Class of monoclonal antibodies having Anti-von willebrand factor effects and use thereof

Abstract

The present invention relates to a class of monoclonal antibodies having anti–von Willebrand factor (VWF) effects, and a use thereof. A pharmaceutical composition of the monoclonal antibodies or antigen-binding fragments having anti–von Willebrand factor effects provided by the present invention can be used to reduce VWF levels in the body, thereby preventing and treating thrombotic microangiopathies having various causes (including thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, and atypical hemolytic-uremic syndrome), cardiovascular disease, stroke, atherosclerosis, senile dementia, vascular dementia, and bacterial and viral infectious diseases.

Description

A class of monoclonal antibodies with anti-von Willebrand factor effects and their uses Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a class of monoclonal antibodies with anti-von Willebrand factor (VWF) effects and their uses. Background Technology

[0002] Von Willebrand factor (VWF) is a large polymeric glycoprotein in plasma that plays a crucial role in hemostasis and thrombosis by mediating platelet adhesion to injured and activated blood vessels. VWF acts as a bridging molecule for platelet adhesion and aggregation after vascular injury. It also serves as a carrier molecule for coagulation factor VIII (FVIII), protecting it from rapid clearance and thus prolonging its plasma half-life. Therefore, VWF is indispensable for both platelet-mediated primary hemostasis and factor-mediated secondary hemostasis; VWF deficiency or dysfunction can lead to bleeding or thrombosis.

[0003] The human VWF gene is located at the tip of the short arm of chromosome 12, consisting of 52 exons, encoding a 240-260 kDa VWF precursor polypeptide (pre-pro-VWF). VWF is synthesized only in megakaryocytes and endothelial cells, with the majority of plasma VWF being synthesized by endothelial cells. Subtle differences exist in VWF synthesis in the vascular endothelium of different organs and tissues; small vascular endothelial cells in the lungs and brain express higher levels of VWF than similar-sized vessels in the liver and kidneys. The pre-pro-VWF produced by gene transcription and translation consists of 2813 amino acid residues, including a signal peptide, a 741-amino acid propeptide, and a mature VWF subunit containing 2050 amino acid residues. The VWF monomeric domain is composed of SP-D1-D2-D'-D3-A1-A2-A3-D4-C1-C6-CK, where SP is the signal peptide, D1 and D2 are VWF propeptide domains that interact with D' and participate in the assembly of VWF dimers; D' and D3 interact with D1 and D2, p-selectin on the surface of platelets and endothelial cells, and coagulation factor VIII during dimer formation and inter-dimer cross-linking. (FVIII) Interactions: The A1 domain binds to GP1b on platelets, collagen in the subendothelial matrix, and several other protein molecules, such as histones, heparin, osteoprotein, and β-2 integrin receptors on leukocytes; the A2 domain is crucial in regulating VWF activity, unfolding to expose other VWF domains when VWF polymers are exposed to shear stress; A2 is also the cleavage site of the protease ADAMTS13 on VWF; the A3 domain binds to extracellular matrix collagen; the C domain binds to fibrin plasma complement proteins and interacts with platelet surface receptors GPIIb / IIIa during platelet adhesion; the CK (cysteine ​​knot) domain plays an important role in VWF polymerization and post-translational folding.

[0004] Prepro-VWF undergoes modification and removal of the (pre-) sequence in the endoplasmic reticulum (ER) and Golgi apparatus. The pro-VWF monomers are linked to dimers via C-terminal disulfide bonds, which are then assembled in the Golgi apparatus into multimeric structures of varying molecular weights via disulfide bonds formed between the N-terminus. The VWF propeptide acts as a VWF-specific disulfide isomerase, promoting disulfide bond formation and VWF multimer assembly, and is subsequently removed from the VWF multimers by furin-like enzymes in the ER and Golgi apparatus. The number of disulfide-linked subunits in the VWF multimers varies, with molecular weights ranging from approximately 500 kDa to over 10,000 kDa, resulting in the largest known protein in human plasma. A portion of VWF is directly secreted into circulation after synthesis and assembly, but the majority is packaged and stored in platelet α-granules and Weibel-Palade bodies (WPBs) of endothelial cells. VWF polymers exist in low (L), medium (I), high (H), and ultra-large (UL) molecular weight forms, with only low, medium, and high molecular weight VWF polymers being continuously secreted into circulation. Stored VWF polymers vary in size, including a large number of ultra-large UL-VWF polymers, which are typically not present in peripheral circulation.

[0005] Each multimeric subunit of VWF has a binding site for the platelet surface receptor GPIb. Simultaneously, VWF subunits can also bind to GPIIb / IIIa on the surface of activated platelets, thereby promoting platelet adhesion and platelet aggregation, respectively. Under normal physiological conditions, VWF multimers can be continuously released from endothelial cells into the bloodstream, or released from endothelial cells or platelets as needed under specific conditions. Once released from the endothelium into circulation, VWF folds into a globular conformation, exposing high-affinity collagen-binding sites in the A3 domain. Upon tissue injury, these collagen-binding sites recruit VWF to the subendothelial extracellular matrix at the site of vascular injury. The GpIbα binding site in the A1 domain of the folded globular VWF molecule in circulation is partially hidden, thus preventing any interaction between resting platelets and VWF in circulation. In the circulating folded conformation of VWF molecules, the ADAMTS13 cleavage site on the A2 domain is also hidden, thus protecting the globular VWF from proteolytic hydrolysis. However, under conditions of vascular injury or high shear stress from blood flow, VWF polymers unfold, exposing their platelet GPIb binding sites within the VWF A1 domain. The role of VWF polymers in hemostasis is closely related to their size. Smaller polymers have fewer binding sites, resulting in lower affinity between low-molecular-weight VWF polymers and platelets, reducing platelet adhesion and aggregation. Conversely, high-molecular-weight VWF polymers are conformationally more sensitive to shear stress, more easily unfolding into long polymer chains and exposing their GPIb binding sites, thereby promoting the binding of VWF to platelets and other molecules. As VWF polymers gradually decrease in size, their hemostatic function also weakens. The collagen-binding capacity of VWF (VWF:CB) and its ability to bind to platelets (restorin cofactor activity; VWF:RCo) both decrease with decreasing VWF polymer size. Analysis of recombinant VWF polymers also shows that the binding affinity of VWF to FVIII (FVIIIB) gradually decreases with decreasing VWF polymer size.

[0006] In the general population, 95% of measurements of von Willebrand factor (VWF) levels (international unit (IU) / dL) fall between 50 and 200 IU / dL. In the normal population, plasma VWF levels (VWF:Ag) and activity (VWF:RCo) increase by approximately 0.17 IU / ml and 0.15 IU / ml per decade, respectively. VWF gene mutations leading to VWF deficiency or functional defects result in hereditary von Willebrand disease (VWD). VWD is the most common known hereditary bleeding disorder in humans. It is inherited in an autosomal dominant manner, affecting males and females with almost equal frequency. Patients have an elevated bleeding tendency, manifesting as excessive and prolonged bleeding after surgery or trauma, mucosal bleeding such as nosebleeds, and menorrhagia. Severe cases may present with hemophilia-like bleeding symptoms, such as joint and muscle bleeding. VWD is classified into three main types: Type 1, characterized by a mild decrease in VWF levels and functional impairment; Type 2, characterized primarily by VWF dysfunction; and Type 3, characterized by a complete lack of VWF. VWD classification is typically determined by measuring VWF functional activity. In 2007, an expert consensus group convened by the National Heart, Lung, and Blood Institute (NHLBI) defined type 1 von Willebrand disease (VWD) as a plasma von Willebrand factor (VWF) level below 30 IU / dL, while levels between 30 and 50 IU / dL were classified as low VWF levels, constituting a risk factor for bleeding. However, some argue that low von Willebrand factor (VWF) levels are not an independent clinical symptom, but rather a physiological phenomenon where VWF levels gradually increase with normal age. Individuals with VWF levels below 30 IU / dL are more likely to carry pathogenic mutations in the VWF gene, and this population also exhibits the most significant familial inheritance.

