Binding protein to human thrombin receptor PAR4

Abstract

To provide PAR4 antagonists that provide an improved therapeutic profile over targeting PAR1 in treatment or prevention of thrombosis.SOLUTION: Provided is a protease activated receptor 4 (PAR4) binding protein, which is an anti-PAR4 recombinant or synthetic or a monoclonal antibody or an antigen-binding fragment thereof, where the binding protein inhibits cleavage of cell surface expressed human PAR4 by equal to or greater than 50% in the presence of thrombin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Embedding by reference All documents cited or referenced herein, and all documents cited or referenced within any documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned herein or incorporated herein by reference, are incorporated herein by reference in their entirety.

[0002] This application claims priority to Australian Patent Application No. 2017903685, filed on 11 September 2017, the entire contents of which are incorporated herein by reference.

[0003] The entire contents of the electronic application for sequence listings are incorporated by reference for all purposes.

[0004] Areas of this disclosure This disclosure relates to human protease-activated receptor 4 (PAR4) binding proteins (e.g., antibodies). More specifically, it relates to anti-PAR4 binding proteins that are human PAR4 antagonists, as well as methods and uses thereof. [Background technology]

[0005] Activated platelets are a key cellular component in arterial thrombosis and are the most common cause of death and disability worldwide (Rosendaal FR et al. (2014) 384:1653-4), accounting for nearly 40% of deaths in many countries (Mozaffarian D et al. (2015) Circulation 131 e29-322), including Australia (Australian Bureau of Statistics, Causes of Death, Australia, 2011 3303 Chapter 4 2011 (2013)). Arterial thrombosis leads to heart attacks and ischemic strokes.

[0006] Platelets are cells that form arterial thrombi. Platelets are activated by a combination of endogenous agonists that cause platelet aggregation and coagulation, and these agonists work together to promote pathological thrombus formation. Therefore, antiplatelet drugs constitute the main drug therapy for preventing arterial thrombosis. However, despite the existence of numerous such drugs, there is a need for rationalization of novel drug targets due to safety and / or efficacy constraints. There is a significant demand for improvements to antiplatelet drugs in a wide range of clinical settings, and there is great interest in the development of new drugs.

[0007] Thrombin is the most potent known human platelet activator and a key effector protease in the coagulation cascade. Thrombin is the body's most potent platelet activator, primarily through protease-activated cell surface receptors (PARs), PAR1 and PAR4 (Vu T-KH et al (1991) Cell 64:1057-68; Coughlin SR (1992) J Clin Invest 89:351-5; Coughlin SR (2000) Nature 407:258-64). These receptors belong to a unique family of seven-transmembrane receptors, G protein-coupled receptors (GPCRs), which are activated by N-terminal proteolysis. Once cleaved, the newly exposed N-terminus acts as a tethering ligand, activating the receptor by binding to extracellular loop 2 (Vu T-KH et al (1991) Cell 64:1057-68). There are four members (PAR1-PAR4) of the PAR family that are expressed and activated by multiple proteases.

[0008] All platelets lack PAR function (PAR4). - / -Mice are protected from thrombosis without spontaneous bleeding (Hamilton J et al. (2004) Thromb Haemost 2:1429-35; Hamilton J et al. (2009) Blood Rev 23:61-5), which demonstrates the potential of targeting such receptors for antithrombotic therapy. Two dominant PARs, PAR1 and PAR4, exist on human platelets. Of these, PAR1 is a higher affinity thrombin receptor and has been a target for antiplatelet drug development. Two PAR1 antagonists, atopaxal (E5555) (Goto S et al. (2010) Eur Heart J 31:2601-13) and borapaxal (Tricoci P et al. (2012) New Engl J Med 366:20-33; Morrow DA et al (2012) New Engl J Med 366:1404-13), are being evaluated in clinical trials. Borapaxal was approved by the US FDA in late 2014 and is scheduled for a TGA in mid-2016 for the prevention of myocardial infarction and peripheral artery disease. However, borapaxal in combination with one or two antiplatelet therapies was associated with a significant increase in intracerebral hemorrhage rates, particularly in patients with a history of stroke or other predisposing factors (Tricoci P et al. (2012) New Engl J Med 366:20-33; Morrow DA et al (2012) New Engl J Med 366:1404-13).

[0009] As a result, there has been great interest in the development of PAR4 antagonists. The functional role of PAR4 has been elucidated mainly in relation to platelets. A key feature characterizing PAR4 is its ability to form heterooligomers with both PAR1 and the ADP receptor P2Y12, which allows PAR4 to influence both thrombin and ADP initiation signaling (Li D et al. (2011) J Biol Chem 286:3805-14). One major difference between the two platelet PARs is the dynamics of intracellular signaling (Holinstat M et al. (2006) J Biol Chem 281:26665-74; Voss B et al. (2007) Mol Pharmacol 71:1399-406; Holinstat M et al. (2007) Mol Pharmacol 71:686-94). Both PAR1 and PAR4 signal via Gq to recruit intracellular calcium, driving platelet function including integrin activation, granule secretion, and phosphatidylserine (PS) exposure. However, PAR4 activation induces a slower but more sustained intracellular calcium signal than PAR1 activation (Covic L et al. (2002) PNAS 99:643-8). This time difference in calcium signaling may be partly due to the anionic C-terminus of the PAR4 cleavage site (Jacques S et al. (2003) Biochem J 376:733-40). The cellular consequences of such sustained platelet activation downstream of PAR4 have not yet been fully characterized, but given that these phenomena depend on sustained elevated intracellular calcium levels, sustained platelet secretion dynamics (Jonnalagadda D et al. (2012) 120:5209-16) and platelet coagulation function may be involved (Williamson P et al. (1995) 34:10448-55; Dachary-Prigent J et al. (1995) Biochemistry 34:11625-34).

[0010] No studies have verified that PAR4 contributes to procoagulant activity in the human thrombus formation setting, likely due to the limited availability of suitable PAR4 antagonists required for such research. The most commonly used PAR4 antagonists are the small molecule YD-3 (Wu CC et al. (2000) Br J Pharmacol 130:1289-96), the peptide mimetic tc-YPGKF-NH2 (Hollenberg MD et al. (2001) 79:439-42), and the peptuducins P4pal-10 and P4pal-il (Leger AJ et al. (2006) Circulation 113:1244-54; Covic L et al. (2002) PNAS 99:643-8; Stampfuss JJ et al. (2003) Nat Med 9:1447). However, these drugs are not widely available in studies using human platelets (e.g., YD-3), or have been reported to lack specificity (e.g., pepducin) and / or efficacy (e.g., tc-YPGKF-NH2) (Stampfuss JJ et al. (2003) Nat Med 9:1447; Holenberg MD et al. (2004) Br J Pharmacol 143:443-54; Wu CC et al. (2002) Thromb Haemost 87:1026-33). PAR4 antagonists (BMS-986141) have been validated in early clinical trials for the treatment of thrombosis, but common PAR4 variants (present in 19-82% of the population depending on the population) make the receptor insensitive to small molecule inhibitors (e.g., BMS-986141).

[0011] Based on the above, it will be apparent to those skilled in the art that the identification of improved human PAR4-binding proteins for the medical treatment of thrombosis would be useful. Furthermore, the treatment of thrombosis with minimal adverse side effects is a significant unmet medical need. Therefore, there is a need in the art for PAR4 antagonists that offer advantages over existing strategies and result in improved therapeutic profiles in PAR1 targeting for the treatment or prevention of thrombosis. [Overview of the Initiative]

[0012] Current clinical programs are developing small molecule orthosteric PAR4 inhibitors. However, it has recently become clear that this approach is completely ineffective in a high percentage of patients. Specifically, a single nucleotide polymorphism (SNP; rs773902) in PAR4 makes the receptor insensitive to orthosteric PAR4 antagonistism (Edelstein LC et al (2014) Blood 124:3450-3458). This small nucleotide polymorphism (SNP) determines whether amino acid 120 is alanine (Ala120) or threonine (Thr120). Pharmacological studies have shown that orthosteric PAR4 antagonistism potently inhibits PAR4-induced platelet activation in patients with the Ala120 genotype, but has no effect whatsoever on platelets from patients with the Thr120 genotype, even at high concentrations. In heterozygotes, only partial inhibition occurs (Edelstein LC et al (2014) Blood 124:3450-3458). The prevalence of inhibitor-resistant Thr120 variants in PAR4 is remarkably high (over 80% in some populations), indicating the significant impact of this antagonistism. The Thr120 allele is racially dimorphic, occurring in 63% of individuals who identify as Black in a cohort of 154 North Americans, compared to 19% of White individuals. Data from the Human Genome Diversity Project (HGDP) show that SNP rs773902 is not region-specific, with up to 80% of people living in sub-Saharan Africa and approximately two-thirds of Papuans and Melanesians possessing the Thr120 PAR4 variant.

[0013] In addition to making PAR4 resistant to orthosteric inhibitors, the SNP rs773902 increases the sensitivity of PAR4 to receptor activation, leading to hyperactive platelets in patients with the Thr120 variant (Edelstein LC et al. (2013) Nat Med 19:1609-1616). This increased PAR4 function persisted in patients treated with standard antiplatelet agents (aspirin and / or P2Y12 inhibitors).

[0014] The inventors developed a monoclonal antibody that specifically binds to human PAR4 and inhibits thrombin-induced PAR4 activation. Therefore, this antibody is an antagonist of thrombin-induced PAR4 cleavage. The antibody identified by the inventors can attenuate or reduce PAR4-mediated events (e.g., thrombosis). Furthermore, the inventors found that targeting PAR4 is less likely to cause bleeding complications than targeting PAR1 due to its different mechanism of action and overall broader safety profile.

[0015] In detail, the antibody identified by the inventors is effective against both PAR4 receptor variants, namely Ala120 and Thr120, which means it is effective in treating thromboembolic disorders in all subjects, not just those with sensitive PAR4 variants. Furthermore, because the antibody specifically binds to PAR4 with minimal cross-reactivity with PAR1, bleeding complications can be minimized or avoided. Thus, the antibody of the present invention is distinguished from prior art PAR1 antagonists and PAR4 small molecule inhibitors.

[0016] In addition, the inventors found that the antibody significantly inhibits thrombin-induced PAR4 cleavage and human platelet aggregation. Furthermore, these effects could be reversed by decompeting the antibody with an immunized peptide.

[0017] Accordingly, this disclosure provides various reagents for diagnosing or predicting thrombosis in subjects. This disclosure also provides methods for treating, preventing, or improving thrombosis or thromboembolic disorders in subjects.

[0018] This disclosure provides a PAR4-binding protein comprising an antigen-binding domain, wherein the antigen-binding domain specifically binds to human PAR4, and the protein attenuates at least one PAR4-mediated event (e.g., thrombosis). In one example, the protein attenuates at least one PAR4-mediated event (e.g., thrombosis) in the presence of thrombin.

[0019] In one example, the antigen-binding domain is from or derived from a non-antibody PAR4-binding protein. In another example, the PAR4-binding protein is not a small molecule antagonist (examples of small molecule antagonists include, for example, imidazothiadiazole derivatives described in WO2013 / 163244, or synthetic peptide analogs described in, for example, US7879792).

[0020] This disclosure also provides a human PAR4-binding protein comprising an antigen-binding domain of an anti-PAR4 antibody, wherein the antigen-binding domain specifically binds to PAR4, and the protein attenuates at least one PAR4-mediated event (e.g., thrombosis) when it comes into contact with a cell expressing PAR4. In one example, the protein attenuates at least one PAR4-mediated event (e.g., thrombosis) in the presence of thrombin.

[0021] In one example, PAR4-binding proteins inhibit the externalization of phosphatidylserine (PS) from the cell surface of PAR4-expressing cells (e.g., platelets). In another example, PAR4-binding proteins reduce thrombus volume as measured by a whole blood thrombosis assay.

[0022] This disclosure provides a protease-activated receptor 4 (PAR4) binding protein which is an anti-PAR4 recombinant, synthetic, or monoclonal antibody or its antigen-binding fragment, and which substantially inhibits thrombin-induced human PAR4 cleavage.

[0023] Those skilled in the art will be able to measure PAR4 cleavage using appropriate in vitro assays. As a non-limiting example, PAR4 cleavage can be evaluated in cell lines expressing a fluorescently labeled tagged PAR4 protein on the cell surface. Loss of FLAG from the cell surface due to PAR4 cleavage in the presence of thrombin can be quantified. In a specific example, PAR4 cleavage can be measured in transfected HEK293 cells containing nucleic acids encoding PAR4 with a fluorescently labeled FLAG tag. Loss of FLAG from the cell surface due to PAR4 cleavage in the presence of thrombin (e.g., 0.1 U / ml) can be quantified using flow cytometry.

[0024] In one example, the binding protein inhibits the cleavage of cell surface-expressed human PAR4 by more than 50% in the presence of thrombin.

[0025] In one example, the PAR4-binding protein specifically binds to an epitope spanning the thrombin cleavage site of human PAR4. In another example, the PAR4-binding protein specifically binds to an epitope containing residues within the sequence represented as GDDSTPSILPAPRGYPGQVC (SEQ ID NO: 2). In yet another example, the peptide consists of SEQ ID NO: 1 or SEQ ID NO: 2. For example, the peptide is shown on the surface of a phage.

[0026] In another example, the epitope contains the sequence APRGY (SEQ ID NO: 42), where the thrombin cleavage site corresponds to RG. In yet another example, the epitope contains or consists of a sequence selected from ILPAPRGY (SEQ ID NO: 43) or APRGYPGQV (SEQ ID NO: 44). In one example, the PAR4-binding protein specifically binds to an epitope containing the sequence indicated as PRGYPG (SEQ ID NO: 1).

[0027] In one example, the PAR4-binding protein specifically binds to either the Ala120 variant or the Thr120 variant of human PAR4. In another example, the PAR4-binding protein binds to both the Ala120 variant and the Thr120 variant of human PAR4.

[0028] In one example, the PAR4-binding protein binds to a thrombin cleavage site within a human PAR4 sequence according to SEQ ID NO: 19. In another example, the PAR4-binding protein binds to a sequence matching residues 35–54 of the human PAR4 sequence shown in SEQ ID NO: 19. In yet another example, the PAR4-binding protein binds to a sequence shown as SEQ ID NO: 2, which optionally additionally contains keyhole limpet hemocyanin (KLH) or other immunostimulatory molecules. In yet another example, the PAR4-binding protein binds to a sequence shown in SEQ ID NO: 4.

[0029] In another example, the PAR4-binding protein binds to the sequence shown as Sequence ID No. 2, which optionally includes an additional C-terminal GGGG and streptavidin-k / biotin (SKB). In one example, the PAR4 protein binds to the sequence shown as Sequence ID No. 7.

[0030] In one example, a PAR4-binding protein does not bind to human PAR3, PAR2, or PAR1, or binds to them substantially.

[0031] In further examples, PAR4-binding proteins do not bind, or substantially do not bind, to PAR sequences selected from the group consisting of SEQ ID NO: 3 (mouse PAR4), SEQ ID NO: 8 (human PAR3), SEQ ID NO: 9 (human PAR2), or SEQ ID NO: 10 (human PAR1).

[0032] In one example, the level of binding is assessed by immobilizing a peptide (e.g., a peptide according to SEQ ID NO: 2 or SEQ ID NO: 7) and bringing the peptide into contact with a PAR4-binding protein.

[0033] Exemplary PAR4-binding proteins having such binding characteristics as described herein include the variable region and / or CDR of an antibody named 5ARC3.F10b.H4b (hereinafter referred to as 5A.RC3) or 5F.RF3.A7b.A1 (hereinafter referred to as 5F.RF3).

[0034] In one example, the PAR4-binding protein binds to peptides or human PAR4 consisting of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7 at a similar or substantially identical level, or with similar or substantially identical affinity, to antibodies named 5A.RC3, 51.RG1, 5F.RG3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, or 5H.RF2.

[0035] In another example, the PAR4-binding protein competitively inhibits the binding of antibodies named 5A.RC3, 51.RG1, 5F.RG3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, and 5H.RF2 to human PAR4. In yet another example, the protein competitively inhibits the binding of antibodies named 5A.RC3, 51.RG1, 5F.RG3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, and 5H.RF2 to peptides consisting of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7.

[0036] In one example, the PAR4-binding protein binds to a peptide consisting of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7, in an amount no more than 75% of the amount bound by an antibody containing VH, which includes the sequence shown in SEQ ID NO: 11, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107, and VL, which includes the sequence shown in SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, or SEQ ID NO: 108.

[0037] In one example, the amount of bound protein or antibody is assessed by contacting the PAR4-binding protein with a peptide consisting of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7, and by the amount of PAR4-binding protein that has come into contact with the peptide (e.g., 10 μg / ml). The amount of PAR4-binding protein bound to the peptide is then measured and compared to the amount of antibody, each containing a VH with the sequence shown in SEQ ID NO: 11, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107, and each containing a VH with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, or SEQ ID NO: 108. In one example, the amount of PAR4-binding protein bound to the peptide is within approximately 80%, 70%, 60%, or 40% of the amount of bound antibody.

[0038] This disclosure also provides PAR4-binding proteins that competitively inhibit the binding of antibodies with the following names: (i) The aforementioned 5A.RC3 antibody containing VH with the sequence shown in SEQ ID NO: 11 and VL with the sequence shown in SEQ ID NO: 12; (ii) 5I.RG1 containing VH with the sequence shown in SEQ ID NO: 45 and VL with the sequence shown in SEQ ID NO: 46; (iii) 5F.RF3 containing VH with the sequence shown in SEQ ID NO: 22 and VL with the sequence shown in SEQ ID NO: 23; (iv) 5G.RA1 containing VH with the sequence shown in SEQ ID NO: 53 and VL with the sequence shown in SEQ ID NO: 54; (v) 5D.RH4 containing VH with the sequence shown in SEQ ID NO: 89 and VL with the sequence shown in SEQ ID NO: 90; (vi) 5H.RH4 containing VH with the sequence shown in SEQ ID NO: 91 and VL with the sequence shown in SEQ ID NO: 92; (vii) 5G.RF6 containing VH with the sequence shown in SEQ ID NO: 93 and VL with the sequence shown in SEQ ID NO: 94; (viii) 5G.RD6 containing VH with the sequence shown in SEQ ID NO: 95 and VL with the sequence shown in SEQ ID NO: 96; (ix) 5H.RA3 containing VH with the sequence shown in SEQ ID NO: 97 and VL with the sequence shown in SEQ ID NO: 98; (x) 5G.RG1 containing VH with the sequence shown in SEQ ID NO: 99 and VL with the sequence shown in SEQ ID NO: 100; (xi) 5H.RG4 containing VH with the sequence shown in SEQ ID NO: 103 and VL with the sequence shown in SEQ ID NO: 104; (xii) 5G.RC5 containing VH with the sequence shown in SEQ ID NO: 103 and VL with the sequence shown in SEQ ID NO: 104; (xiii) 5F.RE6 containing VH containing the sequence shown in SEQ ID NO: 105 and VL containing the sequence shown in SEQ ID NO: 106; or (xiv) 5H.RF2 containing VH with the sequence shown in SEQ ID NO: 107 and VL with the sequence shown in SEQ ID NO: 108, is a peptide containing or consisting of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7, or human PAR4 (e.g., SEQ ID NO: 19).

[0039] In one example, the PAR4-binding protein reduces thrombin-induced cleavage of human PAR4 expressed on the cell surface (e.g., HEK293 cells transfected with PAR4 containing an N-terminal FLAG tag) (i.e., it has PAR4 antagonist activity). In another example, the PAR4-binding protein (e.g., at a concentration of 10 μg / ml) reduces thrombin-induced cleavage of PAR4-expressing HEK293 cells (e.g., approximately 5 × 10⁶). 4 It reduces thrombin-induced cleavage of cells (e.g., 0.1 U / ml). Exemplary antibodies having such activity include antibodies 5A.RC3, 5I.RG1, 5F.RF3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, or 5H.RF2, or antibodies containing a variable region or complementarity-determining region (CDR) of such antibodies.

[0040] In one example, the PAR4-binding protein binds to the peptide containing the thrombin cleavage site of human PAR4 described herein, or to the N-terminal extracellular region of human PAR4, with an affinity dissociation constant (KD) of 2 nM or less (e.g., 1.5 nM or less, e.g., 1 nM or less). In one example, the KD is between approximately 0.01 nM and approximately 2 nM, e.g., between approximately 0.05 nM and approximately 1 nM, e.g., between approximately 0.1 nM and approximately 1 nM, e.g., between approximately 0.3 nM and approximately 1 nM. In one example, the KD is between approximately 0.01 nM and approximately 1 nM, e.g., between approximately 0.05 nM and approximately 0.9 nM, e.g., between approximately 0.09 nM and approximately 0.7 nM, e.g., between approximately 0.1 nM and approximately 0.6 nM.

[0041] In one example, KD is evaluated by using a streptavidin chip to capture a biotin-bound human PAR4 peptide (e.g., a peptide according to SEQ ID NO: 7) on the surface of the chip and passing a PAR4-binding protein through it.

[0042] In one example, KD is evaluated by using a streptavidin chip to capture a biotin-bound human PAR4 peptide (e.g., a peptide according to SEQ ID NO: 7) on the surface of the chip and passing a PAR4-binding protein through it.

[0043] The exemplary PAR4-binding proteins of this disclosure have a KD between approximately 0.01 and 0.61, as assessed by SA-chip biotin peptide SPR. In one example, the PAR4-binding protein has a KD of 0.4 nM (e.g., + / -0.1 nM). In another example, the PAR4-binding protein has a KD shown in Table 4, corresponding to any one of the PAR4-binding proteins described herein.

