polypeptide is due to its ability to suppress blood coagulation (anticoagulant effect), similar to heparin, the ACT of D5-PE / hIgG1-Fc polypeptide was measured. Specifically, four test solutions (PBS [17.8 μL], heparin [1.5 μL of 5,000 units / 5 mL of heparin mixed with 16.3 μL of PBS], D5-PE / hIgG1-Fc polypeptide [17.8 μL of a solution containing 7.3 mg / mL of the polypeptide], and hIgG1-Fc polypeptide [6.6 μL of a solution containing 19.6 mg / mL of the polypeptide mixed with 11.2 μL of PBS]) were each added to a 2.5 mL blood collection syringe, and 2.5 mL of peripheral blood was collected from healthy adults (n=3) per syringe. As a result, the blood concentrations of heparin, D5-PE / hIgG1-Fc polypeptide, and hIgG1-Fc polypeptide were 0.6 U / mL, 130 μg / mL, and 130 μg / mL, respectively. Next, blood was transferred from each syringe to a dedicated tube containing a coagulation activator, and ACT was measured using a coagulation meter (Hemochron Response [Accriva Diagnostics]) according to the product's instructions. The reason for setting the concentration of D5-PE / hIgG1-Fc polypeptide at 130 μg / mL in ACT measurement was to reproduce the in vivo blood concentration of D5-PE / hIgG1-Fc polypeptide, since the maximum blood concentration of the administered D5-PE / hIgG1-Fc polypeptide was estimated to be 80 to 130 μg / mL in the aforementioned experiment using SARS-CoV-2-infected NZW×BXSB F1 mice.

[0074] As a result, it was found that the anticoagulant heparin significantly increased the ACT level compared to the negative control PBS, whereas the D5-PE / hIgG1-Fc polypeptide did not increase the ACT level (see Figure 5).

[0075] This result indicates that the D5-PE / hIgG1-Fc polypeptide, unlike heparin, does not prolong the ACT, and does not have an adverse effect on blood coagulation.

[0076] [Confirmation that D5-PE / hIgG1-Fc polypeptide does not prolong bleeding time] We analyzed whether D5-PE / hIgG1-Fc polypeptide, unlike the anticoagulant low-molecular-weight heparin, does not prolong bleeding time. Specifically, three types of test solutions (200 μL of PBS containing 148 μg of hIgG1-Fc polypeptide, 200 μL of PBS containing 200 μg of D5-PE / hIgG1-Fc polypeptide, and 200 μL of PBS containing 4 IU of low-molecular-weight heparin [dalteparin]) were intraperitoneally administered to four 7-week-old wild-type mice (female BALB / c). The amounts of D5-PE / hIgG1-Fc polypeptide and hIgG1-Fc polypeptide per administration each corresponded to 2.8 nmol. Twenty-five minutes after administration, the mice were intraperitoneally administered the above-mentioned triple anesthetic mixture at a dose of 10 μL / 10 g (body weight). Five minutes later, the tail was cut with a blade at a site 10 mm from the tip of the tail. The tail was held horizontally, and blood from the tip was repeatedly absorbed into filter paper for 5 seconds every 30 seconds. The bleeding time was measured as the time when blood no longer adhered to the filter paper.

[0077] As a result, when mice were administered with dalteparin, a significant prolongation of bleeding time was observed compared to when mice were administered with hIgG1-Fc polypeptide, whereas when mice were administered with D5-PE / hIgG1-Fc polypeptide, no prolongation of bleeding time was observed (see Figure 6).

[0078] This result indicates that the D5-PE / hIgG1-Fc polypeptide, unlike dalteparin, does not prolong the bleeding time, and does not have an adverse effect on blood coagulation.

[0079] The present invention contributes to the prevention or treatment of thromboembolic diseases.

Claims

1. An antithrombotic agent comprising a polypeptide comprising domain V of β2GPI, or a polynucleotide comprising a polynucleotide encoding said polypeptide, wherein domain V of β2GPI has a proline residue at position 46 and a glutamic acid residue at position 76 of the amino acid sequence shown in SEQ ID NO: 1, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The antithrombotic agent according to claim 1, wherein the polypeptide does not contain domain I of β2GPI.

3. The antithrombotic agent according to claim 1, wherein the polypeptide comprises the Fc region of an immunoglobulin.

4. The antithrombotic agent of claim 3, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

7.

5. A preventive or therapeutic agent for thromboembolic diseases, comprising the antithrombotic agent according to any one of claims 1 to 4.

6. A polypeptide comprising domain V of β2GPI and an Fc region of an immunoglobulin, wherein domain V of β2GPI has a proline residue at position 46 and a glutamic acid residue at position 76 of the amino acid sequence shown in SEQ ID NO: 1, and consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

7. The polypeptide of claim 6, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

7.

8. A polynucleotide comprising a polynucleotide encoding the polypeptide of claim 6 or 7.

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