[0007] Endothelial cells, upon exposure to physiological and pharmacological agents such as adrenaline, adenosine diphosphate, collagen, fibrin, histamine, thrombin, complement proteins, and the vasopressin analog desmopressin (DDAVP), release intracellularly stored ultramacromolecules of volatile microfibrils (UL-VWF). Upon stimulation, UL-VWF is secreted from the cell and anchors on the endothelial cell surface, forming a string-like structure. Under normal blood flow shear stress, the VWF polymers elongate from a spherical shape into a string-like form, thereby exposing the cleavage site of the metalloproteinase ADAMTS13 in the A2 domain. Under high fluid shear stress, the endothelial-bound UL-VWF strings are repeatedly cleaved by ADAMTS13 into lower-activity VWF polymers that support normal hemostasis. Besides high shear stress increasing the efficiency of ADAMTS13's cleavage of VWF, FVIII, platelets, and GPIba can also accelerate this cleavage reaction. UL-VWF exhibits stronger platelet aggregation and coagulation activity than smaller circulating VWF polymers. It spontaneously binds to platelets due to the lower shear threshold required to induce ULVWF unfolding and expose binding sites. Under very high shear stress, VWF polymers undergo a conformational change from a native, inactive state to a metastable, active state with an increased unfolding barrier, making them more difficult to cleave by ADAMTS13. This conformational change may be due to shear-induced lateral binding of VWF polymers into a fibrous structure.

[0008] ADAMTS13 regulation of VWF polymer size is crucial for normal hemostasis. Excessive ADAMTS13 proteolytic activity of VWF leads to severe impaired hemostasis due to the absence of high-molecular-weight VWF polymers, resulting in the classic VWD2A pattern. Conversely, ADAMTS13 deficiency leads to the abnormal accumulation of the largest molecular weight ultra-large polymer, UL-VWF polymers. These ULVWF polymers exhibit abnormal adhesion, spontaneously binding to platelets even without vascular injury. The accumulation of UL-VWF polymers on plasma and endothelial cell surfaces induces platelet aggregation and adhesion to the vascular endothelium, resulting in spontaneous platelet aggregation and contributing to various diseases such as thrombotic microangiopathy, inflammation and inflammatory responses, and severe malaria infection. Evidence suggests that some clinical conditions involving microcirculatory thrombosis, such as acute myocardial infarction, sepsis-induced disseminated intravascular coagulation, and diabetic nephropathy, may also be associated with elevated UL-VWF polymer levels, possibly related to ADAMTS13 deficiency.

[0009] Thrombotic microangiopathy (TMA) is a group of diseases characterized by common clinical and laboratory features resulting from thrombosis or occlusion of the microvascular system. TMA is classified into thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), the former further subdivided into "idiopathic" and "secondary" TTP, and the latter into "typical" and "atypical" HUS. Circulating vascular wind-free cells (VWFs) bind to platelets under shear stress, promoting hemostasis. Dysregulation of this process is a major cause of TMA, and its pathogenesis is believed to be due to the accumulation of UL-VWFs caused by hereditary or acquired ADAMTS13 deficiency. Clinically, TTP is the most severe and treatment-required form of TMA. Hereditary / congenital TTP is relatively rare and is caused by mutations in the ADAMTS13 gene. Typically, both alleles must be affected to cause severe ADAMTS13 deficiency (<5% ADAMTS13 activity). Heterozygous carriers of ADAMTS13 mutations are usually asymptomatic and do not have TTP. Symptoms are similar. Acute idiopathic TTP is the most common form of TTP, an autoimmune disease typically characterized by the development of inhibitory autoantibodies, most commonly IgG, and less commonly IgM and / or IgA recognizing ADAMTS13. TTP is a life-threatening disease, characterized by the widespread deposition of VWF and platelet-rich thrombi in the microvascular system. In TTP, severe ADAMTS13 deficiency (acquired or hereditary) leads to the loss of VWF multimer size regulation mechanisms, resulting in the inability to break down ultra-large VWF multimers (UL-VWF). Upon binding to platelets, this leads to thrombocytopenia and microvascular thrombosis, resulting in end-organ ischemia. Due to intraluminal thrombus obstruction, red blood cells are mechanically sheared, ultimately leading to hemolytic anemia, with red blood cell fragments visible on peripheral smears. Microthrombi in TTP patients can... TTP can cause focal neurological deficits, seizures, and even coma. If left untreated, it can lead to vascular occlusion, tissue ischemia, organ failure, and death. Classic presentations of TTP include thrombocytopenia, microangiopathic hemolytic anemia, fluctuating neurological signs, renal impairment, and fever. However, TTP patients often present without a complete pentad of symptoms. Neurological dysfunction caused by TTP can manifest as confusion, headache, paralysis, aphasia, dysarthria, visual problems, and encephalopathy. TTP patients typically exhibit significant thrombocytopenia (platelet count 10-30 × 10⁻⁶). 9 This is due to the formation and retention of platelets and UL-VWF in microvascular thrombi. Microangiopathy-related hemolytic anemia may be caused by the fragmentation of red blood cells as they pass through partially occluded microvessels. Patients have hemoglobin levels as low as 80-100 g / L, fragmented red blood cells in peripheral blood, and elevated lactate dehydrogenase.

[0010] VWF also plays an important role in arterial and venous thrombosis (cardiovascular diseases and stroke, etc.), atherosclerosis, and complications associated with malaria, sepsis, sickle cell disease, and other thrombotic microangiopathy.

[0011] The association between low ADAMTS13 activity and ischemic stroke risk may be due to less cleavage of high molecular weight polymers of VWF, leading to a prothrombotic state and potentially causing thrombus formation at sites of endothelial injury, particularly at sites of high shear stress. Mouse model studies have shown that VWF-deficient mice are less susceptible to thrombotic inflammation in experimental models of myocardial infarction, atherosclerosis, and acute ischemic stroke. In contrast, ADAMTS13-deficient mice are prothrombotic, pro-inflammatory, and prone to thrombotic thrombocytopenic purpura upon Shiga toxin challenge. Furthermore, ADAMTS13-deficient mice are more prone to thrombotic inflammation in experimental models of myocardial infarction, atherosclerosis, and stroke. High VWF levels and low ADAMTS13 levels are both associated with an increased risk of ischemic stroke. Some animal studies suggest that ADAMTS13 plays a pathogenic role in the occurrence or progression of ischemic stroke. Experimental studies on focal cerebral ischemia have shown that cerebral infarction is larger in mice lacking ADAMTS13 than in wild-type mice.

[0012] Acute coronary syndrome (ACS) is caused by atherosclerotic plaque rupture, exposure of subendothelial procoagulant factors, and subsequent thrombosis leading to myocardial ischemia. Vascular fibroblasts (VWF) are a key player in this pathological process; both VWF levels and activity are elevated in ACS patients. VWF mediates platelet adhesion to the subendothelial matrix of the damaged vessel wall and enhances platelet aggregation, promoting fibrin clot formation. The association between VWF levels and the expected incidence of myocardial infarction in patients with cardiovascular disease has been well-established. A large-scale study by the European Coordination Action on Thrombosis and Disability (ECAT) demonstrated that VWF is an independent predictor of recurrent myocardial infarction in patients with angina. In patients with acute myocardial infarction (AMI), plasma VWF concentrations are increased, and shear-induced platelet aggregation is significantly enhanced. VWF levels reflect the typical time course during acute cardiovascular events. In patients with ST-segment elevation myocardial infarction (STEMI), VWF levels rise within 24 hours, peak at 48 to 72 hours, and then return to baseline levels around day 14. The degree of VWF increase also has prognostic value. AMI patients have elevated VWF levels compared to patients with unstable angina. Furthermore, the degree of VWF increase is an independent predictor of short-term adverse clinical outcomes in ACS patients. In addition, studies have confirmed the association between VWF levels and the risk of re-infarction and / or death; moreover, the extent of VWF release (i.e., the difference between baseline and 24-hour VWF values ​​during the exponential event period) is not only associated with the incidence of acute heart failure but also significantly associated with 30-day mortality in STEMI patients.

[0013] Atherosclerotic risk factors such as hypertension, elevated LDL cholesterol, decreased HDL cholesterol, obesity, diabetes, and oxidative stress can induce inflammatory vascular responses, thereby triggering the formation and growth of atherosclerotic plaques. This inflammatory vascular response includes endothelial activation, VWF release, and VWF-mediated platelet adhesion to endothelial cells under high shear stress at atherosclerotic-prone sites in large arteries (such as bifurcation points and areas of turbulent blood flow). Subsequently, platelets are activated and further recruit platelets in various ways, releasing dense granular contents, exposing P-selectin, and attracting additional neutrophils and monocytes to the site. Activated platelets also activate the NOX-1 and NOX-2 systems and produce and release reactive oxygen species that maintain endothelial activation. Studies have shown that VWF-mediated platelet-endothelial adhesion begins to increase in the early stages of a mouse model of atherosclerosis, before plaque formation. VWF knockout reduces the size of atherosclerotic lesions in a mouse model of atherosclerosis. In ApoE knockout mice, the lack of ADAMTS13 accelerates atherosclerosis and increases macrophage infiltration into atherosclerotic lesions. Infusion of ADAMTS13 eliminated VWF-dependent platelet adhesion at atherosclerotic predisposition sites. These findings extend the fundamental characteristics of microvascular thrombosis (including inflammation, endothelial activation, VWF release, and platelet adhesion) to large vessel thrombosis.