[0044] In one example, the PAR4-binding protein of this disclosure specifically binds to human PAR4. In one example, protein binding is evaluated by ELISA and high-throughput antigen microarrays.

[0045] In one example, the PAR4-binding protein binds to the same epitope within human PAR4, or to an epitope within human PAR4 that overlaps with the epitope to which the following antibodies bind: (i) The aforementioned 5A.RC3 antibody containing VH with the sequence shown in SEQ ID NO: 11 and VL with the sequence shown in SEQ ID NO: 12; (ii) 5I.RG1 containing VH with the sequence shown in SEQ ID NO: 45 and VL with the sequence shown in SEQ ID NO: 46; (iii) 5F.RF3 containing VH with the sequence shown in SEQ ID NO: 22 and VL with the sequence shown in SEQ ID NO: 23; (iv) 5G.RA1 containing VH with the sequence shown in SEQ ID NO: 53 and VL with the sequence shown in SEQ ID NO: 54; (v) 5D.RH4 containing VH with the sequence shown in SEQ ID NO: 89 and VL with the sequence shown in SEQ ID NO: 90; (vi) 5H.RH4 containing VH with the sequence shown in SEQ ID NO: 91 and VL with the sequence shown in SEQ ID NO: 92; (vii) 5G.RF6 containing VH with the sequence shown in SEQ ID NO: 93 and VL with the sequence shown in SEQ ID NO: 94; (viii) 5G.RD6 containing VH with the sequence shown in SEQ ID NO: 95 and VL with the sequence shown in SEQ ID NO: 96; (ix) 5H.RA3 containing VH with the sequence shown in SEQ ID NO: 97 and VL with the sequence shown in SEQ ID NO: 98; (x) 5G.RG1 containing VH with the sequence shown in SEQ ID NO: 99 and VL with the sequence shown in SEQ ID NO: 100; (xi) 5H.RG4 containing VH with the sequence shown in SEQ ID NO: 103 and VL with the sequence shown in SEQ ID NO: 104; (xii) 5G.RC5 containing VH with the sequence shown in SEQ ID NO: 103 and VL with the sequence shown in SEQ ID NO: 104; (xiii) 5F.RE6 containing VH containing the sequence shown in SEQ ID NO: 105 and VL containing the sequence shown in SEQ ID NO: 106; or (xiv) 5H.RF2 containing VH with the sequence shown in sequence number 107.

[0046] This disclosure also provides PAR4-binding proteins that specifically bind to human PAR4 and are anti-PAR4 recombinant, synthetic, or monoclonal antibodies or their antigen-binding fragments.

[0047] In one example, the antibody substantially inhibits thrombin-mediated cleavage of PAR4.

[0048] In one example, PAR4 is expressed on human platelets.

[0049] In one example, the PAR4-binding protein is a chimeric antibody containing constant region sequences of human heavy and light chains. In another example, the PAR4-binding protein is a humanized antibody or a fully human antibody.

[0050] In one example, the PAR4-binding protein inhibits cleavage of cell surface-expressed PAR4 by more than 60% in the presence of thrombin or a PAR1 antagonist. In further examples, the protein inhibits PAR4 cleavage by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 8%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 97%, or 100%.

[0051] In another example, thrombin-induced PAR4 cleavage is measured by the loss of the flag tag from flag-tagged PAR4-expressing HEK293 cells. In yet another example, cleavage is measured by flow cytometry.

[0052] In one example, a PAR4-binding protein does not bind to, or substantially does not bind to, human PAR1, PAR2, or PAR3.

[0053] In one example, the PAR4-binding protein contains the variable heavy chain (VH) sequence shown below. JPEG0007895907000001.jpg23152(in the array, X1 is V or I, X2 is either A or V, X3 is either T or A. X4 is L or F. X5 is N or S. X6 is either Y or D. X7 is S or A; X8 is Y or F, X9 is either S or R. X 10 is N or S, X 11 is K or R, X 12 is H or Y, X 13 is A, L, or T, X14 is K or R, X 15 is T or D, X 16 is N or T, X 17 is L or Q, X 18 is Y or F, X 19 is S or I, X 20 is S or T, X 21 is I, S, or A, X 22 is V, I, M, or L, X 23 is E, S, V, or I, X 24 is V, T, R, or G, X 25 is L, R, or G, X 26 is P or V)

[0054] In one example, the PAR4 binding protein further includes the variable light chain (VL) sequence shown below. JPEG0007895907000002.jpg26152 (in the sequence, X1 is K or E, X2 is V or A, X3 is R or G, X4 is A or T, X5 is R or S, X6 is V or I, X7 is N or S, X8 is N or S, X9 is F or Y, X 10 is F or L, X 11 is I or T, X 12 is I or T, X 13 is F or L, X 14 is S or T, X 15 is V or L, X 16 (is N, R, or S)

[0055] In one example, VH contains a CDR1 sequence selected from the following group: (i) GFTLSNYG(sequence number 13); (ii) GFTFSSDG (Sequence ID 59); (iii) GFTFSNYG (Sequence No. 68); (iv) GFTFSSYG (Sequence ID 55); (v) GFAFSSYG (Sequence No. 70); and (vi)GFTLSSYG (sequence number 75).

[0056] In one example, VH contains a CDR2 sequence selected from the following group: (i)IWYDGSNK(sequence number 14); (ii) IWFDGRNK (Sequence ID 60); (iii) IWYDGSNR(sequence number 71); and (iv) IWYDGSSK (Sequence ID 76).

[0057] In one example, VH contains a CDR3 sequence selected from the following group: (i) ARESIVEVLPPFDY(Sequence ID 15); (ii) ARESSISTRPPFDY (Sequence No. 61); (iii)ARETIMVRGVPFD(sequence number 69); (iv)ARETALVRGVPFDY(Sequence ID 56); (v)ARETAMVRGVPFDY(Sequence ID 72); and (vi)ARETILIGGVPFDY(Sequence ID 77).

[0058] In one example, VL contains a CDR1 sequence selected from the following group: (i) QRVRNNY (sequence number 16); (ii) QSVRSSY (sequence number 57); and (iii) QSIRSNY (Sequence ID 78).

[0059] In one example, VL contains the CDR2 sequence GAS (sequence number 28).

[0060] In one example, VL contains a CDR3 sequence selected from the following group: (i) QQYGNSYT (sequence number 18); (ii) QQYGRSYT (sequence number 62); and (iii) QQYGSSYT (Sequence ID 58).

[0061] In another example, the PAR4-binding protein contains the variable heavy chain (VH) sequence shown below.

[0062] In another example, the PAR4-binding protein contains the variable heavy chain (VH) sequence shown below. JPEG0007895907000003.jpg27151(in the array, X1 is either A or S. X2 is either T or A. X3 is V or I, X4 is either Y or S. X5 is either G or S. X6 is L or F. X7 is N, D, or T. X8 is Y or F, X9 is either S or R. X 10 is R or H, X 11 is N or I, X 12 is S or T, X 13 is T or S, X 14 is N or T, X 15is K or N, X 16 is F or L, X 17 is K or N, X 18 is A or K, X 19 is I, F, or V, X 20 is Y or H, X 21 is N or S, X 22 is R, G, or S, X 23 (is V or H)

[0063] In one example, the PAR4-binding protein further includes the variable light chain (VL) sequence shown below. JPEG0007895907000004.jpg30155(in the array, X1 is either V or A. X2 is either V or I. X3 is S or T, X4 is S, Y, or N. X5 is either K or I. X6 is N or K, X7 is either R or S. X8 is R or Q, X9 is T or A, X 10 is T or S, X 11 is Q or R, X 12 is T, S, or N, X 13 is N or N, X 14 (It is E or G)

[0064] In one example, VH contains a CDR1 sequence selected from the following group: (i)GGSLSDYY(array number 86); (iii) SGSFSTYF (sequence number 47); and (iv) GGSFSNYY (Sequence No. 66).

[0065] In one example, VH contains a CDR2 sequence selected from the following group: (i) INHSGTT (sequence number 87); (ii) IIHTGST (Sequence No. 64); or (iii) INHSGST (Sequence No. 48).

[0066] In one example, VH contains a CDR3 sequence selected from the following group: (i)AIEYSNSRGYYYGMDV(Sequence ID 88); (ii)AFEYSSSGGYYYGMDV(Sequence No. 49); and (iii) KVEHSSSSGHYYYGMDV (Sequence ID 65).

[0067] In one example, VL contains a CDR1 sequence selected from the following group: (i) QTISNY (sequence number 109); (ii) QSISSY (sequence number 50); and (iii) QTISYY (Sequence ID 66).

[0068] In one example, VL contains the CDR2 sequence AAS (sequence number 51).

[0069] In one example, VL contains a CDR3 sequence selected from the following group: (i)RQNYNTPLT(sequence number 85); (iii) QQTYSTPLT(sequence number 52); or (iv) QQSYSTPLT (Sequence ID 67).

[0070] This disclosure also provides PAR4-binding proteins comprising variable heavy chains (VHs) having CDR1, CDR2, and CDR3 sequences, each comprising or consisting of the following sequences: (i) Sequence IDs 13, 14, and 15; (ii) Sequence IDs 47, 48, and 49; (iii) Sequence IDs 24, 25, and 26; (iv) Sequence IDs 55, 14, and 56; (v) Sequence IDs 59, 60, and 61; (vi) Sequence IDs 63, 64, and 65; (vii) Sequence ID 68, Sequence ID 14, and Sequence ID 69; (viii) Sequence IDs 70, 71, and 72; (ix) Sequence IDs 55, 73, and 74; (x) Sequence IDs 75, 76, and 77; (xi) Sequence IDs 79, 80, and 81; (xii) Sequence IDs 82, 80, and 83; (xiii) Sequence ID 55, Sequence ID 73, and Sequence ID 74; or (xiv) Sequence IDs 86, 87, and 88.

[0071] This disclosure also provides PAR4-binding proteins comprising variable light chains (VLs) having CDR1, CDR2, and CDR3 sequences, each comprising or consisting of the following sequences: (i) Sequence IDs 16, 17, and 18; (ii) Sequence IDs 50, 51, and 52; (iii) Sequence IDs 27, 28, and 29; (iv) Sequence IDs 57, 28, and 58; (v) Sequence IDs 57, 28, and 62; (vi) Sequence ID 66, Sequence ID 51, and Sequence ID 67; (vii) Sequence IDs 57, 28, and 58; (viii) Sequence ID 57, Sequence ID 28, and Sequence ID 58; (ix) Sequence ID 78, Sequence ID 28, and Sequence ID 62; (x) Sequence ID 84, Sequence ID 51, and Sequence ID 85; (xi) Sequence ID 57, Sequence ID 28, and Sequence ID 58; (xii) Sequence ID 57, Sequence ID 51, and Sequence ID 58; or (xiii) Sequence ID 109, Sequence ID 51, and Sequence ID 85.

[0072] In one example, PAR-4 binding proteins include: (i) A VH containing a sequence that is at least 50% identical to any one of the sequences shown in SEQ ID NOs: 11, 22, 45, 53, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, or a humanized, chimeric, or deimmunized version thereof; and / or (ii) A VL containing a sequence that is at least 85% identical to the sequence shown in SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, or SEQ ID NO: 108, or a humanized, chimeric, or deimmunized version thereof.

[0073] In one example, VH contains sequences that are at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, 97%, 98%, 99%, 97%, 98%, 99%, or 99.5% identical to any one of the following: SEQ ID NO: 11, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107.

[0074] In one example, VL contains sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99%, or 99.5% identical to any one of the following: SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.

[0075] In one example, VH contains sequences that are at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 97%, 98%, 99%, or 99.5% identical to any one of the following sequences: SEQ ID NO: 11, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, 99%, or 99.5% identical to SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107 (excluding CDR sequences).

[0076] In one example, VL contains sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to sequence numbers 12, 23, 46, 54, 90, 92, 94, 96, 98, 100, 102, 104, 106, or 108 (excluding CDR sequences).

[0077] In one example, a CD-R is defined by the IMGT numbering system.

[0078] This disclosure also provides PAR4-binding proteins, including: (i) VH shown in Sequence ID 11 and VL shown in Sequence ID 12; (ii) VH shown in Sequence ID No. 45 and VL shown in Sequence ID No. 46; (iii) VH as shown in Sequence ID No. 22 and VL as shown in Sequence ID No. 23; (iv) VH as shown in Sequence ID 53 and VL as shown in Sequence ID 54; (v) VH as shown in Sequence ID 89 and VL as shown in Sequence ID 90; (vi) VH shown in Sequence ID 91 and VL shown in Sequence ID 92; (vii) VH as shown in sequence number 93 and VL as shown in sequence number 94; (viii) VH as shown in Sequence ID 95 and VL as shown in Sequence ID 96; (ix) VH as shown in Sequence ID 97 and VL as shown in Sequence ID 98; (x) VH shown in sequence number 99 and VL shown in sequence number 100; (xi) VH as shown in Sequence ID 101 and VL as shown in Sequence ID 102; (xii) VH shown in Sequence ID 103 and VL shown in Sequence ID 104; (xiii) VH shown in sequence number 105 and VL shown in sequence number 106; or (xiv) VH shown in sequence number 107 and VL shown in sequence number 108.

[0079] In one example, the PAR4-binding protein antigen-binding fragment is (i) Single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) At least one of (i) and / or (ii) that is bound to the heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3; or (iv) At least one of (i) and / or (ii) is linked to a platelet-binding protein (e.g., von Willebrand factor (vWF)).

[0080] In another example of this disclosure, VL and VH are separate polypeptide chains. For example, the PAR4-binding protein is (i) Diabody; (ii) Triabody; (iii) Tetrabody; (iv)Fab; (v)F(ab′)2; (vi)Fv; or (vii) at least one of (i) to (vi) linked to the heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3; or (viii) At least one of (i) to (vi) is linked to a platelet-binding protein (e.g., vWF).

[0081] This disclosure also provides a chimeric antibody comprising VH and VL as described herein, wherein VH is linked to the human heavy chain constant region and VL is linked to the human light chain constant region.

[0082] It will be apparent to those skilled in the art that, based on this disclosure, the PAR4-binding proteins of this disclosure include human, humanized, synthetically humanized, chimeric, and primate-like proteins.

[0083] The antibodies of this disclosure may belong to any class, including IgM, IgG, IgE, IgA, IgD, or subclasses. Exemplary subclasses of IgG are IgG1, IgG2, IgG3, and IgG4.

[0084] In one example, the PAR4-binding protein is recombinant. In another example, the PAR4-binding protein is synthetic.

[0085] In one example, the PAR4-binding protein or antibody of this disclosure is conjugated with a moiety. For example, the moiety is selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, compounds that increase the half-life of the PAR4-binding protein in a subject, and mixtures thereof.

[0086] This disclosure also provides isolated nucleic acids encoding the PAR4-binding protein or antibody of this disclosure. In one example, the PAR4-binding protein or antibody comprises a VH nucleic acid sequence shown in SEQ ID NO: 20 and / or a VL nucleic acid sequence shown in SEQ ID NO: 21. In another example, the PAR4-binding protein or antibody comprises a VH nucleic acid sequence shown in SEQ ID NO: 30 and / or a VL nucleic acid sequence shown in SEQ ID NO: 31.

[0087] This disclosure further provides an expression construct comprising the nucleic acid of this disclosure ligated to a promoter. Such an expression construct may be in a vector, for example, in a plasmid.

[0088] In an example of this disclosure concerning a single polypeptide PAR4-binding protein, the expression construct may include a promoter ligated to the nucleic acid encoding the polypeptide chain.

[0089] In an example involving multiple polypeptides that form a PAR4-binding protein, the expression construct of the Disclosure comprises a nucleic acid encoding one polypeptide (e.g., VH) ligably linked to a promoter, and a nucleic acid encoding another polypeptide (e.g., VL) ligably linked to another promoter.

[0090] In another example, the expression construct is a bisistronic expression construct that includes components linked together in the following order from 5′ to 3′: (i) promoter; (ii) Nucleic acid encoding the first polypeptide; (iii) Internal ribosome entry sites; and (iv) A nucleic acid encoding a second polypeptide.

[0091] For example, the first polypeptide contains VH and the second polypeptide contains VL, or the first polypeptide contains VL and the second polypeptide contains VH.

[0092] This disclosure also envisions separate expression constructs, one of which encodes a first polypeptide (e.g., VH and optionally a heavy chain constant region or a portion thereof) and the other which encodes a second polypeptide (e.g., VL and optionally a light chain constant region). For example, this disclosure (i) A first expression construct comprising a nucleic acid encoding a polypeptide (for example, a VH ligated to a promoter), and (ii) A second expression construct comprising nucleic acid encoding a polypeptide (for example, a VL ligated to act on a promoter) The present invention also provides a composition comprising the first and second polypeptides, wherein the first and second polypeptides associate to form the PAR4-binding protein of the present disclosure.

[0093] This disclosure further provides isolated cells expressing the PAR4-binding protein or antibody of this disclosure, or recombinant cells genetically modified to express the PAR4-binding protein or antibody of this disclosure. In one example, the cells are isolated hybridomas. In another example, the cells contain the nucleic acid or expression construct of this disclosure, or: (i) A first expression construct comprising a nucleic acid encoding a polypeptide (e.g., VH) ligated to a promoter, and (ii) A second expression construct comprising a nucleic acid encoding a polypeptide (e.g., including VL) that is ligated to the promoter. The present invention comprises a first expression construct and a second expression construct, wherein the first and second polypeptides associate to form the PAR4-binding protein of the present disclosure.

[0094] This disclosure further provides compositions comprising the PAR4-binding protein or nucleic acid or expression construct or cell of this disclosure and a suitable carrier. In one example, the composition comprises the PAR4-binding protein of this disclosure.

[0095] In one example, the carrier is pharmaceutically acceptable.

[0096] The compositions of this disclosure may be administered alone or in combination with other treatments, therapeutic agents, or drugs, either simultaneously or sequentially.

[0097] This disclosure further provides a method for treating or preventing thrombosis or thromboembolic disorder in a subject, comprising administering the subject a PAR4-binding protein or nucleic acid or expression construct or cells or composition of the Disclosure. In one example, the subject is a person at risk of a PAR4-mediated event such as thrombosis. In another example, the subject is a person who has or has had a PAR4-mediated event (e.g., thrombosis).

[0098] In one example, the method involves administering to a subject an antibody comprising a VH containing the sequence shown in any one of SEQ ID NOs: 11, 22, 45, 53, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, or a humanized or deimmunized version thereof, and an antibody containing a VH containing the sequence shown in any one of SEQ ID NOs: 12, 23, 46, 54, 90, 92, 94, 96, 98, 100, 102, 104, 106, or 108, or a humanized or deimmunized version thereof.

[0099] This disclosure further provides PAR4-binding proteins or nucleic acids or expression constructs or cells or compositions for use in the medical field.

[0100] This disclosure further provides PAR4-binding proteins or nucleic acids or expression constructs or cells or compositions for use in the treatment or prevention of PAR4-mediated events (e.g., thrombosis).

[0101] In one example, the present disclosure provides a method for treating, preventing, or improving thrombosis or thromboembolic disorder, comprising administering the PAR4-binding protein or nucleic acid or expression construct or cells or composition of the present disclosure to a subject in need thereof.

[0102] In some examples, the present disclosure provides a method for treating, preventing, or improving thrombosis in a subject requiring treatment, prevention, or improvement of thrombosis, comprising administering the subject requiring treatment, prevention, or improvement of thrombosis to the subject requiring treatment, prevention, or improvement of thrombosis of the present disclosure, comprising administering the subject requiring treatment, prevention, or improvement of thrombosis of the present disclosure, or an expression construct or cell or composition.

[0103] In one example, the present disclosure provides a method for reducing the risk of thrombosis associated with a surgical procedure, comprising administering to a subject either before and / or after the surgical procedure a PAR4-binding protein or nucleic acid or expression construct or cells or composition of the present disclosure. In one example, the surgical procedure is a liver transplant, and the thrombosis is hepatic artery thrombosis.

[0104] In some examples, the Disclosure provides a method for determining whether a dose of a PAR4-binding protein or antibody in accordance with the Disclosure is appropriate. Such a method includes (i) obtaining a blood sample from a subject treated with the PAR4-binding protein or antibody of the Disclosure; (ii) treating platelets from the blood sample in vitro with a PAR4 agonist; (iii) measuring platelet activation; and (iv) comparing the platelet activation in the blood sample after treatment with the PAR4-binding protein or antibody with the platelet activation in the blood sample obtained before treatment with the PAR4-binding protein or antibody.

[0105] Examples of suitable PAR4 agonists are thought to be well known to those skilled in the art. Non-limiting examples include agonist peptides such as AYPGKF-NH2(Tocris).

[0106] In one example, platelet activation is measured according to the method illustrated in the examples herein.

[0107] In some embodiments, the present disclosure includes a method for inhibiting or preventing platelet aggregation, comprising the step of administering a therapeutically effective amount of a PAR4-binding protein according to the present disclosure to a subject (e.g., a human) that requires such inhibition or prevention.

[0108] In some cases, the present disclosure provides a method for treating or preventing thrombosis or thromboembolic disorder, comprising administering to a subject in need (e.g., a human) a therapeutically effective dose of a PAR4-binding protein that inhibits PAR4 cleavage and / or signaling, in accordance with the present disclosure, wherein the subject has a dual PAR1 / PAR4 platelet receptor repertoire.

[0109] Preferably, the subject is a human.

[0110] This disclosure further provides the use of the PAR4-binding protein or nucleic acid or expression construct or cell or composition of the disclosure in the medical field.

