Humanized Anti-glycoprotein ib alpha (gpibalpha) antibody

Abstract

To provide an improved therapeutic agent targeting a platelet GPIb-IX-V complex which would not cause platelet activation, platelet destruction, thrombocytopenia, or significant bleeding complications.SOLUTION: Provided are humanized antibodies specifically recognizing platelet glycoprotein I(b)α (GPIbα). The humanized antibodies lack a Fc portion, and can prevent platelet activation, aggregation, and / or growth of thrombus. The humanized antibodies lack the ability to activate platelets, lack the ability to induce thrombocytopenia, and / or at therapeutic doses, lack the ability to prolong bleeding time.SELECTED DRAWING: Figure 18

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS AND SEQUENCE LISTING) This application claims priority to U.S. Provisional Application No. 62 / 946,086, filed December 10, 2019, which is incorporated herein by reference in its entirety. A sequence listing associated with this application is provided in text format and is incorporated herein by reference. The filename for the text containing the sequence listing is "PCT_-_Sequence_listing_as_filed". The text file is 81.2 Ko, was created and submitted electronically on December 9, 2020.

[0002] (Technical field) The present disclosure relates to humanized antibodies that specifically recognize and bind to platelet glycoprotein I(b)α (GPIbα), and protein constructs comprising same, and related therapeutic uses. [Background technology]

[0003] Platelet adhesion and aggregation at the site of atherosclerotic rupture in coronary or cerebral arteries is usually the key event in acute thrombosis. Therefore, antiplatelet therapy is one of the important treatments to reduce cardiovascular death, including: (i) cyclooxygenase inhibitors such as aspirin, (ii) platelet P2Y12 receptor antagonists such as clopidogrel, prasugrel, and ticagrelor, (iii) αIIbβ3 antagonists such as abciximab, eptifibatide, and tirofiban, and (iv) PAR1 antagonists such as vorapaxar, etc. However, limitations of current antiplatelet therapies such as delayed / weak / poor inhibition of platelet function, excessive bleeding complications, thrombocytopenia, and unexpected platelet activation are major concerns in promoting therapeutic progress. In particular, with regard to acute ischemic stroke, treatment is very limited due to the potential neurotoxicity of current antithrombotic / thrombolytic agents (e.g., recombinant tissue plasminogen activator (tPA)) and / or the risk of intracranial hemorrhage, as well as for patients who have passed the intravenous thrombolytic treatment time window, when it is not available.

[0004] Platelet GPIb-IX-V complex has emerged as a promising antiplatelet target. GPIb-IX-V complex is the key platelet receptor in initiating platelet adhesion and migration to the injured vessel wall, especially at high shear. Platelet adhesion / migration to the subendothelium is mediated by binding of GPIbα subunits to von Willebrand factor (VWF) anchored / immobilized to the injured vessel wall. VWF is a multimeric adhesive blood protein secreted by activated endothelial cells and platelets. GPIbα-VWF binding then triggers a signaling process that results in the release of platelet agonists such as thromboxane A2 and ADP as well as activation of platelet αIIbβ3 integrin, leading to platelet aggregation via αIIbβ3 binding to fibrinogen, VWF, etc. Under high shear conditions, GPIbα-VWF interactions are necessary for the pathological growth (both platelet adhesion and platelet aggregation / adhesion) of occlusive thrombi at sites of arterial stenosis, where blood flows at wall shear rates that can exceed 10,000–40,000 s. On the other hand, under low shear conditions (such as most cases of hemostasis), platelet adhesion can be directly mediated by αIIbβ3-fibrinogen / fibrin and α2β1 / GPVI-collagen interactions, among others. Thus, pharmacological inhibition of GPIbα may lower the risk of systemic bleeding and improve safety compared to other antiplatelet drugs that do not specifically target thrombosis at high shear. GPIbα has been shown to be important for leukocyte recruitment under thromboinflammatory conditions, such as acute ischemic stroke. Furthermore, ischemia-reperfusion of previously hypoxic brain regions (e.g., by thrombolysis or thrombus removal) may increase the proinflammatory function of platelets via GPIbα, which may further promote thromboinflammatory neurological disorders and infarct growth. Furthermore, GPIbα appears to be specifically expressed in platelets and megakaryocytes. Thus, direct platelet GPIbα antagonists have great potential to be developed as effective and safe antiplatelet agents for the treatment of acute thrombotic events such as heart attacks and strokes.

[0005] In particular, novel antiplatelet strategies targeting GPIbα-VWF interactions have been shown as effective therapies to treat acquired thrombotic thrombocytopenic purpura (aTTP), a thrombotic microangiopathy and a life-threatening condition with high mortality if untreated. As autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13), a VWF-cleaving protease that cleaves / reduces the multimer size of VWF, result in severe deficiency of ADAMTS13 activity. These super-large VWF are hyper-adhesive that cause the formation of platelet (GPIbα)-VWF microthrombi in blood vessels, leading to organ ischemia and infarction, reduced platelet counts, and destruction of red blood cells. Therefore, blocking the interaction between VWF and platelet GPIbα may prevent the development of acute TTP by achieving a more rapid normalization of platelet counts and reducing thromboembolic events.

[0006] The nanobody caplacizumab, targeting the VWF A1 domain, had been approved for the treatment of adults experiencing an acute episode of aTTP in combination with plasma exchange (PEX) and immunosuppression for a minimum of 30 days after cessation of daily PEX. However, adverse events related to bleeding were more common with caplacizumab (65% vs. 48%), as were serious bleeding events (11% vs. 1%). Another barrier for caplacizumab was the enormous cost of the drug. The current price of caplacizumab in 2020 is over 8,000 USD for a treatment regimen that recommends daily administration for 30 days after the last plasma exchange with the possibility of continued treatment until restoration of ADAMTS13 activity. Since VWF is consistently released from the activated endothelium, the relatively stable levels of GPIbα on platelets, with a short life span of 7–10 days in humans, seems to be a more attractive and potent target for TTP therapy.

[0007] Patent Document 1 describes a GPIbα-binding snake C-type lectin (snaclec) antiplatelet thrombolytic drug purified from the venom of the snake "Deinagkistrodon acutus". Antiplatelet thrombolysin inhibited ristocetin-induced human platelet aggregation and thrombosis by blocking GPIb-VWF interaction without significantly altering bleeding time or coagulation. Antiplatelet thrombolysin is currently being evaluated in phase II clinical trials in patients with acquired thrombotic thrombocytopenic purpura and ST-segment elevation myocardial infarction. However, it is known that foreign snake proteins may induce immune responses and generate anti-drug antibodies to neutralize the drug and eliminate the therapeutic effect. Furthermore, these antibodies may cause allergic reactions or immune complex formation that may damage the kidneys and joints (arthritis). Furthermore, the necessary re-administration may stimulate memory immune cells and enhance such immune responses and immune responses.

[0008] Patent document 2 describes recombinant GPG-290 or GPIb-290 / 2V-immunoglobulin (Ig) fusion polypeptide, a soluble chimeric protein containing 290 extracellular amino acids of mutant GPIbα N-terminal (G233V and M239V) linked via proline to the Fc fragment of human IgG1. GPG-290 competed with platelet GPIbα for binding to VWF and showed 14-fold higher affinity for VWF compared to wild-type GPIbα. In animal models, GPG-290 dose-dependently prolonged the time to coronary artery occlusion and also inhibited platelet aggregation, thrombosis, and recurrent reduction in coronary circulation without prolonging bleeding time at doses ranging from 50 to 100 μg / kg. However, the higher dose tested (500 μg / kg) induced a 3- to 4-fold increase in bleeding time, presumably due to GPG-290 binding to α-thrombin with high affinity, rendering α-thrombin unavailable for hemostasis.

[0009] Patent document 3 further describes GPIb-290 / 2V / FFFIg variant fusion proteins (Y276F, Y288F, and / or Y297F), which show limited / low affinity binding to α-thrombin, 50% reduced efficacy in suppressing recurrent coronary thrombosis (i.e., inhibiting recurrent coronary circulation loss), and also show prolonged tail bleeding time and increased ADP closure time as assessed by Platelet Function Analyzer-100 (PFA-100) compared to GPIb-290. However, these GPIb-Ig fusion proteins are high molecular weight chimeric proteins (approximately 130 kDa) and target mainly subendothelial immobilized VWF or plasma VWF at high shear rate conditions. Thus, the amount and accessibility of these fusion proteins are less predictable and may be a potential limitation with high amounts of product to be injected. Another limitation may be the risk of anti-drug antibody generation. GPIb-Ig fusion protein is a bioengineered chimeric protein. Both GPIbα polypeptide and Fc fragment are derived from human genes, which can minimize antigenicity, but the mutations of GPIbα variants and the joint region between GPIbα and Fc moieties may generate neoepitopes that induce immune responses. In addition, the fusion protein can generate some conformational neoepitopes that induce anti-drug antibody production.

[0010] The art also described neutralizing monoclonal antibodies against human GPIbα (anti-GPIbα mAbs). However, the majority of currently available anti-GPIbα mAbs are of mouse origin and thus may trigger human anti-mouse responses in clinical use. Furthermore, intact anti-GPIbα mAbs often lead to platelet activation, likely because binding of intact mAbs induces platelet GPIbα-mediated signaling, which may unexpectedly exacerbate platelet aggregation and thrombosis. Furthermore, intact anti-GPIbα mAbs binding to platelets may trigger both Fc-dependent and Fc-independent platelet clearance, leading to thrombocytopenia (i.e., a drop in platelet count). Thus, intact anti-GPIbα mAbs show very limited therapeutic potential.

[0011] Patent document 4 describes the Fab fragment of the chimeric antibody chSZ2, a chimeric mAb against human GPIbα that dose-dependently inhibits ristocetin-induced platelet aggregation in vitro. However, since it is a chimeric antibody that retains the variable region of the mouse antibody and replaces the constant region with a human one, immunogenicity remains a major concern. Importantly, the in vivo function of chSZ2 to prevent / treat thrombotic diseases has not been demonstrated due to the lack of animal models and the possible inability to recognize GPIbα from other animal species as known in the art.

[0012] Patent document 5 describes a murine mAb raised against purified human GPIbα, another Fab fragment of humanized 6B4 (h6B4-Fab). h6B4-Fab reduces or completely abolishes the reduction in circulatory flow in stenosed femoral arteries in baboons. However, the antithrombotic effect of h6B4-Fab was accompanied by prolonged bleeding times. Furthermore, these anti-GPIbα antibodies, together with their corresponding Fab fragments, were generated in wild-type mice (i.e., immunized with human GPIbα) using conventional techniques. These anti-GPIbα antibodies cannot recognize mouse GPIbα (mouse and human GPIbα have significant homology), and there is a limited repertoire of antibodies raised against epitopes present in human GPIbα but absent in mouse GPIbα. Therefore, these mAbs cannot be analyzed and evaluated in rodents or other animal species for significant preclinical pharmacology, toxicology, and pharmacokinetic studies. Whether h6B4-Fab could cause platelet activation has also been of concern, since its precursor mAb6B4 can apparently cause platelet activation and severe thrombocytopenia.

[0013] Thus, there is a need for improved therapeutic agents that target the platelet GPIb-IX-V complex without causing platelet activation, platelet destruction, thrombocytopenia, or serious bleeding complications. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Chinese Patent No. 103263662 [Patent Document 2] U.S. Patent No. 7,049,128 [Patent Document 3] U.S. Patent No. 7,727,535 [Patent Document 4] Chinese Patent No. 102988983 [Patent Document 5] U.S. Patent No. 7,332,162 Summary of the Invention [Means for solving the problem]

[0015] The present disclosure relates to humanized antibodies and protein constructs comprising same that specifically recognize glycoprotein I(b)α (GPIbα) and associated therapeutic uses thereof. The humanized antibodies have the ability to prevent platelet activation, aggregation, thrombus growth (especially at high shear), lack the ability to activate platelets (e.g., do not activate platelets), lack the ability to induce thrombocytopenia, and / or lack the ability to prolong bleeding time at therapeutic doses.

