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 LISTINGS) 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. The 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 and 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 are usually key events in acute thrombosis. Therefore, antiplatelet therapy is one of the important treatments for reducing cardiovascular death, and includes: (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. However, limitations of current antiplatelet therapies, such as delayed / weak / poor inhibition of platelet function, excessive bleeding complications, thrombocytopenia, and unexpected platelet activation, remain major concerns for 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, and for patients who have passed the intravenous thrombolytic treatment time window, when it is not available.

[0004] The platelet GPIb-IX-V complex has emerged as a promising antiplatelet target. The GPIb-IX-V complex is a key platelet receptor that initiates platelet adhesion and migration to injured vessel walls, especially at high shear stresses. Platelet adhesion / migration to the subendothelium is mediated by binding of the GPIbα subunit to von Willebrand factor (VWF), which is 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, and other proteins. Under high-shear conditions, the GPIbα-VWF interaction is required for the pathological growth of occlusive thrombi (both platelet adhesion and platelet aggregation / adhesion) at arterial stenosis sites, where blood flows at wall shear rates potentially exceeding 10,000–40,000 s. On the other hand, under low-shear conditions (such as most hemostasis), platelet adhesion can be directly mediated by αIIbβ3-fibrinogen / fibrin and α2β1 / GPVI-collagen interactions. Therefore, pharmacological inhibition of GPIbα may reduce the risk of systemic bleeding and improve safety compared with other antiplatelet drugs that do not specifically target thrombosis at high shear. GPIbα has also 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 pro-inflammatory function of platelets via GPIbα, which may further promote thromboinflammatory neurological disorders and infarct growth. Furthermore, GPIbα is thought to be specifically expressed in platelets and megakaryocytes. Therefore, direct platelet GPIbα antagonists have great potential for development as effective and safe antiplatelet agents for the treatment of acute thrombotic events such as heart attack and stroke.

[0005] In particular, novel antiplatelet strategies targeting the GPIbα-VWF interaction have been shown to be effective therapeutic approaches for treating acquired thrombotic thrombocytopenic purpura (aTTP), a thrombotic microangiopathy and a life-threatening condition with high mortality rates if untreated. Autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13), a VWF-cleaving protease that cleaves / reduces VWF multimer size, result in severe deficiency of ADAMTS13 activity. These ultra-large VWFs are hyper-adhesive, causing the formation of platelet (GPIbα)-VWF microthrombi within blood vessels, leading to organ ischemia and infarction, reduced platelet counts, and red blood cell destruction. 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] Caplacizumab, a nanobody targeting the VWF A1 domain, was approved for the treatment of adults experiencing an acute episode of aTTP in combination with plasma exchange (PEX) and a minimum of 30 days of immunosuppression after cessation of daily PEX. However, bleeding-related adverse events were more common with caplacizumab (65% vs. 48%), as were serious bleeding events (11% vs. 1%). Another barrier to caplacizumab was the drug's prohibitive cost. As of 2020, the current price of caplacizumab was over $8,000 for a treatment regimen recommended for daily administration for 30 days after the last plasma exchange with the possibility of continued treatment until recovery of ADAMTS13 activity. Because VWF is consistently released from activated endothelium, the relatively stable levels of GPIbα on platelets, with a short lifespan of 7–10 days in humans, appear 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 can induce immune responses, generating antidrug antibodies that neutralize the drug and eliminate its therapeutic effect. Furthermore, these antibodies can cause allergic reactions or immune complex formation, which can damage the kidneys and joints (arthritis). Furthermore, necessary re-administration may stimulate memory immune cells and enhance such immune responses.

[0008] Patent Document 2 describes recombinant GPG-290 or GPIb-290 / 2V-immunoglobulin (Ig) fusion polypeptide, a soluble chimeric protein containing 290 N-terminal extracellular amino acids (G233V and M239V) of mutant GPIbα linked via proline to the Fc fragment of human IgG1. GPG-290 competes with platelet GPIbα for binding to VWF and exhibits 14-fold higher affinity for VWF than wild-type GPIbα. In animal models, GPG-290 dose-dependently prolonged the time to coronary artery occlusion and inhibited platelet aggregation, thrombosis, and recurrent coronary artery flow reduction 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, making α-thrombin unavailable for hemostasis.

[0009] Patent Document 3 further describes GPIb-290 / 2V / FFFIg variant fusion proteins (Y276F, Y288F, and / or Y297F). These exhibit limited / low affinity binding to α-thrombin, a 50% reduction in efficacy in suppressing recurrent coronary thrombosis (i.e., inhibiting recurrent coronary artery circulation loss), and, compared to GPIb-290, a prolonged tail bleeding time and increased ADP closure time as assessed by the Platelet Function Analyzer-100 (PFA-100). However, these GPIb-Ig fusion proteins are high-molecular-weight chimeric proteins (approximately 130 kDa) that primarily target subendothelial-immobilized VWF or plasma VWF under high shear rate conditions. Therefore, the amount and accessibility of these fusion proteins are less predictable, potentially limiting the amount of product to be infused. Another limitation may be the risk of anti-drug antibody generation. GPIb-Ig fusion proteins are bioengineered chimeric proteins. Because both the GPIbα polypeptide and the Fc fragment are derived from human genes, antigenicity can be minimized. However, mutations in GPIbα variants and the joint region between the GPIbα and Fc portions may generate neoepitopes that elicit immune responses. Furthermore, the fusion protein can generate several conformational neoepitopes that induce anti-drug antibody production.