[0111] This disclosure further provides the use of the PAR4-binding protein or nucleic acid or expression construct or cell or composition of this disclosure in the manufacture of a pharmaceutical product for the treatment or prevention of thrombosis or thromboembolic disorder.

[0112] The present disclosure further provides a method for detecting PAR4 in a sample, comprising contacting the sample with a PAR4-binding protein or antibody of the present disclosure to form an antigen-protein complex, and detecting the complex, wherein the detection of the complex indicates PAR4 in the sample.

[0113] This disclosure also provides a vaccine antigen comprising or consisting of a sequence according to Sequence ID No. 4, together with a pharmaceutically acceptable carrier, for generating an antagonist antibody of human PAR4.

[0114] This disclosure also provides PAR-4 binding proteins that do not inhibit, or only partially inhibit, PAR-4 cleavage in the presence of thrombin.

[0115] Therefore, in one example, the present disclosure also provides a PAR4-binding protein including: (i) a VH containing a sequence that is at least 50% identical to the sequence shown in Sequence ID No. 32, or a humanized, chimeric, or deimmunized version thereof; and / or (ii) A VL containing a sequence that is at least 85% identical to the sequence shown in Sequence ID No. 33, or a humanized, chimeric, or deimmunized version thereof. [Brief explanation of the drawing]

[0116] [Figure 1] This diagram shows the extracellular locations of the thrombin cleavage and activation sites of PAR4, as well as a schematic representation of the anti-PAR4 target region of the antibodies described herein. [Figure 2] Flow cytometry quantification shows the percentage of intact PAR4 present on the cell surface of HEK293 cells transfused with human PAR4 containing an N-terminal FLAG tag. Cells were pre-treated with five different hybridoma supernatants (MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BBRH3, and MoB5CRC4) obtained from the first hybridoma screen, and then treated with thrombin (2 U / ml for 10 minutes). A polyclonal anti-PAR4 antibody was used as the positive control (French et al (2016) Journal of Thrombosis and Haemostasis 14:1642-1654). Data are the mean + mean standard error at three individual data points. [Figure 3]Flow cytometry quantification shows the percentage of intact PAR4 present on the cell surface of HEK293 cells transfused with PAR4 containing an N-terminal FLAG tag (measured by FLAG epitope %). Cells were pre-treated with supernatant from two subclones of 5A.RC3 (called B6b and H4b) and then treated with thrombin (2 U / ml for 10 minutes). Data are the mean plus the standard error of the mean across four individual data points. [Figure 4] Flow cytometry quantification shows the percentage of intact PAR4 present on the cell surface of HEK293 cells transfused with either Thr120 or Ala120 of human PAR4 containing an N-terminal FLAG tag (measured by FLAG epitope %). Cells were pre-treated with various concentrations of 5A.RC3 subclones as shown, followed by treatment with thrombin (2 U / ml). [Figure 5] (A) ELISA-based screening of the binding of hybridoma supernatants of 5A.RC3 (dark) and 5B.RB4 (light) to immobilized proteins corresponding to the immunogen PAR4 and the corresponding regions of PAR1, 2, and 3, and (B) Biacore analysis (measured at different concentrations) of the binding affinity of PAR4 antibodies of the 5A.RC3 subclone to human PAR4 peptide. [Figure 6] 5A.RC3 inhibits thrombin cleavage of both Ala120 and Thr120 PAR4 variants. To evaluate thrombin cleavage of PAR4 variants, HEK293T cells were transiently transfused with either PAR4-120Ala or PAR4-120Thr variants containing a FLAG epitope upstream of the thrombin cleavage site. (A) Cells were stimulated with escalating doses (0.1–2 U / mL) of thrombin, and the amount of thrombin cleavage was measured as loss of the FLAG epitope by flow cytometry using a FITC-conjugated anti-FLAG antibody. (B) Pre-incubation of transfused cells with 5A.RC3 before thrombin stimulation resulted in nearly complete thrombin cleavage inhibition of the same degree, regardless of the PAR4 variant. Doses of 5A.RC3 at 1, 10, and 100 μg / ml were compared. [Figure 7] The 5A.RC3 subclone inhibits the upregulated thrombin-induced platelet aggregation response in donors with the PAR4 Thr120 variant. The response to platelet aggregation in PAR4 agonists was evaluated in isolated human platelets of different PAR4 variants. As predicted, the presence of the T allele is associated with higher maximal aggregation in response to (A) PAR4-AP and (B) thrombin in the medium dose range. A similar trend is observed even in the case of thrombin stimulation in the presence of PAR1 blockade by (C) borapaxal (90 nM). (D) The concentration response of 5A.RC3 inhibitory activity to the thrombin-induced platelet aggregation response (i.e., PAR4-dependent) in the presence of a PAR1 antagonist demonstrates efficacy against the Thr120 variant, indicating that antibody-mediated PAR4 inhibition is equally effective for all genotypes. (E) IC50 of 5A.RC3 inhibitory activity in PAR4-dependent thrombin-induced platelet aggregation. [Figure 8] Flow cytometry demonstrates that 5A.RC3 (10 ug / mL) binds to human PAR4 in isolated platelets, as shown against isotype controls. [Figure 9] This study demonstrates that PAR4 genotype is associated with an increased procoagulable platelet phenotype and can be targeted by antibody-mediated inhibition. Procoagulable activity of isolated human platelets was evaluated by measuring phosphatidylserine exposure in response to (A) PAR4-activating peptide (AP) stimulation and (B) thrombin stimulation. Note that the Thr120 variant resulted in increased PS exposure in PAR4-AP stimulated platelets. A similar trend was observed in thrombin-stimulated platelets. (C) Pretreatment with the 5A.RC3 subclone dose-dependently inhibited thrombin-induced phosphatidylserine exposure, regardless of donor genotype. [Figure 10-1]The 5A.RC3 subclone is shown to inhibit thrombosis regardless of donor genotype. The following thrombosis parameters were measured in real time over a 10-minute period in a human whole blood thrombosis assay under coagulation conditions: (A) platelet deposition (PE-conjugated anti-CD9), thrombin activity (FRET-based thrombin probe), fibrin volume (Dylight650-conjugated anti-fibrin antibody), and fibrin-to-thrombus ratio (data shown are at the 10-minute endpoint). Note that the direct thrombin inhibitor hirudin (800 U / mL) eliminated thrombin activity and fibrin volume despite continued platelet deposition. [Figure 10-2] The 5A.RC3 subclone is shown to inhibit thrombosis regardless of donor genotype. The following thrombosis parameters were measured in real time over a 10-minute period in a human whole blood thrombosis assay under coagulation conditions: (B~E) No significant differences in these parameters were observed across all PAR4 genotypes. 5A.RC3 (white bar, 100 μg / mL) did not affect (F) platelet deposition compared to the control (black bar), but significantly inhibited (G) thrombin activity, (H) fibrin volume, and (I) fibrin-to-thrombus volume ratio. Data are mean ± SEM for N=4~6 per genotype (total n=15, color indicates PAR4 genotype: black circle = AA, gray circle = AT, white circle = TT). For (G) and (H), data were normalized to hirudin baseline and expressed as a percentage of the untreated control. Statistical significance was determined by one-way ANOVA (B-E) or Student's t-test (F-G). * indicates P<0.05. [Figure 11] This demonstrates the first functional screening of hybridoma supernatant for platelet aggregation. [Figure 12]The amino acid sequences of VH(A) and VL(B) of MoB5A-RC3.F10b.H4b(5A.RC3) with CDRs identified according to IMGT numbering are shown. VH = heavy chain variable region, VL = light chain variable region, CDR = complementarity-determining region, and FWR = framework region. This antibody showed PAR4 inhibitory activity. [Figure 13] The amino acid sequences of VH(A) and VL(B) of MoB5H-RD2.A7b(5H.RD2) with CDRs identified according to IMGT numbering are shown. VH = heavy chain variable region, VL = light chain variable region, CDR = complementarity-determining region, and FWR = framework region. This antibody bound to PAR4 but did not exhibit inhibitory activity against PAR4. [Figure 14] The amino acid sequences of VH(A) and VL(B) of MoB5F-RF3.A7b.C9(5F.RF3) with CDR identified according to IMGT numbering are shown. VH = heavy chain variable region, VL = light chain variable region, CDR = complementarity-determining region, and FWR = framework region. This antibody showed PAR4 inhibitory activity. [Figure 15] The reactivity of purified mAbs with hPAR4 peptides was demonstrated by ELISA. Purified mAbs bound to the hPAR4 peptide in a dose-dependent manner were detected by anti-mouse Fc conjugated with alkaline phosphatase (Ap). No binding was observed with IC (isotype control) mAbs produced against unrelated non-PAR4 antigens. [Figure 16] ELISA demonstrated that at 10 μg / ml, purified anti-HPAR4 mAb specifically binds to hPAR4 biotinylated peptides and does not react nonspecifically with hPAR1, hPAR2, and hPAR3 (biotinylated peptides). [Figure 17] This study shows the concentration-dependent binding of purified anti-hPAR4 mAbs to isolated human platelets of isotype control, as determined by flow cytometry. Raw data are presented as geometric mean fluorescence intensity. Each mAb binds in a concentration-dependent manner. N=3–5. Data points are mean ± SEM. [Figure 18]This figure shows the concentration-dependent inhibition of human platelet aggregation induced by 0.1 U / ml thrombin by three different anti-hPAR4 mAb clones. Near maximum inhibition was achieved by each clone at the highest concentration tested. The IC50 values ​​(μg / ml) measured from these concentration inhibition curves are also shown in the figure. N=4~8. Data points are mean ± SEM. [Figure 19] This study demonstrates inhibition of human thrombus formation by monoclonal antibodies 5A.RC3 and 5D.RH4. Blood pretreatment with either 5D.RH4 or 5A.RC3 (both at 100 μg / ml) reduced total thrombus volume. Individual data points are shown. Bars represent the mean ± SEM. *P<0.05 (unpaired Student's t-test). [Figure 20] This shows the sequence of the variable heavy chain of an anti-PAR4 mAb, which exhibits a complementarity-determining region (CDR) according to the IMGT numbering system. [Figure 21] This shows the sequence of the variable light chain of an anti-PAR4 mAb, which exhibits a complementarity-determining region (CDR) according to the IMGT numbering system. [Figure 22] This shows synthetic peptides for epitope mapping of anti-hPAR4 mAbs. Duplicate peptides spanning the original human PAR4 antigen were synthesized. These consisted of amino acid residues 1-9, 8-15, and amino acid substitutions 11-20. The thrombin cleavage site sequence is underlined. The C-terminal cysteine ​​residue was removed to prevent multimer formation. [Figure 23] The reactivity of purified hPAR4 mAbs with peptides 1-9, 8-15, and 11-20 is shown. Absorbance values ​​were subtracted from the background level. The non-hPAR4 control mAb did not react with any of the three peptides.

[0117] Explanation of the sequence list Sequence ID 1: Epitope sequence of PAR4 Array 2: hPAR4 array (bare) Array 3: mPAR4 array (bare) Sequence ID 4: Sequence of hPAR4(KLH) used as an immunogen Sequence ID 5: Sequence of mPAR4(KLH) used as an immunogen Sequence ID 6: Sequence of mPAR4 (biotin) Sequence ID 7: hPAR4 (biotin) sequence Sequence ID 8: hPAR3 (biotin) sequence Sequence ID 9: hPAR2 (biotin) sequence Sequence ID 10: hPAR1 (biotin) sequence Amino acid sequence of SEQ ID NO: 11:5A.RC3 VH Amino acid sequence of SEQ ID NO: 12:5A.RC3 VL Sequence ID 13: 5A.RC3 VH CDR1 sequence Sequence ID 14: 5A.RC3 VH CDR2 sequence Sequence ID 15: 5A.RC3 VH CDR3 sequence Sequence ID 16: 5A.RC3 VL CDR1 sequence Sequence ID 17: 5A.RC3 VL CDR2 sequence Sequence ID 18: 5A.RC3 VL CDR3 sequence Sequence ID 19: Sequence of human PAR4 Nucleic acid sequence of sequence number 20:5A.RC3 VH Nucleic acid sequence of sequence number 21:5A.RC3 VL Amino acid sequence of SEQ ID NO: 22:5A.RC3 VH Amino acid sequence of SEQ ID NO: 23: 5F.RF3 VL (5F.RF3) Sequence ID 24: 5F.RF3 VH CDR1 sequence Sequence ID 25: 5F.RF3 VH CDR2 sequence Sequence ID 26: 5F.RF3 VH CDR3 sequence Sequence ID 27: 5F.RF3 VL CDR1 sequence Sequence ID 28: 5F.RF3 VL CDR2 sequence Sequence ID 29: 5F.RF3 VL CDR3 sequence Nucleic acid sequence of SEQ ID NO: 30:5F.RF3 VH Nucleic acid sequence of SEQ ID NO: 31:5F.RF3 VL Amino acid sequence of SEQ ID NO: 32: 5H.RD2 VH Amino acid sequence of SEQ ID NO: 33: 5H.RD2 VL Sequence ID 34: 5H.RD2 VH CDR1 sequence Sequence ID 35: 5H.RD2 VH CDR2 sequence Sequence ID 36: Sequence of 5H.RD2 VH CDR3 Sequence ID 37: Sequence of 5H.RD2 VL CDR1 Sequence ID 38: 5H.RD2 VL CDR2 sequence Sequence ID 39: 5H.RD2 VL CDR3 sequence Sequence ID 40: Nucleic acid sequence of 5H.RD2 VH Nucleic acid sequence of sequence number 41: 5H.RD2 VL Sequence ID 42: Epitope sequence Sequence ID 43: Epitope sequence Sequence ID 44: Epitope sequence Amino acid sequence of SEQ ID NO: 45:5I.RG1 VH Amino acid sequence of SEQ ID NO: 46:5I.RG1 VL Sequence ID 47: 5I.RG1 VH CDR1 Sequence ID 48: 5I.RG1 VH CDR2 sequence Sequence ID 49: 5I.RG1 VH CDR3 sequence Sequence ID 50: 5I.RG1 VL CDR1 sequence Sequence ID 51: 5I.RG1 VL CDR2 sequence Sequence ID 52: 5I.RG1 VL CDR3 sequence Amino acid sequence of SEQ ID NO: 5G.RA1 VH Amino acid sequence of SEQ ID NO: 54: 5G.RA1 VL Sequence ID 55: Sequence of 5G.RA1 VH CDR1 Sequence ID 14: Sequence of 5G.RA1 VH CDR2 Sequence ID 56: Sequence of 5G.RA1 VH CDR3 Sequence ID 57: Sequence of 5G.RA1 VL CDR1 Sequence ID 28: 5G.RA1 VL CDR2 sequence Sequence ID 58: Sequence of 5G.RA1 VL CDR3 Amino acid sequence of SEQ ID NO: 89: 5D.RH4 VH Amino acid sequence of SEQ ID NO: 90:5D.RH4 VL Sequence ID 59: Sequence of 5D.RH4 VH CDR1 Sequence ID 60: Sequence of 5D.RH4 VH CDR2 Sequence ID 61: Sequence of 5D.RH4 VH CDR3 Sequence ID 57: Sequence of 5D.RH4 VL CDR1 Sequence ID 28: Sequence of 5D.RH4 VL CDR2 Sequence ID 62: Sequence of 5D.RH4 VL CDR3 Amino acid sequence of SEQ ID NO: 91: 5H.RH4 VH Amino acid sequence of SEQ ID NO: 92: 5H.RH4 VL Sequence ID 63: Sequence of 5H.RH4 VH CDR1 Sequence ID 64: Sequence of 5H.RH4 VH CDR2 Sequence ID 65: Sequence of 5H.RH4 VH CDR3 Sequence ID 66: Sequence of 5H.RH4 VL CDR1 Sequence ID 51: Sequence of 5H.RH4 VL CDR2 Sequence ID 67: Sequence of 5H.RH4 VL CDR3 Amino acid sequence of SEQ ID NO: 93: 5G.RF6 VH Amino acid sequence of SEQ ID NO: 94: 5G.RF6 VL Sequence ID 68: Sequence of 5G.RF6 VH CDR1 Sequence ID 14: Sequence of 5G.RF6 VH CDR2 Sequence ID 69: Sequence of 5G.RF6 VH CDR3 Sequence ID 57: Sequence of 5G.RF6 VL CDR1 Sequence ID 28: Sequence of 5G.RF6 VL CDR2 Sequence ID 58: Sequence of 5G.RF6 VL CDR3 Amino acid sequence of SEQ ID NO: 95: 5G.RD6 VH Amino acid sequence of SEQ ID NO: 95: 5G.RD6 VL Sequence ID 70: Sequence of 5G.RD6 VH CDR1 Sequence ID 71: Sequence of 5G.RD6 VH CDR2 Sequence ID 72: Sequence of 5G.RD6 VH CDR3 Sequence ID 57: Sequence of 5G.RD6 VL CDR1 Sequence ID 28: Sequence of 5G.RD6 VL CDR2 Sequence ID 58: Sequence of 5G.RD6 VL CDR3 Amino acid sequence of SEQ ID NO: 97: 5H.RA3 VH Amino acid sequence of SEQ ID NO: 98: 5H.RA3 VL Sequence ID 55: 5H.RA3 VH CDR1 sequence Sequence ID 73: 5H.RA3 VH CDR2 sequence Sequence ID 74: 5H.RA3 VH CDR3 sequence Sequence ID 57: Sequence of 5H.RA3 VL CDR1 Sequence ID 28: 5H.RA3 VL CDR2 sequence Sequence ID 58: Sequence of 5H.RA3 VL CDR3 Amino acid sequence of SEQ ID NO: 99:5G.RG1 VH Amino acid sequence of SEQ ID NO: 100: 5G.RG1 VL Sequence ID 75: Sequence of 5G.RG1 VH CDR1 Sequence ID 76: Sequence of 5G.RG1 VH CDR2 Sequence ID 77: Sequence of 5G.RG1 VH CDR3 Sequence ID 78: Sequence of 5G.RG1 VL CDR1 Sequence ID 28: Sequence of 5G.RG1 VL CDR2 Sequence ID 62: Sequence of 5G.RG1 VL CDR3 Amino acid sequence of SEQ ID NO: 101: 5H.RG4 VH Amino acid sequence of SEQ ID NO: 102: 5H.RG4 VL Sequence ID 79: Sequence of 5H.RG4 VH CDR1 Sequence ID 80: Sequence of 5H.RG4 VH CDR2 Sequence ID 81: Sequence of 5H.RG4 VH CDR3 Sequence ID 57: Sequence of 5H.RG4 VL CDR1 Sequence ID 28: 5H.RG4 VL CDR2 sequence Sequence ID 58: Sequence of 5H.RG4 VL CDR3 Amino acid sequence of SEQ ID NO: 103: 5G.RC5 VH Amino acid sequence of SEQ ID NO: 104: 5G.RC5 VL Sequence ID 82: Sequence of 5G.RC5 VH CDR1 Sequence ID 80: Sequence of 5G.RC5 VH CDR2 Sequence ID 83: Sequence of 5G.RC5 VH CDR3 Sequence ID 57: Sequence of 5G.RC5 VL CDR1 Sequence ID 51: Sequence of 5G.RC5 VL CDR2 Sequence ID 58: Sequence of 5G.RC5 VL CDR3 Amino acid sequence of SEQ ID NO: 105: 5F.RE6 VH Amino acid sequence of SEQ ID NO: 106: 5F.RE6 VL Sequence ID 55: 5F.RE6 VH CDR1 sequence Sequence ID 73: 5F.RE6 VH CDR2 sequence Sequence ID 74: 5F.RE6 VH CDR3 sequence Sequence ID 84: 5F.RE6 VL CDR1 sequence Sequence ID 51: 5F.RE6 VL CDR2 sequence Sequence ID 85: 5F.RE6 VL CDR3 sequence Amino acid sequence of SEQ ID NO: 107: 5H.RF2 VH Amino acid sequence of SEQ ID NO: 108: 5H.RF2 VL Sequence ID 86: Sequence of 5H.RF2 VH CDR1 Sequence ID 87: Sequence of 5H.RF2 VH CDR2 Sequence ID 88: Sequence of 5H.RF2 VH CDR3 Sequence ID 109: Sequence of 5H.RF2 VL CDR1 Sequence ID 51: Sequence of 5H.RF2 VL CDR2 Sequence ID 85: Sequence of 5H.RF2 VL CDR3. [Modes for carrying out the invention]

[0118] overview Throughout this specification, unless otherwise specified or the context requires otherwise, any reference to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be interpreted as encompassing one or more (i.e., one or more) of these steps, compositions of a substance, groups of steps, or groups of compositions of a substance.

[0119] Those skilled in the art will understand that variations and modifications are likely to occur in addition to those specifically described herein. It should be understood that this disclosure includes all such variations and modifications. This disclosure includes all steps, features, compositions, and compounds that are individually or collectively referred to or shown herein, as well as any combination or any two or more of such steps or features.

[0120] This disclosure is not limited in scope by the specific examples described herein. Such specific examples are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0121] Any example in this disclosure shall be construed to apply to any other example in this disclosure, with due consideration to any necessary modifications, unless otherwise specified.

[0122] Unless otherwise specifically defined, all technical and scientific terms used herein shall be interpreted in the same way as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0123] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature, for example, in references such as Perbal (1984), Sambrook et al. (1989), Brown (1991), Glover and Hames (1995 and 1996), as well as Ausubel et al. (1988, including all revisions to date), Harlow and Lane (1988), Coligan et al. (including all revisions to date), and Zola (1987).