[0016] According to a first embodiment, the present disclosure provides a humanized antibody that specifically recognizes glycoprotein I(b)α (GPIbα). The humanized antibody lacks an Fc receptor portion. The humanized antibody has the ability to prevent platelet activation, aggregation, and / or thrombus propagation, lacks the ability to activate platelets, lacks the ability to induce thrombocytopenia, and / or lacks the ability to prolong bleeding time at therapeutic doses. According to one embodiment, the humanized antibody has the ability to recognize human GPIbα, mouse GPIbα, dog GPIbα, rat GPIbα, rabbit GPIbα, and / or monkey GPIbα. According to another embodiment, the humanized antibody is an antibody fragment. In one example, the antibody is a F(ab) 2 The antibody fragment is a fragment thereof. For example, the antibody fragment is a Fab antibody fragment. According to another embodiment, the antibody fragment is a single chain variable fragment (scFv). According to one embodiment, the humanized antibody has a heavy chain. In some embodiments, the heavy chain comprises a first CDR having an amino acid sequence of GFTFSSFAMS (SEQ ID NO: 37), a variant thereof or a fragment thereof, a second CDR having an amino acid sequence of SITSAGTPYYPDSVLG (SEQ ID NO: 38), a variant thereof or a fragment thereof, and / or a third CDR having an amino acid sequence of SRGYEDYFDY (SEQ ID NO: 39), a variant thereof or a fragment thereof. In yet another embodiment, the heavy chain comprises a first CDR having an amino acid sequence of human IgG 1 It further comprises the CH1 region of an antibody, for example human IgG 1The CH1 region of the antibody has the amino acid sequence of SEQ ID NO: 40, 47, 54 or 61, a variant thereof or a fragment thereof. In one embodiment, the heavy chain has the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, a variant thereof or a fragment thereof. In another embodiment, the humanized monoclonal antibody has a light chain. In some embodiments, the light chain comprises a first CDR having the amino acid sequence of KSSQSLLNSRNQKNYLA (SEQ ID NO: 65), a variant thereof or a fragment thereof, a second CDR having the amino acid sequence of FTSTRES (SEQ ID NO: 66), a variant thereof or a fragment thereof, and / or a third CDR having the amino acid sequence of QQHYSSPWT (SEQ ID NO: 67), a variant thereof or a fragment thereof. In some embodiments, the light chain further comprises a kappa chain C region of a human IgG1 antibody. In some additional embodiments, the kappa chain C region has the amino acid sequence of SEQ ID NO: 68, 75, 82 or 89, a variant thereof or a fragment thereof. In a further embodiment, the light chain has the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85, a variant thereof or a fragment thereof.In some embodiments, the humanized antibody is selected from the group consisting of a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof; a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 71, a variant or fragment thereof, a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 78, a variant or fragment thereof, a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 85, a variant or fragment thereof, a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof, a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 71, a variant or fragment thereof, a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 78, a variant or fragment thereof, a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 85, a variant or fragment thereof. or a fragment thereof, a heavy chain of SEQ ID NO:50, a variant or a fragment thereof, and a light chain of SEQ ID NO:64, a variant or a fragment thereof, a heavy chain of SEQ ID NO:50, a variant or a fragment thereof, and a light chain of SEQ ID NO:71, a variant or a fragment thereof, a heavy chain of SEQ ID NO:50, a variant or a fragment thereof, and a light chain of SEQ ID NO:78, a variant or a fragment thereof, a heavy chain of SEQ ID NO:50, a variant or a fragment thereof, and a light chain of SEQ ID NO:85, a variant or a fragment thereof, a heavy chain of SEQ ID NO:57, a variant or a fragment thereof, and a light chain of SEQ ID NO:64, a variant or a fragment thereof, a heavy chain of SEQ ID NO:57, a variant or a fragment thereof, and a light chain of SEQ ID NO:71, a variant or a fragment thereof, a heavy chain of SEQ ID NO:57, a variant or a fragment thereof, and a light chain of SEQ ID NO:78, a variant or a fragment thereof, or a heavy chain of SEQ ID NO:57, a variant or a fragment thereof, and a light chain of SEQ ID NO:85, a variant or a fragment thereof.

[0017] According to a second embodiment, the present disclosure provides a chimeric protein comprising a humanized antibody described herein and a carrier protein.

[0018] According to a third embodiment, the present disclosure provides a pharmaceutical composition comprising (i) the humanized antibody or the chimeric protein, and (ii) a pharmaceutical excipient.

[0019] According to a fourth embodiment, the present disclosure provides a method for preventing or limiting an interaction between glycoprotein I(b)α (GPIbα) present on platelets and a GPIbα ligand (e.g., von Willebrand factor (VWF), kininogen, P-selectin, thrombin spondin, etc.). The method comprises contacting a humanized antibody described herein, a chimeric protein described herein, or a pharmaceutical composition described herein with a platelet. In some embodiments, the method is for preventing or limiting activation of the platelet. In one embodiment, the GPIbα ligand is von Willebrand factor (VWF) and / or thrombin. In one specific embodiment, the humanized antibody, chimeric protein, or pharmaceutical composition is contacted with the platelet before, simultaneously with, or after the GPIbα ligand is contacted with the platelet. In a further embodiment, the method is for preventing or limiting an interaction in vivo in a subject. In another embodiment, the method is for preventing the formation or growth of a thrombus in a subject. In another embodiment, the method is for reducing the size of a thrombus or the number of thrombus in a subject. In some embodiments, the method further comprises determining the presence, location, and / or size of a thrombus in the subject. In yet another embodiment, the subject is at risk of experiencing or has experienced pathological thrombosis. In yet another embodiment, the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation. In some embodiments, the method is for reducing or limiting tumor metastasis in a subject in need thereof. In a further embodiment, the tumor metastasis is hepatic tumor metastasis. In yet another embodiment, the method further comprises determining the presence, location, and / or size of tumor metastasis in the subject.

[0020] According to a fifth embodiment, the present disclosure provides a pharmaceutical composition for preventing or limiting the interaction between a humanized antibody described herein, a chimeric protein described herein, or a glycoprotein I(b)α (GPIbα) present on platelets and von Willebrand factor (VWF) and / or thrombin and other GPIbα ligands. The present disclosure also provides the use of a humanized antibody described herein, a chimeric protein described herein, or a pharmaceutical composition in the manufacture of a medicament for preventing or limiting the interaction between a glycoprotein I(b)α (GPIbα) present on platelets and von Willebrand factor (VWF) and / or thrombin and other GPIbα ligands. The contacting step can occur under low or high shear rates. In some embodiments, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing or limiting platelet activation. In one specific embodiment, the humanized antibody, chimeric protein, or pharmaceutical composition is for contacting the platelets before, simultaneously with, or after VWF and / or thrombin and other GPIbα ligands contact the platelets. In further embodiments, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing or limiting in vivo interactions in a subject in need thereof. In another embodiment, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing the formation or growth of thrombi in a subject in need thereof. In another embodiment, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for reducing thrombi size or number of thrombi in a subject in need thereof. In some embodiments, the presence, location, and / or size of thrombi has been pre-determined in the subject. In yet another embodiment, the subject is at risk of experiencing or has experienced pathological thrombosis.In yet another embodiment, the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation. In some embodiments, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for reducing or limiting tumor metastasis in a subject in need thereof. In a further embodiment, the tumor metastasis is hepatic tumor metastasis. In yet another embodiment, the presence, location, and / or size of tumor metastasis is predetermined in the subject.

[0021] According to a sixth embodiment, the present disclosure provides a humanized antibody as described herein, a chimeric protein as described herein, or a pharmaceutical composition for preventing or limiting an interaction between glycoprotein I(b)α (GPIbα) present on platelets and von Willebrand factor (VWF) and / or thrombin. In some embodiments, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing or limiting platelet activation. In one specific embodiment, the humanized antibody, chimeric protein, or pharmaceutical composition is for contacting platelets before, simultaneously with, or after VWF and / or thrombin and other GPIbα ligands contact the platelets. In a particular embodiment, the humanized antibody, chimeric protein, or pharmaceutical composition is for contacting platelets at low or high shear rates. In a further embodiment, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing or limiting an interaction in vivo in a subject in need thereof. In another embodiment, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for preventing the formation or growth of a thrombus in a subject in need thereof. In another embodiment, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for reducing the size or number of thrombi in a subject in need thereof. In some embodiments, the presence, location, and / or size of thrombi has been previously determined in the subject. In yet another embodiment, the subject is at risk of experiencing or has experienced pathological thrombosis. In yet another embodiment, the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation. In some embodiments, the humanized monoclonal antibody, chimeric protein, or pharmaceutical composition is for reducing or limiting tumor metastasis in a subject in need thereof. In a further embodiment, the tumor metastasis is hepatic tumor metastasis.In yet another embodiment, the presence, location, and / or size of tumor metastases has been previously determined in the subject. [Brief description of the drawings]

[0022] Having thus generally described the nature of the invention, preferred embodiments thereof will now be shown, by way of example only, with reference to the accompanying drawings in which:

[0023] [Figure 1] SDS-PAGE of humanized Fab in the supernatant under non-reducing conditions. Different combinations of heavy and light chains are shown above the gel. A molecular weight ladder (KDa) is shown on the left. Arrows point to the humanized Fab. Bovine serum albumin (BSA) was used as a control. [Diagram 2] Western blot results of humanized Fab under non-reducing conditions are shown. Approximately 20 μL of supernatant was loaded per lane. A molecular weight ladder (KDa) is shown on the left. A heavy chain only (HCAb) antibody was used as a control. [Diagram 3] Figures 3A through 3H show the results of SDS-PAGE under non-reducing (labeled "N") and reducing (labeled "R") conditions of purified Fab, which respectively contain: Figure 3A, VH1 and VL2 chains; Figure 3B, VH1 and VL3 chains; Figure 3C, VH2 and VL1 chains; Figure 3D, VH3 and VL2 chains; Figure 3E, VH3 and VL3 chains; Figure 3F, VH4 and VL1 chains; Figure 3G, VH4 and VL2 chains; Figure 3H, VH4 and VL3 chains. [Figure 4] Figures 4A and B show that humanized Fabs H001-H008 (Figure 4A) bind to wild-type mouse platelets but (Figure 4B) do not bind to GPIbα- / - mouse platelets (5 μg / mL). The "*" shown in Figure 4B indicates a control signal. [Diagram 5]Figures 5A-E show that purified Fabs H001 (△) and H002 (▽) bind to (Figure 5A) mouse, (Figure 5B) dog, (Figure 5C) human, (Figure 5D) rat, and (Figure 5E) rabbit platelets. (Note: In the original text, "△" and "▽" are filled in black.) The platelet-binding humanized Fabs were tested in vitro by flow cytometry assay. [Figure 6] Flow cytometry results of purified Fabs H001 and H002 binding to monkey platelets are shown. [Figure 7] In surface plasmon resonance (SPR) assay, purified FabH001 antibody is shown to bind to recombinant GPIbα. Figure 7A shows SPR data of 25 μL injection of 500, 100, 50, and 10 nM H001, and fits to a kinetic binding model, giving ka (on-rate) = 2.61 × 107 s-1, kd (off-rate) = 1.1 × 10-1 s-1, and kd (dissociation or association constant) = 4.4 nM. Figure 7B shows the dose-response curve of SPR response of 25 μL injection of 500, 100, 50, and 10 nM purified FabH001 plotted against ligand concentration. Fitting the curve to a one-site ligand binding model gives R2 = 0.9929 and Kd = 8.0 ± 2.1 nM. [Figure 8] Figures 8A-D show standard aggregometry traces demonstrating that purified Fabs (Figure 8A) H001, (Figure 8B) H002, (Figure 8C) H005, and (Figure 8D) H008 did not induce platelet activation in platelet-rich plasma. [Figure 9] Figures 9A to D show standard aggregometry traces showing that purified Fab (Figures 9A and 9D) H001 and (Figures 9B, 9C, and 9D) H002 inhibited platelet aggregation induced by ristocetin (A, B, and D) or low dose thrombin (C). Platelets were obtained from healthy volunteers (A to C) or patients with peripheral vascular disease (D). [Figure 10]Figures 10A-D show that humanized Fab H001 (Figures 10A and 10B) and H002 (Figures 10C and 10D) antibodies inhibited clot formation from human whole blood under both low shear (300 s-, Figures 10A and 10C) and high shear (1800 s-, Figures 10B and 10D) conditions. Figure 10A shows representative photographs showing platelet clot formation after 1, 2, and 3 min perfusion of heparinized human whole blood and treatment with control PBS buffer (top panel) and humanized Fab H001 antibody (5 μg / mL, bottom panel) under low shear (300 s-) conditions. Figure 10B shows representative photographs showing platelet thrombus formation after perfusion of heparinized human whole blood for 1, 2, and 3 minutes and treatment with control PBS buffer (top panel) and humanized Fab H001 antibody (2.5 μg / mL, middle panel, and 5 μg / mL, bottom panel) at high shear rate (1800 s-) conditions. Figure 10C shows representative photographs showing platelet thrombus formation after perfusion of heparinized human whole blood for 1, 2, and 3 minutes and treatment with control PBS buffer (top panel) and humanized Fab H002 antibody (5 μg / mL, bottom panel) at low shear rate (300 s-) conditions. FIG. 10D shows representative photographs demonstrating platelet thrombus formation after perfusion of heparinized human whole blood for 1, 2, and 3 min and treatment with control PBS buffer (top panel) and humanized Fab H002 antibody (2.5 μg / mL, middle panel, and 5 μg / mL, bottom panel) at high shear rate (1800 s-) conditions. [Figure 11] Figures 11A and B show that humanized Fab H001 and H002 antibodies prolonged vascular occlusion time in an in vivo FeCl3-induced mesenteric arteriolar thrombosis model. Figure 11A is a histogram showing the time to occlusion (in minutes) for the antibody or dose used. Figure 11B is a representative photograph of arterioles treated with control (upper panel), humanized Fab H001 antibody (5 μg / mouse, middle panel), and humanized Fab H002 antibody (5 μg / mouse, lower panel) at different times after FeCl3-induced vascular injury. *P<0.05, **P<0.01. [Figure 12]Figures 12A-C show that humanized Fab H001 and H002 antibodies inhibited thrombus formation in a laser-induced cremaster arteriolar thrombosis model in vivo. Figure 12A is a histogram showing platelet mean fluorescence intensity (MFI; shaded areas indicate SD) with respect to time at laser injury when animals received control treatment (top) or H001 antibody (bottom, 5 μg dose). Figure 12B is a histogram showing platelet mean fluorescence intensity (MFI) with respect to time at laser injury when animals received control treatment (top) or H002 antibody (bottom, 5 μg dose). Figure 12C is a histogram showing platelet mean fluorescence intensity (MFI) with respect to time at laser injury when animals received control treatment (top) or H002 antibody (bottom, 10 μg dose) 24 hours prior to injury. [Figure 13] Figures 13A-C show that while injected humanized Fab H001 was able to bind to platelets in vivo, it did not induce an increase in the expression of P-selectin or phosphatidylserine (PS). The results are shown as flow cytometry results for (Figure 13A) platelets, (Figure 13B) P-selectin, and (Figure 13C) phosphatidylserine. [Figure 14] Figures 14A and B show that humanized Fab H001 and H002 antibodies prevented or prolonged vascular occlusion in an in vivo FeCl3-induced carotid artery thrombosis model. Figure 14A is a representative photograph showing mouse carotid artery flow (mL / min) when animals were treated with control (upper panel), Fab H001 antibody (middle panel, 10 μg dose), or Fab H002 antibody (lower panel, 10 μg dose) 5 min prior to injury. Arrows indicate time of vascular occlusion. Figure 14B is a histogram showing time to vascular occlusion (in minutes) for the antibody or dose used. * P<0.05, # P<0.05, ** P<0.01. [Figure 15]Figures 15A-C show that humanized Fabs H001 and H002 dramatically reduced ischemic brain infarct size without increasing the risk of intracerebral hemorrhage in a mouse model of cerebral ischemia and reperfusion injury (transient middle cerebral artery occlusion (tMCAO) model). Figure 15A is a histogram showing the ischemic brain infarct area for the antibody or dose used immediately after induction of tMCAO. Figure 15B is a histogram showing the ischemic brain infarct area for humanized Fab H002 (100 μg dose) treatment 1 hour after tMCAO. Figure 15C shows representative photographs of multiple 2 mm thick coronal brain sections cut from whole brains 24 hours after tMCAO induction in sham (no filament inserted) control group, or in control groups treated with PBS control (200 μL) or treatment groups treated with humanized Fab H001 antibody (100 μg / mouse) or H002 antibody (100 μg / mouse and 50 μg / mouse), respectively, immediately after tMCAO. White areas indicate infarcted brains. *P<0.05, **P<0.01. [Figure 16]Figures 16A-B show that prophylactic treatment of ADAMTS13- / - mice with humanized Fab H001 or humanized C100-scFv fused to human albumin (C100-scFv-HSA) effectively suppressed ionophore-induced VWF-mediated microvascular thrombosis in a mouse model of TTP. Figure 16A shows representative photographs showing the accumulation of platelet thrombi in ADAMTS13- / - mice with or without H001 or C100-scFv-HAS treatment. ADAMTS13- / - mice were injected with fluorescently labeled platelets from mice of the same genotype, and mesenteric vessels were exposed and treated with calcium ionophore to induce VWF secretion. Platelet accumulation in the vessels was monitored by microscopy. Serial images were taken at the indicated times after application of calcium ionophore or prophylactic treatment of H001 or C100-scFv-HSA in ADAMTS13- / - mice (control). Figure 16B is a histogram showing the number of emboli (platelet thrombi >20 μm in diameter) in ionophore-induced ULVWF-mediated microvascular thrombosis in a mouse model of TTP. Figure 16C is a histogram showing the time to recovery of normal blood flow for the antibody or dose used. *P<0.05, **P<0.01. [Figure 17] Humanized Fab H001 and H002 antibodies did not induce thrombocytopenia. Results are shown as time (in hours) and % change in platelet count with respect to antibody treatment: IVIG (○), NIT-B1 (◆), H001 (△) or H002 (▽). (Note: In the original text, "▽" is blackened.) [Figure 18] Figure 2 shows that humanized Fab H001 and H002 antibodies did not prolong bleeding time, whereas murine NIT-B significantly increased bleeding time. Results are shown as bleeding time (in minutes) for treatment and dose (shown below the x-axis). [Figure 19]Figures 19A-D show that humanized C100-scFv and humanized C100-scFv fused to human albumin (C100-scFv-HSA) bind to (Figure 19A) wild-type mouse platelets, (Figure 19B) but not to GPIBA- / - mouse platelets, and (Figure 19C) human platelets at the indicated doses. The "*" in Figures 19A and C indicates the control signal. [Figure 20] 1 shows standard aggregometry traces demonstrating that humanized C100-scFv inhibited ristocetin-induced platelet aggregation. [Figure 21] FIG. 1 shows standard aggregometry traces demonstrating that humanized C100-scFv did not induce platelet activation in platelet-rich plasma. [Figure 22] Figure 1 shows that humanized C100-scFv-HSA inhibited clot formation from human whole blood at high shear (1200s-) rate conditions. Representative photographs showing platelet clot formation after 1, 2, and 3 min perfusion of heparinized human whole blood, which was treated with control PBS buffer (upper panel) and humanized C100-scFv-HSA (10 μg / mL, lower panel) at high shear (1200s-) conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (Anti-GPIbα antibody) US Patent No. 8,323,652 describes that mouse NITmAbs (NIT-A1, NIT-B1, and NIT-F1), generated by immunizing GPIbα-deficient BALB / c mice with wild-type platelets, could specifically recognize both human and mouse GPIbα and significantly inhibited ristocetin-induced platelet aggregation and thrombus formation. However, as shown in the following examples, these intact mAbs can induce severe thrombocytopenia. Moreover, because they are of murine origin, they are immunogenic in humans.

[0025] The present disclosure provides specific antibodies against GPIbα polypeptides. An antibody is considered to be "specific" for a GPIbα polypeptide because it has a higher affinity for the GPIbα polypeptide than for other polypeptides (e.g., other platelet surface polypeptides). The antibodies of the present disclosure can recognize and bind to human GPIbα polypeptides (described in Gene ID:2811), mouse GPIbα polypeptides (described in Gene ID:110331805 and Gene ID:110304274), rat GPIbα polypeptides (as described in Gene ID:691992), monkey GPIbα polypeptides (Gene ID:721584), dog GPIbα polypeptides (Gene ID:403638), and / or rabbit GPIbα polypeptides (Gene ID:100349951). According to one embodiment, the antibodies of the present disclosure are capable of recognizing and binding to human GPIbα polypeptide (described in Gene ID:2811), mouse GPIbα polypeptide (described in Gene ID:110331805 and Gene ID:110304274), rat GPIbα polypeptide (described in Gene ID:691992), monkey GPIbα polypeptide (Gene ID:721584), dog GPIbα polypeptide (Gene ID:403638), and / or rabbit GPIbα polypeptide (Gene ID:100349951).

[0026] In one embodiment, the humanized antibodies of the present disclosure have a dissociation constant (K D In some embodiments, the dissociation constant of the humanized antibody with human GP1bα (K D In some embodiments, the dissociation constant (K) of the humanized antibody with human GP1bα is 9, 8, 7, 6, 5, 4, 3, 2, 1 μM or less. D In some embodiments, the dissociation constant (K) of the humanized antibody with human GP1bα is 9, 8, 7, 6, 5, 4, 3, 2, 1 nM or less. D) is 9, 8, 7, 6, 5, 4, 3, 2, 1 pM or less.

[0027] The antibodies of the present disclosure are "humanized" antibodies because they contain both portions derived from human antibodies or immunoglobulins and portions derived from non-human antibodies or immunoglobulins. Humanizing an antibody involves replacing portions of a non-human antibody with the corresponding portions of a human antibody. For example, a humanized antibody as used herein may contain a non-human variable region (e.g., a region derived from a murine (e.g., mouse) antibody) capable of specifically recognizing GPIbα and a human framework region derived from a human antibody. In another example, a humanized immunoglobulin may contain a heavy chain and a light chain, in which the light chain contains one or more complementarity determining regions (or CDRs) derived from an antibody of non-human origin that binds to a GPIbα polypeptide and a framework region (or FR) derived from a light chain of human origin. And the heavy chain contains a complementarity determining region derived from an antibody of non-human origin that binds to a GPIbα polypeptide and a framework region derived from a heavy chain of human origin. A "complementarity determining region" or "CDR" refers to a region of an immunoglobulin that is located in the variable portion of a polypeptide and specifically binds an epitope. The combination of the CDRs constitutes the paratope of the antibody.

[0028] The human regions of the humanized antibody may be derived from an IgG, IgM, IgA, IgE, or IgD isotype. In some embodiments, the human regions of the humanized antibody may be derived from an IgG isotype, e.g., an IgG1, IgG2, IgG3, or IgG4 subclass. In some specific embodiments, the human regions of the humanized antibody may be derived from an IgG1 subclass. As shown below, because humanized antibodies are monovalent antibodies, the human regions of the humanized antibody may be derived from the CH 1 Area and / or V H CH from the region (excluding CDR) 2 and / or C.H. 3 The human region of a humanized antibody may be a heavy chain that does not contain a C region. L Area and / or V LThe human region of the humanized antibody may comprise a light chain derived from the region (excluding the CDRs). The human region of the humanized antibody comprises a light chain that may be of the kappa or lambda type. In one specific embodiment, the human region of the humanized antibody comprises a light chain derived from the kappa type.

[0029] The humanized antibodies of the present disclosure do not comprise (e.g., lack) an Fc portion. For example, a humanized antibody portion may comprise a fragment of a multivalent antibody antigen-binding region, F(ab) 2 F(ab) 2 The fragment is a dimer of two molecular entities (a light chain fragment and a heavy chain fragment), which is composed of a single antigen-binding site and contains one constant domain and one variable domain from each of the heavy and light chains of the antibody, which are linked together by disulfide bonds. F(ab) 2 Each chain of three V L Domains and the three Vs H Contains the domain F(ab) 2 The antibody portion may be fully or partially glycosylated when compared to the parent multivalent antibody.

[0030] In some embodiments, the antibodies of the present disclosure are "monovalent" antibodies. As used in the context of this disclosure, a "monovalent" antibody comprises a single antigen-binding site. A monovalent antibody moiety comprises no more than one associated (covalently or not) variable light domain (V L ) and up to one corresponding variable heavy domain (V H ) which has at least two antigen binding sites and multiple V H and multiple V's L The monovalent antibody portion is different from the multivalent full-length antibody containing the domain. The monovalent antibody portion may be fully or partially glycosylated when compared to the parent multivalent antibody from which it can be derived. In some cases, the monovalent antibody portion is not glycosylated. The monovalent antibody portion has the ability to compete for the binding site recognized by the corresponding multivalent antibody (e.g., NIT-B1 in some embodiments). The monovalent antibody portion does not include the crystallizable fragment (Fc fragment) of the multivalent antibody from which it is derived.

[0031] In some cases, the monovalent antibody is a single chain variable fragment (scFv) derived from one or more multivalent antibodies. An scFv is a single molecular entity (fusion protein) consisting of a single antigen-binding region, connected to a linker (usually a short peptide linker) and connected to the V from the multivalent antibody. H Domain and V L Therefore, scFv consists of a single antigen-binding region and has one V H Domain and one V L The scFvs can be obtained, for example, from screening a synthetic library of scFvs, such as a phage display library of scFvs. The scFvs of the present disclosure include, for example, the V H Domain and V L In some embodiments, one or more GGGGS (SEQ ID NO: 92) linkers may be included between the V domains. L The carboxy terminus of the domain is V H In another embodiment, the V H The carboxy terminus of the domain is V L The scFv may be linked to the amino terminus of the domain. In some embodiments, the scFvs of the present disclosure may include a purification tag (such as a 6X His tag) that can be removed when the scFv is purified. In some additional embodiments, the scFv may be (covalently) linked to a carrier protein to form a chimeric protein. In such embodiments, the carrier protein may be linked at the amino or carboxy terminus of the scFv. In some embodiments, the scFv does not include a purification tag or has been treated to remove the purification tag.

[0032] In another example, a monovalent antibody is a fragment antigen-binding region (Fab) of a multivalent (or, in some embodiments, monoclonal) antibody. A Fab fragment contains two molecular entities (a light chain fragment and a heavy chain fragment), consists of a single antigen-binding site, and contains one constant domain and one variable domain from each of the heavy and light chains of an antibody bound to each other by disulfide bonds. A Fab contains a single VL Domains and a single V H Includes the domain.

[0033] In a further example, the monovalent antibody is a single domain antibody or nanobody. Single domain antibodies comprise a single monomeric variable antibody domain that comprises at least three complementarity determining regions (CDRs). Single domain antibodies are derived from camelids (V H H antibodies, etc.), fish (V NAR Single domain antibodies can be obtained from humanized or phage display. Single domain antibodies can be derived from heavy or light chains. Single domain antibodies can be humanized.