[0010] Neutralizing monoclonal antibodies against human GPIbα (anti-GPIbα mAb) have also been described in the art. However, most currently available anti-GPIbα mAbs are derived from mice and may therefore provoke a human anti-mouse response in clinical use. Furthermore, intact anti-GPIbα mAb often leads to platelet activation, likely because binding of intact mAb induces platelet GPIbα-mediated signaling, potentially exacerbating platelet aggregation and thrombosis. Furthermore, intact anti-GPIbα mAb binding to platelets can trigger both Fc-dependent and Fc-independent platelet clearance, potentially resulting in thrombocytopenia (i.e., a decrease in platelet count). Therefore, intact anti-GPIbα mAb exhibits very limited therapeutic potential.

[0011] Patent Document 4 describes the Fab fragment of the chimeric antibody chSZ2, a chimeric mAb against human GPIbα, which inhibits ristocetin-induced platelet aggregation in vitro in a dose-dependent manner. However, because it is a chimeric antibody that retains the variable region of a mouse antibody and replaces the constant region with a human one, immunogenicity remains a major concern. Importantly, the in vivo function of chSZ2 for preventing / treating thrombotic diseases has not been demonstrated due to the lack of animal models and the potential 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 reduced or completely eliminated 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, along 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 share significant homology), and the repertoire of antibodies raised against epitopes present in human GPIbα but absent in mouse GPIbα is limited. Therefore, these mAbs cannot be analyzed and evaluated in rodents or other animal species for important preclinical pharmacology, toxicology, and pharmacokinetic studies. Whether h6B4-Fab can cause platelet activation is also of concern, as its precursor mAb 6B4 can apparently cause platelet activation and severe thrombocytopenia.

[0013] Therefore, improved therapeutic agents that target the platelet GPIb-IX-V complex without causing platelet activation, platelet destruction, thrombocytopenia, or serious bleeding complications are needed. [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 that specifically recognize glycoprotein I(b)α (GPIbα) and protein constructs comprising same, and related therapeutic uses. The humanized antibodies have the ability to prevent platelet activation, aggregation, and thrombus growth (especially at high shear), but lack the ability to activate platelets (e.g., do not activate platelets), induce thrombocytopenia, and / or 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 formation, and lacks the ability to activate platelets, induce thrombocytopenia, and / or prolong bleeding time at therapeutic doses. According to one embodiment, the humanized antibody has the ability to recognize human GPIbα, mouse GPIbα, canine 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 fragment. 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 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. In yet another embodiment, the heavy chain further comprises a CH1 region of a human IgG1 antibody. For example, the CH1 region of a human IgG1 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 further embodiments, 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 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. or a fragment thereof, the heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 50, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 85, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 64, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 71, a variant thereof or a fragment thereof, the heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 78, a variant thereof or a fragment thereof, or the heavy chain of SEQ ID NO: 57, a variant thereof or a fragment thereof, and the light chain of SEQ ID NO: 85, a variant thereof 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 the 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 platelets with a humanized antibody described herein, a chimeric protein described herein, or a pharmaceutical composition described herein. In some embodiments, the method is for preventing or limiting platelet activation. 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 platelets before, simultaneously with, or after contacting the GPIbα ligand with the platelets. In a further embodiment, the method is for preventing or limiting the 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 or number of thrombi 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 or has experienced pathological thrombosis. In yet another embodiment, the subject is at risk of 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 liver 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 glycoprotein I(b)α (GPIbα) present on platelets and von Willebrand factor (VWF) and / or thrombin and other GPIbα ligands using a humanized antibody described herein, a chimeric protein described herein, or a pharmaceutical composition. 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 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 platelets before, simultaneously with, or after contacting VWF and / or thrombin and other GPIbα ligands with 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 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 predetermined 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 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 liver tumor metastasis. In yet another embodiment, the presence, location, and / or size of tumor metastasis has been previously determined in the subject.

[0021] According to a sixth embodiment, the present disclosure provides a humanized antibody described herein, a chimeric protein described herein, or a pharmaceutical composition for preventing or limiting the 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 a specific embodiment, the humanized antibody, chimeric protein, or pharmaceutical composition is for contacting platelets before, simultaneously with, or after contact of VWF and / or thrombin and other GPIbα ligands with the platelets. In certain embodiments, 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 the 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 predetermined in the subject. In yet another embodiment, the subject is at risk of or has experienced pathological thrombosis. In yet another embodiment, the subject is at risk of 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 liver tumor metastasis.In yet another embodiment, the presence, location, and / or size of tumor metastases has been previously determined in the subject. [Brief explanation of the drawings]