[0014] Besides its role in thrombosis and hemostasis, vascular endothelial cells (VWFs) are also ligand-binding sites for bacteria that can cause systemic infections, such as Staphylococcus aureus and Streptococcus pneumoniae. The interaction between bacteria and VWFs has significant medical and scientific implications because it is directly related to specific clinical manifestations and long-term complications of infectious diseases. The binding of Staphylococcus aureus and Streptococcus pneumoniae to VWF polymers is controlled by hydrodynamic flow conditions, while shear-mediated bacterial adhesion to VWFs is directly related to coagulation and typical disease symptoms. Currently, the believed pathological mechanisms by which VWFs participate in bacterial infectious diseases include: VWF polymer binding and mediating bacterial attachment to the endothelial surface in the bloodstream, promoting bacterial colonization, inflammation, and spread; VWF binding to bacteria can also prevent bacterial clearance through immune thrombosis; and intravascular VWF recruitment induces bacterial aggregate formation, leading to capillary occlusion and impaired blood supply.

[0015] However, it is worth noting that in the aforementioned pathological conditions, ADAMTS13 levels were normal or near normal in some patients. Therefore, ADAMTS13 lysis is not the only mechanism that can regulate VWF adhesion function, and there may be other unexplained mechanisms.

[0016] Epidemiological studies have also shown that higher VWF levels and lower ADAMTS13 activity are associated with an increased risk of dementia, but differences in time course and lack of synergistic effects may partly indicate that these two factors are independent underlying mechanisms for dementia risk.

[0017] Therefore, inhibiting or eliminating VWF and reducing VWF activity levels are important treatment strategies for treating and preventing the occurrence and development of the aforementioned diseases. TTP is a fatal disease, and therapeutic plasma exchange (TPE) was once the only effective treatment. TPE uses fresh frozen plasma (FFP) to replace ADAMTS13 in TTP patients and remove anti-ADAMTS13 autoantibodies and ultra-large von Willebrand factor multimers (UL-VWF) that cause acquired TTP. Randomized controlled trials have shown that TPE is superior to plasma transfusion in treating TTP patients; therefore, TPE has become the standard treatment for TTP and is applied to all TMA-related diseases. Performing TPE requires sufficient FFP, specialized equipment, and trained professionals, and may cause complications including hypocalcemia, hypotension, muscle cramps, headache, and perioral and finger sensory abnormalities caused by urticaria. TPE and immunosuppressive therapy have a slow onset of action and cannot immediately resolve the pathophysiological platelet aggregation that leads to microthrombus formation. Even with treatments including daily plasma exchange and immunosuppression (such as glucocorticoids and rituximab), approximately one-sixth of patients remain refractory, characterized by no increase or very slow increase in platelet count, which is associated with adverse outcomes. Another major challenge in TTP treatment is reducing the risk of persistent, potentially life-threatening relapses. TTP is termed an acute exacerbation if it occurs within 30 days of the last plasma exchange, and a relapse if it occurs more than 30 days after the last plasma exchange. Most relapses occur within the first one to two years, but can also occur 10 or 20 years after a TTP episode. Persistent or recurrent ADAMTS13 deficiency is a strong risk factor for relapse. Therefore, although plasma exchange significantly reduces TTP mortality, acquired TTP still carries a considerable risk of death and morbidity, and new treatment methods are urgently needed to better manage this disease.

[0018] Blocking the binding of UL-VWF to the GpIb-IX-V receptor on the platelet surface is a novel strategy for treating diseases caused by VWF multimer-mediated thrombosis, including TTP. Currently, various drugs, including nanobodies, monoclonal antibodies, and nucleic acid ligands, have been developed to target VWF or its binding site on the platelet surface, the GpIb-IX-V receptor, thereby inhibiting VWF multimer-mediated platelet thrombosis. The humanized anti-VWF monoclonal antibody AJW200 and the anti-VWF nanobody caplacizumab can both bind tightly to multimer VWF and block the interaction between multimer VWF and the platelet GpIb-IX-V receptor, thus immediately blocking the further formation and accumulation of UL-VWF-platelet-mediated microthrombi. The nucleic acid aptamer ARC1779, which recognizes the A1 domain of VWF, can also produce a similar effect, interfering with platelet-VWF binding. Clinical trials have shown that blocking the binding of the A1 domain of VWF to the GpIb-IX-V receptor on the platelet surface can increase platelet counts in patients with TTP, and combining it with plasma exchange therapy yields better TTP treatment outcomes. The addition of cappraziquantel significantly reduces the risk of relapse and disease progression in refractory immune thrombotic thrombotic thrombotic purpura (iTTP) / acquired thrombotic thrombotic thrombotic purpura (aTTP), while shortening the treatment response time, but increasing the likelihood of disease relapse. As a first-line treatment, although this drug can reduce the risk of refractory disease, accelerate treatment response, and improve the rate of disease progression, it does not significantly reduce all-cause mortality compared to standard treatment and carries a higher risk of relapse and bleeding. Nanobodies have a small molecular weight (15kDa) and can be cleared from the body via glomerular filtration; therefore, the biggest drawback of nanobodies compared to conventional antibodies is their very short plasma half-life. Following a single intravenous injection of 10 mg caprasizumab in healthy volunteers, the mean clearance, measured using a non-compartmental model, was 769 ± 343 mL / h, with a mean terminal half-life of 19.2 ± 7.5 hours. Pharmacokinetics of caprasizumab after subcutaneous administration showed that absorption was a rate-limiting step. With a single subcutaneous injection of 10 mg, the mean apparent clearance in healthy volunteers was 386 ± 160 mL / h, and the terminal half-life was prolonged to 38.5 ± 22.2 hours. Therefore, frequent administration of caprasizumab is necessary for adult acquired thrombotic thrombocytopenic purpura (aTTP). In the recommended dose-time schedule, an 11 mg dose is administered intravenously at least 15 minutes before the start of plasma exchange; after the first day of plasma exchange, another 11 mg dose is administered subcutaneously. Subsequent treatment during daily plasma exchange: After the daily plasma exchange, continue with an 11 mg dose administered subcutaneously. Treatment after plasma exchange: 11 mg subcutaneously daily for 30 days, starting from the last daily plasma exchange.If underlying disease signs persist after initial treatment (e.g., persistent ADAMTS13 activity inhibition), the treatment period may be extended by up to 28 days. Because capprazitumab cannot eliminate pathogenic high molecular weight VWF multimers, aTTP patients still require plasma exchange therapy.

[0019] In other diseases involving VWF multimers, reducing VWF levels and activity is also an important means of improving treatment efficacy. Cerebral embolism caused by large artery atherosclerosis is a major cause of stroke. Recent data show that the early risk of recurrent stroke is high after a minor stroke or transient ischemic attack (TIA), with large artery disease being the highest risk. Aspirin can reduce the risk of stroke recurrence, but it cannot prevent 80% of recurrences. Dipyridamole combination therapy or clopidogrel monotherapy is more effective, but similarly, it cannot prevent many recurrences. Therefore, it is suggested that a stronger antiplatelet therapy regimen is needed in the early stages after stroke or TIA, and inhibiting the binding of von Willebrand factor (VWF) to platelets is one feasible treatment strategy. After vascular wall damage, VWF multimers bound to subendothelial collagen or the surface of activated endothelial cells recruit platelets to the damaged arterial wall by binding to platelet surface receptor glycoprotein (GPIb), thereby activating and aggregating platelets. Recent studies have shown that while GPIb or VWF deficiency can prolong thrombin time in coagulation tests, it can still protect mice from cerebral infarction without inducing cerebral hemorrhage. This suggests that the GPIb-VWF axis may represent a suitable target for stroke prevention. The anti-VWF aptamer ARC1779 inhibits isolated platelet aggregation in myocardial infarction and suppresses VWF activity and VWF-dependent platelet aggregation in healthy volunteers, but has no significant effect on the coagulation system and other platelet activation pathways. This indicates that inhibiting the interaction of VWF and GPIb can reduce thrombus formation on activated atherosclerotic plaques and reduce subsequent cerebral embolism.