[0124] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies, and fragments thereof herein may be further clarified by consideration in the numbering of Kabat (1987 and / or 1991), Bork et al. (1994), and / or Chothia and Lesk (1987 and / or 1989), or Al-Lazikani et al. (1997), or Lefranc M.-P. (1997) Immunology 5 Today 18, 509, the IMGT numbering.

[0125] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", are to be understood to mean including the stated element, integer, or step, or group of elements, integers, or steps, but not excluding any other element, integer, or step, or group of elements, integers, or steps.

[0126] As used herein, the term "derived from" is to be construed to indicate that the specified entity can be obtained from a particular source (although not necessarily directly from that source).

[0127] The present invention uses conventional molecular biology, microbiology, and recombinant DNA techniques within the skill range of those skilled in the art. See, for example, Sambrook et al “Molecular Cloning” A Laboratory Manual (1989).

[0128] Selected definition As used herein, unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents. The terms “a” (or “an”), as well as the terms “one or more” and “at least one,” may be used interchangeably herein.

[0129] Furthermore, as used herein, “and / or” shall be construed to specifically disclose two specified features or components, with or without the other. Thus, the term “and / or” when used in an expression such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), as well as “B” (alone). Similarly, the term “and / or” when used in an expression such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); as well as C (alone).

[0130] The term “about” is used herein to mean approximately, roughly, around, or within the region of. When the term “about” is used in conjunction with a numerical range, “about” modifies that range by extending the boundaries above and below the recited values. Generally, the term “about” is used herein to modify a numerical value by an amount that varies above or below (higher or lower) the recited value by up to ten percent (%).

[0131] It will be understood that the PAR4-binding proteins and antibodies, nucleic acids, cells, and vectors described herein are in isolated forms. “Isolated” means polypeptides, antibodies, polynucleotides, vectors, or cells in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, or cells include those purified to a degree that they are no longer found in nature. In some embodiments, isolated antibodies, polynucleotides, vectors, or cells are substantially pure. In some embodiments, isolated antibodies, polynucleotides, vectors, or cells are “recombinant.”

[0132] As used herein, the term “protease-activated receptor (PAR4)” means all or part of a vertebrate cell surface protein that is specifically activated by thrombin or a thrombin agonist, thereby activating PAR4-mediated signaling events (e.g., phosphoinositide hydrolysis, Ca efflux, platelet aggregation). This polypeptide is characterized by having the ligand-activating properties (including agonist-activating and antagonist-inhibiting properties) and tissue distribution described herein. The term includes these PAR4 moieties capable of binding to thrombin or the PAR4 receptor moieties shown in SEQ ID NO: 2.

[0133] The term "PAR4 antagonist" refers to a platelet aggregation inhibitor that binds to PAR4 and inhibits PAR4 cleavage and / or signaling. Typically, PAR4 activity is dose-dependently reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such activity in control cells. Control cells are cells not treated with the compound. PAR4 activity is measured by any standard method in the art, including the methods described herein (e.g., platelet activation assays measuring calcium mobilization, platelet aggregation, e.g., calcium mobilization, p-selectin, or CD40L release in PAR4-expressing cells, or thrombosis and hemostasis models).

[0134] The term "hPAR4," or "human PAR4," refers to fully human antibodies. For nominal and non-restrictive purposes, the amino acid sequence of hPAR4 is shown in SEQ ID NO: 19.

[0135] As used herein, the term "mAb" is intended to refer to a monoclonal antibody containing a mouse constant region sequence and a human variable region sequence.

[0136] The term “antibody” refers to immunoglobulins that are naturally occurring, partially or completely synthetically produced, or recombinantly produced. This term also encompasses any polypeptide or protein that has an antibody-binding domain, or a binding domain homologous to an antibody-binding domain. CDR-transplant antibodies are also intended within this term. “Antibody” is any immunoglobulin that binds to a specific epitope, and includes antibodies and fragments thereof. This term encompasses polyclonal antibodies, monoclonal antibodies, multivalent antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Furthermore, the term “antibody” also refers to at least two immunoglobulin heavy (H) chains and two immunoglobulin light (L) chains interconnected by disulfide bonds, or proteins containing these antigen-binding moieties. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). CH typically consists of three domains, CH1, CH2, and CH3 (for example, IgM has an additional domain CH4). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The CL consists of one domain and can be of the lambda or kappa type. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWRs). Each VH and VL consists of three CDRs and four FWRs, arranged from the amino terminus to the carboxyl terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. In some embodiments, both VH and VL contain a binding domain that interacts with the antigen. In other embodiments, a single VH domain or a single VL domain may specifically interact with the antigen. The CH domain of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), cells lining blood vessel walls, other cell-expressed receptors for the CH domain of immunoglobulins, and the first component (C1q) of the classical complementation system.Antibody molecules may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). As used herein, the term “antibody” includes “chimeric” antibodies in which portions of the heavy and / or light chain are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). The basic antibody structures in vertebrate systems are well understood. For example, see Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press). Any "antigen-binding fragment" is also included in the meaning of the term "antibody."

[0137] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PAR4). Fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, i.e., monovalent fragments consisting of VL and CL, VH and CH1 domains; (ii) F(ab)2 fragments, i.e., bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and CL domains of a single arm of the antibody; (v) single-domain antibody fragments or dAb consisting only of the VH domain or VL domain (Ward et al., Nature 341:544-546 (1989)); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VH and VL, are encoded by separate genes, they can be linked using recombination or synthesis methods, for example, by synthetic linkers that enable the creation of a single protein chain. In this case, the VH and VL regions pair up to form a monovalent molecule (known as single-stranded Fv (scFv)) (e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci.USA 85:5879-5883). scFv is also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for usefulness in the same manner as intact antibodies.

[0138] As used herein, “antibody variable region” refers to the light and heavy chain portions of an antibody molecule, including the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework region (FWR). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. According to the method used in this invention, the amino acid positions assigned to the CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or Chotia and Lesk 1987 J. Mol Biol. 196:901-917), or according to the IMGT numbering system.

[0139] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule with a single molecular composition. Monoclonal antibodies exhibit single-binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced from any animal, such as mice, rats, rabbits, or pigs, or they can be produced synthetically and may be partially or completely human sequences.

[0140] As used herein, the term “polyclonal antibody” refers to a mixture of antibodies purified from the serum of a mammal injected with an antigen to produce antibodies against that antigen. Polyclonal antibodies can be produced from any mammal, such as mice, rats, rabbits, pigs, or humans, or they can be produced synthetically, for example, as VH and VL genephage display libraries.

[0141] The term "chimeric antibody" refers to an antibody in which portions of the heavy chain and / or light chain are identical or homologous to a corresponding sequence in an antibody originating from a particular species (e.g., a rodent) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical or homologous to a corresponding sequence in an antibody originating from another species (e.g., a primate) or belonging to another antibody class or subclass, and also refers to fragments of such antibodies insofar as they exhibit the desired biological activity.

[0142] The term “humanized antibody” is generally understood to refer to a chimeric molecule prepared using recombinant techniques, having an epitope-binding site derived from an immunoglobulin from a non-human species, and the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site preferably comprises a complementation-determining region (CDR) from a non-human antibody transplanted into a suitable framework region within the variable domain of the human antibody, and the remaining region from the human antibody.

[0143] When used herein in conjunction with antibody molecules and binding proteins, the term “human antibody” refers to both variable antibody regions (e.g., VH, VL, CDR, and FR regions) and constant antibody regions derived from or corresponding to sequences found in humans (e.g., in human germ cells or somatic cells).

[0144] "IMGT numbering," as used herein, refers to a numbering system used to identify the CDR and FWR sequences of the antibody variable region. IMGT unique numbering is defined for comparing variable domains in any antigen receptor, chain type, or species (Lefranc M.-P., Immunology 5 Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., ThouveninContet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)). In IMGT unique numbering, conserved amino acids are always in the same position, for example, cysteine ​​23 (1 st CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2 ndThis will be CYS, phenylalanine, or tryptophan 118 (J-PHE or J-TRP). IMGT unique numbering provides standardized boundaries for framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: 66-104, and FR4-IMGT: 118-128) and complementarity determination regions (CDR1-IMGT: 27-38, CDR2-IMGT: 56-65, and CDR3-IMGT: 105-117). Since gaps represent unoccupied positions, the CDR-IMGT length is deterministic information. IMGT unique numbering is used in the 2D diagrammatic representation called IMGT Colliers de Perles (Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)) and the 3D structure in IMGT / 3Dstructure-DB (Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)).

[0145] As used herein, the term “specifically binds” should be interpreted to mean that the proteins of this disclosure react with or associate with a particular cell or substance more frequently, rapidly, for a longer duration, and / or with higher affinity than alternative cells or substances. Furthermore, by reading this definition, it should be understood that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. Therefore, “specific binding” does not necessarily require exclusive binding or undetectable binding to another antigen, which is what the term “selective binding” implies.

[0146] "Transferred" and "transferred cells" refer to cells into which a DNA molecule encoding PAR4 (or a DNA encoding a biologically active fragment or analog thereof) has been introduced using genetic engineering. Such DNA molecules are "positioned for expression." This means that the DNA molecule is positioned next to the DNA sequence that directs the transcription and translation of the sequence (i.e., drives the production of the PAR4 protein or its fragment or analog).

[0147] The term "identity" and its grammatical variations mean that two or more entities mentioned are the same. Therefore, if two antibody sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. If two nucleic acid sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. Identity may also be limited to a defined area (region or domain) of the sequence. Polynucleotide identity % is measured by GAP (Needleman and Wunsch, J. Mol Biol. 48:444-453. 1970) analysis (GCG program), with a gap creation penalty of 5 and a gap extension penalty of 0.3. Unless otherwise stated, the query sequence is at least 45 nucleotides long, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 100 nucleotides long, and the GAP analysis aligns the two sequences over a region of at least 100 nucleotides. Most preferably, the two arrays are aligned along their entire length.

[0148] As used herein, the term "pharmaceutical composition" means any composition that contains at least one therapeutically or biologically active agent and is suitable for administration to a patient. Any such formulations can be prepared by methods well known and accepted in the art. See, for example, Gennaro, A.R., ed., Remington: The Science and Practice of Pharmacy, 20th Edition, Mack Publishing Co., Easton, Pa. (2000).

[0149] As used herein, the expression "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical evaluation, without excessive toxicity, irritation, allergic response, and / or other problems or complications, in accordance with a reasonable benefit / risk ratio.

[0150] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) an undesirable physiological change or disorder (e.g., the progression of thromboembolism (e.g., acute coronary syndrome)). Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, attenuation of disease severity, stabilization of the disease state (i.e., not getting worse), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (partial or total), including both detectable and undetectable ones. "Treatment" may also mean extending the survival period compared to the expected survival period in the case of not receiving treatment. Those who need treatment include those who already have a condition or disorder, as well as those who tend to have a condition or disorder, or those who need to prevent a condition or disorder.

[0151] As used herein, “prevention” or “prevention” refers to preventive treatment for asymptomatic disease conditions in mammals, particularly humans, aimed at reducing the likelihood of a clinical disease condition developing. Preventive therapy is selected based on factors known to increase the patient’s risk of developing a clinical disease condition compared to the general population. “Preventive” therapy is classified into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in subjects who have not yet presented a clinical disease condition, while secondary prevention is defined as preventing the secondary occurrence of the same or similar clinical disease condition.

[0152] The term "therapeutic effective dose" shall be interpreted to mean a sufficient amount of PAR4-binding protein or antibody to reduce or inhibit one or more symptoms of PAR4 activation to below a level observed and accepted as clinically specific to the disorder. Those skilled in the art will recognize that such amounts vary depending on the particular antibody, fragment, and / or the particular target and / or the type of severity or level of disorder. Therefore, this term should not be interpreted to limit the invention to a specific amount.

[0153] As used herein, the term "PAR4 antagonist therapy" means treatment of a subject with a PAR4 antagonist.

[0154] "Subject" means any subject, in particular mammalian subject, for whom diagnosis, prognosis, or therapy is desired. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, bears, chickens, amphibians, reptiles, etc. As used herein, expressions such as "subject with a PAR4-mediated condition or disorder" include subjects that would benefit from administration of a PAR4 antagonist, e.g., mammalian subjects.

[0155] As used herein, the reference to binding being at a “similar” level should be understood to mean that an antibody binds to an antigen at a level within approximately 30%, 25%, or 20% of the level at which it binds to another antigen. This term also means that one antibody binds to an antigen at a level within approximately 30%, 25%, or 20% of the level at which another antibody binds to the same antigen.

[0156] As used herein, the statement that binding is “substantially the same level” should be understood to mean that an antibody binds to an antigen at a level within approximately 15%, 10%, or 5% of the level at which it binds to another antigen. This term also means that an antibody binds to an antigen at a level within approximately 5%, 4%, or 3% of the level at which another antibody binds to the same antigen.

[0157] The term “competitively inhibiting” is understood to mean that the protein of this disclosure reduces or prevents the binding of the listed production antibody (e.g., 5A.RC3) to the thrombin cleavage site of PAR4 or its fragment. From the above, it will be apparent that the protein does not need to completely inhibit antibody binding, but rather it is sufficient if it reduces binding by a statistically significant amount, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Methods for measuring competitive inhibition of binding are known in the art and / or described herein. For example, an antibody is exposed to PAR4 or its fragment either in the presence or absence of the protein. If antibody binding is less in the presence of the protein than in the absence of the protein, then the protein is considered to competitively inhibit antibody binding. In one example, the protein and antibody are exposed to PAR4 substantially simultaneously. Further methods for measuring competitive inhibition of binding are apparent to those skilled in the art and / or described herein. In one example, the antigen-binding domain of a protein competitively inhibits antibody binding.

[0158] In the context of two epitopes, "duplication" is interpreted to mean that the two epitopes share a sufficient number of amino acid residues, thereby allowing an antibody binding to one epitope to competitively inhibit the binding of an antibody binding to the other epitope. For example, the epitopes share at least one, two, three, four, five, six, seven, eight, nine, or ten amino acids.

[0159] As used herein, the term “undetectable binding” shall be understood to mean that a protein (e.g., an antibody) binds to a candidate antigen at a level less than 10%, 8%, 6%, or 5% above the background. The background may be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected by immobilizing the protein and contacting it with the antigen using biosensor analysis (e.g., Biacore).

[0160] antibody To avoid misunderstanding, it should be noted that the monoclonal antibody mAb ARC3 is synonymous with other names for this antibody, such as MoB5A-RC3, as shown in the examples. This antibody has been further subcloned to produce the derived monoclonal antibody MoB5-ARC3.F10b.H4b. This subclone is also called mAb ARC3.H4b. The sequences corresponding to this antibody can be found in SEQ ID NOs: 11-18.

[0161] Functionally equivalent antibodies This disclosure also envisions anti-PAR4 antibodies or antigen-binding fragments thereof that involve one or more amino acid additions, deletions, or substitutions in the heavy and light chain variable region sequences of the antibody mAb ARC3.H4b, but that still retain the function of mAb ARC3.H4b. In some examples, the PAR4-binding protein contains 10 or fewer conserved amino acid substitutions, e.g., 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 conserved amino acid substitution. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain and / or hydrophobic and / or hydrophilic. Hydrophobic and hydrophilic indices are described, for example, Kyte and Doolittle (1982), and hydrophilic indices are described, for example, US4554101.

[0162] Such modifications may be planned, for example, through site-directed mutagenesis, or they may be accidental, for example, modifications acquired through mutations within a host expressing an antibody.

[0163] Mutant (modified) polypeptides can be prepared using any technique known in the art. For example, the polynucleotides of this disclosure may be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include subcloning the polynucleotide in a suitable vector, transforming the vector in a “mutagenetic” strain, such as E. coli XL-1 red (Stratagene), and growing the transformed bacteria for a suitable number of generations. Products derived from mutant / modified DNA can be readily screened using the techniques described herein to determine whether they have receptor binding and / or inhibitory activity.

[0164] When designing amino acid sequence variants, the location and nature of the mutation site depend on the feature(s) being modified. The mutation site can be modified individually or sequentially, for example, by (1) first substituting with a conserved amino acid choice and then substituting with a more radical choice depending on the desired outcome, (2) deleting a target residue, or (3) inserting another residue adjacent to the site where the mutation occurred.

[0165] The range of amino acid sequence deletions is generally about 1 to 15 residues, more preferably about 1 to 10 residues, and typically about 1 to 5 consecutive residues.

[0166] Substitutional mutants involve the removal of at least one amino acid residue within the antibody and / or immunoglobulin chain molecule, including the variable region, and the insertion of a different residue in its place. Sites of most interest in substitutional mutagenesis include those identified as important for antigen binding. Such sites, particularly those that fall within the sequence of at least three other equally conserved sites in the human antibody and / or immunoglobulin chain, are preferably substituted in a relatively conserved manner. Such conservative substitutions are shown in Table 1 under the heading "Exemplary Substitutions."

[0167] Conservative amino acid substitutions are also intended in this invention. Such substitutions are interpreted as meaning the amino acid substitutions shown in the table below.

[0168] [Table 1]

[0169] The amino acids described herein preferably take the "L" isomer form. However, residues in the D isomer may be substituted for any L-amino acid residue, provided that the desired functional properties of immunoglobulin binding are maintained by the polypeptide. Modifications also include structural and functional analogs, such as synthetic or non-natural amino acids or amino acid analogs and peptide mimes having derivatized forms.

[0170] This disclosure also intends to address non-conservative amino acid changes. Of particular interest, for example, is the substitution of charged amino acids with other charged amino acids and neutral or positively charged amino acids. In some examples, PAR4-binding proteins contain 10 or fewer non-conservative amino acid substitutions, for example, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0171] Mutant forms of PAR4-binding proteins described herein, following any example, retain the ability to specifically bind to PAR4. Methods for measuring specific binding to PAR4 are described herein. For example, a labeled PAR4-binding protein is brought into contact with immobilized PAR4 or a peptide containing a thrombin cleavage site of PAR4 (e.g., as shown in SEQ ID NO: 2). After washing, the bound label is detected. Alternatively, a labeled PAR4-binding protein is brought into contact with immobilized PAR4 and related proteins or mutant forms of PAR4 or fragments of PAR4 as discussed above, and after washing, the bound label is detected. If a label is detected that binds to PAR4 but not to related PAR4 proteins (e.g., PAR1, PAR2, or PAR3) or mutant proteins or fragments, this indicates that the mutant PAR4-binding protein retains the ability to specifically bind to PAR4.

[0172] In one example, the mutation(s) occur within the FWR of the PAR4-binding protein of this disclosure. In another example, the mutation(s) occur within the CDR of the PAR4-binding protein of this disclosure.

[0173] antibody generation Methods for generating antibodies are known in the art and / or described in Harlow and Lane (1988) or Zola (1987). Generally, such methods involve the administration of cells (i.e., immunogens) containing, or expressing, the Fnl4 protein or its immunogenic fragment or epitope, formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient at the discretion of the administration of non-human animals, such as mice, chickens, rats, rabbits, guinea pigs, dogs, horses, cattle, goats, or pigs. The immunogens can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0174] The production of polyclonal antibodies can be monitored by sampling the blood of immunized animals at various time points after immunization. If necessary to achieve the desired antibody titer, one or more additional immunizations may be performed. The process of additional immunization and titration is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, blood is collected from the immunized animals, serum is isolated and stored, and / or the animals are used for the production of monoclonal antibodies (mAbs).

[0175] Monoclonal antibodies are exemplary antibodies as intended by this disclosure. The terms “monoclonal antibody,” “mAb,” or “MAb” refer to a homogeneous population of antibodies capable of binding to the same antigen(s), for example, the same epitope within the antigen. The terms are not intended to be limited to the source of the antibody or the method of producing the antibody.

[0176] For mAb production, any one of several known techniques can be used, for example, the procedures exemplified in US4,196,265, or Harlow and Lane (1988) Antibodies: A laboratory manual Cold Spring Harbor Laboratory, or Zola (1987) Monoclonal antibodies: A manual of techniques.

[0177] For example, a suitable animal is immunized with an effective amount of cells expressing the protein or its immunogenic fragment or epitope, or the protein, under conditions sufficient to stimulate antibody-producing cells. Rodents such as rabbits, mice, and rats are exemplary animals, with mice being the most commonly used. Mice genetically engineered to express human immunoglobulin proteins and not murine immunoglobulin proteins can also be used to produce the antibodies of this disclosure (for example, as described in WO2002 / 066630).

[0178] After immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells), are selected for use in mAb generation protocols. Such cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from peripheral blood samples. B cells from immunized animals are then fused with immortalized myeloma cells. These myeloma cells generally originate from the same species as the animal immunized with the immunogen. B cells and immortalized cells are fused by incubating a mixture of multiple cell types in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. Fusion methods using Sendai virus are described in Kohler and Milstein, (1975) and Kohler and Milstein, (1976). Methods using polyethylene glycol (PEG) (e.g., 37% (v / v) PEG) are described in Gefter et al, (1977) Somatic Cell Genet. 3(2):231-6. Fusion methods using electrical induction are also appropriate.

[0179] The hybrids are amplified by culturing them in a selective medium containing an agent that blocks the novel synthesis of nucleotides in the tissue medium. Exemplary agents include aminopterin, methotrexate, and azacerin.

[0180] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer by, for example, flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.). This disclosure also intends to include subcloning of antibody-producing cells, as illustrated herein.

[0181] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) can be used to produce mAb-secreting cell lines (e.g., as described in Kumar et al, (1999) Immunol Lett. 65(3):153-9).

[0182] Antibodies can also be produced or isolated using display libraries, such as phage display libraries, e.g., those described in US6300064, EP0368684, and / or US5885793.