[0034] The antibodies of the present disclosure may have the ability to prevent platelet activation and aggregation. The phrase "having the ability to prevent platelet activation and aggregation" refers to the ability of the humanized antibodies of the present disclosure to avoid platelet activation and platelet aggregation in the presence of platelets and platelet agonists. Platelet activation occurs primarily at the initiation of hemostasis or thrombosis. Upon activation, platelets will change their shape and release the contents of their granules. Activated platelets modulate their expression of membrane proteins (e.g., P-selectin), lipids (e.g., phosphatidylserine), and conformational changes in platelet αIIbβ3 integrin that result in platelet aggregation. Platelet activation and aggregation can be measured, for example, by determining the shape of the platelets, the level of platelet aggregation (e.g., using an aggregometer), the expression of surface proteins or lipids, and the like. Platelets can be activated with the following agonists (activators), thrombin, ADP, collagen, and the like. Ristocetin can also bind von Willebrand factor to the platelet receptor GPIbα. To determine whether a humanized antibody prevents platelet activation and aggregation, platelets (e.g., obtained in the form of platelet-rich plasma or gel-filtered platelets) can first be contacted with a humanized antibody and then with an agonist. Then, it is necessary to determine whether the platelets are activated / aggregated by methods known in the art. Antibodies that prevent platelet activation and aggregation are considered to be antibodies of the present disclosure.

[0035] Furthermore, the humanized antibody of the present disclosure can lack the ability to induce platelet activation. The phrase "lacking the ability to induce platelet activation" refers to one of the characteristics of the humanized antibody of the present disclosure, namely, not activating platelets in the absence of a known platelet agonist. To determine whether a humanized antibody lacks the ability to induce platelet activation, platelets (which can be obtained, for example, in the form of platelet-rich plasma or gel-filtered platelets) can be placed in contact with the antibody (in the absence of a known platelet agonist), and then whether the platelets are activated should be determined by methods known in the art. An antibody that cannot induce platelet activation is considered to be an antibody of the present disclosure.

[0036] The antibody of the present disclosure may lack the ability to induce thrombocytopenia. The phrase "lacking the ability to induce thrombocytopenia" refers to one of the characteristics of the humanized antibody of the present disclosure, namely, not causing a substantial and pathological deficiency in the total number of platelets. In humans, less than 50,000 platelets per μL in the blood results in thrombocytopenia requiring emergency treatment. To determine whether a humanized antibody lacks the ability to induce thrombocytopenia, it is administered to a test subject (e.g., a mouse) and the level of platelets is monitored using techniques known in the art to determine whether the antibody causes a decrease in the number of platelets (if so, a substantial or pathological decrease in the number of platelets). An antibody that does not induce thrombocytopenia is considered to be an antibody of the present disclosure.

[0037] The antibodies of the present disclosure may lack the ability to prolong bleeding time (at therapeutic doses). The phrase "lacking the ability to prolong bleeding time" refers to one of the properties of the humanized antibodies of the present disclosure, namely, that they do not substantially and pathologically increase the time it takes to stop bleeding. To determine whether a humanized antibody lacks the ability to prolong bleeding time, a cut (standardized width and depth) is made on the tail of a subject (e.g., a mouse) and the time it takes for bleeding to stop (e.g., the minimum time for blood flow to stop) is determined using techniques known in the art (in some embodiments, the Ivy or Duke method) to see whether the antibody causes an increase in bleeding time (if so, a substantial increase in bleeding time) compared to the standard. Antibodies that fail to prolong bleeding time at the indicated doses are considered to be antibodies of the present disclosure.

[0038] The antibodies of the present disclosure may also have the ability to antagonize the biological activity of GPIbα polypeptides, which are platelet surface membrane glycoproteins that function as receptors for von Willebrand factor (VWF), thrombin, and other ligands. Thus, by antagonizing their biological activity, the antibodies of the present disclosure may be used to limit or prevent platelet activation and aggregation (especially under high shear conditions).

[0039] The antibodies of the present disclosure may be derived from monoclonal antibodies. An antibody specific for a single epitope on a GPIbα polypeptide is considered a monoclonal antibody (also called mAb). In some embodiments, a monoclonal antibody is produced from a single clone of immune cells. Monoclonal antibodies can be produced by techniques known in the art, such as by using cell culture by fusing myeloma cells with spleen cells from a subject (such as a mouse or human) immunized with an antigen that contains an epitope of a GPIbα polypeptide. Monoclonal antibodies can also be obtained by phage display, by screening a library of monoclonal antibodies with an antigen that contains an epitope of a GPIbα polypeptide. Further techniques for making monoclonal antibodies include, but are not limited to, single B cell culture, amplification of a single cell from a population of B cells. The monoclonal antibodies of the present disclosure can be derived from various sources (e.g., mouse or human) and can contain two identical light chains and two identical heavy chains, each chain containing three CDRs. Monoclonal antibodies may be made of any isotype, including, but not limited to, immunoglobulin A (IgA), IgD, IgE, IgG (including subtypes IgG1, IgG2, IgG3, or IgG4), or IgM. In one embodiment, monoclonal antibodies may be made of the IgG isotype.

[0040] In one embodiment, an antibody of the present disclosure has at least one complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, a variant thereof or a fragment thereof. In the context of the present disclosure, particularly when referring to the amino acid sequence of a CDR, the term "consisting essentially of" indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, but that additional non-essential amino acid residues may be added to the amino or carboxyl termini of these sequences (as long as these amino acid residues do not substantially alter the affinity of the antibody for or its ability to antagonize the biological activity of the GPIbα polypeptide).

[0041] In one embodiment, an antibody of the present disclosure has at least two complementarity determining regions that comprise or consist essentially of the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, a variant thereof or a fragment thereof. In yet another embodiment, an antibody of the present disclosure has at least three complementarity determining regions that comprise or consist essentially of the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, a variant thereof or a fragment thereof. In yet another embodiment, an antibody of the present disclosure has at least four complementarity determining regions that comprise or consist essentially of the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, a variant thereof or a fragment thereof. In yet another embodiment, an antibody of the present disclosure has at least five complementarity determining regions that comprise or consist essentially of the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67, a variant thereof or a fragment thereof. In yet another embodiment, the antibodies of the disclosure have complementarity determining regions comprising or consisting essentially of the amino acid sequences of SEQ ID NOs: 37, 38, 39, 65, 66 and 67, variants thereof or fragments thereof.

[0042] In some embodiments, the antibodies of the disclosure have a complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 37, 38, and 39 (including variants and fragments), and at least one complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 65, 66, or 67 (including variants and fragments). In some additional embodiments, the antibodies of the disclosure have a complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 37, 38, and 39 (including variants and fragments), and at least two complementarity determining regions that comprise or consist essentially of the amino acid sequence of SEQ ID NO: 65, 66, or 67 (including variants and fragments). In some further embodiments, the antibodies of the disclosure have a complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 65, 66, or 67 (including variants and fragments), and at least one complementarity determining region that comprises or consists essentially of the amino acid sequence of SEQ ID NO: 37, 38, and 39 (including variants and fragments). In some additional embodiments, the antibodies of the disclosure have a complementarity determining region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66 or 67 (including variants and fragments), and at least two complementarity determining regions comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 37, 38 and 39 (including variants and fragments).

[0043] The antibodies of the present disclosure may include functional variants of CDRs having the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67. A variant CDR comprises at least one amino acid difference compared to the amino acid sequence of the CDR. As used herein, variant refers to a change in amino acid sequence that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for GPIbα polypeptide). In some embodiments, the overall charge, structure, or hydrophobic / hydrophilic properties of the antibody can be altered without adversely affecting the biological activity. Thus, the amino acid sequence of the CDR can be altered, for example, to make the antibody more hydrophobic or hydrophilic without adversely affecting the biological activity of the antibody. A CDR variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs described herein. As known in the art, the term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences determined by comparing the sequences. The level of identity can be conventionally determined using known computer programs. Identity can be easily calculated by known methods, including but not limited to those described below.Computational Molecular Biology (Lesk, A.M., ed., Oxford University Press, New York, 1988), Biocomputing: Informatics and Genome Projects (Smith, D.W., ed., Academic Press, New York, 1993), Computer Analysis of Sequence Data, Part I (Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994), Sequence Analysis in Molecular Biology (von Heinje, G., ed., Academic Press, 1987), and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are written into publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the Megalign program of the "LASERGENE" bioinformatics computing suite (DNASTAR Inc., Madison, Wisconsin). Multiple alignments of the sequences disclosed herein were performed using the Clustal alignment method (Higgins and Sharp, 1989, CABIOS, 5:151-153) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5.

[0044] A CDR variant may be (i) one in which one or more amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), which may or may not be one encoded by the genetic code, or (ii) one in which one or more amino acid residues include a substituent group. A "variant" of a CDR may be a conservative variant or an allelic variant.

[0045] The antibodies of the present disclosure may comprise functional fragments of CDRs having the amino acid sequence of SEQ ID NO: 37, 38, 39, 65, 66 or 67. A fragment of a CDR comprises at least one less amino acid residue compared to the amino acid sequence of the CDR. A CDR fragment comprises several consecutive amino acid residues of the amino acid sequence of the CDR of SEQ ID NO: 37, 38, 39, 65, 66 or 67. A CDR fragment has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a CDR described herein.

[0046] In another embodiment, an antibody of the disclosure comprises a heavy chain, wherein the heavy chain comprises at least one CDR comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 37, 38 or 39, functional variants thereof and functional fragments thereof. In a further embodiment, the heavy chain comprises at least two CDRs comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 37, 38 or 39, functional variants thereof and functional fragments thereof. In yet another embodiment, the heavy chain comprises at least three CDRs comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 37, 38 or 39, functional variants thereof and functional fragments thereof.

[0047] In another embodiment, the heavy chain comprises a CH1 region of a human IgG1 antibody and comprises the amino acid sequence of SEQ ID NO: 40, 47, 54 or 61, functional variants thereof, and functional fragments thereof. As used in the context of this disclosure, a functional variant of a CH1 region of a human IgG1 antibody refers to an amino acid sequence change that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional variant of a CH1 region of a human IgG1 antibody has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a CH1 region described herein (such as having the amino acid sequence of SEQ ID NO: 40, 47, 54 or 61). As used in the context of the present disclosure, a functional fragment of a CH1 region of a human IgG1 antibody refers to a fragment that contains at least one less amino acid residue compared to the amino acid sequence of the CH1 region of a human IgG1 antibody so as not to adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional fragment of a CH1 region of a human IgG1 antibody has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a CH1 region described herein (e.g., having the amino acid sequence of SEQ ID NO: 40, 47, 54 or 61).

[0048] In some embodiments, the heavy chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, functional variants thereof and functional fragments thereof. In the context of this disclosure, particularly when referring to the amino acid sequence of the heavy chain, the phrase "consists essentially of" indicates that the heavy chain necessarily comprises the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, but additional non-essential amino acid residues may be added to the amino or carboxyl termini of these sequences (as long as these amino acid residues do not substantially alter the affinity of the antibody for the GPIbα polypeptide or its ability to antagonize the biological activity of the GPIbα polypeptide). As used in the context of this disclosure, a functional variant of a heavy chain antibody refers to an amino acid sequence change that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for the GPIbα polypeptide). In one embodiment, a functional variant of a heavy chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain described herein (such as having an amino acid sequence of SEQ ID NO: 36, 43, 50 or 57). As also used in the context of this disclosure, a functional fragment of a heavy chain contains at least one fewer amino acid residue compared to the amino acid sequence of the heavy chain that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional fragment of a heavy chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain described herein (such as having the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57).

[0049] In another embodiment, the antibody comprises a light chain, wherein the light chain comprises at least one CDR comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66 or 67, functional variants thereof and functional fragments thereof. In a further embodiment, the light chain comprises at least two CDRs comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66 or 67, functional variants thereof and functional fragments thereof. In yet another embodiment, the light chain comprises at least three CDRs comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66 or 67, functional variants thereof and functional fragments thereof.

[0050] In another embodiment, the light chain comprises a human IgG1 kappa chain C region, which may have, for example, the amino acid sequence of SEQ ID NO: 68, 75, 82, or 89, a variant thereof, or a fragment thereof. As used in the context of this disclosure, a functional variant of a human IgG1 kappa chain C region refers to an amino acid sequence change that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional variant of a human IgG1 kappa chain C region has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a human IgG1 kappa chain C region described herein (e.g., having the amino acid sequence of SEQ ID NO: 68, 75, 82, or 89). As used in the context of this disclosure, a functional fragment of a human IgG1 kappa chain C region refers to a fragment that contains at least one less amino acid residue compared to the amino acid sequence of a human IgG1 kappa chain C region so as not to adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional fragment of a human IgG1 kappa chain C region has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% 97%, 98% or 99% identity to a CH1 region described herein (e.g., having the amino acid sequence of SEQ ID NO: 68, 75, 82 or 89).

[0051] In another embodiment, the light chain comprises or essentially consists of the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85, functional variants thereof and functional fragments thereof. In the context of the present disclosure, particularly when referring to the amino acid sequence of the light chain, the phrase "essentially consists of" indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85, but additional non-essential amino acid residues may be added to the amino or carboxyl termini of these sequences (as long as these amino acid residues do not substantially alter the affinity of the antibody for the GPIbα polypeptide or its ability to antagonize the biological activity of the GPIbα polypeptide). As used in the context of the present disclosure, a functional variant of a light chain antibody refers to an amino acid sequence change that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for the GPIbα polypeptide). In one embodiment, a functional variant of a light chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a light chain described herein (e.g., having an amino acid sequence of SEQ ID NO: 64, 71, 78 or 85). As used in the context of this disclosure, a functional fragment of a light chain contains at least one less amino acid residue compared to the amino acid sequence of the heavy chain so as not to adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional fragment of a light chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a light chain described herein (such as having the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85).