[0022] Having thus generally described the principles of the present 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 is shown. Various combinations of heavy and light chains are indicated 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. [Figure 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. [Figure 3] Figures 3A to 3H show the results of SDS-PAGE under non-reducing (labeled "N") and reducing (labeled "R") conditions of purified Fab, each containing: 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 4B show that humanized Fabs H001-H008 bind to wild-type mouse platelets (Figure 4A) but not to GPIbα- / - mouse platelets (5 μg / mL) (Figure 4B). The "*" in Figure 4B indicates a control signal. [Figure 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 solid.) The platelet-binding humanized Fabs were tested in vitro by flow cytometry assay. [Figure 6] Flow cytometry results for purified Fabs H001 and H002 binding to monkey platelets are shown. [Figure 7] Surface plasmon resonance (SPR) assays demonstrate that purified FabH001 antibody binds to recombinant GPIbα. Figure 7A shows SPR data for 25 μL injections of 500, 100, 50, and 10 nM H001. Fitting to a kinetic binding model yields k (on-rate) = 2.61 × 10 s, k (off-rate) = 1.1 × 10 s, and k (dissociation or association constant) = 4.4 nM. Figure 7B shows dose-response curves of the SPR response for 25 μL injections of 500, 100, 50, and 10 nM purified FabH001 plotted against ligand concentration. Fitting the curve to a one-site ligand binding model yields R = 0.9929 and K = 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 9D show standard aggregometry traces demonstrating 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 thrombus formation after heparinized whole blood was perfused for 1, 2, and 3 minutes and treated with control PBS buffer (upper panel) and humanized Fab H001 antibody (5 μg / mL, lower panel) under low shear (300 s) conditions. Figure 10B shows representative photographs demonstrating platelet thrombus formation after perfusion of heparinized whole blood for 1, 2, and 3 minutes and treatment with control PBS buffer (upper panel) and humanized Fab H001 antibody (2.5 μg / mL, middle panel, and 5 μg / mL, lower panel) at high shear rate (1800 s-). Figure 10C shows representative photographs demonstrating platelet thrombus formation after perfusion of heparinized whole blood for 1, 2, and 3 minutes and treatment with control PBS buffer (upper panel) and humanized Fab H002 antibody (5 μg / mL, lower panel) at low shear rate (300 s-). Figure 10D shows representative photographs demonstrating platelet thrombus formation after heparinized human whole blood was perfused for 1, 2, and 3 minutes and treated 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). [Figure 11] Figures 11A and 11B 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 each 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), or 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 an in vivo laser-induced cremaster arteriole thrombosis model. 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 injected humanized Fab H001 was able to bind to platelets in vivo but did not induce increased 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 14B 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 a control (upper panel), Fab H001 antibody (middle panel, 10 μg dose), or Fab H002 antibody (lower panel, 10 μg dose) 5 minutes before injury. Arrows indicate the time of vascular occlusion. Figure 14B is a histogram showing the time to vascular occlusion (in minutes) for each antibody or dose used. * P<0.05, # P<0.05, ** P<0.01. [Figure 15]Figures 15A to 15C 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 cerebral infarct area for each antibody or dose used immediately after induction of tMCAO. Figure 15B is a histogram showing the ischemic cerebral 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 a sham (no filament inserted) control group, a control group 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-HSA 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 microscopically. Figure 16B shows serial images taken at the indicated times after calcium ionophore application or prophylactic treatment with H001 or C100-scFv-HSA in ADAMTS13- / - mice (control). Figure 16B shows a histogram showing the number of emboli (platelet thrombi greater than 20 μm in diameter) in ionophore-induced ULVWF-mediated microvascular thrombosis in a mouse model of TTP. Figure 16C shows a histogram showing the time to recovery of normal blood flow for each 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 percent change in platelet count with antibody treatment: IVIG (○), NIT-B1 (◆), H001 (△), or H002 (▽). (Note: In the original text, "▽" is blacked out.) [Figure 18] The results show 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 to 19D 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 a standard aggregometry trace demonstrating that humanized C100-scFv inhibited ristocetin-induced platelet aggregation. [Figure 21] 1 shows a standard aggregometry trace 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 under high shear (1200 s-) conditions. Representative photographs show platelet clot formation after perfusion of heparinized human whole blood for 1, 2, and 3 minutes, which were treated with control PBS buffer (upper panel) and humanized C100-scFv-HSA (10 μg / mL, lower panel) under high shear (1200 s-) conditions. DETAILED DESCRIPTION OF THE INVENTION

[0024] (Anti-GPIbα antibody) U.S. Patent No. 8,323,652 describes mouse NIT mAbs (NIT-A1, NIT-B1, and NIT-F1) generated by immunizing GPIbα-deficient BALB / c mice with wild-type platelets. These mAbs specifically recognize both human and mouse GPIbα and significantly inhibit ristocetin-induced platelet aggregation and thrombus formation. However, as shown in the following examples, these intact mAbs can induce severe thrombocytopenia. Furthermore, 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 "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 polypeptide (described in Gene ID: 2811), mouse GPIbα polypeptide (described in Gene ID: 110331805 and Gene ID: 110304274), rat GPIbα polypeptide (as 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). According to one embodiment, the antibodies of the present disclosure can recognize and bind 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) with human GP1bα of 10 μM, 10 nM, 10 pM, or lower. D In some embodiments, the dissociation constant of the humanized antibody with human GP1bα (K D ) is 9, 8, 7, 6, 5, 4, 3, 2, 1 μM or less. In some embodiments, the dissociation constant (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 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. The light chain contains one or more complementarity-determining regions (or CDRs) derived from a non-human antibody that binds to a GPIbα polypeptide and a framework region (or FR) derived from a human light chain. The heavy chain contains a complementarity-determining region derived from a non-human antibody that binds to a GPIbα polypeptide and a framework region derived from a human heavy chain. A "complementarity-determining region" or "CDR" refers to the region of an immunoglobulin located in the variable portion of the polypeptide that specifically binds an epitope. The combination of CDRs constitutes the paratope of the antibody.