[0020] Given the recognized role of VWF-platelet interactions in normal hemostasis, bleeding risk is a safety concern associated with anti-VWF drugs. Currently, in vivo and in vitro data on Caplacizumab, ARC1779, and other anti-VWF antagonists have not demonstrated a significant effect of functional VWF neutralization on bleeding tendency, and the effectiveness of VWF inhibition in cases of low platelet counts has not been thoroughly evaluated. Technical issues

[0021] The technical problem to be solved by this invention is to provide a class of monoclonal antibodies with anti-von Willebrand factor effects and their uses. These antibodies can be used to reduce VWF levels in the body and reduce ultra-high molecular weight VWF polymers, thereby preventing and treating thrombotic microangiopathy (including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome and atypical hemolytic uremic syndrome), congenital ADAMTS13 deficiency (Upshaw-Schulman syndrome), acquired ADAMTS13 deficiency, cardiovascular disease, stroke, atherosclerosis, Alzheimer's disease, vascular dementia, and bacterial and viral infectious diseases caused by various reasons. Technical solutions

[0022] This invention provides a monoclonal antibody or its antigen-binding fragment with an anti-von Willebrand factor effect, wherein the antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the antibody comprises a complementarity determining region (CDR): heavy chain variable region 1 CDR (HCDR1), heavy chain variable region 2 CDR (HCDR2), and heavy chain variable region 3 CDR (HCDR3), and the light chain variable region comprises a complementarity determining region: light chain variable region 1 CDR (LCDR1), light chain variable region 2 CDR (LCDR2), and light chain variable region 3 CDR (LCDR3), wherein:

[0023] The amino acid sequences of HCDR1 are shown in SEQ ID NO:5, HCDR2 in SEQ ID NO:6, and HCDR3 in SEQ ID NO:7; and

[0024] The amino acid sequences of LCDR1 are shown in SEQ ID NO:8, LCDR2 in SEQ ID NO:9, and LCDR3 in SEQ ID NO:10.

[0025] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0026] This invention also provides another monoclonal antibody or its antigen-binding fragment having an anti-von Willebrand factor effect, wherein the antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the antibody comprises a complementarity determining region (CDR): heavy chain variable region 1 CDR (HCDR1), heavy chain variable region 2 CDR (HCDR2), and heavy chain variable region 3 CDR (HCDR3), and the light chain variable region comprises a complementarity determining region: light chain variable region 1 CDR (LCDR1), light chain variable region 2 CDR (LCDR2), and light chain variable region 3 CDR (LCDR3), wherein:

[0027] The amino acid sequence of HCDR1 is shown in SEQ ID NO:15, the amino acid sequence of HCDR2 is shown in SEQ ID NO:16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17.

[0028] The amino acid sequences of LCDR1 are shown in SEQ ID NO:18, LCDR2 in SEQ ID NO:19, and LCDR3 in SEQ ID NO:20.

[0029] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0030] Furthermore, the heavy chain constant region of the antibody is the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, or IgE type antibodies; the light chain constant region is the constant region of κ-type and λ-type light chains.

[0031] Furthermore, the monoclonal antibody or its antigen-binding fragment with anti-von Willebrand factor effect is selected from Fab, Fab', F(ab')2, Fv, complementarity-determining region fragment or single-chain antibody.

[0032] Furthermore, the monoclonal antibody or its antigen-binding fragment having an anti-von Willebrand factor effect includes a non-CDR region, and the non-CDR region is derived from a human antibody.

[0033] Furthermore, the present invention also provides a nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment that has an anti-von Willebrand factor effect.

[0034] Furthermore, the nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region of a monoclonal antibody with anti-von Willebrand factor effect is shown in SEQ ID NO:3, and the nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; the nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region of a monoclonal antibody with anti-von Willebrand factor effect is shown in SEQ ID NO:13, and the nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14.

[0035] The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof having an anti-von Willebrand factor effect.

[0036] The present invention also provides a host cell comprising a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof having an anti-von Willebrand factor effect, or the recombinant vector thereof.

[0037] The present invention also provides a pharmaceutical composition comprising an effective amount of the monoclonal antibody or its antigen-binding fragment having an anti-von Willebrand factor effect.

[0038] Furthermore, it also includes one or more pharmaceutically acceptable excipients.

[0039] One object of this invention is to provide a class of human monoclonal antibodies or antigen-binding fragments thereof with an anti-von Willebrand factor (VWF) effect, their amino acid sequences and corresponding encoding nucleotide sequences; a method for producing monoclonal antibodies; a class of pharmaceutical combinations with monoclonal antibodies or antigen-binding fragments thereof as active ingredients; a class of therapeutic drugs for reducing VWF levels with monoclonal antibodies or antigen-binding fragments thereof as active ingredients; a drug containing monoclonal antibodies or antigen-binding fragments thereof as active ingredients for treating VWF-mediated thrombotic diseases; and methods for treating such diseases, which may include administering to an individual a composition with any of the monoclonal antibodies or antigen-binding fragments thereof described herein as active ingredients at a therapeutically effective dose. The monoclonal antibody or antigen-binding fragment pharmaceutical combinations of this invention with an anti-VWF effect can be used to reduce VWF levels in the body, reduce ultra-high molecular weight VWF polymers, thereby preventing and treating thrombotic microangiopathy caused by various reasons, congenital ADAMTS13 deficiency (Upshaw-Schulman thrombosis), etc. Syndrome), acquired ADAMTS13 deficiency, cardiovascular disease, stroke, atherosclerosis, Alzheimer's disease, vascular dementia, bacterial and viral infectious diseases.

[0040] This invention provides a novel class of compositions containing human monoclonal antibodies or antigen-binding fragments that reduce VWF levels and remove oversized VWF multimers, specifically exerting an effect against VWF multimers of different molecular weights composed of different numbers of subunits in humans or other non-human animals. These antibodies can be any of the five known classes of immunoglobulins, including IgA, IgD, IgE, IgG, and IgM. Antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, dimers, linear antibodies, nanobodies, or multispecific antibodies formed from antibody fragments. Antibody fragments F(ab′)2, Fab′, and Fab can be obtained by digesting the aforementioned monoclonal antibodies with proteolytic enzymes (such as trypsin, papain, and pepsin) or by recombinant expression in prokaryotic or eukaryotic cells followed by purification; these fragments can also be used to reduce VWF levels, provided they possess the same properties as the aforementioned monoclonal antibodies.

[0041] The types or forms of pharmaceutical combinations prepared with antibodies or antigen-binding fragments having an anti-VWF effect in this invention include, for example, injections, sublingual tablets, plasters, tablets or pills, capsules, granules, syrups, suppositories, ointments, and drops. Injections, sublingual tablets, and plasters are preferred. Depending on the type of pharmaceutical preparation, the anti-VWF monoclonal antibody or antigen-binding fragment can be mixed with pharmaceutically permissible excipients, such as lactose, potato starch, calcium carbonate, and sodium alginate. The pharmaceutical combinations prepared with antibodies or antigen-binding fragments having an anti-VWF effect in this invention can be administered in any manner known to those skilled in the art, including but not limited to oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, percutaneous, nasopharyngeal, intralesional, intratumoral, intradermal, or mucosal absorption. In the case of injection, the pharmaceutical agent used as the injection solvent includes water for injection, physiological saline, and Ringer's solution. In addition, antibodies or antigen-binding fragments with anti-VWF effects can be used together with other antithrombotic components.

[0042] An effective therapeutic dose refers to the amount of a composition sufficient to achieve the therapeutic purpose when administered with the combination of antibodies or antigen-binding fragments of the present invention that have an anti-VWF effect to reduce VWF levels, decrease large VWF polymers, and prevent or treat, including but not limited to, thrombotic microangiopathy. The dosage of the formulations or compositions of the present invention for reducing VWF levels and decreasing large VWF polymers will be determined based on the disease and its severity, as well as the age, weight, physical condition, and responsiveness of the patient to be treated. Generally, in the case of intravenous or subcutaneous administration, the present invention uses a monoclonal antibody as the active ingredient, and it is anticipated that this can be administered in the range of 0.1 μg / kg to 1000 mg / kg, more preferably 1 μg / kg to 100 mg / kg for a single adult dose.

[0043] The present invention also provides the use of the antibody or its antigen-binding fragment having an anti-VWF effect in the preparation of a medicament for reducing VWF levels in vivo, reducing super-large VWF polymers, thereby preventing and treating, but not limited to, thrombotic microangiopathy, congenital ADAMTS13 deficiency (Upshaw-Schulman syndrome), acquired ADAMTS13 deficiency, cardiovascular disease, stroke, atherosclerosis, Alzheimer's disease, vascular dementia, and bacterial and viral infectious diseases caused by various reasons.