[0183] Chimeric antibodies and proteins One example of an antibody or PAR4-binding protein in this disclosure is a chimeric antibody. That is, a PAR4-binding protein is a chimeric protein. The term "chimeric protein" refers to a protein in which the antigen-binding domain VH or VL is identical or homologous to a corresponding sequence in a protein derived from a particular species (e.g., a murid such as a mouse or rat) or in a protein belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in a protein derived from another species (e.g., a primate such as a human) or in a protein belonging to another antibody class or subclass. In one example, the chimeric protein is a chimeric antibody containing VH and VL from a non-human antibody (e.g., a murid antibody), with the rest of the antibody being from a human antibody. The production of such chimeric proteins is known in the art and can be achieved by standard means (e.g., those described in US6331415; US5807715; US4816567; and US4816397). The production of such chimeric antibodies is known in the art and can be achieved by standard means (e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, (1989) J.Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; No. 4,816,567; and No. 4,816,397). Furthermore, it is intended that the human constant region of the chimeric antibody of the present invention may be selected from the IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19 constant regions.

[0184] Humanization and human antibodies / proteins The PAR4-binding proteins of this disclosure may be humanized or human.

[0185] The term “humanized protein” is understood to refer to a protein containing a human-like variable region (including CDRs) from an antibody of a non-human species, transplanted or inserted into the FR from a human antibody (this type of antibody is also called a “CDR-transplanted antibody”). Humanized proteins also include proteins in which one or more residues of a human protein are modified by one or more amino acid substitutions, and / or one or more FR residues of a human protein are replaced by corresponding non-human residues. Humanized proteins may also contain residues not found in either human or non-human antibodies. Any further region of the protein (e.g., the Fc region) is human. Humanization can be carried out using methods known in the art, e.g., US5225539, US6054297, US7566771, or US5585089. The term “humanized protein” also includes super-humanized proteins, e.g., those described in US7732578.

[0186] In one example, the humanized protein includes regions between 26 and 33, between 51 and 58, and between 97 and 110, as well as between 27 and 33, between 51 and 53, and between 90 and 97 within the heavy chain sequence disclosed herein (numbering follows the IMGT numbering system).

[0187] As used herein, the term “human protein” refers to a protein having a variable antibody region and, optionally, a constant antibody region derived from or corresponding to a sequence found in humans (e.g., in human germline or somatic cells). “Human” antibodies may include amino acid residues not encoded by human sequences, e.g., mutations introduced in vitro by random or site-directed mutations (more specifically, mutations involving a small number of residues in the protein, e.g., conservative substitutions or mutations of 1, 2, 3, 4, or 5 residues in the protein). Such “human antibodies” do not actually need to be produced as a result of a human immune response, but rather can be produced using recombinant means (e.g., screening of phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding human antibody constant and / or variable regions, and / or by guided selection (e.g., as described in US5565332). The term also encompasses affinity-matured forms of such antibodies. It should also be considered that human proteins may include proteins containing FRs from human antibodies, or FRs containing sequences from consensus sequences of human FRs, where one or more CDRs are random or semi-random as described in US6300064 and / or US6248516.

[0188] Human proteins or antibodies that recognize selected epitopes can also be generated using a technique called "inducible selection." In this approach, selected non-human monoclonal antibodies (e.g., mouse antibodies) are used to induce the selection of fully human antibodies that recognize the same epitope (Jespers LS et al, (1988) Biotechnology 12(9):899-903).

[0189] The human PAR4-binding protein of this disclosure includes a variable region of a human antibody.

[0190] Synthetic humanized and primate-like proteins The PAR4-binding proteins of this disclosure may also be synthetic humanized proteins. The term "synthetic humanized protein" refers to a protein prepared by the method described in WO2007 / 019620. A synthetic humanized PAR4-binding protein includes a variable region of an antibody in which the variable region comprises a FR from a New World primate antibody variable region and a CDR from a non-New World primate antibody variable region. For example, a synthetic humanized PAR4-binding protein includes a variable region of an antibody in which the variable region comprises an FWR from a New World primate antibody variable region and a CDR from a mouse antibody (e.g., as described herein). In one example, a synthetic humanized PAR4-binding protein is a PAR4-binding antibody in which one or both of the variable regions are synthetically humanized.

[0191] The PAR4-binding protein of this disclosure may also be a primate-forming protein. A “primate-forming protein” includes one variable region from an antibody produced after immunization of a non-human primate (e.g., a macaque or cynomolgus monkey). Optionally, the variable region of a non-human primate antibody is linked to a human constant region to produce a primate-forming antibody. An exemplary method for producing a primate-forming antibody is described in US6113898.

[0192] Deimmunized antibodies and proteins This disclosure also intends to describe immunized antibodies or PAR4-binding proteins. Immuneized antibodies reduce the likelihood of a target eliciting an immune response to an antibody or protein by removing (i.e., mutating) one or more epitopes, such as B-cell epitopes or T-cell epitopes. Methods for producing immunized antibodies and proteins are known in the art and are described, for example, in WOOO / 34317, WO2004 / 108158, and WO2004 / 064724.

[0193] Methods for introducing suitable mutations and expressing the resulting proteins for assay will be apparent to those skilled in the art based on the description herein.

[0194] The antibody variable region containing proteins. Single-domain antibody In some instances, the PAR4-binding proteins of this disclosure are single-domain antibodies (this designation is used interchangeably with the terms “domain antibody” or “dAb”). A single-domain antibody is a single polypeptide chain containing all or part of the heavy chain variable region of the antibody. In some instances, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; e.g., US6248516; WO90 / 05144; and / or WO2004 / 058820).

[0195] Diabody, Triabody, Tetrabody Exemplary PAR4-binding proteins containing antibody-antigen-binding domains are diabodies, triabodies, tetrabodies, and higher-order protein complexes, such as those described in WO98 / 044001 and WO94 / 007921.

[0196] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL (wherein VL is the antibody light chain variable region, VH is the antibody heavy chain variable region, and X is a linker containing an incomplete residue to allow VH and VL within a single polypeptide chain to associate (or form an Fv), with VH of one polypeptide chain binding to VL of the other polypeptide chain to form an antigen-binding site, i.e., forming an Fv molecule that can specifically bind to one or more antigens). VL and VH may be the same within each polypeptide chain, or they may be different within each polypeptide chain to form a bispecific diabody (i.e., containing two Fvs with different specificities).

[0197] Single chain Fv (scFv) fragment Those skilled in the art will recognize that scFv contains a VH region and a VL region within a single polypeptide chain. The polypeptide chain further includes a polypeptide linker between VH and VL, which enables scFv to form a structure desirable for antigen binding (i.e., desirable for the VH and VL of the single polypeptide chain to associate with each other to form Fv). For example, the linker contains more than 12 amino acid residues together with (Gly4Ser)3, which is one of the more preferred linkers for scFv.

[0198] This disclosure also envisions disulfide-stabilized Fv (or diFv or dsFv), in which a single cysteine ​​residue is introduced into the FR of VH or VL, and the cysteine ​​residue is linked by a disulfide bond to yield a stable Fv (see, for example, Brinkmann et at, (1993) Proc Natl Acad Sci USA 90:547-551).

[0199] Alternatively or additionally, the Disclosure provides a protein comprising a dimeric scFv, i.e., two scFv molecules linked by non-covalent or covalent bonds, for example, by a leucine zipper domain (e.g., derived from Fos or Jun) (see, e.g., Kruif and Logtenberg, 1996). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow the formation of both scFvs and their binding to an antigen, for example, as described in US20060263367.

[0200] For an overview of scFv, see Ahmad ZA et al., (2012) Clinical and Developmental Immunology doi:10.1155 / 2012 / 980250.

[0201] Mini body Those skilled in the art will recognize that the minibody includes the VH and VL domains of an antibody fused with the (CH2 domain and / or (CH3 domain). Optionally, the minibody includes a hinge region between VH and VL, and this conformation is sometimes referred to as a Flex minibody. The minibody does not include CH1 or CL. In one example, the VH and VL domains are fused with the hinge region and CH3 domain of the antibody. At least one of the variable regions of the minibody binds to PAR4 in the manner of the present disclosure. Exemplary minibodies and methods for generating them are described, for example, in WO94 / 09817.

[0202] Other antibody variable regions, including proteins This disclosure also intends to include other variable regions, including PAR4-binding proteins, such as the following: (i) The “key and hole” bispecific protein described in US5,731,168; (ii) Heteroconjugated proteins (e.g., those described in US4,676,980); (iii) Heteroconjugated proteins produced using chemical crosslinkers (e.g., those described in US4,676); (iv) Fab'-SH fragments (e.g., those described in Shalaby (1992)j Exp Med 1;175(1):217-25); (v) Single-stranded Fab; or (vi) Fab3 (for example, the one described in EP19930302894).

[0203] Non-antibody-based antigen-binding domain-containing protein Immunoglobulins and immunoglobulin fragments One example of the compounds of this disclosure is a protein containing a variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., IgNA, a camelid antibody).

[0204] The term "immunoglobulin" is understood to include any antigen-binding protein containing an immunoglobulin domain. An exemplary immunoglobulin is an antibody. Additional proteins that the term "immunoglobulin" encompasses include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., novel immunoglobulin antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs contain VH but lack VL and are often called heavy-chain immunoglobulins. Other "immunoglobulins" include T cell receptors.

[0205] Heavy chain immunoglobulin Heavy-chain immunoglobulins are structurally different from many other forms of immunoglobulins (e.g., antibodies) in that they contain heavy chains but not light chains. Therefore, such immunoglobulins are also called "heavy-chain-only antibodies." Heavy-chain immunoglobulins are found, for example, in camelids and cartilaginous fish (also known as IgNARs).

[0206] The variable regions present within natural heavy-chain immunoglobulins are distinguished from the heavy-chain variable regions (called "VH domains") and light-chain variable regions (called "VL domains") present within conventional four-chain antibodies, and are generally referred to as "VHH domains" in camelid Ig and V-NARs in IgNARs.

[0207] Heavy chain immunoglobulins do not require the presence of a light chain to bind to the relevant antigen with high affinity and specificity. This means that single-domain binding fragments can be derived from heavy chain immunoglobulins that are readily expressed and generally stable and soluble. A general description of heavy chain immunoglobulins and their variable regions from camelid animals, as well as methods for their production and / or isolation and / or use, can be found, in particular, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0208] A general description of heavy chain immunoglobulins and their variable regions from cartilaginous fishes, as well as methods for their production and / or isolation and / or use, can be found, in particular, in WO2005 / 118629.

[0209] V-like protein One example of a PAR4-binding protein in this disclosure is the T cell receptor. The T cell receptor has two V domains, which combine to form a structure similar to the antibody's Fv module. Novotny et al, Proc Natl Acad Sci USA 88:8646-8650, 1991, describes how the two V domains of the T cell receptor (referred to as alpha and beta) can fuse to be expressed as a single-chain polypeptide, and further, how surface residues can be modified to reduce the hydrophobicity directly similar to that of the antibody scFv. Other publications describing the production of single-chain or multimeric T cell receptors containing two V-alpha and V-beta domains include WO1999 / 045110 and WO2011 / 107595.

[0210] Other non-antibody proteins containing antigen-binding domains include proteins with V-like domains, which are generally monomeric. Examples of such V-like domain-containing proteins include CTLA-4, CD28, and ICOS. Further disclosures of such V-like domain-containing proteins are included in WO1999 / 045110.

[0211] Adnectin In one example, the PAR4-binding protein of this disclosure is adnectin.

[0212] Adnectin is based on the 10Fn3 domain of human fibronectin, in which the loop region is modified to confer antigen binding. For example, three loops at one end of the β-sandwich of 10Fn3 can be manipulated to allow adnectin to specifically recognize antigens. For further details, see US20080139791 or WO2005 / 056764.

[0213] Anticharin In a further example, the PAR4-binding protein of this disclosure is antikalin. Antikalin is derived from lipocalin, a family of extracellular proteins that transport hydrophobic small molecules (e.g., steroids, bilines, retinoids, and lipids). Lipocalin has a rigid β-sheet secondary structure with multiple loops at the open end of a conical structure, and these loops can be manipulated to bind to an antigen. Such manipulated lipocalin is known as antikalin. For a further description of antikalin, see US7250297B1 or US20070224633.

[0214] Affibody In a further example, the PAR4-binding protein of this disclosure is an affibody. The affibody is a scaffold derived from the Z domain (antigen-binding domain) of protein A of Staphylococcus aureus, which can be manipulated to bind to an antigen. The Z domain consists of a three-helix bundle of approximately 58 amino acids. The library is generated by randomization of surface residues. For further details, see EP1641818.

[0215] Abimar In a further example, the PAR-binding proteins of this disclosure are avimers. Avimers are multi-domain proteins derived from the A-domain scaffold family. The native domain, approximately 35 amino acids long, adopts a defined disulfide bond structure. Diversity is generated by shuffling the native variant forms shown by the A-domain family. For further details, see WO2002088171.

[0216] DARPin In a further example, the PAR4-binding protein of this disclosure is a designed ankyrin repeat protein (DARPin). DARPin is derived from ankyrin, a family of proteins that mediate the attachment of complex membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and one β-turn. DARPin can be engineered to bind to different target antigens by randomizing the residues in the first α-helices and β-turns of each repeat. The binding interface of DARPin can be increased by increasing the number of modules (affinity maturation method). For further details, see US20040132028.

[0217] Other non-antibody polypeptides Other non-antibody proteins containing binding domains include those based on human γ-crystallin and human ubiquitin (affilin), the Knitz-type domain of human protease inhibitors, the PDZ domain of the Ras-binding protein AF-6, scorpion venom (caribudotoxin), and C-type lectin domains (tetranectin).

[0218] Steady-state region This disclosure encompasses PAR4-binding proteins that include variable regions and constant regions or domains (e.g., CH2 and / or CH3). Those skilled in the art will understand the meaning of the terms constant region and constant domain based on the disclosure herein and the references discussed herein.

[0219] The constant region sequences useful for the production of the PAR4-binding protein of this disclosure can be obtained from several different sources. In some examples, the constant region or portion of the PAR4-binding protein is derived from a human antibody. Furthermore, the constant domain or portion may be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgGl, IgG2, IgG3, and IgG4. In one example, the human isotype IgGl is used.

[0220] Various constant region gene sequences are available in the form of publicly accessible deposits, or their sequences are available from publicly accessible databases. Constant regions may be selected to have (or lack) specific effector functions, or to have specific modifications to reduce immunogenicity.

[0221] In one example, the proteins of this disclosure have or exhibit effector functions that promote or enable at least partial, substantial, or disappearance of cells expressing PAR4. Such effector functions may include enhanced binding affinity to the Fc receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0222] In one example, PAR4-binding proteins can induce an enhancement of effector function levels.

[0223] In one example, the level of effector function induced by the constant region is enhanced compared to the wild-type Fc region of the IgG1 antibody or compared to the wild-type Fc region of the IgG3 antibody.

[0224] In another example, a constant region is modified to increase the level of effector function it can induce compared to an unmodified constant region. Such modifications may be at the amino acid level and / or the secondary and / or tertiary structure level, and / or to glycosylation of the Fc region.

[0225] Those skilled in the art will understand that greater effector functions can be manifested in multiple ways, for example, as a greater level of effect, a more sustained effect, or a faster effect. Exemplary constant region modifications include amino acid substitutions, e.g., S239D / I332E (numbering according to Kabat's EU index) or S239D / A330L / I332E (numbering according to Kabat's EU index).

[0226] Further amino acid substitutions that increase the effector function-inducing ability of the Fc region are known in the art and / or described, for example, in US6737056 or US7317091.

[0227] In one example, glycosylation of the constant region is modified to increase the ability to induce effector enhancement. In several examples, the Fc region according to this disclosure includes a carbohydrate structure in which fucose attached (directly or indirectly) to the Fc region is absent, i.e., the Fc region is "non-fucosylated". Such variants may have improved ADCC-inducing ability. Methods for producing non-fucosylated antibodies include expressing Fnl4-binding protein in cell lines that cannot express al,6-fucosyltransferase (FUT8) (e.g., as described by Yumane-Ohnuki et al. ah, 2004). Other methods include using cell lines that inherently produce antibodies capable of inducing effector enhancement (e.g., duck embryo-derived stem cells for viral vaccine production (WO2008 / 129058); production of recombinant protein in tori EBX® cells (WO2008 / 142124)).

[0228] PAR4-binding proteins can also contain Fc regions capable of inducing enhanced CDC levels. For example, IgG1 and IgG3 hybrids produce antibodies with enhanced CDC activity (Natsume et al., 2008).

[0229] Methods for measuring the effector function-inducing ability of an antibody or its antigen-binding fragment are known in the art and / or described herein.

[0230] In another example, a protein contains one or more amino acid substitutions that increase the half-life of a PAR4-binding protein. For example, a PAR4-binding protein contains a constant region containing one or more amino acid substitutions that increase the affinity of the constant region to the neonatal Fc region (FcRn). For example, the constant region has increased affinity for FcRn at lower pH (e.g., around pH 6.0), promoting Fc / FcRn binding within endosomes. In one example, the constant region has increased affinity for FcRn at around pH 6 compared to its affinity at around pH 7.4, thereby promoting Fc re-release into the bloodstream after cell recycling. Such amino acid substitutions are useful in extending the half-life of a protein by reducing clearance from the blood.

[0231] Examples of amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784. The neutralizing PAR4-binding proteins in this disclosure may include an IgG4 constant region or a stabilized IgG4 constant region. The term “stabilized IgG4 constant region” should be understood as an IgG4 constant region modified to reduce the tendency to undergo Fab arm exchange, or to undergo Fab arm exchange or semi-antibody formation, or to reduce the tendency to form semi-antibodies. “Fab arm exchange” refers to one type of protein modification in human IgG4 in which one IgG4 heavy chain and attached light chain (half a molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Therefore, the IgG4 molecule can acquire two distinct Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs spontaneously in vivo and can also be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. The "half-antibody" form is formed when the IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0232] In one example, the stabilized IgG4 constant region contains proline at position 241 of the hinge region according to the Kabat system. This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is generally serine. After the substitution of serine for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that connects to the Fc and Fab regions, conferring mobility to the two Fab arms of the antibody. The hinge region contains a cysteine ​​residue involved in the disulfide bond between the heavy chains. The hinge region is defined as extending from Glu226 to Pro243 in human IgG1 according to the Kabat numbering system. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same position (see, for example, WO2010 / 080538).

[0233] Modified proteins This disclosure provides a PAR4-binding protein having at least 80% identity to the sequence of this disclosure and having the same functional features as described or claimed herein.

[0234] In one example, the PAR4-binding protein of this disclosure comprises a sequence having at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with the VL sequence disclosed herein (e.g., SEQ ID NO: 11).

[0235] In another example, the PAR4-binding protein of this disclosure comprises a sequence having at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with respect to the VH disclosed herein (e.g., SEQ ID NO: 12).

[0236] This disclosure also provides nucleic acids encoding the aforementioned proteins, or nucleic acids that hybridize with those nucleic acids under medium to high stringency conditions.

[0237] This disclosure also includes nucleic acids encoding proteins that, as a result of genetic code degeneracy, include sequences shown in SEQ ID NOs: 11 and 12, which are different from the sequences exemplified herein.

[0238] Nucleic acid or polypeptide identity % is measured by GAP (Needleman and Wunsch, 1970) analysis (GCG program), with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. In one example, the two sequences are aligned over their entire length.

[0239] The glycosylation pattern of an antibody may be modified from the original glycosylation pattern of a reference antibody. Modification means the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites that were not present in the antibody. Antibody glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the binding of the asparagine residue of the carbohydrate moiety to the side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzymatic binding of the asparagine side chain of the carbohydrate moiety. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used). The addition of a glycosylation site to an antibody is conveniently carried out by modifying the amino acid sequence to include the aforementioned tripeptide sequence (for the N-linked glycosylation site). The modification can also be performed by adding or substituting one or more serine or threonine residues to the original antibody sequence (for the O-linked glycosylation site).

[0240] The modified glycoforms of the antibodies of the present invention may be useful for a variety of purposes, including, but are not limited to, enhancing or reducing effector function and / or modifying the antibody half-life (see, e.g., WO / 2007 / 010401). Such modifications may result in a decrease or increase in C1q binding and CDC, or a decrease or increase in FcγR binding and ADCC. Substitutions may be made, for example, at one or more amino acid residues in the heavy chain constant region, thereby altering effector function while retaining antigen-binding ability compared to the modified antibody (see US5,624,821 and US5,648,260). The manipulated glycoforms can be produced by any method known to those skilled in the art, for example, by the use of manipulated or variant expression strains, co-expression with one or more enzymes (e.g., β(l,4)-N-acetylglucosaminyltransferase III (GnTII 1)), expression of antibodies or their fragments in or from cell lines of various organisms, or modification of the carbohydrate(s) after expression of the antibody or fragment. Methods for purifying manipulated glycoforms are known in the art and include, but are not limited to, the following: Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2007 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) US Pat. No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCT No. WO00 / 61739A1; PCT No. WO01 / 292246A1; PCT No. WO02 / 311140Al; PCT No. WO02 / 30954A1; Potelligent® Technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ Glycosylation Engineering Technology (GLYCART biotechnology AG, Zurich, Switzerland).For example, see WO00061739;EA01229125;US20030115614;Okazaki et al.,2004,JMB,336:1239-49.