[0028] The human region of a humanized antibody may be derived from an IgG, IgM, IgA, IgE, or IgD isotype. In some embodiments, the human region of a humanized antibody may be derived from an IgG isotype, e.g., an IgG1, IgG2, IgG3, or IgG4 subclass. In some specific embodiments, the human region of a humanized antibody may be derived from an IgG1 subclass. As described below, because humanized antibodies are monovalent antibodies, the human region of a humanized antibody may be derived from the CH1 region and / or V H The human region of a humanized antibody may comprise a heavy chain derived from a region (excluding the CDRs) and not including the CH2 and / or CH3 regions. 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, the humanized antibody portion is a fragment antigen-binding region of a multivalent antibody, F(ab)2. The F(ab)2 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 an antibody, which are linked to each other by disulfide bonds. Each chain of F(ab)2 contains three V L Domains and the Three Vs H The F(ab)2 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 a multivalent full-length antibody containing a domain. The monovalent antibody portion may be fully or partially glycosylated compared to the parent multivalent antibody from which it is 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 domain, connected to a linker (usually a short peptide linker) and connected to the V fragment from the multivalent antibody. H Domains and V L scFv has only one domain, so it consists of a single antigen-binding region and 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 can comprise, for example, a V H Domains 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 comprise a purification tag (such as a 6X His tag) that can be removed once 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 comprise 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. The 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 the antibody, linked together by disulfide bonds. Fab is 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 comprising 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 human antibodies (such as antibodies), 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 be capable of preventing platelet activation and aggregation. The phrase "capable of preventing platelet activation and aggregation" refers to the ability of the humanized antibodies of the present disclosure to prevent platelet activation and aggregation in the presence of platelets and a platelet agonist. Platelet activation primarily occurs at the onset of hemostasis or thrombosis. Upon activation, platelets change their shape and release the contents of their granules. Activated platelets regulate the expression of membrane proteins (e.g., P-selectin), lipids (e.g., phosphatidylserine), and conformational changes in the platelet αIIbβ3 integrin, which result in platelet aggregation. Platelet activation and aggregation can be measured, for example, by determining platelet shape, the level of platelet aggregation (e.g., using an aggregometer), the expression of surface proteins or lipids, and the like. Platelets can be activated by the following agonists (activators): thrombin, ADP, collagen, etc. 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 from platelet-rich plasma or gel-filtered platelets) can first be contacted with the humanized antibody and then with an agonist. It is then 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 antibodies 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 properties of the humanized antibodies 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 contacted 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 antibodies of the present disclosure can lack the ability to induce thrombocytopenia. The phrase "lacking the ability to induce thrombocytopenia" refers to one of the properties of the humanized antibodies of the present disclosure, namely, not causing a substantial and pathological decrease in the total number of platelets. In humans, a blood count of less than 50,000 platelets per μL constitutes 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 platelet levels are monitored using techniques known in the art to determine whether the antibody causes a decrease in platelet count (and, if so, a substantial or pathological decrease in platelet count). Antibodies that do not induce thrombocytopenia are considered to be antibodies of the present disclosure.

[0037] The antibodies of the present disclosure can 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 in 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 determine whether the antibody causes an increase in bleeding time compared to the reference (if so, a substantial increase in bleeding time). Antibodies that fail to prolong bleeding time at the indicated dose are considered to be antibodies of the present disclosure.

[0038] The antibodies of the present disclosure may also be capable of antagonizing the biological activity of GPIbα polypeptides. GPIbα polypeptides are platelet surface membrane glycoproteins that function as receptors for von Willebrand factor (VWF), thrombin, and other ligands. Therefore, by antagonizing their biological activity, the antibodies of the present disclosure can 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 a mAb). In some embodiments, a monoclonal antibody is produced from a single clone of immune cells. Monoclonal antibodies can be produced using 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 containing 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 containing an epitope of a GPIbα polypeptide. Additional techniques for producing monoclonal antibodies include, but are not limited to, single B cell culture and amplification of a single cell from a B cell population. 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 containing three CDRs. Monoclonal antibodies can 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 can be made of the IgG isotype.

[0040] In one embodiment, an antibody of the present disclosure has at least one complementarity determining region comprising or consisting 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 phrase "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 comprising or consisting 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 comprising or consisting 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 comprising or consisting 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 comprising or consisting 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 present 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, antibodies of the disclosure have a complementarity determining region comprising or consisting 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 comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66, or 67 (including variants and fragments). In some additional embodiments, antibodies of the disclosure have a complementarity determining region comprising or consisting 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 comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, 66, or 67 (including variants and fragments). In some further embodiments, 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 one complementarity determining region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 37, 38, and 39 (including variants and fragments). In some additional embodiments, antibodies of the present 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] Antibodies of the present disclosure may comprise functional variants of CDRs having the amino acid sequences of SEQ ID NOs: 37, 38, 39, 65, 66, or 67. A variant CDR contains at least one amino acid difference compared to the amino acid sequence of the CDR. As used herein, variant refers to changes in the amino acid sequence that do not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In some embodiments, the overall charge, structure, or hydrophobic / hydrophilic properties of an antibody can be altered without adversely affecting biological activity. Thus, the amino acid sequence of a CDR can be altered, for example, to make the antibody more hydrophobic or hydrophilic without adversely affecting the antibody's biological activity. A CDR variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a CDR described herein. As known in the art, the term "percent identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be conventionally determined using known computer programs. Identity can be readily 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 in 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 can 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), where such substituted amino acid residues may or may not be those encoded by the genetic code, or (ii) one or more amino acid residues include a substituent group. A "variant" of a CDR can be a conservative variant or an allelic variant.

[0045] 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 CDR fragment comprises at least one fewer amino acid residue than 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, or functional variants 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, or functional variants 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, or functional variants and functional fragments thereof.

[0047] In another embodiment, the heavy chain comprises a CH1 region of a human IgG1 antibody and includes 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 the present 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 (e.g., 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 than 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 "essentially consisting of" indicates that the heavy chain necessarily comprises the amino acid sequence of SEQ ID NO: 36, 43, 50, or 57, but that additional, non-essential amino acid residues may be added to the amino or carboxyl terminus of these sequences (so long as these amino acid residues do not substantially alter the antibody's affinity for or ability to antagonize the biological activity of a GPIbα polypeptide). As used in the context of this disclosure, a functional variant of a heavy chain antibody refers to changes in the amino acid sequence that do 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 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 the amino acid sequence of SEQ ID NO: 36, 43, 50, or 57). As used in the context of the present 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 (e.g., 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 the present 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 the present 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 than the amino acid sequence of a human IgG1 kappa chain C region, such that the fragment does not adversely affect the biological function of the antibody (e.g., providing specificity and affinity for a GPIbα polypeptide). In one embodiment, the 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 consists essentially of the amino acid sequence of SEQ ID NO: 64, 71, 78, or 85, or functional variants 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 "consisting essentially of" indicates that the CDRs necessarily comprise the amino acid sequence of SEQ ID NO: 64, 71, 78, or 85, 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). As used in the context of the present disclosure, a functional variant of a light chain antibody refers to changes in the amino acid sequence that do 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 the amino acid sequence of SEQ ID NO: 64, 71, 78, or 85). As used in the context of the present disclosure, a functional fragment of a light chain contains at least one fewer amino acid residue compared to the amino acid sequence of the heavy chain, such that it 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 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 the amino acid sequence of SEQ ID NO: 64, 71, 78 or 85).