[0044] Furthermore, the thrombotic microangiopathy includes thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, atypical hemolytic uremic syndrome, and thrombotic microangiopathy associated with malaria, sepsis, and sickle cell disease; congenital ADAMTS13 deficiency (Upshaw-Schulman syndrome) and acquired ADAMTS13 deficiency; cardiovascular and cerebrovascular diseases include transient ischemic attack, unstable angina, cerebral infarction, myocardial infarction and peripheral artery occlusive disease, PTCA re-occlusion and coronary artery bypass graft occlusion, coronary valve replacement and essential thrombocytosis, deep vein thrombosis, pulmonary embolism, atrial fibrillation, coronary artery thrombosis, intracardiac thrombosis, postoperative thrombosis, cancer-induced thrombosis, cancer-related thrombin expression, infection, disseminated intravascular coagulation, or thrombosis related to arterial thrombosis (including cerebral arteries and coronary arteries). In some implementation plans, patients who need to prevent blood clots may have atrial fibrillation or be at risk of deep vein thrombosis, stroke, heart attack, or pulmonary embolism.

[0045] The combination of antibodies or antigen-binding fragments of the present invention, which have anti-VWF effects, can not only prevent and treat thrombosis by reducing VWF levels, but can also be used to dissolve existing thrombi, thereby reducing or "destroying" existing blood clots.

[0046] The combination of antibodies or antigen-binding fragments of the present invention with anti-VWF effect can also reduce platelet aggregation function as detected by RIPA (ritoxetine-induced platelet aggregation), BIPA (botoxetine-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) tests by reducing VWF levels.

[0047] The antibody or antigen-binding fragment with anti-VWF effect described in this invention can also be used to treat or prevent viral, bacterial and parasitic infections.

[0048] Preferably, the virus is an enveloped virus or a non-enveloped virus, including vaccinia virus, Pitchind virus, cytomegalovirus, Lassa virus, Lenti virus, dengue virus, Ebola virus, and Marburg virus, as well as one or more of SV40, hepatitis A virus, and poliovirus.

[0049] Preferably, the bacteria are Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium marineum, Chlamydia, Candida albicans, Acinetobacter baumannii, Staphylococcus epidermidis, Citrobacter brucellosis, Klebsiella pneumoniae, Klebsiella acidogenetica, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Citrobacter freundii, Candida glabrata, Streptococcus galline hydrophila, Corynebacterium urealyticum, Staphylococcus aureus, Candida parapsilosis, Candida krusei, Citrobacter keloidea, and others. One or more of the following: Citrobacter, Oral Streptococcus, Staphylococcus ludens, Morganella morganii, Candida glabrata, Acinetobacter pieterii, Candida lucida, Proteus vulgaris, Proteus mirabilis, Acinetobacter jumbo, Candida tropicalis, Pseudomonas japonicus, Staphylococcus hemolyticus, Enterococcus faecalis, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Streptococcus agalactiae, Flavorful fungi, Streptococcus constellations, Streptococcus pharyngitis, Burkholderia cepacia, Gardnerella vaginalis, Enterobacter cloacae, and Serratia marcescens.

[0050] The antibodies or antigen-binding fragments with anti-VWF effects described in this invention can also be used for neurological diseases, including but not limited to vascular dementia and Alzheimer's disease.

[0051] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the definition of the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0053] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0054] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using the hybridoma technique first reported by Kohler et al. (G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but can also be obtained using recombinant DNA techniques (see US Patent 4,816,567).

[0055] As used herein, the term "single-chain fragment variable (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker. The VL and VH domains pair to form a monovalent molecule by enabling them to produce linker pairs as a single polypeptide chain (see, e.g., Birdet et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0056] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0057] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0058] As used herein, a host cell refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0059] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10⁻⁵ M, for example less than about 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or less.

[0060] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies bind antigens with a dissociation equilibrium constant (KD) of less than about 10⁻⁵ M, for example, less than about 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or even smaller. KD can be determined using methods known to those skilled in the art.

[0061] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0062] As used herein, the terms "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier and / or excipient" refer to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0063] Beneficial effects

[0064] Von Willebrand factor (VWF) is a large multimeric glycoprotein synthesized by endothelial cells and platelets. After synthesis, VWF is linked by disulfide bonds to form a series of VWF multimers with different subunits, including low (L), medium (I), high (H), and ultra-large (UL) molecular weight forms, which are stored intracellularly or released into circulation. The metalloproteinase ADAMTS13 in peripheral blood further processes the UL-VWF multimer downstream, cleaving it to produce VWF multimers of different sizes.

[0065] VWF participates in primary hemostasis by binding to platelets. VWF multimer subunits contain binding sites for GPIb receptors on platelets; simultaneously, VWF can also bind to GPIIb / IIIa receptors on activated platelets. Under normal physiological conditions, circulating VWF multimers exist in a loosely coiled conformation, concealing their binding domains to platelet GPIb receptors and subendothelial collagen. Under vascular injury or high shear stress, the VWF conformation changes, exposing its binding sites for platelet GPIb and collagen, thereby binding to platelets and damaged vascular subendothelial tissue. The hemostatic function of VWF is closely related to multimer size; high molecular weight VWF multimers are most effective in supporting platelet adhesion and aggregation at sites of vascular injury and high shear stress. As the size of VWF multimers gradually decreases, VWF function is lost. The presence of VWF multimers and their hemostatic potential are closely controlled at different stages of VWF storage, release, and degradation. ADAMTS13 plays a crucial role in regulating the size of vascular endothelial cell (VWF) multimers, essential for normal hemostasis. Increased VWF synthesis and secretion due to various causes, along with absolute or relative ADAMTS13 deficiency, leads to the accumulation of ultra-large VWF multimers in plasma and on the surface of endothelial cells. This, in turn, induces spontaneous platelet aggregation and adhesion to the vascular endothelium, ultimately forming thrombi. This accumulation of ultra-large VWF multimers is associated with various diseases, such as thrombotic microangiopathy due to congenital or acquired ADAMTS13 deficiency, inflammation and inflammatory responses, and severe malaria infection. Evidence suggests that some clinical conditions involving microcirculatory thrombosis, such as acute myocardial infarction, stroke, sickle cell anemia, sepsis-induced disseminated intravascular coagulation, diabetic nephropathy, vascular dementia, Alzheimer's disease, and viral and bacterial infections, may also be associated with elevated levels of ultra-large VWF multimers.

[0066] The antibodies or antigen-binding fragments with anti-VWF effects described in this invention can act on VWF multimers, clearing ultra-large VWF multimers upon binding to VWF, thereby reducing VWF antigen and activity levels. Specifically, after applying the antibodies with anti-VWF effects of this invention, the level of circulating VWF antigen (VWF:Ag) is reduced, and VWF multimers, especially ultra-large VWF multimers, are reduced or eliminated in VWF multimer analysis. VWF biological activity measured by different detection methods is reduced. This includes, but is not limited to, VWF collagen-binding capacity (VWF:CB) and VWF platelet-binding capacity (restorin cofactor activity; VWF:RCo), VWF's ability to promote platelet aggregation, and its binding affinity to coagulation factor VIII (FVIII).

[0067] By applying the antibodies or antigen-binding fragments with anti-VWF effects described in this invention to interact with VWF polymers of different molecular weights, ultra-large VWF polymers can be cleared in vivo, reducing VWF antigen and activity levels. This effectively assists or replaces plasma exchange in the prevention and treatment of thrombotic microangiopathy (including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, atypical hemolytic uremic syndrome, and thrombotic microangiopathy associated with malaria, sepsis, and sickle cell disease) and cardiovascular and cerebrovascular diseases (including transient ischemic attacks). The following conditions are considered as potential risks: angina pectoris, unstable angina, cerebral infarction, myocardial infarction and peripheral artery occlusive disease, PTCA re-occlusion and coronary artery bypass graft occlusion, coronary valve replacement and essential thrombocytosis, deep vein thrombosis, pulmonary embolism, atrial fibrillation, coronary artery thrombosis, intracardiac thrombosis, postoperative thrombosis, cancer-induced thrombosis, cancer-related thrombin expression, infection, disseminated intravascular coagulation or arterial thrombosis-related thrombosis; neurological diseases (including Alzheimer's disease and vascular dementia) and bacterial and viral infections.

[0068] The application of drug combinations containing antibodies or antigen-binding fragments of the present invention that have an anti-VWF effect can clear ultra-large VWF multimers, reduce VWF antigen and activity levels, and improve the prognosis of patients with thrombotic microangiopathy, including thrombotic thrombocytopenic purpura. It also provides benefits to patients with stroke, myocardial infarction, atherosclerosis or other thrombotic diseases (such as sickle cell disease), as well as infectious diseases and neurological diseases.