[0241] Modifications to the effector function of antibodies as described herein are considered desirable, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region to enable the formation of interchain disulfide bonds within this region. Homodimeric antibodies thus produced may have improved internal migration ability and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1 191-195 (1992) and Shopes, BJImmunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinking agents as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, antibodies that are thought to have enhanced complement lysis and ADCC capabilities due to the presence of a double Fc region may be manipulated. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

[0242] To increase the serum half-life of an antibody, salvage receptor-binding epitopes, such as those described in U.S. Patent No. 5,739,277, may be incorporated into the antibody (particularly the antibody fragment). As used herein, the term “salvage receptor-binding epitope” means an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that plays a role in increasing the in vivo serum half-life of the IgG molecule. D. Alternatively, the antibody half-life can be increased by pegylation.

[0243] affinity maturation In a further example, the existing PAR4-binding proteins of this disclosure have matured affinity to produce antibodies capable of binding to PAR4 with increased affinity. For example, sequences encoding VL and / or VH are isolated, and the CDR-coding region (e.g., the region encoding CDR3 of VL and / or VH) is mutated to introduce one or more amino acid substitutions. The resulting mutant PAR4-binding proteins are then screened for binding to PAR4, for example, by a competitive assay.

[0244] The PAR4-binding proteins according to this disclosure may be soluble secreted proteins, fusion proteins on the surface of cells, or particles (e.g., phages or other viruses, ribosomes, or spores). Exemplary phage display methods are described, for example, in US5821047;US6248516; and US6190908. The phage-displayed particles produced using these methods are then screened to identify the displayed PAR4-binding proteins that have a conformation sufficient to bind to a target antigen (e.g., PAR4).

[0245] Protein production In one example, the PAR4-binding protein of this disclosure is produced by culturing a cell line (e.g., a hybridoma under conditions sufficient for the production of the protein, as described herein and / or known in the art).

[0246] Recombinant expression In the case of recombinant proteins, the nucleic acid encoding the recombinant protein is placed in one or more expression constructs (e.g., expression vectors) and then transmigrated into host cells (e.g., cells capable of disulfide crosslinking or binding, e.g., E. coli cells, yeast cells, insect cells, or mammalian cells). Exemplary mammalian cells include monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. Molecular cloning techniques for achieving such objectives are known in the art and are described, for example, by Ausubel or Sambrook. A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see US4816567, US7923221, and US7022500.

[0247] The nucleic acids encoding the proteins of this disclosure are, after isolation, inserted into an expression construct or a replicable vector for further cloning (DNA amplification) or expression in a cell-free system or within cells. For example, the nucleic acid is ligated to a promoter. As used herein, the term “promoter” is to be interpreted in its broadest sense and includes the transcriptional regulatory sequence of a genomic gene (including the TATA box or initiation element required for proper transcription initiation), and may or may not include additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that modify the expression of the nucleic acid in response to development and / or external stimuli or in a tissue-specific manner. Also in the context of the present invention, the term “promoter” is used to describe a recombinant, synthetic, or fused nucleic acid or derivative that confers, activates, or enhances the expression of a ligated nucleic acid. An exemplary promoter may include one or more further copies of specific regulatory elements to further enhance the expression of the nucleic acid and / or modify spatial and / or temporal expression.

[0248] As used herein, the term “actably linked to ~” means that the promoter is positioned relative to the nucleic acid so that the expression of the nucleic acid is controlled by the promoter.

[0249] Cell-free expression systems are also intended in this disclosure. For example, the nucleic acids encoding the Fnl4 binding protein of this disclosure are ligated to a suitable promoter (e.g., the T7 promoter), and the resulting expression construct is exposed to conditions sufficient for transcription and translation. Typical expression vectors for in vitro or cell-free expression are described, including, but are not limited to, the TNT T7 and T3 TNT systems (Promega), and the pEXPl-DEST and pEXP2-DEST vectors (Invitrogen).

[0250] Many vectors are available for intracellular expression. Generally, vector components include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding the Fnl4 binding protein of this disclosure (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. For example, exemplary signal sequences include prokaryotic secretory signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II), yeast secretory signals (e.g., invertase reader, factor reader, or acid phosphatase reader), or mammalian secretory signals (e.g., herpes simplex gD signal).

[0251] Exemplary promoters include those active in prokaryotes (e.g., the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter system, and hybrid promoters, such as the tac promoter).

[0252] Exemplary promoters of activity in mammalian cells include the cytomegalovirus early promoter (CMV-IE), human elongation factor 1-oc promoter (EF1), micronuclear RNA promoters (Ula and Ulb), oc-myosin heavy chain promoter, monkey virus 40 promoter (SV40), Roussarcoma virus promoter (RSV), adenovirus main late promoter, β-actin promoter; and hybrid regulatory elements including the CMV enhancer / β-actin promoter or immunoglobulin promoter or its active fragments. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7, Australian Cell Bank CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth under suspension culture); baby hamster kidney cells (BHK, Australian Cell Bank CCL 10); or Chinese hamster (CHO).

[0253] Typical promoters suitable for expression in yeast cells (e.g., yeast cells selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, and S. pombe) include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUPl promoter, PH05 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0254] Means for introducing isolated nucleic acid molecules or gene constructs containing such nucleic acid molecules into cells are known to those skilled in the art. The technique used for a given cell depends on known techniques that have yielded favorable results. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transduction, liposome-mediated transduction using, for example, lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, viral transduction (e.g., transduction using lentiviruses), and particle impaction (e.g., particle impaction using, among other things, DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA)).

[0255] The host cells used to produce the PAR4-binding protein of this disclosure can be cultured in a variety of media depending on the cell type used. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0256] Protein isolation The PAR4-binding protein of this disclosure can be isolated or purified.

[0257] Methods for purifying the PAR4-binding protein of this disclosure are known in the art and / or described herein.

[0258] When recombinant techniques are used, the PAR4-binding protein of this disclosure may be produced intracellularly, within the periplasmic space, or secreted directly into the culture medium. When the protein is produced intracellularly, the first step is to remove host cells or lysed fragments, which are particulate debris, by, for example, centrifugation or ultrafiltration. When the protein is secreted into the culture medium, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). A protease inhibitor such as PMSF may be included in one of the aforementioned steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of exogenous contaminants.

[0259] Proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof. These methods are known in the art and are described, for example, in W099 / 57134 or Zola (1997).

[0260] Those skilled in the art will also recognize that the PAR4-binding proteins of this disclosure may be modified to include tags for facilitating purification or detection, such as polyhistidine tags, such as hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or simian virus type 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. For example, this tag is a hexa-his tag. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-his tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively or additionally, ligands or antibodies that bind to the tags may be used in affinity purification methods.

[0261] combination This disclosure also provides PAR4-binding protein conjugates described herein, subject to any example. Examples of compounds to which proteins can be conjugated are selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, compounds that increase the half-life of proteins in a subject, and mixtures thereof. Exemplary therapeutic agents include, but are not limited to, anti-angiogenic agents, anti-neoangiogenic agents and / or other angiogenic agents, antiproliferative agents, apoptotic promoters, chemotherapeutic agents, or therapeutic nucleic acids. Toxins include any agent that is harmful to cells (e.g., that kills cells). For a description of the class of drugs known in the art and their mechanisms of action, see Goodman et al., (1990). Further techniques suitable for the preparation of immunoglobulin-immunotoxin conjugates are shown, for example, in US5194594. Exemplary toxins include diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, diansine protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes. See, for example, W093 / 21232.

[0262] In one example, a PAR4-binding protein described herein, following any example, is bound or ligated to another protein, such as an immunomodulator, or a half-life-extending protein or peptide, or in particular another protein that binds to serum albumin. Exemplary serum albumin-binding peptides or proteins are described in US20060228364 or US20080260757.

[0263] In another example, the protein may be conjugated with a "receptor" (e.g., streptavidin) for use in cell pre-targeting, in which case the conjugate is administered to the patient, the unconjugated conjugate is removed from the blood circulation using a scavenging agent, and then the "ligand" (e.g., avidin) conjugated with a therapeutic agent (e.g., radionucleotide) is administered.

[0264] The PAR4-binding proteins of this disclosure can be modified to include further non-protein moieties known and readily available in the art. For example, suitable moieties for protein derivatization are physiologically acceptable polymers, such as water-soluble polymers. Such polymers are useful for increasing stability and / or reducing clearance (e.g., by the kidney) and / or reducing the immunogenicity of the Fnl4-binding proteins of this disclosure. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propropylene glycol (PPG).

[0265] In one example, a PAR4-binding protein described herein, according to any example, includes one or more detectable markers to facilitate detection and / or isolation. For example, compounds include fluorescent labels such as fluorescein (FITC), 5,6-carboxymethylfluorescein, Texas Red, nitrobenzo-2-oxa-1,3-diazole-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4′-6-diamidino-2-phenylindole (DAPI), and cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), and rhodamine (5,6-tetramethylrhodamine). The maximum absorption and emission values ​​for these fluorescent substances are as follows: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm), and Cy7 (755 nm; 778 nm). Alternatively or additionally, the Fnl4-binding proteins described herein, according to any example, may be labeled with, for example, fluorescent semiconductor nanocrystals (e.g., as described in US6,306,610).

[0266] Alternatively or additionally, PAR4-binding proteins may be labeled with magnetic or paramagnetic compounds such as iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous iron-chromium-cobalt, nickel-ferrous iron, cobalt-platinum, or strontium ferrite.

[0267] Protein fixation In one example, a PAR4-binding protein is immobilized on a solid or semi-solid matrix. The term “immobilization” should be understood to include various methods and techniques for immobilizing a protein on a particular matrix, such as those described, for example, in W099 / 56126 or WO02 / 26292. For example, immobilization can help stabilize a protein so that its activity is not reduced or adversely modified by biological, chemical, or physical exposure, particularly during storage or single-batch use. Various methods for immobilizing proteins on a matrix are known in the art, such as crosslinking, binding to a carrier, and retention within a semipermeable matrix. Exemplary matrices include porous gels, aluminum oxide, bentonite, agarose, starch, nylon, or polyacrylamide.

[0268] Assay of the activity of the binding protein of this disclosure Binding assay One form of such assay is the antigen-binding assay, as described, for example, in Scopes (1994) Protein Purification: principles and practice Springer-Verlag. Such methods generally involve labeling a PAR4-binding protein and contacting the antigen or fragment immobilized with the protein with a protein containing the extracellular portion of biotin-fused PAR4 (e.g., as shown in SEQ ID NO: 6). After washing to remove nonspecific binding proteins, the amount of labeling and, consequently, the binding protein is detected. Naturally, PAR4-binding proteins can be immobilized and antigens labeled. Panning assays can also be used. The examples herein describe a binding assay based on flag-tagged PAR4 that can be expressed on the surface of HEK cells. Inhibition of PAR4 cleavage by PAR4-binding proteins in the presence of thrombin can be measured by flow cytometry.

[0269] The PAR4-binding proteins of the present invention that competitively inhibit the binding of PAR4 antibodies to epitopes can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0270] Competitive binding assay Assays for measuring PAR4-binding proteins that competitively inhibit the binding of the antibody of this disclosure (e.g., mAb ARC3.H4b) will be apparent to those skilled in the art. For example, the antibody of this disclosure is conjugated with a detectable label, such as a fluorescent or radioactive label. The labeled antibody and the test PAR4-binding protein are then mixed and brought into contact with PAR4 or its extracellular domain fused to the Fc region of a peptide containing the antibody or its epitope. The level of the labeled antibody is then measured and compared to the level measured when the labeled antibody was brought into contact with PAR4 or a peptide containing the PAR4-Fc region or its epitope in the absence of the PAR4-binding protein. If the level of the labeled antibody in the presence of the test PAR4-binding protein is reduced compared to the absence of the PAR4-binding protein, then that PAR4-binding protein competitively inhibits the binding of the antibody.

[0271] Optionally, the test PAR4-binding protein is conjugated with a label different from the antibody. This allows for the detection of the level of binding of the test PAR4-binding protein to a protein or epitope.

[0272] In another example, a test PAR4-binding protein is conjugated to a peptide containing PAR4 or a PAR4-Fc region or its epitope, and then the peptide containing PAR4 or a PAR4-Fc region or its epitope is contacted with the antibody described herein. If the amount of bound antibody in the presence of the PAR4-binding protein is reduced compared to the absence of the PAR4-binding protein, it indicates that the PAR4-binding protein competitively inhibits the binding of the antibody to PAR4. Alternatively, a cross-assay can be performed using a labeled PAR4-binding protein, in which case the antibody is first conjugated to a peptide containing PAR4 or a PAR4-Fc region or its epitope. In this case, if the amount of labeled PAR4-binding protein bound to the peptide containing PAR4 or a PAR4-Fc region or its epitope is reduced compared to the absence of the antibody, it indicates that the PAR4-binding protein competitively inhibits the binding of the antibody to PAR4.

[0273] Epitope Mapping Assay In another example, the epitopes to which the PAR4-binding proteins described herein bind are mapped. The method of epitope mapping will be apparent to those skilled in the art. For example, a series of duplicate peptides, e.g., peptides containing 10-15 amino acids, are produced that span a PAR4 sequence or region containing the target epitope. The PAR4-binding protein is then brought into contact with each peptide or combination thereof to determine the peptide(s) to which the protein binds. This allows for the determination of the peptide(s) containing the epitope to which the PAR4-binding protein binds. If the PAR4-binding protein binds to multiple discontinuous peptides, the PAR4-binding protein may bind to a structural epitope.

[0274] In one example, random fragments of PAR4 are expressed on the surface of phages, and the phages are brought into contact with a PAR4-binding protein. The antibody-bound phages are then isolated, and the amino acid sequence of the expressed peptide can be estimated from the coding nucleic acid contained within the phage. By isolating a series of phages with duplicate peptides, peptides containing the PAR4 region, including residues in the epitope, can be identified.

[0275] Alternatively or additionally, amino acid residues within PAR4 are mutated, for example, by alanine scanning mutagenesis, to determine mutations that reduce or prevent the binding of PAR4-binding proteins. Any mutations that reduce or prevent the binding of PAR4-binding proteins may be located within the epitope to which the PAR4-binding protein binds.

[0276] A further method involves binding PAR4 or its region to an immobilized PAR4-binding protein of the present disclosure, and digesting the resulting complex with a protease. The peptide, still bound to the immobilized PAR4-binding protein, is then isolated and its sequence is determined by analysis, for example, using mass spectrometry.

[0277] A further method involves converting hydrogen in PAR4 or its region to a deuterium nucleus (deutron), and then binding the resulting protein to an immobilized PAR4-binding protein of the disclosure. The deuterium nucleus is then converted back to hydrogen, the PAR4 or its region is isolated, digested enzymatically, and analyzed, for example, by mass spectrometry, to identify the region containing the deuterium nucleus. Such regions are thought to be protected from conversion to hydrogen by the binding of the PAR4-binding protein described herein.

[0278] In the above paragraph, the reference to PAR4 includes recombinant PAR4 and its extracellular domain.

[0279] affinity assay Optionally, the dissociation constant (Kd), association constant (Ka), or binding constant (KD, i.e., Ka / Kd) of a PAR4-binding protein to PAR4 or an epitope-containing peptide is measured. One example of how these constants for PAR4-binding proteins are measured is by an emitting or fluorescently labeled PAR4 binding assay. This assay equilibrates the PAR4-binding protein with a minimum concentration of labeled PAR4 in the presence of unlabeled PAR4 in a titration series. After washing to remove unbound PAR4, the amount of labeling is measured. In another example, the coefficients are measured by using a surface plasmon resonance assay, for example, by using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized PAR4 or a region thereof.

[0280] Protein detection assay One example of the present disclosure is the detection of the presence of PAR4 or cells expressing PAR4 (e.g., platelets). The quantity, level, or presence of the protein or cells is measured using any of the various techniques known to those skilled in the art, for example, using techniques selected from the group consisting of flow cytometry, immunohistochemistry, immunofluorescence, immunoblotting, Western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), electrospray ionization (ESI), tandem mass spectrometry (including mass spectrometry, LC MS / MS), biosensor techniques, evanescent fiber optic techniques, or protein chip techniques.

[0281] In one example, the assay used to measure the amount or level of protein is a semi-quantitative assay. In another example, the assay used to measure the amount or level of protein is a quantitative assay.

[0282] For example, proteins are detected by immunoassays, using assays selected from the group consisting of, for example, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), Western blotting, radioimmunoassay (RIA), biosensor assays, protein chip assays, and immunostaining assays (e.g., immunofluorescence).

[0283] Standard solid-phase ELISA or FLISA formats are particularly useful for measuring protein concentrations from a variety of samples.

[0284] In one form, ELISA or FLISA involves immobilizing the PAR4-binding protein of this disclosure or a protein that binds to different epitopes of PAR4 onto a solid matrix, for example, a membrane, polystyrene or polycarbonate microwells, polystyrene or polycarbonate dipsticks, or a glass support. A sample is then subjected to physical contact with the immobilized protein, and the PAR4 is bound or “captured.” The bound PAR4 is then detected using a second labeled compound that binds to different epitopes of PAR4. Alternatively, a third labeled antibody that binds to a second (detection) antibody may be used. It will be apparent to those skilled in the art that the assay forms described herein are applicable in high-throughput formats, such as automated screening processes or microarray formats. Furthermore, variations of the above assays, such as competitive ELISA, will also be apparent to those skilled in the art.

[0285] In alternative examples, polypeptides are detected intracellularly or on cells using methods known in the art, such as immunohistochemistry or immunofluorescence. Methods using immunofluorescence are exemplary because they are quantitative or at least semi-quantitative. Methods for quantifying the degree of fluorescence of stained cells are known in the art and are described, for example, in Cuello (1984).

[0286] Biosensor devices are generally integrated into the device with an assay substrate (e.g., as described in US5567301) using an electrode surface combined with a current or impedance measuring element. The PAR4-binding protein of this disclosure is incorporated onto the surface of the biosensor device, and a biological sample is brought into contact with the device. A change in current or impedance detected on the biosensor device indicates protein binding to the PAR4-binding protein. Some forms of biosensors known in the art also rely on surface plasmon resonance (SPR) to detect protein interactions, where a change in reflection on the surface plasmon resonance surface indicates a protein bound to a ligand or antibody (US5485277 and US5492840).

[0287] Biosensors are particularly useful in high-throughput analysis because such systems can be easily adapted to the micro or nanoscale. Furthermore, these systems are conveniently adapted to incorporate several detection reagents, enabling the redundancy of diagnostic reagents within a single biosensor unit. This allows for the simultaneous detection of several proteins or peptides in small amounts of bodily fluids.

[0288] The binding of proteins to PAR4 can also be detected using flow cytometry, as described in the examples herein.

[0289] Generation and selection of anti-PAR4 antibodies Alternative techniques for generating and selecting useful antibodies as described herein include in vitro exposure of lymphocytes to (e.g., PAR4 proteins or PAR4 peptides) and selection of antibody display libraries on phages or similar vectors (e.g., using immobilized or labeled PAR4 proteins or peptides). Genes encoding polypeptides with promising PAR4 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phages (phage display) or bacteria such as E. coli. Nucleotide sequences encoding polypeptides can be obtained in several ways, for example, by random mutagenesis and random polynucleotide synthesis. Such random peptide display libraries can be used to screen peptides that interact with known targets, which may be proteins or polypeptides (e.g., ligands or receptors), biological or synthetic macromolecules, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the field (Ladner et al., U.S. Patent No. 5,223,409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al., U.S. Patent No. 5,403,484; and Ladner et al., U.S. Patent No. 5,571,698), and random peptide display libraries and kits for screening such libraries are commercially available from, for example, Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the PAR4 sequences disclosed herein to identify proteins that bind to PAR4.Such "binding proteins" that interact with PAR4 polypeptides can be used for cell tagging, isolation of homologous polypeptides by affinity purification, and can be directly or indirectly complexed with drugs, toxins, radionucleotides, etc. Such binding proteins can also be used, for example, in analytical methods for screening expression libraries and neutralizing activity. Furthermore, binding proteins can be used in diagnostic assays to measure circulating polypeptide levels, or for detecting or quantifying soluble polypeptides as markers of underlying pathological conditions or diseases. Such binding proteins can also act as PAR4 "antagonists" to block PAR4 binding and signaling in vitro and in vivo. Such anti-PAR4 binding proteins are thought to be useful in inhibiting the cellular response to protease-activated PAR4.

[0290] Antibodies that specifically bind to PAR4 proteins or peptides can be detected using various assays known to those skilled in the art. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include simultaneous immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), blot or Western blot assays, inhibitory or competitive assays, and sandwich assays. In addition, antibodies can be screened for binding to wild-type vs. mutant PAR4 proteins, polypeptides, or fragments.

[0291] Assay of the functional characteristics of PAR4-binding proteins The antithrombotic activity of PAR4-binding proteins against all PAR4 variants can be validated by ex vivo platelet aggregation assays using blood from individuals identified as homozygous Ala120 or Thr120, or heterozygous, to confirm the efficacy of all variants.

[0292] The usefulness of PAR4-binding proteins as antithrombotic agents is similar to that of inhibitors of existing antiplatelet drug pathways (aspirin (50 μM), P2Y). 12 The antithrombotic effect can be verified by measuring the antithrombotic effect in the absence and presence of the inhibitor 2-MeSAMP (50 μM or 100 μM), and the PAR1 inhibitor borapaxal (100 mM) is also used in ex vivo platelet aggregation assays for comparative studies of its antithrombotic effect with that of PAR4-binding proteins. If the inventors indicate that thrombosis occurs independently of these mechanisms, high shear conditions (3000 s) can be used. -1 (This includes Neeves KB et al. (2008) J Thromb Haemost 6:2193-2201).