[0052] 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 "essentially consisting of" indicates that the CDRs necessarily comprise the amino acid sequence of SEQ ID NO: 36, 43, 50, or 57, 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 antibody's affinity for or ability to antagonize the biological activity of a GPIbα polypeptide). As used in the context of this disclosure, a functional variant of a heavy chain antibody refers to changes in the amino acid sequence that do 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 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 the amino acid sequence of SEQ ID NO: 36, 43, 50, or 57). As used in the context of the present 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, such that it 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 (e.g., having the amino acid sequence of SEQ ID NO: 36, 43, 50 or 57).

[0053] In yet another embodiment, an antibody may comprise both a heavy chain and a light chain. In such an embodiment, a humanized antibody may have 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. In another embodiment, a humanized antibody may have 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. In yet another embodiment, a humanized antibody may have 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. In yet another embodiment, a humanized antibody may have 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. In another embodiment, a humanized antibody may have 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. In yet another embodiment, a humanized antibody may have 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. In yet another embodiment, a humanized antibody may have 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. In yet another embodiment, a humanized antibody may have 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. In a further embodiment, a humanized antibody may have 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. In a further embodiment, a 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: 64, a variant thereof, or a fragment thereof. In a further embodiment, a 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: 71, a variant thereof, or a fragment thereof. In yet another embodiment, a 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: 78, a variant thereof, or a fragment thereof.In yet another embodiment, a 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, a 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, a 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, a 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, a 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 can include leader sequences that are cleaved upon secretion from the cell. For example, the amino acid sequence of SEQ ID NO: 35 includes the amino acid sequence of SEQ ID NO: 36 plus 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 can be linked to a carrier (e.g., directly or indirectly via a linker, such as one or more GGGGS (SEQ ID NO: 92) linkers) at any amino acid residue(s), provided that this linkage 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 specific binding to GPIbα and inhibiting 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 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] When 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 genetic engineering techniques. Alternatively, the covalent bond between the two portions can 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 noted above, the bond between two entities (e.g., a humanized antibody portion and a carrier portion) can be non-covalent. Exemplary non-covalent bonds include, but are not limited to, biotin-streptavidin / avidin systems. 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 either 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 non-covalent system, the first entity is designed to be non-covalently bound to the second entity only upon administration to the intended recipient. This embodiment is particularly useful when the carrier is a protein present in the recipient's blood. For example, a humanized antibody portion can be linked (covalently or non-covalently) to a second antibody, lectin, or fragment thereof (referred to herein as an antibody-derived linker) that is capable of non-covalently binding 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 simultaneously bind to multiple sites on activating Fc receptors), alpha-1-acid glycoprotein, transferrin, or lipoprotein). 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 embodiments, the second antibody, lectin, or fragment thereof acts as 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 proteins) 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 contain naturally occurring introns, genic regions, nongenic regions, and regulatory regions. Furthermore, such genomic DNA may 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 appropriate 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 host cells, tissues, or organisms. In one embodiment, the nucleotide molecules can be codon-optimized for expression in a particular host. In some embodiments, the nucleotide molecule may 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. In some embodiments, the nucleotide molecule of the present disclosure may 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 Because the interaction between platelet GPIbα and its ligands (such as VWF) is recognized as a key player in the pathogenesis of various diseases, humanized antibodies can be used to prevent and / or treat ischemic stroke, acute myocardial infarction, restenosis, angina pectoris, acute coronary syndrome, atherothrombosis, vascular inflammation, venous thrombosis, peripheral vascular disease, thrombotic thrombocytopenic purpura, sepsis, and / or tumor metastasis. Humanized antibodies or chimeric proteins can be used in subjects with platelets specifically recognized by the humanized antibody (or the humanized antibody portion of the chimeric protein). Thus, humanized antibodies can be used in mammalian subjects, such as 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 involves contacting a humanized antibody, chimeric protein, or pharmaceutical composition described herein with platelets (expressing GPIbα on their surface) under conditions that allow binding of the humanized antibody / humanized antibody portion to GPIbα. As shown in the examples below, the humanized antibodies and chimeric proteins of the present disclosure have the ability to bind to GPIbα and antagonize its biological activity under low and high shear stress. Therefore, this method can be used to bind to GPIbα regardless of the applied shear stress. This method can be used in vitro or in vivo in a subject in need thereof. This method can be used at low or high shear rates.

[0062] When 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, platelets are first contacted with a humanized antibody (optionally presented as a chimeric protein or pharmaceutical composition) before the ligand is located or discovered in the vicinity of the platelets. In such embodiments, binding of the humanized antibody of the present disclosure is understood to prevent physical association between GPIbα and its ligand and ultimately prevent or limit 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 simultaneously with or after contact between GPIbα and its ligand has occurred. Platelets are then contacted with the humanized antibody (optionally presented as a chimeric protein or pharmaceutical composition) simultaneously with or after the ligand is located or discovered 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, prevent or limit platelet activation and aggregation.

[0064] Humanized antibodies (optionally in chimeric form or within 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 can prevent platelet activation and aggregation (at least in the examples below), 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 monoclonal antibodies from which they are derived). As used in the context of this disclosure, the term "pathological thrombosis" refers to a condition in which a thrombus (blood clot) forms in a blood vessel and causes damage to 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 lung (pulmonary embolism). In some embodiments, the humanized antibody or chimeric protein is used to prevent, treat, or alleviate symptoms associated with pathological thrombosis under conditions of high shear stress. 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 prevention of thrombus formation or growth is warranted, 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 the subject should receive additional doses to achieve the desired therapeutic effect.