[0052] In some embodiments, the heavy chain comprises or essentially consists of the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, functional variants thereof and functional fragments thereof. In the context of this disclosure, particularly when referring to the amino acid sequence of the heavy chain, the term "essentially consists of" indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, but additional non-essential amino acid residues may be added to the amino or carboxyl termini of these sequences (as long as these amino acid residues do not substantially alter the affinity of the antibody for the GPIbα polypeptide or its ability to antagonize the biological activity of the GPIbα polypeptide). As used in the context of this disclosure, a functional variant of a heavy chain antibody refers to an amino acid sequence change that does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for the GPIbα polypeptide). In one embodiment, a functional variant of a heavy chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain described herein (e.g., having an amino acid sequence of SEQ ID NO: 36, 43, 50 or 57). As used in the context of this disclosure, a functional fragment of a heavy chain contains at least one fewer amino acid residue compared to the amino acid sequence of the heavy chain so as not to adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, a functional fragment of a heavy chain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain described herein (such as having the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57).

[0053] In yet another embodiment, the antibody may comprise both a heavy chain and a light chain. In such an embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof. In another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 71, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 78, a variant or fragment thereof. In another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 36, a variant or fragment thereof, and a light chain of SEQ ID NO: 85, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 71, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 78, a variant or fragment thereof. In a further embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 43, a variant or fragment thereof, and a light chain of SEQ ID NO: 85, a variant or fragment thereof. In a further embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 50, a variant or fragment thereof, and a light chain of SEQ ID NO: 64, a variant or fragment thereof. In a further embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 50, a variant or fragment thereof, and a light chain of SEQ ID NO: 71, a variant or fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 50, a variant or fragment thereof, and a light chain of SEQ ID NO: 78, a variant or fragment thereof.In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof. In one embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof. In yet another embodiment, the humanized antibody may have a heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof.

[0054] The heavy and light chains of the antibodies of the present disclosure may include a leader sequence that is cleaved upon secretion from a cell. For example, the amino acid sequence of SEQ ID NO: 35 includes the amino acid sequence of SEQ ID NO: 36 and additional amino acid residues at the N-terminus that act as a leader sequence. Leader sequences that can be included in the heavy and / or light chains include, but are not limited to, the amino acid sequence of SEQ ID NO: 91.

[0055] In some embodiments, the antibodies of the present disclosure may be further modified or engineered into chimeric proteins (including carrier proteins) to, among other things, increase their circulating half-life. The humanized antibody portion may be attached to a carrier (e.g., directly or indirectly through a linker, such as one or more GGGGS (SEQ ID NO: 92) linkers) at any amino acid residue(s), provided that this attachment does not prevent the humanized antibody portion from binding to GPIbα and inhibiting its biological activity. In one embodiment, the linker comprises three copies of the GGGGS (SEQ ID NO: 92) linker. In one embodiment, the linker comprises four copies of the GGGGS (SEQ ID NO: 92) linker. In some examples, the linker (if present) or carrier is associated with one or more amino acid residues of the humanized antibody portion and is not involved in the specific binding to GPIbα and inhibition of its biological activity. In some examples, the linker or carrier is associated with a single amino acid residue of the humanized antibody portion. The linker or carrier can be attached to any amino acid residue of the humanized antibody portion, including the amino acid residue located at the amino terminus of the humanized antibody portion or the carboxyl terminus of the humanized antibody portion. In some embodiments, the carrier protein can be located upstream (amino terminus) or downstream (carboxy terminus) of the humanized antibody portion. When the linker and carrier are proteinaceous, the humanized antibody portion can be attached to any amino acid residue of the linker or carrier, including the amino acid residue located at the amino terminus of the linker or carrier or the carboxyl terminus of the linker or carrier. In one embodiment, the amino acid residue located at the amino terminus of the linker or carrier is attached to the amino acid residue located at the carboxyl terminus of the humanized antibody portion. In yet another embodiment, when a linker is present and is proteinaceous, its amino terminus is attached to the carboxyl terminus of the humanized antibody and its carboxyl terminus is attached to the amino terminus of the carrier. In one embodiment, the carrier protein is albumin (e.g., human serum albumin). In one embodiment, the carrier comprises one or more additional antibodies or antibody fragments.

[0056] If a covalent bond is required between the humanized antibody portion and the carrier, the bond between the two entities can be a peptide bond. Such an embodiment is particularly useful for chimeric proteins in which at least two entities are both proteinaceous and are intended to be produced as a fusion protein in an organism (prokaryotic or eukaryotic) using recombinant gene technology. Alternatively, the covalent bond between the two portions may be mediated by other types of chemical covalent bonds. In some examples, the chimeric protein is designed to be less susceptible to cleavage into two portions in the systemic circulation (e.g., in plasma).

[0057] As indicated above, the bond between the two entities (e.g., the humanized antibody portion and the carrier portion) can be non-covalent. Exemplary non-covalent bonds include, but are not limited to, the biotin-streptavidin / avidin system. In such systems, a label (biotin) is covalently attached to one entity / moiety and a protein (streptavidin or biotin) is covalently attached to the other entity / moiety. In such embodiments, biotin can be attached to the humanized antibody portion or the carrier, provided that the other entity in the system is attached to streptavidin or avidin.

[0058] In a further system of non-covalent binding, the first entity is designed to be non-covalently bound to the second entity only upon its administration to the intended recipient. This embodiment is particularly useful when the carrier is a protein present in the blood of the recipient. For example, the humanized antibody portion can be linked (in a covalent or non-covalent manner) to a second antibody, lectin or fragment thereof (herein referred to as an antibody-derived linker) that has the ability to non-covalently bind to the carrier once administered to the intended recipient. For example, the second antibody, lectin or fragment thereof can be specific for any blood / plasma protein present in the intended recipient (e.g., serum albumin, immunoglobulin fragments (provided that these fragments do not directly bind to activating Fc receptors or cause the chimeric protein to bind to multiple sites of activating Fc receptors simultaneously), alpha-1-acid glycoprotein, transferrin, or lipoproteins). The second antibody, lectin or fragment thereof can be attached to the humanized antibody portion, preferably covalently, at any amino acid residue of the humanized antibody portion, preferably at the amino or carboxyl terminus of the humanized antibody portion. In such an embodiment, the second antibody, lectin or fragment thereof is similar to a linker between the humanized antibody portion and the carrier. Upon administration of the humanized antibody portion of this embodiment to a recipient, the carrier (e.g., blood or plasma protein) binds to the second antibody, lectin or fragment thereof to form a chimeric protein in the body. In one specific embodiment, the second antibody is an antibody that specifically recognizes albumin (e.g., an antibody that specifically recognizes human albumin).

[0059] The present disclosure also provides nucleotide molecules encoding the antibodies described herein. The nucleotide molecules can be provided in isolated form and can be derived from a variety of sources, including DNA, cDNA, synthetic DNA, synthetic RNA, derivatives, mimetics, or combinations thereof. Such sequences can include genomic DNA, which may or may not include naturally occurring introns, genic regions, non-genic regions, and regulatory regions. Furthermore, such genomic DNA can be obtained in association with promoter regions or poly(A) sequences. The sequences, genomic DNA, or complementary DNA (cDNA) can be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from cells and used to generate cDNA by reverse transcription or other means. The nucleotide molecules described herein are used in specific embodiments of the methods of the present disclosure for the production of RNA, proteins, or polypeptides via incorporation into a host cell, tissue, or organism. In one embodiment, the nucleotide molecules can be codon-optimized for expression in a particular host. The nucleotide molecule may, in some embodiments, include one or more promoter sequences and / or one or more terminator sequences. The nucleotide molecule may be included in an expression vector in a recombinant host. The nucleotide molecule of the present disclosure may, in some embodiments, include the nucleic acid sequence of SEQ ID NO: 41, 48, 55, 62, 69, 76, 83, and / or 90. In one embodiment, the nucleotide sequence of the present disclosure includes the nucleic acid sequence of SEQ ID NO: 41 and 69, 41 and 76, 41 and 83, or 41 and 90. In another embodiment, the nucleotide sequence of the present disclosure includes the nucleic acid sequence of SEQ ID NO: 48 and 69, 48 and 76, 48 and 83, or 48 and 90. In another embodiment, the nucleotide sequence of the present disclosure includes the nucleic acid sequence of SEQ ID NO: 55 and 69, 55 and 76, 55 and 83, or 55 and 90. In yet another embodiment, the nucleotide sequence of the present disclosure includes the nucleic acid sequence of SEQ ID NO: 62 and 69, 62 and 76, 62 and 83, or 62 and 90.

[0060] Therapeutic Uses of Humanized Antibodies Since the interaction of platelet GPIbα with its ligands (such as VWF) is recognized as a key player in the pathogenesis of various diseases, the humanized antibodies can be used to prevent and / or treat ischemic stroke, acute myocardial infarction, restenosis, angina, acute coronary syndrome, atherothrombosis, vascular inflammation, venous thrombosis, peripheral vascular disease, thrombotic thrombocytopenic purpura, sepsis and / or tumor metastasis. The humanized antibodies or chimeric proteins can be used in subjects having platelets that are specifically recognized by the humanized antibodies (or the humanized antibody portion of the chimeric proteins). Thus, the humanized antibodies can be used in mammalian subjects, such as, for example, humans, monkeys, mice, rabbits, and / or dogs.

[0061] The present disclosure provides a method for preventing or limiting the physical interaction between GPIbα and its cognate ligand. The method includes contacting a humanized antibody, chimeric protein, or pharmaceutical composition described herein with a platelet (expressing GPIbα on its surface) under conditions that allow binding of the humanized antibody / humanized antibody portion to GPIbα. As shown in the examples below, the humanized antibody and chimeric protein of the present disclosure have the ability to bind to GPIbα and antagonize its biological activity under low and high shear stress. Therefore, the method can be used to bind to GPIbα regardless of the applied shear stress. The method can be used in vitro or in vivo in a subject in need thereof. The method can be used at low or high shear rates.

[0062] If it is desired to prevent the interaction between GPIbα and its ligand (such as VWF), a humanized antibody or chimeric protein can be used prior to contacting GPIbα with its ligand. In this way, the platelets are first contacted with a humanized antibody (optionally presented as a chimeric protein or pharmaceutical composition) before the ligand is located or found in the vicinity of the platelets. In such an embodiment, it is understood that binding of the humanized antibody of the present disclosure prevents physical association of GPIbα with its ligand, and ultimately prevents or limits platelet activation and aggregation.

[0063] When it is desired to limit the interaction between GPIbα and its ligand (such as VWF), a humanized antibody can be used at the same time or after contact between GPIbα and its ligand has occurred. The platelets are then contacted with the humanized antibody (optionally presented as a chimeric protein or pharmaceutical composition) at the same time or after the ligand is placed or found in the vicinity of the platelets. In such embodiments, binding of the humanized antibody of the present disclosure is understood to limit the physical association between GPIbα and its ligand, and in some embodiments, to prevent or limit platelet activation and aggregation.

[0064] The humanized antibodies (optionally in chimeric form or in pharmaceutical compositions) can be used to prevent, treat, or alleviate symptoms associated with pathological thrombosis in subjects in need thereof. Because the humanized antibodies of the present disclosure (at least in the examples below) can prevent platelet activation and aggregation, they can be used to prevent pathological thrombosis in subjects susceptible to pathological thrombosis. Furthermore, because the humanized antibodies of the present disclosure do not induce thrombocytopenia or prolonged bleeding, their use is safer (e.g., compared to the original monoclonal antibodies from which they are derived). As used in the context of the present disclosure, the term "pathological thrombosis" refers to a condition in which a thrombus (blood clot) forms in a blood vessel and causes damage to the surrounding tissue. Pathological thrombosis can occur in veins or arteries. Pathological thrombosis can occur or be observed in the cavernous sinus, renal vein, deep vein, or lungs (pulmonary embolism). In some embodiments, the humanized antibody or chimeric protein is used to prevent, treat or alleviate symptoms associated with pathological thrombosis under high shear stress conditions. Near occluded or partially occluded blood vessels, shear stress is high and the interaction between GPIbα and VWF is important for vascular occlusion.

[0065] In embodiments where it is warranted to prevent the formation or growth of thrombi, the humanized antibody or chimeric protein can be used in subjects at risk of forming or growing thrombi. In one embodiment, the method can include determining (using methods and assays known in the art) whether the subject is at risk of forming or growing thrombi prior to administration of the antibody. The humanized antibody can be used in subjects previously determined to be at risk of forming or growing thrombi. In another embodiment, the method can include determining whether the subject has at least one thrombus, and in some further embodiments, the size of the thrombus, after at least one administration of the humanized antibody or chimeric protein. Such a determination can help determine whether additional doses should be administered to the subject to achieve a desired therapeutic effect.