[0293] Additionally or alternatively, mouse in vivo thrombosis experiments (Lee H et al. (2012) Brit J Pharmacol 166:2188-2197; Mountford JK et al. (2015) Nat Commun 6:6535) may be used to validate the functionality of PAR4-binding proteins. To ensure the inclusion of a positive control for anti-PAR4 activity in such mouse experiments, the antithrombotic effect of the generated antibodies can be validated by screening using antigens corresponding to either mouse or human receptor sequences (Table 2) as outlined above. It should be noted that primates are the only species known to have platelets expressing only the combination of PAR1 and PAR4. Mouse platelets express PAR3 and PAR4, with only PAR4 being functional. Therefore, while such studies are limited to in vivo mechanism demonstration, they represent the most appropriate in vivo validation of the antithrombotic activity of PAR4-binding proteins outside of human trials and preclinical studies in non-human primates. Electrolyte injury to the carotid artery in anesthetized mice can be used to investigate the effects of PAR4-binding protein on in vivo thrombus formation and stability (Lee H et al. (2012) Brit J Pharmacol 166:2188-2197; Lee H et al. (2012) Thromb Haemost 107). Blood flow is recorded using a Doppler flow probe. Endpoints can be evaluated to assess thrombus formation (time to arterial occlusion) and stability (number and severity of recanalization events after occlusion), as well as total blood flow through the injured artery and thorough examination of thrombotic tissue features by Carstair staining of paraffin-embedded arterial sections.

[0294] PAR4 activation can be studied by measuring phosphoinositide hydrolysis after protease stimulation. The epitope-tagged PAR4 assay described herein can also be used to verify PAR4 cleavage and activation by PAR4-binding proteins.

[0295] Mammalian cells transfected with the PAR4 construct or PAR4 polymorphic variant (e.g., HEK293T cells) are a useful system for studying PAR4 antagonists. PAR4 transfected cells are used for screening ligands for the receptor, as well as for screening antagonists of native ligands. In summary, this approach involves combining the cDNA or receptor-encoding gene with other genetic elements necessary for its expression (e.g., transcription promoters), and inserting the resulting expression vector into host cells. Cells expressing the DNA and producing a functional receptor are selected and used in various screening systems.

[0296] Cells expressing functional PAR4 are used in screening assays. Various suitable assays are known in the art. Such assays are based on detecting the biological response in target cells. An increase in metabolism above the control value indicates a test compound that modulates PAR4 activity or response. One such assay is a cell proliferation assay. Cells are cultured in or without the test compound, and cell proliferation is detected, for example, by measuring the uptake of tritium-labeled thymidine or by a colorimetric assay based on the metabolic degradation of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65:55-63, 1983). Additional assay methods include measuring the effect of a test compound on receptor (+) cells that have the target receptor on their cell surface and receptor (-) cells that do not express the target receptor. Such cells can be manipulated to express a reporter gene. The reporter gene is linked to a promoter or response element that is responsive to the receptor ligation pathway, and the assay detects the transcriptional activity of the reporter gene. Suitable response elements include the cyclic AMP response element (CRE), hormone response element (HRE), insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci.USA 87:5273-77, 1990), and serum response element (SRE) (Shaw et al., Cell 56:563-72, 1989). The cyclic AMP response element is outlined in Roestler et al., J. Biol. Chem. 263(19):9063-66; 1988; and Habener, Molec. Endocrinol. 4(8):1087-94; 1990. The hormone response element is outlined in Beato, Cell 56:335-44;1989. In this regard, the preferred promoter element is the serum response element, i.e., SRE (see, e.g., Shaw et al., Cell 56:563-72, 1989).A preferred reporter gene of this type is the luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Luciferase gene expression is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activity assay kits are commercially available, for example, from Promega Corp. (Madison, Wis). This type of target cell line can be used to screen libraries of chemicals, cell conditioning media, fungal broths, soil samples, water samples, etc. This type of assay detects compounds that directly block PAR4 ligand binding and compounds that further block processes in the cellular pathway following receptor-ligand binding. In an alternative form, compounds or other samples can be tested for direct blocking of PAR4 binding using a portion tagged with a detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, etc.). Within this type of assay, the ability of the test sample to inhibit activated PAR4 is an indicator of inhibitory activity, which can be confirmed by a secondary assay. The ability of the test sample to stimulate PAR4 activity can also be measured and confirmed by a secondary assay.

[0297] Assay systems and commercially available biosensor devices (BIAcore, Pharmacia Biosensor, Piscataway, NJ) using ligand-binding receptors or antibodies or their binding fragments may be advantageously used. Such receptors, antibodies, or fragments are immobilized on the surface of a receptor chip. The use of this device is disclosed in Karlsson, J. Immunol. Methods 145:229-40, 1991; and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. The receptor, antibody, or fragment is covalently attached to dextran fibers attached to a gold film in a flow cell using amine or sulfhydryl chemistry. The test sample is passed through cells. If a ligand or epitope is present in the sample, the ligand or epitope binds to the immobilized receptor or antibody, respectively, causing a change in the refractive index of the medium, which is detected as a change in the surface plasmon resonance of the gold film. This system enables the measurement of on-velocity and off-velocity (from which bond affinity can be calculated) and the evaluation of bond stoichiometry.

[0298] Ligand-binding receptor polypeptides can also be used in other assay systems known in the art. Such systems include Statchard analysis for measuring binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and colorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0299] The FLIPR assay is an exemplary in vitro assay for measuring the activity of the PAR4 antagonist of the present invention. In this assay, intracellular calcium mobilization in PAR4-expressing cells is induced by the PAR4 agonist, and calcium mobilization is monitored.

[0300] The PAR4-binding proteins of this disclosure can be tested in vitro for their ability to inhibit gammathrombin-induced platelet aggregation. Gammathrombin is a proteolytic product of alphathrombin that no longer interacts with PAR1 and selectively cleaves and activates PAR4 (Soslau, G. et al, “Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein lb”, J. Biol. Chem., 276:21173-21183 (2001)). Platelet aggregation can be monitored in the form of a 96-well microplate aggregation assay or using a standard platelet agglutinator. The aggregation assay can be used to test the selectivity of compounds for inhibiting platelet aggregation induced by PAR4 agonist peptides, ADP, or the thromboxane analog U46619.

[0301] Another example is the alpha-thrombin-induced platelet aggregation assay, as shown in the examples herein. Alpha-thrombin activates both PAR1 and PAR4. The platelet aggregation inhibitory ability of selective PAR4 antagonists can be measured using a standard optical agglutinator.

[0302] Another example is the tissue factor-induced platelet aggregation assay. The conditions in this assay mimic the physiological events during thrombus formation. In this assay, platelet aggregation in human PRP is initiated by the addition of tissue factor and CaCl2. Tissue factor, an initiator of the extrinsic coagulation cascade, is very high in human atherosclerotic plaque. Exposure of blood to tissue factor in atherosclerotic sites activates robust thrombin production, inducing the formation of occlusive thrombi.

[0303] The efficacy of the PAR4-binding protein of the present invention in preventing thrombosis can also be measured by various in vivo assays. Exemplary mammals that can provide models of thrombosis and hemostasis for testing the efficacy of the PAR4 antagonist of the present invention as an antithrombotic agent include, but are not limited to, guinea pigs and primates. Applicable efficacy models include, but are not limited to, electrolyte injury carotid thrombosis, FeCl3-induced carotid thrombosis, and arteriovenous shunt thrombosis. Models for measuring kidney bleeding time, renal bleeding time, and other bleeding times can be used to assess bleeding risk.

[0304] PAR4-binding proteins can be tested in an in vivo model of arterial thrombosis in cynomolgus monkeys. In this model, PAR4-binding proteins can be tested for their ability to inhibit thrombus formation induced by electrolyte damage in the carotid artery.

[0305] Platelet aggregation assay Platelet aggregation can be measured using microplate-based platelet light transmission aggregation assay (French et al (2016) Journal of Thrombosis and Haemostasis 14:1642-1654).

[0306] This test (Born GV (1962) Nature 194:927-929) evaluates in vitro interplatelet clamp formation in a glycoprotein (GP) IIb / IIIa-dependent manner, i.e., aggregation, the most important function of platelets. The assay is based on measuring the increase in light transmittance through optically dense samples of platelet-rich plasma (PRP) or washed platelets after the addition of an exogenous platelet agonist. During the assay, the PRP or washed platelet preparation becomes clearer after agonist addition due to the precipitation of platelet aggregates. This determines the increase in light transmittance through the plasma sample. The device records this percentage and maximum percentage of increase using a photometer from 0% (maximum optical density of PRP or washed platelets) to 100% (no optical density of autologous platelet-poor plasma or Tyrode's buffer, respectively). This signal is automatically converted into a graph curve corresponding to the increase in light transmittance during platelet aggregation. The available agglutinators are user-friendly devices with automatic settings (100% and 0%), software for storing results, and disposable cuvettes with stirring rods. Curve slope, maximum degree of agglutination (%), and latency (induction period) are automatically measured parameters, allowing for a graphical visualization of changes in shape and primary and secondary agglutination. Different agonists are added to PRP or washed platelet samples to stimulate different platelet activation pathways and obtain information about several characteristics of platelet function. Born's platelet agglutination assay is the most widely used methodology for detecting platelet dysfunction and monitoring antiplatelet therapy.

[0307] In vivo analysis of platelet function after administration of PAR4-binding protein can be measured using bleeding time (BT) (Duke WW et al (1910) JAMA 55:1185-1192). BT evaluates platelet hemostatic ability by recording the time required for platelets to occlude an in vivo skin wound to stop bleeding.

[0308] Impedance whole blood agglutination assay (WBA) allows for the evaluation of platelet function without any sample preparation by using anticoagulated whole blood (WB) as the environment (Mackie IJ, et al. (1984) J Clin Pathol. 37:874-878). This assay is based on the principle that activated platelets adhere via surface receptors to the artificial surfaces of two electrodes positioned at a predetermined distance from each other within the WB sample. Platelet aggregation is evaluated by detecting the increase in electrical impedance generated by the aggregation of other platelets on platelets fixed to the electrodes. Therefore, reducing the current intensity increases the electrical impedance. The degree of impedance increase is recorded in ohms.

[0309] Lumiaggregometry enables the simultaneous measurement of adenine nucleotide release from platelet granules and platelet aggregation (Holmsen H, et al. (1966) Anal Biochem. 17:456-47). This method is based on the evaluation of adenosine triphosphate (ATP) released from platelets activated by different agonists using luminescence techniques in platelets (PRP), washed platelets (WP), or Western blotting (WB). The assay is based on the conversion of ADP released from high-density platelet granules to ATP through reaction with a luciferin-luciferase reagent. The emitted light is proportional to the ATP concentration and is quantified by a luminogram.

[0310] Further platelet function studies are outlined in Paniccia R et al (2015) Vasc Health Risk Manag. 11:133-148.

[0311] Calcium signaling assay Calcium flow can be measured in isolated platelets by a dual-dye ratiometric microimaging assay (Nesbitt WS et al. (2012) Methods Mol Biol 788:73-89).

[0312] Animal models In vivo animal models of thrombosis are available to those skilled in the art for further or additional screening, evaluation, and / or confirmation of the antibodies or fragments thereof, including further in vivo evaluation of PAR4 activation or antithrombotic effects. Such animal models include, but are not limited to, models of carotid artery electrolyte injury, where thrombus formation (time to arterial occlusion), stability (number and extent of recanalization events after occlusion), and total blood flow through the injured artery.

[0313] The exemplary or preferred mouse model is the PAR4- / - mouse (Sambrano GR et al. (2001) Nature 2000 407:258-64; Mao Y et al. (2010) J Cereb Blood Flow Metab. 30(5):1044-1052).

[0314] Pharmaceutical composition The PAR4-binding protein (synonymous with the active ingredient) of this disclosure is useful for formulation into pharmaceutical compositions for parenteral, topical, oral, or localized administration, aerosol administration, or transdermal administration for prophylactic or therapeutic treatment. The pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. For example, suitable unit dosage forms for oral administration include powders, tablets, pills, capsules, and lozenges.

[0315] The pharmaceutical compositions of this disclosure are useful for parenteral administration, such as intravenous or subcutaneous administration.

[0316] The administration composition will generally consist of a solution of the PAR4-binding protein of this disclosure dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, can be used. The composition may also contain pharmaceutically acceptable carriers, such as pH adjusters and buffers, to approximate physiological conditions as needed, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the PAR4-binding protein of this disclosure in such formulations can vary widely and is selected mainly based on liquid volume, viscosity, body weight, etc., depending on the specific administration mode chosen and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Non-aqueous solvents such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The solvent may contain small amounts of additives, such as buffers and preservatives, to enhance isotonicity and chemical stability.

[0317] The PAR4-binding proteins of this disclosure can be formulated for parenteral administration, for example, for infusion via intravenous, intramuscular, subcutaneous, transdermal, or other such routes (including peristaltic administration into tumors or disease sites and direct intracavitary infusion). The preparation of aqueous compositions containing the compounds of this disclosure as active ingredients will be known to those skilled in the art.

[0318] Preferred pharmaceutical compositions according to this disclosure generally include a mixture of a certain amount of the PAR4-binding protein of this disclosure with a pharmaceutical carrier (e.g., a sterile aqueous solution) to yield a range of final concentrations depending on the intended use. Techniques for preparation are widely known in the art, as exemplified in Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company, 1980.

[0319] During formulation, the compounds of this disclosure are administered in a manner suitable for formulation of drug doses and in amounts that are therapeutically / prophylactically effective. The preferred drug dose of the compounds of this disclosure varies depending on the specific compound, the condition being treated, and / or the subject being treated. For example, determining a preferred drug dose by starting with a dose below the optimal dose and increasing the dose in order to determine the optimal or useful dose is within the scope of the skill of those skilled in the art.

[0320] Exemplary drug doses and timings of administration will be apparent to those skilled in the art based on the disclosure herein. The preferred dose of a PAR4 antagonist is the bioactive dose, which is a dose that inhibits PAR4 cleavage and / or signaling and has an antithrombotic effect. Preferably, the PAR4 antagonist has the ability to reduce PAR4 activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% from the level of an untreated control. The level of PAR4 in platelets is measured by any method known in the art, including, for example, receptor binding assays, platelet aggregation, platelet activation assays (e.g., p-selectin expression by FACS), Western blotting, or ELISA analysis. Alternatively, the bioactivity of PAR4 is measured by evaluating intracellular signaling induced by PAR4 (e.g., calcium mobilization or other secondary messenger assays).

[0321] In some examples, the therapeutically effective dose of the PAR4 compound is preferably less than about 100 mg / kg, 50 mg / kg, 10 mg / kg, 5 mg / kg, 1 mg / kg, or less than 1 mg / kg. In a more preferred embodiment, the therapeutically effective dose of the PAR4 compound is less than 5 mg / kg. In the most preferred embodiment, the therapeutically effective dose of the PAR4 compound is less than 1 mg / kg. The effective dose varies depending on the route of administration and the use of excipients, as will be recognized by those skilled in the art.

[0322] In some cases, liposomes and / or nanoparticles may also be used in conjunction with PAR4-binding proteins. The formation and use of liposomes are widely known to those skilled in the art. Liposomes can be formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs can generally have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small single-layer vesicles (SUVs) with a diameter in the range of 200 to 500 angstroms and containing an aqueous solution in the center. When dispersed in water, phospholipids can form various structures other than liposomes depending on the molar ratio of lipids to water. Liposomes at low ratios are a preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar substances, but at high temperatures, their permeability changes significantly through a phase transition. Phase transitions involve a change from a densely packed, ordered structure known as a gel state to a loosely packed, less ordered structure known as a fluid state.

[0323] The composition may be administered alone, or in combination with other treatments, therapeutics, or drugs, either simultaneously or sequentially. Other treatments, therapeutics, or drugs include, but are not limited to, the following: (i) Anticoagulants, such as Fxa inhibitors, FXIa inhibitors like apixaban or rivaroxaban, or thrombin inhibitors like dabigatran; (ii) Antiplatelet agents, such as aspirin or P2Y12 antagonists (e.g., clopidogrel, ticagrelol, or prasugrel); (iii) Angioplasting agents, such as angiogenesis inhibitors.

[0324] Treatment method As discussed herein, the PAR4-binding proteins of this disclosure can be used to treat, prevent, or improve thrombosis or thromboembolic disorders in subjects.

[0325] Thrombosis refers to the formation or presence of blood clots (thrombus; plural: thrombi) within blood vessels that can cause ischemia or infarction of the tissue supplied by those blood vessels.

[0326] Thromboembolic disorders are characterized by the sudden blockage of an artery by a blood clot (e.g., embolus formation) or foreign body carried by the bloodstream to a site of accumulation. "Thromboembolism" refers to the occlusion of a blood vessel by thrombotic material carried by the bloodstream from a site of origin that blocks another blood vessel. The term "thromboembolic disorder" encompasses both "thrombotic" and "embolic" disorders (as defined above).

[0327] Thromboembolic disorders include arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the ventricular or peripheral circulation. As used herein, the term “thromboembolic disorder” also includes, but is not limited to, certain disorders selected from, unstable angina or other acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, sudden ischemic death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary thrombosis, cerebral arterial thrombosis, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. Medical implants or devices include, but are not limited to, prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygen supply devices, shunts, vascular access ports, ventricular assist devices and artificial hearts or ventricles, and vascular grafts. Procedures include, but are not limited to, cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis. In another embodiment, the term “thromboembolic disorder” includes acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism.

[0328] As used herein, the term “stroke” refers to an embolic stroke or atherothrombotic stroke resulting from occlusive thrombosis in the common carotid communis, internal carotid arteries, or intracerebral arteries.

[0329] kit This disclosure also provides therapeutic / preventive / diagnostic kits containing the compounds of this disclosure for use in the detection / diagnosis / prognosis / treatment / prevention methods of the present invention. Such kits generally contain the PAR4-binding proteins of this disclosure in a suitable containment means. The kits may also contain other compounds, for example, for detection / isolation / diagnosis / imaging or combination therapy. For example, such kits may contain one or more of a range of anticoagulants or antiplatelet agents.

[0330] In one example, the kit is for the treatment or prevention of a condition. In such a kit, the PAR4-binding protein may be provided in solution or lyophilized form, optionally with a solution for resuspension. The PAR4-binding protein may be conjugated with a therapeutic compound, or the kit may contain a therapeutic compound for conjugation.

[0331] Those skilled in the art will understand that numerous variations and / or modifications can be applied to the present invention, as shown in the specific embodiments, without departing from the broadly described scope of the invention. Therefore, the embodiments of the present invention should be considered in all respects as illustrative and non-limiting.

[0332] The following specific embodiments should be interpreted as illustrative only and should not be construed as limiting the remainder of this disclosure in any way. Those skilled in the art will likely be able to make the most of the present invention based on the above description without further detail.

[0333] The present invention is further illustrated by the following non-limiting embodiments. [Examples]

[0334] method Antibody production HumAb mice (Regeneron Pharmaceuticals) were immunized with the C-terminal KLH (keyhole limpet hemocyanin) conjugated peptides listed in Table 2 below to produce anti-PAR4 monoclonal antibodies. The immunized peptides correspond to the N-terminal thrombin cleavage and activation sites of hPAR4 (SEQ ID NO: 2). The cleavage sites are indicated by RG and are underlined in the following human PAR4 sequences. Screening was performed on JPEG0007895907000006.jpg1475, a naked peptide (SEQ ID NO: 3).

[0335] Mice were immunized intraperitoneally three times at two-week intervals with a combination of 16 μg of antigen and an immunoadjuvant (Sigma Aldrich catalog number S6322) containing methylated CpG. Serum samples were collected from the immunized mice, and their reactivity to the antigen was tested by ELISA at 1:250 and 1:1250 dilutions and compared with samples from before immunization. A difference of more than a threefold increase in serum titer between pre-immunization and post-immunization dilutions was observed at 1:250 and 1:1250 dilutions.

[0336] The mouse with the highest titer was selected for fusion.

[0337] Immunotherapy peptides Immunotherapy peptides and SKB-labeled peptides (shown in Table 2) were synthesized using solid-phase synthesis at Auspep (Melbourne, Australia).

[0338] Biotin was attached to the peptide at its C-terminus using serine (S) and lysine (K) linkers (SK) (see Table 2). By chemically conjugating biotin to the C-terminal lysine residue, SKBs as shown in Table 2 were generated.

[0339] Mice were immunized with a human PAR4 keyhole limpet hemocyanin (KLH) peptide containing the sequence GDDSTPSILPAPRGYPGQVC-KLH.

[0340] Hybridoma proliferation To generate hybridoma cells, mouse spleens were removed, isolated, and prepared as single-cell suspensions. These suspensions were then fused with Sp2 / 0-Ag14 myeloma cells using polyethylene glycol. The resulting hybridoma cells were grown in azaserine hypoxanthine-containing medium in 20 × 96 well tissue culture plates.

[0341] Hybridoma colonies were grown for 10 days, and the number of hybridoma colonies at that time (expressed as fusion efficiency) was measured. After a further 3 days of incubation, aliquots of the antibody supernatant were collected for screening. The reactivity of the supernatant to antigens and any screening samples was assayed first by microarray, and then by ELISA of any IgG-positive clone.

[0342] Next, the most responsive ELISA-positive clones were grown in 24-well tissue culture plates for 3–4 days, at which point they were grown in 6-well tissue culture plates. Cells were seeded in a ratio of 1:5 (supernatant wells) and 1:25 (cell wells). Once the cell wells reached 80% confluence, the cells were extracted and frozen in liquid nitrogen in 10% DMSO, and the supernatant from the supernatant wells was pooled and frozen at -20°C.