[0066] In subjects with multiple thrombi, humanized antibodies or chimeric proteins can be used to reduce the size and / or number of thrombi. In one embodiment, the method may 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 administering the antibody. Humanized antibodies can be used in subjects who have previously determined that they have multiple thrombi and, optionally, the size of the thrombi. In another embodiment, the method may include determining the presence, number, and size of thrombi after at least one administration of the humanized antibody or chimeric protein. Such a determination may help determine whether the subject should receive additional doses to achieve the desired therapeutic effect.

[0067] In some embodiments, the methods of the present disclosure include determining whether a subject is at risk of or has experienced pathological thrombosis. A positive determination that the subject is at risk of or has experienced pathological thrombosis 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 or chimeric protein to a subject determined to be at risk of or have experienced pathological thrombosis. The humanized antibodies and chimeric proteins of the present disclosure may be used on subjects determined to be at risk of 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 can include administering a humanized antibody to a subject determined to be at risk of 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 can be used in subjects determined to be at risk of 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] Because the interaction between GPIbα and VWF is important for the spread of tumor metastasis, the humanized antibodies or chimeric proteins of the present disclosure can be used to reduce or limit tumor metastasis in a subject in need thereof. In some embodiments, the humanized antibodies or chimeric proteins can be used to reduce the number and / or size of tumor metastases. In one embodiment, tumor metastasis is associated with liver cancer (e.g., hepatocarcinoma or adenocarcinoma), and the humanized antibodies can be used to reduce or limit liver tumor metastasis.

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

[0071] The humanized antibody or chimeric protein comprising the same can be formulated as a pharmaceutical composition for administration together with an additive. An additive or "pharmaceutical excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmacologically inert vehicle, typically liquid, for delivering one or more chimeric proteins to a subject. The pharmaceutical excipient is generally selected to provide the desired volume, 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 corn 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), disintegrants (e.g., starch, sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).

[0072] The humanized antibody or chimeric protein comprising the same can be formulated with pharmaceutically acceptable additives in a unit dosage form or as a pharmaceutical composition for administration. Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administering such compositions to a subject. 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 a liquid solution or suspension. Methods well known in the art for preparing 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 pharmaceutically effective amount. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (dosage) effective to treat 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 "pharmaceutically effective amount" may be interpreted as an amount that provides the desired therapeutic effect, either administered alone or in combination with other therapeutic agents, either in a single dose or at 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, and other ranges according to the present invention include 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 10-20 mg, about 20-30 mg, about 30-40 mg, and about 40-50 mg. In one embodiment, the chimera is administered at a dosage of between about 40-80 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 with the International Depositary Authority of Canada on October 7, 2008, 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 CDR and annotated human sequences in the NCBI database.

[0078] Briefly, the variable domain sequences of the parent antibodies were searched against a human germline database using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ). For each heavy and light chain, four diverse human receptors (i.e., human variable domains with high homology to the parent antibodies) 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 were prepared. These scFvs contained both VH1 and VL2. These included a (GGGGS (SEQ ID NO: 92)) x 4 linker between VH1 and VL2 (arranged as VH1-(G4S)4-VL2). To facilitate purification, the scFvs contained a 6xHis tag and a TEV cleavage site, ENLYFQG, before VH1. However, the tag was removed using TEV protease before further testing. The scFvs were also incorporated into chimeric proteins (scFv-HSA) with human serum albumin (HSA). In these examples, the scFv-HSA contained an additional linker, GGGGS (SEQ ID NO: 92), before the HSA. The chimeric scFv-HSA was produced in a stable form and did not form aggregates during its production or purification.

[0080] Size: DNA sequences encoding the humanized heavy and light chains were synthesized and inserted into the pTT5 vector to construct Fab expression plasmids. Sixteen humanized Fabs were transiently expressed in HEK293 or CHO3E7 cell cultures, followed by cell spindown. 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 analyzed by Capture select. TM Kappa XL affinity matrix resin (Capture select TM The purified humanized Fab was purified using Kappa XL Affinity Matrix resin. The purified humanized Fab was buffer exchanged into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by 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 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 300 g for 7 min. 200 μL of PRP was transferred to 10 mL of PBS and washed by subsequent centrifugation at 800 g for 10 min. The supernatant was then removed, and the platelets were resuspended in 200 μL of PBS. 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 an FITC-labeled anti-human Fab antibody. All eight of these 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: 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 BDLSR 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) a second purification by SEC-FPLC chromatography was added to washed cynomolgus monkey platelets (2 × 10 6 ) for 30 minutes. Then, an FITC-labeled anti-human kappa chain secondary antibody (Ab) was incubated for 30 minutes. Humanized Fab binding to monkey platelets was detected by flow cytometry assay. Figure 6 shows that Fab H001 and H002 antibodies bound to monkey platelets.

[0086] The affinity between H001 and recombinant GPIbα was measured by surface plasmon resonance (SPR) assay. To prepare the SPR biosensor, a bare gold-coated SPR biosensor (Biosensing Instrument Inc., Arizona, USA) was washed for 2 hours in a solution of 0.5 M sodium borohydride dissolved in 1:1 absolute ethanol:ddH2O. The biosensor was then rinsed with a large amount of absolute ethanol and subsequently incubated for 16 hours in a solution of 1 mM mercaptopropionic acid in dimethylformamide. After incubation, the SPR biosensor was rinsed with a large amount of dimethylformamide, followed by absolute ethanol, and finally ddH2O. The biosensor was then incubated for 1 hour in 40 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 20 mM N-hydroxysuccinimide dissolved in ddH2O. The sensor was then washed with copious amounts of ddH2O and incubated with 100 mM Nα,Nα-bis(carboxymethyl)-L-lysine hydrate for 4 hours. After incubation, the biosensing surface was rinsed with ddH2O. The final step before the SPR experiment was to expose the functionalized biosensing surface to 100 mM NiCl2 for 1 hour.