[0069] By applying therapeutic doses of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention, ultra-large VWF multimers can be cleared, and VWF levels reduced, thus preventing and treating thrombotic microangiopathy (TMA) caused by thrombosis in the microvascular system. In patients with severe ADAMTS13 deficiency due to hereditary or acquired pathological factors, ultra-large VWF multimers (high molecular weight VWF multimers) accumulate due to reduced cleavage, leading to microthrombosis and inducing thrombotic thrombocytopenic purpura (TTP). The application of therapeutic doses of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention, used in combination with or alone with treatments such as plasma exchange, can clear large VWF multimers, reduce VWF levels, and decrease thrombus formation in the microcirculation. This prevents and treats organ dysfunction in patients with thrombotic thrombocytopenic purpura (TTP), such as thrombocytopenia, microangiopathic hemolytic anemia, fluctuating neurological signs, renal impairment, and fever. It also prevents and alleviates focal neurological deficits, seizures, and coma caused by microthrombi in TTP patients; and avoids vascular occlusion, tissue ischemia, organ failure, and death caused by microthrombi mediated by large VWF multimers. The application of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention can also reduce the need for plasma exchange in TMA treatment.

[0070] "Transplant-related microangiopathy" (TMA), or transplant-related microangiopathy, is a pathological condition specifically occurring during organ transplantation. TMA develops after organ transplantation and is often associated with immune rejection, donor-recipient mismatch, and drug toxicity. It is most common in kidney transplantation but can also occur in other transplant types such as liver and heart transplantation. TMA is a serious complication following kidney and other organ transplants, often manifesting as thrombocytopenia, hemolytic anemia, and acute renal failure. Timely reduction and discontinuation of nephrotoxic drugs and individualized immunomodulatory therapy are crucial. Early identification and treatment are of great significance in saving the transplanted kidney and the patient's life. The application of therapeutic doses of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention can clear ultra-large VWF multimers, reduce VWF levels, and also have a preventive and therapeutic effect on TMA.

[0071] Applying therapeutic doses of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention to clear ultra-large VWF multimers and reduce VWF levels can prevent and treat cardiovascular and cerebrovascular diseases. Pathophysiological evidence suggests that VWF is not only a biomarker of cardiovascular events but also a participant in the development and progression of disease. Various stimuli associated with acute ischemic syndrome, such as hypoxia, inflammatory cytokines, thrombin, and adrenaline, lead to the production and release of VWF by vascular endothelial cells. VWF is crucial for the initiation of platelet adhesion and subsequent aggregation under high shear rates found in arterial circulation, and due to its function in binding to chaperone proteins of subendothelial collagen and coagulation factor VIII, it can promote fibrin formation after arterial injury.

[0072] VWF is an independent risk factor for recurrent myocardial infarction in patients with angina pectoris, and the association between high VWF levels and the risk of myocardial infarction (MI) in patients with vascular disease has been well established. Applying the antibody or antigen-binding fragment with anti-VWF effect of this invention to clear ultra-large VWF multimers and reduce VWF levels will have a preventive and therapeutic effect on myocardial infarction. Platelet aggregation is the main pathophysiological process in the occurrence and progression of AMI. Currently available AMI treatment interventions include PCI, GPIIb / IIIa antagonism, or thrombolysis. Studies have shown that shear-induced platelet aggregation and VWF binding to platelets are significantly enhanced in the plasma of patients with acute myocardial infarction (AMI). Applying the antibody or antigen-binding fragment with anti-VWF effect of this invention to clear ultra-large VWF multimers and reduce VWF levels will reduce platelet aggregation caused by VWF binding to platelet receptors (GPIb and GPIIb / IIIa) and reduce coronary artery thrombosis in vivo, thereby achieving the goal of treating and preventing MI.

[0073] Percutaneous coronary intervention (PCI) is a treatment for acute coronary syndrome (ACS) and has become the preferred reperfusion therapy for patients with ST-segment elevation myocardial infarction (STEMI) or high-risk non-STEMI acute myocardial infarction. The application of antibodies or antigen-binding fragments with anti-VWF effects of this invention can clear VWF release from endothelial injury after PCI and stent implantation (multiple coronary stenting procedures), reducing VWF levels in the coronary circulation, thereby alleviating the prethrombotic state and abnormal myocardial perfusion in ACS patients, reducing infarct size, improving left ventricular ejection fraction, and increasing survival rate.

[0074] Ischemic stroke is a leading cause of death in humans. Studies on its pathogenesis have shown an association between high VWF levels and the risk of ischemic stroke. Low ADAMTS13 activity has also been revealed to be associated with the risk of ischemic stroke, and the mechanism is related to reduced cleavage of high molecular weight VWF. The accumulation of high molecular weight VWF polymers, which strongly promote platelet aggregation and thrombus formation, can enhance the prothrombotic state even in patients with lower levels than those with total thrombosis (TTP), promoting thrombosis locally at the site of vascular injury, especially at sites of high shear stress. The antibody or antigen-binding fragment of this invention with anti-VWF effect can reduce VWF levels by clearing high molecular weight VWF polymers, thereby achieving the effect of preventing and treating stroke.

[0075] Cerebral embolism caused by large artery atherosclerosis is a major cause of stroke. Data shows that the early risk of recurrent stroke is high after a minor stroke or transient ischemic attack (TIA), with large artery disease being the highest risk factor. Aspirin and similar drugs can reduce the risk of recurrent stroke, but cannot completely prevent recurrence in stroke patients. Other antiplatelet drugs, such as dipyridamole in combination or clopidogrel, also fail to prevent stroke recurrence. Therefore, stronger antiplatelet therapy regimens, especially early application after stroke or TIA, will help reduce the risk of stroke recurrence. During arterial thrombosis, platelets initially bind to the damaged arterial wall through a reversible interaction between the receptor glycoprotein (GPIb) and subendothelial collagen or VWF polymers on the surface of activated endothelial cells, subsequently activating and aggregating. Therefore, the interaction between platelet receptor GPIb and VWF constitutes a suitable intervention target for stroke prevention. The application of the antibody or antigen-binding fragment with anti-VWF effect of the present invention can reduce VWF levels by clearing high molecular weight VWF polymers, thereby inhibiting platelet aggregation activity mediated by GPIb binding to VWF, and thus reducing thrombus formation on activated atherosclerotic plaques and subsequent cerebral embolism.

[0076] VWF-mediated platelet aggregation and vascular inflammation play a crucial role in the development of atherosclerosis. In a mouse model of atherosclerosis, knocking out the VWF gene and reducing VWF levels significantly slowed and reduced the progression and severity of atherosclerosis. The application of antibodies or antigen-binding fragments with anti-VWF effects of this invention can reduce VWF levels by clearing high molecular weight VWF polymers, thereby preventing and slowing the progression of atherosclerosis and reducing the risk of arterial thrombotic diseases.

[0077] In bacterial infections such as Staphylococcus aureus and Streptococcus pneumoniae, VWF polymers interact with bacteria and mediate bacterial attachment to the endothelial surface in the bloodstream, promoting bacterial colonization, inflammation, and spread. VWF binding to bacteria can also prevent bacterial clearance through immune thrombosis. Intravascular VWF recruitment induces bacterial aggregate formation, leading to capillary occlusion and impaired blood supply. The antithrombotic agents of this invention, consisting of antibodies or antigen-binding fragments with anti-VWF effects, can be used to reduce VWF levels in the body, thereby preventing bacterial colonization and proliferation, promoting the clearance of infectious pathogens, and achieving the purpose of preventing and treating bacterial infectious diseases.

[0078] Higher VWF levels are associated with an increased risk of dementia. The combination of antibodies or antigen-binding fragments with anti-VWF effects of the present invention can be applied to reduce VWF levels in the body, thereby reducing the risk of dementia.

[0079] In the normal population, VWF levels range from 50 to 200 IU / dL. In patients with von Willebrand disease (VWD), VWF activity or antigen levels are decreased. In the most common type, VWD 1, plasma VWF levels are below 30 IU / dL, while individuals with low VWF levels have levels between 30 and 50 IU / dL, posing a risk factor for bleeding. By applying appropriate doses of the antibodies or antigen-binding fragments with anti-VWF effects described in this invention, VWF levels can be partially reduced. This achieves the therapeutic effect of VWF-mediated thrombotic diseases while maintaining the VWF levels necessary for normal physiological hemostasis, thereby avoiding bleeding risks similar to those experienced by VWD patients, such as excessive bleeding of the skin and mucous membranes, including menorrhagia, epistaxis, easy bruising, prolonged bleeding from minor wounds and the mouth, gastrointestinal bleeding, and bleeding during dental procedures, childbirth, and postoperative periods, or even severe muscle and joint bleeding.