[0066] In subjects with multiple thrombi, the humanized antibody or chimeric protein can be used to reduce the size and / or number of thrombi. In one embodiment, the method can include determining (using methods and assays known in the art) whether the subject has one or more thrombi, and optionally the size of the thrombi, before administration of the antibody. The humanized antibody can be used in subjects that have previously determined to have multiple thrombi, and optionally the size of the thrombi. In another embodiment, the method can include determining the presence, number, and size of thrombi after at least one administration of the humanized antibody or chimeric protein. Such a determination can help determine whether the subject should be administered additional doses to achieve a desired therapeutic effect.

[0067] In some embodiments, the method of the present disclosure includes determining whether a subject is at risk of experiencing or has experienced pathological thrombosis. A positive determination that a subject is at risk of experiencing or has experienced pathological thrombosis indicates that the subject would benefit from receiving a humanized antibody of the present disclosure. Thus, the method of the present disclosure may include administering a humanized antibody or chimeric protein to a subject determined to be at risk of experiencing or have experienced pathological thrombosis. The humanized antibody and chimeric protein of the present disclosure may be used for subjects determined to be at risk of experiencing or have experienced pathological thrombosis.

[0068] In some further embodiments, the methods of the present disclosure include determining whether a subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation. A positive determination that the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation indicates that the subject would benefit from receiving a humanized antibody of the present disclosure. Thus, the methods of the present disclosure may include administering a humanized antibody to a subject determined to be at risk of experiencing or have experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation. The humanized antibodies of the present disclosure may be used in subjects determined to be at risk of experiencing or have experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation.

[0069] Since the interaction between GPIbα and VWF is important for the spread of tumor metastasis, the humanized antibody or chimeric protein of the present disclosure can be used to reduce or limit tumor metastasis in a subject in need thereof.In some embodiments, the humanized antibody or chimeric protein can be used to reduce the number and / or size of tumor metastasis.In one embodiment, tumor metastasis is associated with liver cancer (e.g., hepatoma or adenocarcinoma), and the humanized antibody can be used to reduce or limit liver tumor metastasis.

[0070] In some embodiments, the methods of the disclosure include determining the presence, location and / or size of tumor metastases before and / or after providing one or more doses of a humanized antibody or chimeric protein. Such an assessment can be useful to determine whether additional doses of a humanized antibody should be administered to achieve a desired therapeutic outcome in the subject.

[0071] The humanized antibody or the chimeric protein comprising it can be formulated as a pharmaceutical composition for administration with an additive. An additive or "pharmaceutical excipient" is a pharmaceutical acceptable solvent, suspending agent or other pharmacologically inert vehicle, typically liquid, for delivering one or more chimeric proteins to a subject. Pharmaceutical excipients are generally selected to provide the desired bulk, consistency, etc., when combined with the components of a given pharmaceutical composition, taking into account the intended mode of administration. Typical pharmaceutical excipients include, but are not limited to, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0072] The humanized antibody or chimeric protein comprising it can be formulated with pharma- ceutically acceptable additives in unit dosage form or as pharmaceutical composition for administration. Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administering such compositions to subjects. Intravenous administration is preferred, but any suitable route can be used, for example, oral, enteral, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, or aerosol administration. Therapeutic formulations can be in the form of liquid solutions or suspensions. Methods well known in the art for making formulations are described, for example, in Remington: The Science and Practice of Pharmacy (19th ed., AR Gennaro AR, 1995, Mack Publishing Company, Easton, Pennsylvania).

[0073] Furthermore, in some embodiments, the humanized antibody or chimeric protein may be administered in a pharmacologic effective amount. The term "pharmacologic effective amount" or "therapeutically effective amount" refers to an amount (dosage) effective for treating a subject suffering from or suspected of suffering from a thrombotic, metastatic or inflammatory condition or disorder. It should also be understood that, as used herein, a "pharmacologic effective amount" may be interpreted as an amount that provides the desired therapeutic effect, administered alone or in combination with other therapeutic agents, either in a single dose or in any dosage or route.

[0074] The therapeutically effective amount or dosage of the humanized antibody or chimeric protein comprising same or pharmaceutical composition disclosed herein may range from about 0.001 to 30 mg / kg body weight, with other ranges according to the present invention including about 0.01 to 25 mg / kg body weight, about 0.025 to 10 mg / kg body weight, about 0.3 to 20 mg / kg body weight, about 0.1 to 20 mg / kg body weight, about 1 to 10 mg / kg body weight, 2 to 9 mg / kg body weight, 3 to 8 mg / kg body weight, 4 to 7 mg / kg body weight, 5 to 6 mg / kg body weight, and 20 to 50 mg / kg body weight. In other embodiments, the therapeutically effective amount or dosage may range from about 0.001 to 50 mg total, with other ranges of the invention including about 0.01 to 10 mg, about 0.3 to 3 mg, about 3 to 10 mg, about 6 mg, about 9 mg, about 10 to 20 mg, about 20 to 30 mg, about 30 to 40 mg, and about 40 to 50 mg. In one embodiment, the chimera is administered at a dosage of between about 40 to 80 mg / kg (e.g., 60 mg / kg).

[0075] Example I: Humanization of murine NIT-B1 antibody The variable domains of the murine NIT-A1 and NIT-B1 antibodies (described in U.S. Pat. No. 8,323,652 and deposited on Oct. 7, 2008 with the International Depositary Authority of Canada under accession numbers 071008-01 (NIT A1 clone) and 071008-02 (NIT B1 clone) respectively) have been sequenced. The murine NIT-A1 antibody has a heavy chain of SEQ ID NO: 1 (comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 4 and CDR3 of SEQ ID NO: 5) and a light chain of SEQ ID NO: 11 (comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 15). The murine NIT-B1 antibody has a heavy chain of SEQ ID NO:6 (comprising CDR1 of SEQ ID NO:8, CDR2 of SEQ ID NO:9 and CDR3 of SEQ ID NO:10) and a light chain of SEQ ID NO:16 (comprising CDR1 of SEQ ID NO:18, CDR2 of SEQ ID NO:19 and CDR3 of SEQ ID NO:20).

[0076] The murine NIT-B1 antibody was further developed as chimeric C100-Fab by fusing the NIT-B1 heavy chain variable domain (SEQ ID NO:6) with the human IgG1 constant region CH1 (SEQ ID NO:26; https: / / www.uniprot.org / uniprot / P01857) and the NIT-B1 light chain variable domain (SEQ ID NO:16) with the human Ig kappa light chain constant region (SEQ ID NO:33; http: / / www.uniprot.org / uniprot / P01834).

[0077] Example II: Characterization of humanized anti-GPIBALPHA antibodies Using the grafting method (Safdari et al., 2013), four human heavy chains (VH1 of SEQ ID NO: 35, VH2 of SEQ ID NO: 42, VH3 of SEQ ID NO: 49, VH4 of SEQ ID NO: 56) and four human light chains (VL1 of SEQ ID NO: 63, VL2 of SEQ ID NO: 70, VL3 of SEQ ID NO: 77, VL4 of SEQ ID NO: 84) were synthesized based on framework homology to the CDRs and annotated human sequences in the NCBI database.

[0078] Briefly, NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ) was used to search the variable domain sequences of the parent antibodies in the human germline database. For each heavy and light chain, four diverse human receptors (i.e., human variable domains with high homology to the parent antibody) were selected. The CDRs of the human receptors were replaced with their mouse counterparts, resulting in humanized variable domain sequences.

[0079] Humanized single chain variable fragments (scFv) and related chimeric proteins: scFvs containing both VH1 and VL2 were prepared, which contained a (GGGGS (SEQ ID NO:92)) x 4 linker between VH1 and VL2 (VH1-(G 4S)4-VL2). To facilitate purification, the scFvs had a 6xHis tag and a TEV cleavage site ENLYFQG in front of VH1. However, the tag was removed using TEV protease before another test. The scFvs were also included in chimeric proteins (scFv-HSA) with human serum albumin (HSA). In these instances, the scFv-HSA contained an additional linker GGGGS (SEQ ID NO: 92) in front of the HSA. The chimeric scFv-HSA was generated in a stable form and did not form aggregates during its production or purification.

[0080] Size: DNA sequences encoding humanized heavy and light chains were synthesized and inserted into pTT5 vector to construct expression plasmids for Fabs. Sixteen humanized Fabs were transiently expressed in HEK293 or CHO3E7 cell cultures, and then the cells were spun down. The supernatants were filtered and evaluated by SDS-PAGE and Western blot analysis. The molecular weight of the humanized Fabs is approximately 47 kDa under non-reducing conditions (Figures 1 and 2).

[0081] Next, the supernatants of the eight selected humanized Fabs (see Table 1) were subjected to Capture Select TM Kappa XL affinity matrix resin (Capture select TM The purified humanized Fab was buffer exchanged into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were evaluated using OD 280 and SDS-PAGE (approximately 2 μg of protein was loaded in each lane). As shown in Figure 3, the purified humanized Fab migrated as an approximately 47 kDa band on SDS-PAGE under non-reducing conditions and as approximately 24 kDa and approximately 23 kDa bands under reducing conditions.

[0082] Table 1: Description of humanized Fabs [Table 1]

[0083] Specificity: Human and mouse platelet-rich plasma (PRP) was prepared by centrifugation at 300g for 7 min and washed by transferring 200 μL of PRP to 10 mL of PBS followed by centrifugation at 800g for 10 min. The supernatant was then removed and the platelets were resuspended in 200 μL of PBS. The washed platelets (10 μL) were incubated with various antibodies (2.5-5 μg / mL) in a 200 μL system at room temperature for 30 min and detected with a FITC-labeled anti-human Fab antibody. All of these eight selected humanized Fabs H001-H008, scFvs, and chimeric proteins bound to both human and wild-type mouse platelets but not to GPIbα-deficient mouse platelets. This indicates that the humanized Fabs are specific for platelet GPIbα (Figures 4 and 19).

[0084] Platelets (2 × 10 ) from mouse, dog, human, rat, and rabbit PRP. 5 ) were transferred to 200 μL of PBS containing a series of concentrations of Fab H001 or H002 at 50, 16.7, 5.6, 1.8, 0.6, 0.2 nM (for mouse and dog), 200, 67, 22, 7.4, 2.5, 0.8, 0.3, 0.1 nM (for human and rat) and 500, 100, 20, 4, 0.8 nM (for rabbit). After 30 min of incubation, detection antibody (FITC-labeled anti-human kappa chain, 1:200) was added and incubated for 15 min in the dark. Flow cytometry was performed using a BD LSR Fortessa TM This was performed using X-20. H001 and H002 also bound to rat, dog, and rabbit platelets (Figure 5).

[0085] Humanized Fab with (purified H001 / H002, 5 μg / mL) and without (non-purified H001 / H002, 5 μg / mL) second purification by SEC-FPLC chromatography was incubated with washed cynomolgus monkey platelets (2 × 10 6) for 30 min. Then, FITC-labeled anti-human kappa chain secondary antibody (Ab) was incubated for 30 min. Humanized Fab binding to monkey platelets was detected by flow cytometry assay. Figure 6 shows that FabH001 and H002 antibodies bound to monkey platelets.

[0086] The affinity between H001 and recombinant GPIbα was measured by surface plasmon resonance (SPR) assay. For the preparation of SPR biosensors, SPR bare gold-coated biosensors (Biosensing Instrument Inc., Arizona, USA) were diluted with 1:1 absolute ethanol:ddH 2 The biosensor was washed for 2 h in a solution of 0.5 M sodium borohydride dissolved in ddHO. The biosensor was rinsed with a large volume of absolute ethanol and subsequently incubated in a solution of 1 mM mercaptopropionic acid in dimethylformamide for 16 h. After incubation, the SPR biosensor was rinsed with a large volume of dimethylformamide, followed by absolute ethanol and finally ddHO. 2 The biosensor was then rinsed with ddHO. 2 The sensor was incubated for 1 h in 40 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 20 mM N-hydroxysuccinimide dissolved in 20H2O. The sensor was then diluted with a large volume of ddHO. 2 The biosensing surface was washed with ddHO and incubated with 100 mM Nα,Nα-bis(carboxymethyl)-L-lysine hydrate for 4 h. After incubation, the biosensing surface was rinsed with ddHO. The final step before the SPR experiment was to incubate the functionalized biosensing surface with 100 mM NiCl 2 The subjects were exposed to the same temperature for one hour.

[0087] SPR experiments were performed on a Biosensing Instrument 4000SPR. The functionalized biosensor was loaded onto the instrument and the biosensor was equilibrated with running buffer (10 mM tris(hydroxymethyl)aminomethane, 140 mM NaCl, 20 mM imidazole pH=7.4) at a flow rate of 40 μL / min. For binding measurements, 25 μL of 500 nM hexahistidine-tagged GPIbα was injected onto the biosensing surface, immobilizing GPIbα on the SPR sensor surface. After allowing the baseline to equilibrate, 25 μL of the desired concentration of ligand (Fab H001 or control) was injected. The biosensing surface was equilibrated with 100 μL of 500 mM imidazole in running buffer, followed by 100 μL of 100 mM NiCl. 2 The ligand injections were regenerated between injections by injecting 0.01% ethanol. The data obtained were analyzed using the SPR analysis software included with the instrument.