[0087] SPR experiments were performed on a Biosensing Instrument 4000SPR. The functionalized biosensor was loaded onto the instrument and 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 baseline equilibration, 25 μL of the desired concentration of ligand (Fab H001 or control) was injected. The biosensing surface was regenerated between ligand injections by injecting 100 μL of 500 mM imidazole into the running buffer, followed by 100 μL of 100 mM NiCl. The acquired data 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 antibody 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 The dissociation constant (Kd) of 8.0 ± 2.1 nM was obtained. The data clearly demonstrate that the Fab H001 antibody binds tightly to recombinant GPIbα with a low nanomolar dissociation constant and a fast binding kinetics.

[0089] In vitro inhibition of agonist-induced platelet aggregation: To evaluate whether humanized Fabs can induce abnormal platelet activation and their role in platelet aggregation, an in vitro platelet aggregation assay was performed. Human PRP from healthy volunteers and patients with peripheral vascular disease was prepared from sodium citrate-anticoagulated whole blood by centrifugation at 300 g for 7 minutes. Platelet aggregation in PRP was induced by the addition of 5 μg / mL of humanized Fab clones and monitored using a computerized Chrono-log aggregometer (Chrono-log, USA). Platelet aggregation in PRP with or without humanized Fabs 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 a type I collagen-coated surface 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, Nichodt, Linz, Austria). Anticoagulated (heparin 15 U / mL) whole blood from healthy donors was fluorescently labeled with DiOC6 (1 μM, 10 min, 37°C, Sigma). Next, whole blood treated with control or humanized Fab or scFv-HSA was perfused over 300 s using a syringe pump (Harvard Apparatus, USA). -1 , 1200s -1 , and 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 (60x / 0.90 NA water objective). Quantitative dynamics of platelet fluorescence intensity were acquired using SlideBook software.

[0092] Humanized Fabs H001 and H002 correspond to venular / aortic and arteriolar blood flow, respectively, for 300 s -1 and 1800s -1 wall shear rate (but preferably 1800 s -1 The chimeric protein significantly inhibited thrombus formation at wall shear rates of 1200 s (Fig. 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 investigate whether humanized Fab affects thrombus growth in vivo, we utilized two complementary intravital 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 using a Zeiss Axiovert 135 inverted fluorescence microscope (Zeiss, Germany). Briefly, blood was collected from donor-matched mice into acid citrate dextrose (ACD) solution (anticoagulant). Gel-filtered platelets were prepared and labeled with calcein AM (1 mg / mL, Invitrogen, Canada) for 20 minutes at room temperature. Next, platelets were injected into experimental mice via the tail vein along 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 measuring 100-120 μm in diameter 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, thrombus growth and vascular occlusion induced by FeCl3 injury were significantly inhibited by injection of the humanized Fab H001 and H002 antibodies (compared to control saline injection).

[0095] Table 2: Number of mice without obstruction 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 inserted to facilitate respiration. The cremaster muscle was prepared under a dissecting microscope and perfused with prewarmed bicarbonate-buffered saline throughout the experiment. Platelet antibodies, control (saline buffer), humanized, and monovalent H001 and H002 antibodies (5 or 10 μg / mouse), were administered into the jugular vein cannula as indicated. Platelets were labeled by injection of a rat anti-mouse CD41 antibody (Leo.A1, EMFRET Analytics, Germany, 0.1 μg / g). Multiple independent upstream injuries were induced in the cremaster arteriole with a pulsed nitrogen dye laser using an Olympus BX51WI microscope. The dynamic accumulation of fluorescently labeled platelets within the developing thrombus was captured and analyzed using Slidebook software. In this cremaster arteriole intravital microscopy thrombosis model (which uses mild laser-induced vascular injury and does not involve oxidative stress), thrombus growth was almost completely abolished after intravenous injection of the humanized and monovalent H001 and H002 antibodies (Figure 12). These results suggest that the humanized Fabs inhibit thrombus growth and promote thrombus dissolution, and have great potential for development as novel antithrombotic agents.

[0097] Before and after the intravital microscopy thrombosis model experiment, blood was collected from the mice, 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 abnormal platelet activation in vivo, as it did not induce platelet P-selectin expression or phosphatidylserine (PS) exposure. 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 or older, 25–30 g) were anesthetized and intravenously injected with Fab H001 or H002 antibody (5, 10, or 20 μg / mouse) or an equal volume (200 μL) of PBS 5 minutes before inducing arterial injury. The left common carotid artery was incised and monitored with a miniature Doppler flow probe (TS420 transit-time perivascular flowmeter, Transonic Systems Inc., USA). Baseline blood flow was measured for 30 seconds. Next, carotid artery injury was induced for 3 minutes 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 antibodies in ischemic stroke, we performed a cerebral ischemia and reperfusion injury model (transient middle cerebral artery occlusion (tMCAO)). Male mice (25 g) were anesthetized with inhaled isoflurane. A midline neck incision was made, and the soft tissue was dissected free. The left common carotid artery (LCCA) was carefully isolated from surrounding nerves (without damaging the vagus nerve), and a ligature was made using a 5.0 string. Next, the left external carotid artery (LECA) was isolated, and a second ligature was made. Next, the left internal carotid artery (LICA) was isolated, and a ligature was made with a 6.0 filament. After visualizing the left internal carotid artery (LICA) and left pterygopalatine artery (LPA), 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 in the circle of Willis. A third ligature on the LICA was closed to secure the filament in place. After 1 hour, the third ligature was opened and the filament was withdrawn. Antibodies were administered intravenously either immediately after filament insertion or 1 hour after the filament was withdrawn.