[0080] Unlike nanobodies, IgG antibodies typically have a longer half-life. This is because of their larger molecular weight and their ability to specifically bind to FcRn. FcRn protects the antibody from lysosomal degradation; after being transported to the cell membrane surface with FcRn, the antibody loses its binding to FcRn and is released back into the plasma, thus prolonging its half-life. Since the typical half-life of IgG is approximately 23 days, the application of appropriate doses of the anti-VWF antibody described in this invention can reduce VWF levels and pathogenic hypermacromers for a longer period, thereby providing more effective and sustained treatment and prevention of VWF-mediated intravascular thrombotic diseases. Attached Figure Description

[0081] Figure 1 shows the binding of antibodies A / B with anti-VWF effect to VWF as determined by enzyme-linked immunosorbent assay (ELISA).

[0082] Figures 2A-F show the antigen levels (VWF:Ag) and activity levels (VWF:CB) in the supernatant after immunoprecipitation (IP) of purified VWF with antibodies A / B that have an anti-VWF effect, as well as the eluted VWF.

[0083] Figures 3A-F show the antigen levels (VWF:Ag) and activity levels (VWF:CB) of VWF remaining in the supernatant and eluted after immunoprecipitation (IP) of normal mixed plasma (NPP) with antibodies A / B that have an anti-VWF effect (VWF:Ag) and the activity levels evaluated by collagen binding capacity (VWF:CB).

[0084] Figures 4A-D show the analysis of VWF polymers remaining in the supernatant and eluted after immunoprecipitation (IP) of purified VWF from plasma and antibodies A / B with anti-VWF effect. Embodiments of the present invention

[0085] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0086] Example 1

[0087] This embodiment provides an antibody or antigen-binding fragment thereof with an anti-VWF effect, denoted as Antibody A with an anti-VWF effect. The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0088] The amino acid sequences of HCDR1 are shown in SEQ ID NO:5, HCDR2 in SEQ ID NO:6, and HCDR3 in SEQ ID NO:7; and

[0089] The amino acid sequences of LCDR1 are shown in SEQ ID NO:8, LCDR2 in SEQ ID NO:9, and LCDR3 in SEQ ID NO:10.

[0090] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0091] The nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0092] This embodiment provides another antibody or antigen-binding fragment thereof with an anti-VWF effect, denoted as Antibody B with an anti-VWF effect. The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0093] The amino acid sequence of HCDR1 is shown in SEQ ID NO:15, the amino acid sequence of HCDR2 is shown in SEQ ID NO:16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17.

[0094] The amino acid sequences of LCDR1 are shown in SEQ ID NO:18, LCDR2 in SEQ ID NO:19, and LCDR3 in SEQ ID NO:20.

[0095] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0096] The nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14.

[0097] Example 2

[0098] Enzyme-linked immunosorbent assay (ELISA) is used to measure the binding of antibodies with anti-VWF effects to VWF:

[0099] 1. In the double-antibody sandwich ELISA assay, antibodies A and B with anti-VWF effect and an equal volume of total human mixed antibody (immunoglobulin) separated from human mixed plasma using Protein A agarose gel were diluted to 10 μg / ml with carbonate coating buffer (50 mM carbonate buffer, pH 9.4). 50 μl was added to each well of an ELISA plate and the plate was incubated overnight at 4°C for antibody coating.

[0100] 2. Wash the antibody-coated 96-well plate three times with phosphate-buffered saline (PBST, pH 7.4) containing 0.05% TWEEN 20 to remove unbound antigens and impurities. Block each well with 100 μL of 1% BSA-PBS at 37°C for 1 h, and wash the 96-well plate twice with PBST.

[0101] 3. The von Willebrand factor (VWF) purified from human plasma was diluted to 0.075 IU / mL with 0.3% BSA-PBS and then serially diluted. The samples were added to 96-well plates according to the concentration gradient (50 μL per well), incubated at 37°C for 1 h, and the 96-well plates were washed 3 times with PBST to thoroughly wash away unbound VWF.

[0102] 4. Add 50 μL of rabbit anti-human VWF polyclonal antibody to each well of a 96-well plate and incubate at 37°C for 1 hour. Wash three times with PBST to thoroughly remove unbound antibody.

[0103] 5. Add 50 μL of horseradish peroxidase (HRP)-labeled goat anti-rabbit polyclonal antibody to each well and incubate at 37°C for 1 hour. Wash four times with PBST to thoroughly remove unbound enzyme-labeled antibody.

[0104] 6. Add 50 μL of TMB chromogenic solution to each well and react for 3 min. Stop the chromogenic reaction by adding TMB stop solution. Measure the absorbance at 450 nm using a microplate reader. Plot the absorbance (OD) of antibodies A and B coated with anti-VWF antibodies and equal volumes of mixed antibodies isolated from human mixed plasma after incubation with different concentrations of VWF against the VWF gradient concentration.

[0105] ELISA results (Figure 1) showed that antibodies A and B, coated with anti-VWF antibodies, could effectively bind to von Willebrand factor (VWF) molecules isolated from plasma, while mixed antibodies isolated from human blood under the same experimental conditions could not specifically bind to VWF.

[0106] Example 3

[0107] Immunoprecipitation (IP) measures the binding characteristics of antibodies with anti-VWF effects to VWF:

[0108] 1. The PBS buffer, 0.1M glycine solution (pH 2.8), and 1M Tris-HCl (pH 8.5) buffer required for the experiment were all filtered.

[0109] 2. Take an equal volume of 250 μL of thoroughly mixed Protein A agarose gel suspension and place it in a 1.5 mL EP tube. Centrifuge at 1000 rpm for 1 min and discard the supernatant. Add 400 μL of PBS buffer to resuspend the Protein A agarose gel, centrifuge at 1000 rpm for 1 min, discard the supernatant, and repeat twice. Dilute the recombinant anti-VWF effect monoclonal antibody to be tested to 400 μL with PBS buffer according to the required volume. Add solutions containing different concentrations of antibody to the above equilibrated Protein A agarose gel, resuspend thoroughly, and place in a rotary mixer at room temperature for 30 min to ligate the Protein A agarose gel with the antibody to be tested. After the ligation reaction is complete, centrifuge at 1000 rpm for 1 min and discard the supernatant. Add 400 μL of PBS buffer to resuspend the antibody-ligated Protein A agarose gel and wash it. Centrifuge at 1000 rpm for 1 min and discard the supernatant. Repeat twice. An equal volume of human mixed total antibodies (immunoglobulins) isolated from human mixed plasma using Protein A agarose gel electrophoresis and subjected to the same treatment was used as a control.

[0110] 3. Add 250 μL of normal plasma (NPP) or purified VWF from plasma to the antibody-conjugated Protein A agarose gel suspension. Gently pipette the antigen and antibody-conjugated Protein A agarose gel and incubate at room temperature in a rotary mixer for 1 hour to allow VWF and antibody to fully bind. Centrifuge the Protein A agarose gel containing VWF and antibody at 1000 rpm for 1 minute and collect the supernatant for analysis.

[0111] 4. Resuspend the Protein A agarose gel containing VWF and antibody in 400 μL of PBS buffer, centrifuge at 1000 rpm for 1 min, discard the supernatant, and repeat this operation twice. Combine the two 400 μL wash buffers for subsequent detection. Add 0.9 mL of 0.1 M glycine solution (pH 2.8), mix thoroughly, rotate on a rotary mixer for 10 min at room temperature, centrifuge at 1000 rpm for 1 min, and aspirate the supernatant to mix with 1 / 10 of the elution buffer volume (100 μL) of 1 M Tris-HCl (pH 8.5) buffer.

[0112] 5. The antigen level (VWF:Ag) and activity level (VWF:CB) assessed by collagen binding capacity were detected by ELISA at different stages of the immunoadsorption reaction. Rabbit anti-human VWF antibody (1:3000 dilution) and type III collagen (1:100 dilution) were added to 50 μL per well and incubated overnight at 4°C in 96-well ELISA plates. After washing three times with PBST, the plates were blocked with 1% BSA-PBS at 37°C for 1 h. The plates were then washed three times with PBST. Normal mixed plasma (NPP) was diluted 1:200 with PBS, and four serial dilutions were used to determine the VWF standard curve. The supernatant was diluted 1:200, and the eluent was diluted 1:50. 50 μL of each well was added and incubated at 37°C for 1 h. The plates were washed three times with PBST. 50 μL of HRP-labeled anti-human VWF polyclonal antibody was added and incubated at 37°C for 1 h. The plates were washed four times with PBST. Add 50 μL of TMB chromogenic solution to each well and react for 3 min. Stop the chromogenic reaction by adding TMB stop solution. Measure the absorbance at 450 nm using a microplate reader. Calculate the corresponding VWF antigen level (VWF:Ag) and activity level (VWF:CB) as evaluated by collagen binding capacity by comparing with a standard curve. Using the VWF level in the supernatant after incubating the test sample with uncoated Protein A agarose gel as 100%, calculate the relative amounts of residual and eluted VWF in the supernatant.