[0088] GPIbα-immobilized SPR biosensors were exposed to 500, 100, 50, or 10 nM of Fab H001 antibody, and 100 nM of a control antibody (PSI E1, an antibody against GPIIbIIIa). The control did not bind to immobilized GPIbα and completely dissociated from the biosensor before the end of the injection (data not shown). In contrast, the Fab H001 antibody clearly bound to GPIbα, resulting in a clear SPR shift with only partial dissociation after the end of the injection (Figure 7A). The SPR shifts were fitted to a kinetic binding model, yielding an on-rate (k a ) is 2.61 × 10 7 s -1 , off rate (k d )=1.1×10 -1 s -1 , the dissociation constant (Kd) was found to be 4.4 nM (Figure 7B). To determine the dissociation constant, the magnitude of the SPR shift was plotted against the Fab H001 antibody concentration and fitted to a one-site binding model with a goodness of fit R of 0.9929. 2 A dissociation constant (Kd) of 8.0 ± 2.1 nM was obtained. The data clearly show that the Fab H001 antibody binds tightly to recombinant GPIbα with a low nanomolar dissociation constant and fast binding kinetics.

[0089] In vitro inhibition of agonist-induced platelet aggregation: To evaluate whether humanized Fabs can induce aberrant platelet activation and their role in platelet aggregation, in vitro platelet aggregation assays were performed. Human PRP from healthy volunteers and patients with peripheral vascular disease were prepared from sodium citrate-anticoagulated whole blood by centrifugation at 300 g for 7 min. Platelet aggregation in PRP was induced by addition of 5 μg / mL of humanized Fab clones and monitored by a computerized Chrono-log aggregometer (Chrono-log, USA). Platelet aggregation in PRP with or without humanized Fab was induced by ristocetin (1 mg / mL) and in gel-filtered platelets by thrombin (0.05 U / ml) using a computerized Chrono-log aggregometer (Chrono-log, USA).

[0090] Fab H001, H002, H005, H008 and scFv antibodies did not induce platelet activation. Furthermore, H001 and H002 significantly inhibited ristocetin-induced human platelet aggregation in PRP and low-dose thrombin-induced platelet aggregation in gel-filtered platelets (Figures 8, 9, 20 and 21).

[0091] Inhibition of thrombus formation at low and high shear rates: To measure platelet adhesion, aggregation, and thrombus formation at different shear rates, heparinized whole blood from 30 healthy volunteers was perfused over type I collagen-coated surfaces using an extracorporeal perfusion chamber system under real-time fluorescence microscopy. Briefly, rectangular microcapillary tubes (ibidi channel slides, ibidi GmbH) were coated with Horm collagen (100 μg / mL, overnight, 4 °C, Niecod Linz, Austria). Anticoagulated (heparin 15 U / mL) whole blood from healthy donors was perfused with DiOC 6(1 μM, 10 min, 37 °C, Sigma). Then, the whole blood treated with control or humanized Fab or scFv-HSA was pumped for 300 s using a syringe pump (Harvard Apparatus, USA). -1 , 1200s -1 , and the 1800s -1 Platelets were perfused over the collagen-coated surface at a shear rate of 100 s for 3 min. Platelet accumulation and thrombus formation were recorded in real time with a Zeiss Axiovert 135 inverted fluorescence microscope (60× / 0.90 NA water objective). Quantitative dynamics of platelet fluorescence intensity were acquired using SlideBook software.

[0092] Humanized Fabs H001 and H002 were used to measure 300 s stimuli, which correspond to venular / aortic and arteriolar blood flow, respectively. -1 and the 1800s -1 Both wall shear rates (but preferably 1800 s -1 The chimeric protein significantly inhibited thrombus formation at a wall shear rate of 1200s (Figure 10). -1 (Figure 22) These in vitro results suggest that the humanized C100-Fab, scFv, and chimeric proteins are significant inhibitors of thrombosis under both low and high shear conditions.

[0093] Inhibition of thrombus growth and vascular occlusion in vivo: To examine whether humanized Fab affects thrombus growth in vivo, we utilized two complementary in vivo microscopy thrombosis models and an aortic thrombosis model.

[0094] Thrombus formation in mesenteric arterioles was monitored in 3-4 week-old C57BL / 6 wild-type mice. Mice were injected with donor-matched fluorescently labeled platelets and visualized with a Zeiss Axiovert 135 inverted fluorescent microscope (Zeiss, Germany). Briefly, blood was collected from donor-matched mice into acid citrate dextrose solution (ACD; as anticoagulant). Gel-filtered platelets were prepared and labeled with calcein AM (1 mg / mL, Invitrogen, Canada) for 20 min at room temperature. Platelets were then injected into experimental mice via the tail vein together with control saline buffer, Fab H001, or H002 (2.5 or 5 μg / mouse). Mice were then anesthetized and the mesentery was externalized. A single mesenteric arteriole with a diameter of 100–120 μm was selected and injury was induced by topical application of 30 μL of 250 mM ferric chloride. The time to complete vascular occlusion was recorded. Images of thrombus formation and dissolution were visualized by fluorescence microscopy. As shown in Figure 11 and Table 2, FeCl 3 Injury-induced thrombus growth and vascular occlusion were significantly inhibited by injection of humanized Fab H001 and H002 antibodies (when compared with control saline injection).

[0095] Table 2: Number of mice without occlusion according to treatment received. [Table 2]

[0096] For the laser-induced cremaster arteriole thrombosis model, C57B / 6 wild-type mice (male, 6–8 weeks old) were anesthetized and a tracheal tube was inserted to facilitate breathing. The cremaster muscle was prepared under a dissecting microscope and perfused throughout the experiment with pre-warmed bicarbonate-buffered saline. Platelet antibodies, control (saline buffer), humanized as well as monovalent H001 and H002 antibodies (5 or 10 μg / mouse) were administered as indicated into the jugular vein cannula. Platelets were labeled by rat anti-mouse CD41 antibody (Leo.A1, EMFRET Analytics, Germany, 0.1 μg / g) injection. Multiple independent upstream injuries were induced in cremaster arterioles by a pulsed nitrogen dye laser using an Olympus BX51WI microscope. The dynamic accumulation of fluorescently labeled platelets within the growing thrombus was captured and analyzed using Slidebook software. In this cremaster arteriole intravital microscopy thrombosis model (which does not involve oxidative stress, since mild vascular damage is induced by laser) thrombus growth was almost completely abolished after intravenous injection of humanized and monovalent H001 and H002 antibodies (Figure 12). These results indicate that humanized Fab inhibits thrombus growth and promotes thrombus lysis, and has great potential for development as a novel antithrombotic agent.

[0097] Before and after the thrombosis model experiment by intravital microscopy, blood of the mice was collected, and platelets were isolated and characterized using flow cytometry with FITC-labeled mouse anti-human CD62P antibody and Annexin V-Alexa Fluor® 647. As shown in Figure 13, the humanized monovalent H001 antibody did not induce platelet abnormal activation in vivo, as it did not cause platelet P-selectin expression or exposure to phosphatidylserine (PS). Similar results were obtained with the humanized FabH002 antibody (data not shown).

[0098] For the ferric chloride-induced large carotid artery thrombosis model, C57BL / 6J wild-type mice (both sexes, ≥8 weeks old, 25–30 g) were anesthetized and intravenously injected with Fab H001 or H002 antibodies (5, 10, or 20 μg / mouse) or an equal volume (200 μL) of PBS 5 min before inducing arterial injury. The left common carotid artery was dissected and held with a miniature Doppler flow probe (TS420 transit-time perivascular flowmeter, Transonic Systems Inc., USA). Baseline blood flow was measured for 30 s. Carotid artery injury was then induced for 3 min with a strip of Whatman filter paper saturated with 7.5% ferric chloride. Blood flow was monitored until complete vascular occlusion was observed. Fab H001 and H002 antibodies significantly inhibited thrombus growth and prevented stable vascular occlusion (Figure 14).

[0099] In vivo reduction of ischemic brain infarct size without increasing the risk of intracerebral hemorrhage: To investigate the therapeutic potential of the antibody in ischemic stroke, a cerebral ischemia and reperfusion injury model (transient middle cerebral artery occlusion (tMCAO)) was performed. Male mice (25 g) were anesthetized with inhaled isoflurane. A midline cervical incision was made and the soft tissue was dissected. The left common carotid artery (LCCA) was carefully detached from the surrounding nerves (without damaging the vagus nerve) and a ligature was created using a 5.0 string. Next, the left external carotid artery (LECA) was isolated and a second ligature was created. Next, the left internal carotid artery (LICA) was isolated and a ligature was created with a 6.0 filament. After the left internal carotid artery (LICA) and the left pterygopalatine artery (LPA) were well visualized, both arteries were clipped using a microvascular clip. A small hole was made in the LCCA before it bifurcated into the LECA and LICA. A standardized silicone rubber-coated 6.0 nylon monofilament (6021, Doccol Corp, Redlands, CA) was introduced into the LICA until it was clipped. The clipped artery was opened while the filament was inserted into the LICA to occlude the origin of the LMCA at the Circle of Willis. A third ligature on the LICA was closed to secure the filament in place. After 1 h, the third ligature was opened and the filament was withdrawn. Antibodies were administered intravenously either immediately after the filament was inserted or 1 h after the filament was withdrawn.

[0100] To measure the volume of cerebral infarction, mice were euthanized 24 h after tMCAO induction. To visualize cerebral infarction, multiple 2 mm-thick coronal brain sections cut from whole brains were stained with 2% 2,3,5-triphenyl-tetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO). The presence of cerebral hemorrhage was assessed by naked eye. To measure neurological function 24 h after tMCAO induction, mice were subjected to a modified Bederson test and a grip test to evaluate overall neurological and motor function, respectively. The results showed that blockade of GPIbα with humanized Fab H001 and H002 antibodies and scFv-HSA significantly reduced infarct size in the brain and improved functional outcomes after tMCAO without increasing the risk of intracerebral hemorrhage (Figure 15).

[0101] In vivo protection of TTP: To test the therapeutic efficacy of antibodies in TTP, we used an ionophore-induced ultra-large VWF (ULVWF)-mediated microvascular thrombosis model. ADAMTS13 - / -Mice were anesthetized and intravenously injected with fluorescently labeled platelets purified from donor mice of the same genotype, and ionophore-induced microvascular thrombosis in mesenteric veins was monitored in real time under intravital microscopy. For platelet preparation, mice (6–8 weeks old) were anesthetized by intraperitoneal injection of ketamine / xylazine (100 mg / kg and 10 mg / kg body weight, respectively), and whole ocular blood was collected from the retroorbital plexus using a heparin-coated glass capillary tube. Blood was collected into tubes containing citrate-dextrose solution (38 mmol / L citric acid, 75 mmol / L trisodium citrate, 100 mmol / L dextrose). Platelet-rich plasma was obtained by centrifuging the whole blood at 300 g for 7 min. Gel-filtered platelets were then separated from platelet-rich plasma using a Sepharose 2B column in PIPES buffer (PIPES 5 mmol / L, NaCl 1.37 mmol / L, KCl 4 mmol / L, and glucose 0.1%, pH 7.0). Platelet counts were determined using a Hemovet (HV950, Drew Scientific). Fluorescent labeling of gel-filtered platelets was achieved by incubating platelets with calcein-acetoxymethyl ester (1 μg / mL) for 15 min at room temperature. Prior to use for in vivo imaging, the efficacy of fluorescent labeling of platelets was determined by fluorescence microscopy.

[0102] For intravital microscopy, 4-week-old mice were anesthetized and incubated with fluorescently labeled platelets (1.25 × 10 from mice of the same genotype) 6Mesenteric vessels were surgically prepared and monitored under an inverted fluorescence microscope (Zeiss Axio Observer Z1 Advanced Marianas Microscope) using a 25x oil objective (Zeiss). Approximately 2.5 mm sections of mesenteric veins (diameter 100–150 μmol / L) were treated locally with 10 μL of 10 μmol / L calcium ionophore to induce Weibel-Palade body secretion of ULVWF from the endothelium, resulting in the immediate adhesion of fluorescently labeled platelets and the formation of platelet thrombi anchored to the vessel wall. For each mouse, the process of thrombus formation and resolution of the thrombus were monitored and recorded for 20 min in addition to the prerecording. Fab H001 or scFv-HSA chimeric proteins inhibited ADAMTS13 - / - The drug was administered via a tail vein catheter 10 min (prophylactically) before the onset of calcium ionophore-stimulated thrombosis in the mesenteric microvasculature of mice. The kinetics of platelet accumulation in selected vessel segments was quantitatively analyzed by (1) the number of emboli (platelet thrombi >20 μm in diameter) and (2) the time to restore normal blood flow (defined as the time required for platelet fluorescence to return to approximately baseline after topical application).