[0100] To measure the cerebral infarct volume, mice were euthanized 24 hours after tMCAO induction. To visualize the cerebral infarct, multiple 2-mm-thick coronal brain sections cut from the whole brain were stained with 2% 2,3,5-triphenyl-tetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO). The presence of cerebral hemorrhage was assessed visually. To measure neurological function 24 hours after tMCAO induction, mice were subjected to a modified Bederson test and a grip test to assess 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 cerebral infarct size 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 genotype-matched donor mice. 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. The 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 minutes. 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 the platelets with calcein-acetoxymethyl ester (1 μg / mL) for 15 minutes at room temperature. The efficacy of fluorescent labeling of platelets was assessed by fluorescence microscopy before use in in vivo imaging.

[0102] For intravital microscopy, 4-week-old mice were anesthetized and fluorescently labeled platelets (1.25 × 10 from mice of the same genotype) were added. 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 locally treated 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 thrombus resolution were monitored and recorded for 20 minutes in addition to the pre-recording. Fab H001 or scFv-HSA chimeric proteins stimulated 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 rats. The kinetics of platelet accumulation in selected vessel segments was quantitatively analyzed by (1) the number of emboli (platelet thrombi greater than 20 μm in diameter) and (2) the time to restore normal blood flow (defined as the time required for platelet fluorescence to return to near baseline after topical application).

[0103] As shown in Figure 16, all ADAMTS13 antibodies treated with saline (control), Fab H001 antibody, or scFv-HSA chimeric protein - / - Before the ionophore was applied 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 single platelet strings attached to the endothelium in the direction of blood flow. Within 1 minute, multiple large thrombi (diameter >20 μm) 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 separated from the vessel wall and downstream from the embolus. Platelet adhesion to the vessel wall and embolic thrombosis in control mice continued for more than 10 minutes, but the mice eventually died over time, eventually restoring normal blood flow to the affected portion of the vessel. ADAMTS13 - / - The thrombotic response in 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 to the control group, the number of large embolic thrombi was significantly lower in both Fab H001 antibody or scFv-HSA chimeric protein treatment groups, and the time to restore normal blood flow in mesenteric venules was shorter (Figure 16). These results support the suppression of ADAMTS13 by Fab H001 antibody or scFv-HSA chimeric protein. - / - We showed that prophylactic treatment of mice effectively inhibited ionophore-induced VWF-mediated microvascular thrombosis and mimicked 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 antibody (10 μg / mouse, n=3 per group), or NIT-B1 antibody (5 μg / mouse, n=2). Serial blood samples were collected from the medial saphenous vein of the mice at different time points (0, 30 min, 1, 2, 4, 8 h, 1, 2, 3, 4, 5, 6, and 7 days). 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 stark contrast to the NIT-B1 antibody.

[0105] Bleeding time: BALB / c mice were intravenously injected with PBS (n=4), Fab H001, or H002 antibody (5-10 μg / mouse, n=3) 120 minutes before injury. Mice were anesthetized with 2.5% avertin (18 mL / kg body weight, i.p.) and maintained on a 37°C heating pad. The tip of the tail (2 mm) was cut with a sharp scalpel, and the wound was gently 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, 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] While the present invention has been described in connection with particular 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 May prevent platelet activation, aggregation, and / or thrombus growth, lacks the ability to activate platelets, lack the ability to induce thrombocytopenia, and / or lacks the ability to prolong bleeding time at therapeutic doses, A humanized antibody characterized by:

[0109] [Appendix 2] A humanized antibody according to Appendix 1, which is 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] 4. The humanized antibody of claim 3, which is a F(ab)2 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] 6. 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 appendices 1 to 7, having a heavy chain.

[0116] [Appendix 9] The heavy chain 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 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; 9. The humanized antibody of claim 8, comprising:

[0117] [Appendix 10] 10. The humanized antibody of claim 8 or 9, wherein the heavy chain further comprises a CH1 region of a human IgG1 antibody.

[0118] [Appendix 11] 11. The humanized antibody of claim 10, wherein the CH1 region of the human IgG1 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 appendices 1 to 12, having a light chain.

[0121] [Appendix 14] The light chain 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; 14. The humanized antibody of claim 13, comprising:

[0122] [Appendix 15] 15. The humanized antibody of claim 13 or 14, wherein the light chain further comprises a kappa chain C region of a human IgG1 antibody.

[0123] [Appendix 16] 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 appendices 1 to 17, comprising:

[0126] [Appendix 19] 19. A chimeric protein comprising a humanized antibody of any one of appendices 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] 23. The method of 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 blood clot in a subject in need thereof.

[0135] [Appendix 28] 27. The method of claim 26, for reducing the size or 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 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 clauses 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 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 has or lacks an Fc portion, and can prevent platelet activation, aggregation, and / or thrombus growth; lacks the ability to activate platelets, lacking 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 (except when the humanized antibody or fragment thereof has a heavy chain having the amino acid sequence of SEQ ID NO: 36 or 57 and a light chain having the amino acid sequence of SEQ ID NO: 71 or 78); A humanized antibody or fragment thereof characterized in that:

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

3. The humanized antibody or fragment thereof according to claim 1 or 2, which is an antibody fragment.

4. The humanized antibody or fragment thereof of claim 3, which is an F(ab)2 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 according to 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 further comprises a CH1 region of the antibody. １ The humanized antibody or fragment thereof of claim 1, wherein the CH1 region of the antibody has the 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, wherein the kappa chain C region has the 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 of claim 1.

11. A pharmaceutical composition comprising (i) the humanized antibody or fragment thereof of claim 1, or the chimeric protein of claim 10, and (ii) a pharmaceutical additive.

12. 1. A pharmaceutical for use in a method for preventing or limiting the 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 comprises 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 carried out under any shear rate.

16. The medicament according to 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 of 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. 18. 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 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.

Images