[0113] 6. VWF multimer electrophoresis analysis (low resolution: 0.9% stacking gel / 1.5% separating gel). Prepare a 1.5% separating gel (14 ml Minigel buffer + 0.21 g Seakem HGT Agarose) and a 0.9% stacking gel (5 ml Minigel buffer + 0.045 g Seakem HGT Agarose). Crosslink the protein A agarose gel with NPP / or plasma-derived VWF sample supernatant adsorbed with anti-VWF monoclonal antibody, dilute 2:8 with PBS buffer, dilute the elution buffer 8:2 with PBS buffer, add an equal volume of loading buffer, and incubate at 60°C for 20 min. Add 10 μL of sample to the sample wells of the electrophoresis gel, electrophores at 70-80 V for 25 min to press the sample into a single line, and then electrophores at 120 V for approximately 2.5 h. After removing the electrophoresis gel, wash with ddH2O for approximately 10 min; wash 3 times with TBS (10 min each time). Place the gel on a GelBond Film for Agarose gels and air dry overnight. Block with 5% milk at room temperature for 2 hours; wash twice with TBST-0.1% Tween (5 min / wash). Incubate with rabbit anti-human VWF primary antibody diluted 1:3000 with TBST for 2 hours, then incubate with goat anti-rabbit HRP-labeled secondary antibody diluted 1:5000 at room temperature for 1 hour. Wash 5 times with TBST-0.1% Tween (5 min / wash). Develop with ECL (500 μL of solution A + 500 μL of solution B, one whole membrane).

[0114] As shown in Figures 2 and 3, antibodies A and B, which exhibit anti-VWF effects, were cross-linked onto Protein A agarose gel and incubated with NPP or VWF purified from plasma. They specifically bound to VWF, causing VWF in the sample to be cleared as the antibody concentration increased (0, 75, 200, 400, 600, 800 ug / mL). Both the antigenic level of VWF (VWF:Ag) and the activity level (VWF:CB), assessed by collagen-binding ability, showed a gradient decrease. The control antibody, however, did not exhibit this effect. The amount of VWF bound to antibodies A and B, which are cross-linked with Protein A agarose gel and have an anti-VWF effect, increases with the increase of antibody concentration. The antigen level of VWF in the eluent (VWF:Ag) and the activity level evaluated by collagen binding ability (VWF:CB) both show a gradient increase, while the control antibody does not have this effect (Figures 2A, B, C and 3A, B, C show the VWF antigen levels in the supernatant and eluent after incubation of different concentrations of antibodies A and B with anti-VWF effect and control antibody on Protein A agarose gel with purified VWF and NPP from plasma; Figures 2D, E, F and 3D, E, F show the VWF activity levels in the supernatant and eluent after incubation of different concentrations of antibodies A and B with anti-VWF effect and control antibody on Protein A agarose gel with purified VWF and NPP from plasma with purified VWF and NPP).

[0115] Because antibodies A and B, which have an anti-VWF effect, specifically bind to VWF after being cross-linked onto Protein A agarose gel and incubated with VWF purified from plasma, VWF in the sample is cleared as the antibody concentration increases (0, 75, 200, 400, 600, 800 ug / mL), as shown in Figure 4. In the multimer electrophoresis, the number of VWF multimers of different molecular weights remaining in the supernatant is reduced, but the reduction is more significant for large molecular weight multimers (Figure 4A and C are the analysis of VWF multimers remaining in the supernatant after incubation of antibodies A and B, which have an anti-VWF effect, on Protein A agarose gel with VWF purified from plasma, respectively; the antibody concentrations of bands 1-6 are 0, 75, 200, 400, 600, and 800 ug / mL, respectively; and bands 7 and 8 are the results after incubation with control antibodies of 75 and 800 ug / mL, respectively). Conversely, in the eluent after incubation, the number of VWF polymers of different molecular weights gradually increased in the polymer electrophoresis (Figures 4B and D show the analysis of VWF polymers in the eluent after incubation of antibodies A and B with anti-VWF effect on Protein A agarose gel with VWF purified from plasma; bands 1-6 show antibody concentrations of 0, 75, 200, 400, 600, and 800 μg / mL, respectively; bands 7 and 8 show the results after incubation with control antibodies of 75 and 800 μg / mL, respectively). Therefore, the antibody with anti-VWF effect of the present invention can specifically bind to VWF and clear circulating VWF in a concentration-dependent manner, thereby reducing VWF levels and preventing and treating thrombotic diseases (such as TTP caused by ultra-high molecular weight VWF polymers) caused by elevated VWF levels or activity.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof having an anti-factor VIII effect, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region in the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 7; and the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

10.

2. The monoclonal antibody or antigen-binding fragment thereof having anti-factor VIII effect according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

2.

3. A monoclonal antibody or antigen-binding fragment thereof having an anti-factor VIII effect, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region in the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 15, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 16, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 17, and the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 18, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 19, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

20.

4. The monoclonal antibody or antigen-binding fragment thereof having anti-factor VIII effect according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

12.

5. The monoclonal antibody or antigen-binding fragment thereof having anti-factor VIII effects according to any one of claims 1 to 4, characterized in that: The monoclonal antibody with anti-Factor VIII effect is an antibody of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secreted IgA, IgD or IgE type; the light chain constant region of the monoclonal antibody with anti-Factor VIII effect is a constant region of kappa type and lambda type light chain.

6. The monoclonal antibody or antigen-binding fragment thereof having anti-factor VIII effects according to any one of claims 1 to 4, characterized in that: The monoclonal antibody with anti-Factor VIII effect or the antigen binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, complementarity determining region fragment or single chain antibody.

7. The monoclonal antibody or antigen-binding fragment thereof having anti-factor VIII effects according to any one of claims 1 to 4, characterized in that: The monoclonal antibody with anti-Factor VIII effect or the antigen binding fragment thereof comprises a non-CDR region, and the non-CDR region is from a human antibody.

8. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody with anti-Factor VIII effect or the antigen binding fragment thereof in claim 1 or 2 is as shown in SEQ ID NO:

3.

9. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody with anti-Factor VIII effect or the antigen binding fragment thereof in claim 1 or 2 is as shown in SEQ ID NO:

4.

10. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the heavy chain variable region amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof having anti-von Willebrand factor effect according to claim 3 or 4 is shown as SEQ ID NO:

13.

11. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the light chain variable region amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof having anti-von Willebrand factor effect according to claim 3 or 4 is shown as SEQ ID NO:

14.

12. A recombinant vector comprising the nucleic acid molecule of any one of claims 8-11.

13. A host cell comprising the nucleic acid molecule of any one of claims 8-11 or the recombinant vector of claim 12.

14. An antibody having at least 90% sequence identity to the amino acid sequence of the heavy chain variable region or the light chain variable region of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12 in the antibodies according to claims 2 and 4 and having von Willebrand factor VWF binding activity.

15. A pharmaceutical composition comprising an effective amount of the monoclonal antibody or antigen-binding fragment thereof having anti-von Willebrand factor effect according to any one of claims 1-4.

16. The pharmaceutical composition of claim 15, wherein: one or more pharmaceutically acceptable adjuvants.

17. Use of a monoclonal antibody or antigen-binding fragment thereof having an anti- von Willebrand Factor effect according to any one of claims 1 to 4 for the manufacture of a medicament for reducing the level of VWF, characterized in that: The composition reduces the components of various molecular weight VWF multimers including ultra-high molecular weight, high molecular weight VWF, and reduces the VWF antigen and activity levels.

18. Use of the monoclonal antibody or antigen-binding fragment thereof having an anti- factor VIII effect according to any one of claims 1 to 4 for the treatment and prevention of thrombotic microangiopathy, cardiovascular and cerebrovascular diseases, neurological diseases and infectious diseases, characterized in that: The prevention and treatment include, but are not limited to, thrombotic microangiopathy caused by various reasons; congenital ADAMTS13 deficiency and acquired ADAMTS13 deficiency; cardiovascular and cerebrovascular diseases; neurological diseases and bacterial and viral infectious diseases.

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