[0103] As shown in FIG. 16, all ADAMTS13 - / - Before the application of the ionophore to the mice, no platelet adhesion to the mesenteric vessel wall was detected under intravital microscopy. - / -Immediately after topical application of calcium ionophore to mesenteric vessels in mice, platelet adhesion was observed in mesenteric venules, manifesting as the formation of a single platelet string attached to the endothelium in the direction of blood flow. Within 1 min, multiple large thrombi (diameter >20 μm) were formed, some of which grew to 50% of the diameter of the ionophore-treated vessels. The platelet strings and thrombi were visually very loose and easily detached from the vessel wall and emboli downstream. Platelet adhesion to the vessel wall and embolic thrombosis in control mice continued for more than 10 min, but over time, the mice died and eventually restored normal blood flow in the affected parts of the vessels. ADAMTS13 - / - The thrombotic response of mice was dramatically suppressed by prophylactic treatment with both Fab H001 antibody or scFv-HSA chimeric protein (Figure 16). Platelet adhesion to the vessel wall and the formation of large thrombi were strongly inhibited by both Fab H001 antibody or scFv-HSA chimeric protein treatment (Figure 16). Compared with the control group, the number of large embolic thrombi was significantly lower and the time to restore normal blood flow in mesenteric venules was shorter in both Fab H001 antibody or scFv-HSA chimeric protein treatment groups (Figure 16). These results support the enhanced inhibition of ADAMTS13 by Fab H001 antibody or scFv-HSA chimeric protein. - / - We show that prophylactic treatment of mice effectively inhibits ionophore-induced VWF-mediated microvascular thrombosis and mimics platelet accumulation on novel free endothelium-bound ULVWF in TTP.

[0104] Suppression of thrombocytopenia: C57BL / 6 mice were intravenously injected with intravenous immunoglobulin (IVIg), humanized Fab H001 or H002 antibodies (10 μg / mouse, n=3 for each group), or NIT-B1 antibody (5 μg / mouse, n=2). Serial blood samples at different time points (0, 30 min, 1, 2, 4, 8 h, 1, 2, 3, 4, 5, 6, and 7 days) were collected from the medial saphenous vein of the mice. At each time point, 10 μL of blood sample was collected and added to 240 μL of 1% PBS-EDTA (pH 7.4) to prevent clotting. To count platelets, 50 μL of blood sample (in PBS-EDTA) was transferred to 10 mL of diluent (Isoton II, Coulter Corporation) and the platelet count was measured using a Coulter counter (Beckman Z2, Coulter Corporation). As shown in Figure 17, the humanized and monovalent H001 or H002 antibodies did not induce a significant decrease in platelet counts in the first 24 hours after administration, in clear contrast to the NIT-B1 antibody.

[0105] Bleeding time: BALB / c mice were injected intravenously with PBS (n=4), Fab H001 or H002 antibody (5-10ug / mouse, n=3) 120 minutes prior to injury. Mice were anesthetized with 2.5% Avertin (18mL / kg body weight, ip) and maintained on a 37°C heating pad. The tip of the tail (2mm) was cut with a sharp scalpel and the wound was tapped every 15 seconds using tissue paper. Bleeding time was recorded as the time until blood flow stopped (bleeding stopped for more than 10 seconds). If the tail was still bleeding, the analysis was terminated after 15 minutes. As shown in Figure 18, administration of H001 or H002 antibody did not prolong the bleeding time.

[0106] (References) Yaghoub Safdari, Safar Farajnia, Mohammad Asgharzadeh & Masoumeh Khalili (2013), "Antibody humanization methods - a review and update", Biotechnology and Genetic Engineering Reviews, 29:2, 175-186.

[0107] Although the present invention has been described in connection with specific embodiments thereof, it is to be understood that the claims should not be limited by the preferred embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0108] [Note] [Appendix 1] A humanized antibody that specifically recognizes platelet glycoprotein I(b)α (GPIbα), The humanized antibody lacks an Fc portion, and Prevents platelet activation, aggregation, and / or thrombus growth; Lacking the ability to activate platelets lack the ability to induce thrombocytopenia, and / or lack of ability to prolong bleeding time at therapeutic doses, A humanized antibody characterized by the following structure:

[0109] [Appendix 2] A humanized antibody described in Appendix 1, capable of recognizing human GPIbα, mouse GPIbα, dog GPIbα, rat GPIbα, rabbit GPIbα, and / or monkey GPIbα.

[0110] [Appendix 3] 3. The humanized antibody of claim 1 or 2, which is an antibody fragment.

[0111] [Appendix 4] F(ab) 2 A humanized antibody described in Appendix 3, which is a fragment.

[0112] [Appendix 5] The humanized antibody of claim 3, wherein the antibody is a monovalent antibody.

[0113] [Appendix 6] 6. The humanized antibody of claim 5, which is a Fab antibody fragment.

[0114] [Appendix 7] The humanized antibody of claim 5, which is a single-chain variable fragment (scFv).

[0115] [Appendix 8] 8. A humanized antibody according to any one of claims 1 to 7, having a heavy chain.

[0116] [Appendix 9] The heavy chain comprises: A first CDR having the amino acid sequence of GFTFSSFAMS (SEQ ID NO: 37), a variant thereof or a fragment thereof; A second CDR having the amino acid sequence of SITSAGTPYYPDSVLG (SEQ ID NO: 38), a variant thereof or a fragment thereof, and / or A third CDR having the amino acid sequence of SRGYEDYFDY (SEQ ID NO: 39), a variant thereof or a fragment thereof; The humanized antibody of claim 8, comprising:

[0117] [Appendix 10] The heavy chain is human IgG 1 10. The humanized antibody of claim 8 or 9, further comprising an antibody CH1 region.

[0118] [Appendix 11] The human IgG 1 11. The humanized antibody of claim 10, wherein the CH1 region of the antibody has the amino acid sequence of SEQ ID NO: 40, 47, 54 or 61, a variant thereof or a fragment thereof.

[0119] [Appendix 12] 12. The humanized antibody of claim 11, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57, a variant thereof or a fragment thereof.

[0120] [Appendix 13] 13. A humanized antibody according to any one of claims 1 to 12, having a light chain.

[0121] [Appendix 14] The light chain comprises: A first CDR having an amino acid sequence of KSSQSLLNSRNQKNYLA (SEQ ID NO: 65), a variant thereof or a fragment thereof; A second CDR having the amino acid sequence of FTSTRES (SEQ ID NO: 66), a variant thereof or a fragment thereof, and / or A third CDR having the amino acid sequence of QQHYSSPWT (SEQ ID NO: 67), a variant thereof or a fragment thereof; 14. The humanized antibody of claim 13,

[0122] [Appendix 15] The light chain is human IgG 1 15. The humanized antibody of claim 13 or 14, further comprising a kappa chain C region of the antibody.

[0123] [Appendix 16] The humanized antibody of claim 15, wherein the kappa chain C region has the amino acid sequence of SEQ ID NO: 68, 75, 82 or 89, a variant thereof or a fragment thereof.

[0124] [Appendix 17] 17. The humanized antibody of claim 16, wherein the light chain has the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85, a variant thereof or a fragment thereof.

[0125] [Appendix 18] A heavy chain of SEQ ID NO: 36, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 36, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 36, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 36, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 43, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 43, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 43, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 43, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof; A heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof, or A heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and a light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof; 18. The humanized antibody of any one of claims 1 to 17, comprising:

[0126] [Appendix 19] 19. A chimeric protein comprising a humanized antibody according to any one of claims 1 to 18 and a carrier protein.

[0127] [Appendix 20] A pharmaceutical composition comprising (i) a humanized antibody according to any one of appendices 1 to 18 or a chimeric protein according to appendix 19, and (ii) a pharmaceutical excipient.

[0128] [Appendix 21] A method for preventing or limiting the interaction between glycoprotein I(b)α (GPIbα) present on platelets and GPIbα ligands, comprising contacting platelets with a humanized antibody described in any one of appendices 1 to 18, a chimeric protein described in appendix 19, or a pharmaceutical composition described in appendix 20.

[0129] [Appendix 22] 22. The method of claim 21, for preventing or limiting platelet activation.

[0130] [Appendix 23] The method according to claim 21 or 22, wherein the GPIbα ligand is von Willebrand factor (VWF) and / or thrombin.

[0131] [Appendix 24] 24. The method of any one of claims 21 to 23, wherein the contacting is caused under low or high shear rate.

[0132] [Appendix 25] 25. The method of any one of claims 21 to 24, wherein the humanized antibody, the chimeric protein, or the pharmaceutical composition is contacted with the platelets before, simultaneously with, or after the GPIbα ligand is contacted with the platelets.

[0133] [Appendix 26] 26. The method of any one of claims 21 to 25, for preventing or limiting in vivo interactions in a subject in need thereof.

[0134] [Appendix 27] 27. The method of claim 26 for preventing the formation or growth of a thrombus in a subject in need thereof.

[0135] [Appendix 28] 27. The method of claim 26, for reducing the size of thrombi or the number of thrombi in a subject in need thereof.

[0136] [Appendix 29] 29. The method of any one of claims 26 to 28, further comprising determining the presence, location, and / or size of a thrombus in the subject.

[0137] [Appendix 30] 29. The method of any one of claims 26 to 28, wherein the subject is at risk of experiencing or has experienced a pathological thrombosis.

[0138] [Appendix 31] 29. The method of any one of claims 26 to 28, wherein the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation.

[0139] [Appendix 32] 29. The method of any one of claims 26 to 28, for reducing or limiting tumor metastasis in a subject in need thereof.

[0140] [Appendix 33] 33. The method of claim 32, further comprising determining the presence, location, and / or size of tumor metastases in the subject.

Claims

1. A humanized antibody or a fragment thereof that specifically recognizes platelet glycoprotein I(b)α (GPIbα), the fragment comprises an antigen-binding region of the humanized antibody, The humanized antibody or fragment thereof may have or lack an Fc portion, and Preventing platelet activation, aggregation, and / or thrombus growth; Lacking the ability to activate platelets lacks the ability to induce thrombocytopenia, and / or At therapeutic doses, it lacks the ability to prolong bleeding time, Furthermore, the humanized antibody or fragment thereof has a heavy chain having the amino acid sequence of SEQ ID NO: 93 or 94 and a light chain having the amino acid sequence of SEQ ID NO: 95 or 96. A humanized antibody or a fragment thereof.

2. The humanized antibody or fragment thereof according to claim 1, which is capable of recognizing human GPIbα, mouse GPIbα, canine GPIbα, rat GPIbα, rabbit GPIbα, and / or monkey GPIbα.

3. The humanized antibody or fragment thereof described in claim 1 or 2, which is an antibody fragment.

4. F(ab) ２ The humanized antibody or fragment thereof of claim 3 which is a fragment.

5. The humanized antibody or fragment thereof of claim 3, wherein the antibody is a monovalent antibody.

6. The humanized antibody or fragment thereof described in claim 5, which is a Fab antibody fragment.

7. The humanized antibody or fragment thereof of claim 5, which is a single-chain variable fragment (scFv).

8. The heavy chain is human IgG １ The human IgG １ The humanized antibody or fragment thereof of claim 1, wherein the CH1 region of the antibody has an amino acid sequence of SEQ ID NO: 40, 47, 54 or 61, or a variant or fragment of said amino acid sequence having at least 90% identity to said amino acid sequence.

9. The light chain is human IgG １ The humanized antibody or fragment thereof of claim 1, further comprising a kappa chain C region of the antibody, the kappa chain C region having an amino acid sequence of SEQ ID NO: 68, 75, 82 or 89, or a variant or fragment of said amino acid sequence having at least 90% identity to said amino acid sequence.

10. A chimeric protein comprising the humanized antibody or fragment thereof described in claim 1.

11. A pharmaceutical composition comprising (i) a humanized antibody or fragment thereof according to claim 1, or a chimeric protein according to claim 10, and (ii) a pharmaceutical excipient.

12. 1. A pharmaceutical for use in a method for preventing or limiting an interaction between glycoprotein I(b)α (GPIbα) present on platelets and a GPIbα ligand, comprising: The pharmaceutical is a pharmaceutical comprising the humanized antibody or fragment thereof according to claim 1, a pharmaceutical comprising the chimeric protein according to claim 10, or a pharmaceutical composition according to claim 11, The method includes contacting platelets with the medicament.

13. The medicament according to claim 12 for preventing or limiting platelet activation.

14. The pharmaceutical composition according to claim 12, wherein the GPIbα ligand is von Willebrand factor (VWF), P-selectin, kininogen, thrombospondin, and / or thrombin.

15. The pharmaceutical composition of claim 12, wherein the contacting is effected under any shear rate.

16. The method of claim 12, wherein the medicament is contacted with the platelets before, simultaneously with, or after the GPIbα ligand is contacted with the platelets.

17. The pharmaceutical composition of claim 12 for preventing or limiting interactions within the body in a subject in need thereof.

18. 18. The pharmaceutical composition of claim 17, for preventing the formation or growth of thrombi in a subject in need thereof, for reducing the size or number of thrombi in a subject in need thereof, or for removing occluding thrombi in a subject in need thereof.

19. The method of claim 17, wherein the method further comprises determining the presence, location, and / or size of a thrombus in the subject.

20. The method of claim 17, wherein the subject is at risk of experiencing or has experienced pathological thrombosis.

21. The pharmaceutical of claim 17, wherein the subject is at risk of experiencing or has experienced ischemic stroke, thrombotic thrombocytopenic purpura, myocardial infarction, acute coronary syndrome, atherothrombosis, peripheral vascular disease, deep vein thrombosis, sepsis, and / or vascular inflammation.

22. The pharmaceutical composition of claim 17 for reducing or limiting tumor metastasis in a subject in need thereof.

23. The method of claim 22, wherein the method further comprises determining the presence, location, and / or size of tumor metastases in the subject.

24. The humanized antibody or fragment thereof of claim 1, wherein the humanized antibody or fragment thereof lacks an Fc portion.

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