[0343] Clones selected for subcloning were subjected to at least two rounds of serial dilution. After each dilution stage, cells were grown for 4–5 days, and single colonies producing antibodies positive for the antigen were identified by supernatant ELISA. The top-performing clones were then grown for further rounds. The final monoclonal cell lines were grown in 6-well tissue culture plates for 4–5 days, the supernatant was extracted, and frozen with the cells.

[0344] The supernatant of subclonal cell lines was tested using a commercially available assay kit to determine the isotype of the monoclonal antibody produced.

[0345] Microarray assay Screening of hybridoma supernatants using microarrays was performed according to standard techniques.

[0346] ELISA screening of monoclonal antibodies A 96-well ELISA plate was coated with 50 μl of antigen diluted to a concentration of 4 μg / ml in coating buffer (0.1 M sodium bicarbonate (NaHCO3) (Merck, #1.06329.0500)). The plate was incubated overnight at 4°C.

[0347] Next, the wells were washed in an automated plate washer (300 μl, 1×PBS, 3 times). The wells were blocked in 200 μl of blocking buffer (3% BSA / 1×PBS (BSA:(Sigma,#1001647742)) at room temperature for 1 hour, and then washed as described above.

[0348] 50 μl of undiluted hybridoma cell culture supernatant was added to the appropriate wells and incubated at room temperature for 1 hour, then washed as described above. The secondary antibody (alkaline phosphatase-conjugated AffiniPure goat anti-mouse IgG (H+L)) (Jackson ImmunoResearch Laboratories, Inc. #115-055-003)) was diluted 1:1000 in 1×PBS (8% sodium chloride (NaCl, Merck #1.06404.5000), 0.2% potassium chloride (KCl, Merck #1.04936.0500), 1.44% disodium hydrogen phosphate (Na2HPO4, Merck #1.06586.0500), 0.24% potassium dihydrogen orthophosphate (KH2PO4, Merck #1.04873.0500)), and 50 μl of the diluted antibody was added to each well and incubated at room temperature for 1 hour. The wells were washed as described above. A set of substrate tablets (1× silver, 1× gold) was added to 20 ml of ddH2O by vigorously shaking on a thermomixer at room temperature for 6 minutes. 50 μl of substrate (SIGMAFAST™ p-nitrophenyl phosphate (Sigma-Aldrich, N2770-50SET) tablets) was added to each well and incubated at room temperature for 20-25 minutes. 50 μl of stop solution (2 M sodium hydroxide, solid (NaOH) (Merck, Cas#1310-73-2)) was added to each well to stop the reaction. Absorbance was read at 405 nm using an ELISA reader "RdrOle4" immediately after adding the stop solution.

[0349] Human blood samples Blood was collected from healthy adults (men and women aged 21-50) who had not taken antiplatelet drugs for the past 10 years, after obtaining informed consent. Blood was collected from the precubital vein using a 19-gauge butterfly needle. For platelet isolation, it was collected in a syringe containing 1 / 7 volume of acidic dextrose citrate (ACD) (7:1 v / v final concentration), or for whole blood flow experiments as previously described (Mountford JK et al (2015) Nat Commun 6:6535), it was collected in a syringe containing 1 / 10 volume of trisodium citrate (0.32% w / v final concentration).

[0350] mouse HumAb mice (Murphy AJ et al. (2014) Proc Natl Acad Sci USA 111:5153-5158) were obtained from Regeneron Pharmaceuticals. HumAb mice are genetically engineered to generate human antibody responses by replacing the 3Mb segments of the mouse heavy variable Ig locus and κ light variable Ig locus with human counterparts (Murphy AJ et al. (2014) Proc Natl Acad Sci USA 111:5153-5158). HumAb mice exhibit normal variable segment rearrangement, somatic hypermutation, and class switching, and demonstrate robust humoral responses that result in a wide variety of monoclonal antibodies, making them a platform for producing fully human monoclonal antibodies against a range of targets used by Regeneron.

[0351] Detection of PAR4 by flow cytometry Washed human or mouse platelets (5 x 10) 7 The suspension (0.1 mg / ml) was incubated with anti-PAR4 antibody (0.1 mg / ml) at 37°C for 30 minutes, and then fixed with paraformaldehyde (1% v / v final concentration). The suspension was then centrifuged at 1000 × g for 2 minutes to obtain a platelet pellet, which was then resuspended in modified Tyrode's buffer (12 mM NaHCO3, 10 mM HEPES pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5.5 mM D-glucose, 1 mM CaCl2) containing a 1:50 dilution of FITC-conjugated anti-rabbit IgG. After 30 minutes at room temperature, the sample was centrifuged again, and the platelet pellet was resuspended in modified Tyrode's buffer and analyzed using a flow cytometer (FACSCalibur, BD Biosciences).

[0352] PAR4 thrombin cleavage assay 1x10 6HEK293T cells (in Dulbecco's modified Eagle medium + 10% fetal bovine serum) were seeded in 12-well plates 24 hours before translocation. Once confluent, 1 μg of DNA from one of the PAR4 variants (pBJ-FLAG-PAR4-120A-296F or pBJ-FLAG-PAR4-120T-296F; Edelstein et al (2014) Blood 124(23):3450-8) in addition to 4 μL of Lipofectamine 2000 was translocated according to the manufacturer's instructions. 48 hours after translocation, cells were harvested, washed twice with PBS, and then resuspended to 1 × 10⁶ cells. 6 The count was set to / mL. Next, cells (50μL assay / 0.5x10 5 Cells (conditions) were pre-treated at 37°C for 15 minutes with either 1, 10, or 100 μg / ml of 5RC3 or subclone 5A,RC3,F10b,H4b, or 100 μg / ml of a matched isotype control (mouse IgG1). Cells were then stimulated with 2 U / mL thrombin for 10 minutes. The reaction was stopped by adding 4 U / mL of hirudin. Cells were then washed once, resuspended in PBS containing a 1:200 dilution of FITC anti-FLAG antibody (Sigma, clone M2), and incubated in the dark at room temperature for 1 hour. Cells were then fixed with 1% paraformaldehyde (final concentration) and read using a FACSCalibur flow cytometer to measure the percentage of FC1 / FITC positive events. Data were normalized to resting samples (100% untreated with thrombin).

[0353] Surface plasmon resonance (SPR) assay Surface plasmon resonance (SPR) is a biosensor technology that enables real-time measurement of protein-protein interactions without labeling. SPR binding analysis of proteins and antibodies was performed using standard techniques with the Bio-Rad ProteOn XPR36 array system or the Biacore T200 (GE system).

[0354] Method for measuring the dynamics of 5A.RC3 ProteOn is an SPR biosensor with a multi-channel module and interaction array sensor chip for analyzing up to 36 protein interactions in a single injection step. The analysis was performed using the ProteOn NLC biosensor chip, which includes a surface made of NeutrAvidin bound to an alginate polymer for the capture of biotinylated proteins and peptides.

[0355] The NLC tip was prepared with 50 mM NaOH, and then with 1 M NaCl. Both preparations were performed at a flow rate of 30 μl / min. Preparation was carried out in both horizontal and vertical directions (channels). A biotinylated peptide (ligand sample) at a concentration of 25 μg / ml was captured on the tip in the vertical channel at a flow rate of 30 μl / min (see Table 1 for the ligand sample set up on the biosensor tip). To ensure stable capture of the peptide ligand before injection of the analyte, running buffer (1 × PBS, 0.005% Tween®, pH 7.4) was injected into the entire vertical channel. The tip was then rotated horizontally, and a dilution series of mAb5ARC3.F10b.H4b (samples) was injected into the entire channels A1-A6 at a flow rate of 100 μl / min (see Table 1 for the concentrations of the tested analytes). Note: The mAb concentration of 6.25 nM was excluded from the final analysis because it showed a higher value than the usual reading.

[0356] [Table 2]

[0357] Method for measuring the kinetics of purified hPAR4 mAb with respect to hPAR4 Biacore analysis was performed on the Biacore T200 using one of two different methods: an anti-mouse Fc capture approach, or a similar method using the ProteoOn system.

[0358] Antibody capture method Series S CM5 sensor chips were activated by standard EDC / NHS amine coupling chemistry using the manufacturer's recommended protocol with goat anti-mouse IgG Fc. Anti-hPAR4 mAb was captured at 1–2 ug / ml for 2 minutes on flow cell 2 (FC2) at 10 ul / m min, with flow cell 1 (FC1) used as the reference channel. Dilutions of hPAR4 peptide were prepared in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween® 20, and 0.1% BSA) at typically 1000, 500, 250, 125, and 62.5 nM and flowed through both FC1 and FC2 at 30 ul / m for 60–100 seconds, with dissociation monitored for 80–120 seconds. Blank injections of buffer alone were also performed. All injections were performed in 2-way and random order. After each capture / injection / dissociation cycle, the tip was regenerated by injecting 0.1M glycine pH 2.0 for 30 seconds.

[0359] Streptoavidin capture method Series S sensor tips SA were prepared using the manufacturer's recommended protocol and immobilized with 1 ug / ml hPAR4-biotinylated peptide on FC2 and hPAR1-biotinylated peptide (control) on FC1. Dilutions of anti-hPAR4 were prepared in running buffer at typically 10, 5, 2.5, 1.25, 6.25, and 0 nM and flowed through both FC1 and FC2 at a flow rate of 100 ul / min for 60 seconds, with dissociation monitored for 200 seconds. Blank injections of buffer alone were also performed. All injections were performed in pairs and in a random order. After each capture / injection / dissociation cycle, the tips were regenerated by injecting 0.1 M glycine pH 2.0 for 30 seconds.

[0360] Platelet aggregation assay Platelet aggregation was measured using light transmission agglutination assay in a 96-well plate format. (2 × 10⁶ human isolated platelets) 8Platelets were pre-treated at 37°C for 10 minutes with dimethyl sulfoxide (DMSO) (1% v / v), PAR1 antagonist borapaxal (90 nM), anti-PAR4 antibody 5RC3 (20-100 μg / ml), or a combination of borapaxal and 5RC3. Platelets were treated with thrombin (0.1 U / ml), and aggregation was analyzed at 37°C for 50 minutes using a 595 nm excitation filter in a FLUOstar OPTIMA plate reader (BMG Labtech) (10 reading cycles with a 5-minute dual-orbit shaking period between each reading). Optical density was normalized to the blank (maximum value) and unstimulated platelets (minimum value) and expressed as the maximum value %.

[0361] Whole blood thrombosis assay Human whole blood collected in citrate (3.2%) was pre-incubated at 37°C for 15 minutes with PE-conjugated anti-CD9 antibody (4 μg / mL) and anti-fibrin antibody (5 μg / mL), as well as one of the following: hirudin (800 U / mL), DMSO (1% v / v), PAR-1 antagonist E5555 (1 μM), anti-PAR4 antibody (0.2 mg / mL), or a combination of both PAR inhibitors. Whole blood was remineralized with 5–7.5 mM CaCl2 (final concentration) to initiate coagulation, and then transferred to glass microslides (1 × 0.1 mm inner diameter) coated with bovine type 1 collagen (250 μg / mL) at a fixed flow rate of 0.06 ml / min for 600 s. -1 The wall shear velocity was obtained. Two-color confocal fluorescence images were recorded with excitations at 488 and 561 nm and acquired through a 40× water immersion objective lens. The confocal z-stack was continuously recorded for 2 minutes before correction, and calcium-free Tyrode's buffer was flowed over the thrombus, recording the z-stack encompassing the entire thrombus field for a period of 10 minutes. Platelet thrombi were defined using anti-CD9-PE, and fibrin volume was quantified using the mean fluorescence of the thrombus field. Data were normalized to hirudin baseline and expressed as a percentage of the control.

[0362] PCR-based SNP genotyping assay for PAR4 SNPs Genomic DNA (gDNA) was extracted from the buffy coat of human whole blood using the QIAamp Blood Kit-Mini according to the manufacturer's instructions. The Taqman SNP genotyping assay (Life Technologies, Carlsbad, CA, USA) was used according to the manufacturer's instructions, and 10 ng of DNA was used to determine the rs773902 genotype of the DNA sample. PCR was performed on a Roche 96-well plate lightcycler under the following thermal cycling conditions: 15 seconds at 95°C, followed by 60 seconds at 60°C for 40 cycles. Endpoint genotyping analysis using Mygo Pro software was used for allele differentiation. Ratiometric analysis of relative fluorescence signals accumulated at 465-510 nm ("A" allele) / 533-580 nm ("T" allele) was used.

[0363] statistical analysis Statistical analysis was performed using GRAPHPAD PRISM (version 6.0, La Jolla, CA, USA). Significance was defined as P<0.05, determined by either an unpaired two-tailed Student's t-test or a one-way ANOVA with Fisher's LSD test for multiple comparisons.

[0364] Antibody Nomenclature Unless otherwise indicated, the following nomenclature is used to refer to the antibodies mentioned herein, as shown in Table 2 below. For example, a brief reference to 5A.RC3 refers to the monoclonal antibody subclone 5A.RC3.F10b.H4b, unless otherwise indicated.

[0365] [Table 3]

[0366] [Example 1: Development of an antagonist monoclonal antibody against human PAR4] The inventors worked to develop a monoclonal antibody that targets human PAR4. At the Monash Antibody Technology Facility (MATF), Professor Mark Sleeman, the facility's supervisor, performed antibody production and screening using Regeneron Pharmaceuticals' proprietary HuMab mouse (VelocImmune®), and produced a high-affinity antibody against PAR4, as will be described in further detail later.

[0367] (i) Immunization Human PAR4 (hPAR4) was cleaved at its N-terminal thrombin site, and a KLH-conjugated peptide was generated and used to immunize HumAb mice. Three additional immunizations were then performed according to a standard protocol. Serum titers were measured by ELISA using the naked hPAR4 peptide (Table 3).

[0368] The peptide was used to administer three separate immunization programs to a total of 12 HumAb mice. The sequences of the peptides used for immunization are shown in Table 3. These sequences included additional C-terminal cysteine ​​in some cases (as indicated by the underlined portion). The peptides were conjugated with keyhole limpet hemocyanin (KLH) at the C-terminus or with biotin (SKB) via cysteine-lysine.

[0369] [Table 4]

[0370] (ii) Formation of hybridomas Fusion was performed using a standard fusion protocol with SP2 / O Ag14 as the fusion partner (Yokoyama, W. Production of monoclonal antibodies. In: Coligan J, Kruisbeek A, Marguiles D, Shevach EM, Strober W., editors. Current Protocols in Immunology. Vol.1. New York, NY: John Wiley & Sons; 1994. pp.2.5.2-2.5.17.), and the cells were cultured in 20 96-well plates. The cells were grown in a medium containing HAT as a selection marker.

[0371] Hybridomas were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 μM beta-mercaptoethanol, and 1 mM sodium pyruvate.

[0372] (iii) Screening of hybridoma clones The inventors screened thousands of hybridoma supernatants for high-affinity specific antigen-positive strains. The monoclonal antibodies contain a human Ig variable region linked to a mouse constant region (and are therefore referred to as "mAb" in this specification to refer to such chimeric antibodies).

[0373] First, binding to the immunized PAR4 peptide sequence (GDDSTPSILPAPRGYPGQVC-KLH) was screened in the hybridoma supernatant using an antigen microarray. Binding was measured by fluorescence intensity above the background (signal from the medium alone). The top 46 clones per spleen fusion were selected for further ELISA-based screening of binding to PAR4 antigens (both native and KLH-linked; binding multiplier of native vs. KLH-linked PAR4 peptide). Clones showing more than 3 times binding to the native PAR4 peptide compared to the KLH-linked PAR4 peptide were selected for similar ELISA-based specificity screening (binding to the native PAR4 peptide vs. binding to the PAR1 peptide (SKATNATLDPRSFLLRNP), PAR2 peptide (SCSGTIQGTNRSSKGRSL), and PAR3 peptide (SCSGTIQGTNRSSKGRSL) corresponding to the equivalent region; binding multiplier of PAR4 peptide vs. PAR1, 2, or 3 peptide). Clones that showed binding to PAR4 peptides more than three times more than other PAR peptides were grown and tested using functional bioassays (inhibition of PAR4 cleavage and inhibition of PAR4-induced platelet activation / aggregation) (Figures 2 and 12).

[0374] Clones that demonstrated both binding and function were selected for subcloning and retesting. After each round of subcloning, the binding of the clone to the native PAR4 peptide (compared to the medium alone) was again tested by ELISA to confirm the retention of the binding antibody within the clone.

[0375] [Table 5] JPEG0007895907000011.jpg237151JPEG0007895907000012.jpg25249

[0376] [Table 6] JPEG0007895907000014.jpg221120

[0377] [Example 2: Anti-PAR4 hybridoma clone blocks thrombin-induced cleavage of PAR4] We screened monoclonal antibody hybridoma supernatants (MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BRH3, and MoB5CRC4) for their ability to cleave intact PAR4 present on the surface of HEK293 cells transfused with human PAR4 containing an N-terminal FLAG tag. Cleavage was quantified by flow cytometry and is shown in Figure 2.

[0378] Transplanted HEK293 cells were incubated for 10 minutes at room temperature in the presence of thrombin (0.1 U / ml) with either an anti-PAR4 polyclonal antibody (described in French SL et al. (2016) J Thromb Haemost 14, 1642-1654, used as a positive control) or a monoclonal antibody clone MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BRH3, or MoB5CRC4, along with an untreated negative control. Thrombin-induced PAR4 cleavage was measured by flow cytometry, specifically by the loss of the FLAG tag from PAR4-expressing HEK293 T cells.

[0379] Cell treatment with thrombin (2 U / ml for 10 minutes) resulted in cleavage of approximately 50% of total PAR4 (negative control). Pretreatment with polyclonal anti-PAR4 antibody was found to almost completely block thrombin-induced cleavage (positive control) (Figure 2). Initial screening of the supernatants of five hybridomas showed that MoB5ARC3 almost completely blocked thrombin-induced cleavage of PAR4, while supernatants from the other four hybridomas (B5A.RC3, B5.BRB4, B5.BRC6, B5.BRH3, and B5.CRC4) showed limited and variable responses.

[0380] Clone B5A.RC3 blocked at least 90% of thrombin-induced cleavage of PAR4. Clone B5.BRB4 blocked approximately 60% of thrombin-induced cleavage, B5BRC6 blocked approximately 50%, B5BRH3 blocked approximately 65%, and B5CRC4 blocked approximately 50%.

[0381] Clone 5A.RC3 was further subcloned by limiting dilution according to a standard protocol, and its ability to block thrombin-induced cleavage of PAR4 was tested by flow cytometry, as shown in Figure 3. Both hybridoma supernatants from 5A.RC3 (subclone H4b) and 5A.RC3 (subclone B6b) blocked thrombin-induced cleavage of PAR4 on HEK293T cells (100 μl cell suspension) to a remarkable degree of over 90% in 100 μl supernatant.

[0382] Figure 4 shows that the 5A.RC3.H4b subclone effectively inhibited the cleavage of PAR4 expressed on the surface of HEK293 cells in a dose-dependent manner, and that this cleavage was effective against both the Ala120 and Thr120 variants of the human PAR4 receptor.

[0383] [Example 3: Binding specificity of H4b and B6b subclones of mAb-5RC3] To measure the specificity of anti-hPAR clones for PAR4, ELISA screening was performed as described above. Figure 5A shows the results of the binding specificity of clones mAb-5ARC3 (5A.RC3) and mAb-5BRB4 (5B.RB4) for hPAR1, hPAR2, hPAR3, and hPAR4.

[0384] 5A.RC3 showed 16-fold selectivity for human PAR4 peptides compared to PAR1, PAR2, and PAR3. mAb 5B.RB4 bound similarly to all four human PAR peptides, but its affinity for PAR4 was lower than that of 5A.RC3.

[0385] The inventors performed surface plasmon resonance (SPR) analysis using Bio-Rad Proteon XPR36 to gain insights into the binding affinity (rates of association and dissociation) and specificity of 5A.RC3.F10b.H4b. All biotin-bound human PAR peptides (Table 1) were captured on the surface using a streptavidin chip and passed through different concentrations of purified 5A.RC3. Clones 5A.RC3.F10b.H4b were observed to have a dissociation constant (KD) of approximately 0.4 nM by SA chip SPR (Figure 5B).

[0386] [Example 4: The anti-PAR4 hybridoma mAb-5RC3.F10b.H4b (hereinafter 5A.RC3) blocks thrombin-induced cleavage of both human PAR4 variants.] One limitation of prior art PAR4 inhibitors is that they are specific to particular variants of PAR4, and therefore can only successfully inhibit platelet aggregation in individuals possessing the relevant PAR4 receptor variant.

[0387] To determine whether anti-PAR4 clones can inhibit the effect of thrombin on PAR4 cleavage, an in vitro inhibition assay was performed. HEK293T cells were transiently transfused with PAR4-120Ala(A) or PAR4-120Thr(T) variants containing a FLAG epitope upstream of the thrombin cleavage site. Thrombin-mediated PAR4 cleavage was measured by flow cytometry, specifically by the loss of the Flag tag from PAR4-expressing HEK293 cells.

[0388] Cells were stimulated with gradually increasing doses of thrombin (0.1–2 U / ml), and the amount of thrombin cleavage was measured as loss of the FLAG epitope by flow cytometry using a FITC-conjugated anti-FLAG antibody. As shown in Figure 6A, the inhibition was dose-dependent.

[0389] Figure 6B shows that pre-incubating the transmissible cells with 5A.RC3 (100 μg / ml) before thrombin stimulation resulted in nearly complete thrombin cleavage inhibition to the same degree, regardless of the PAR4 variant.

[0390] Figure 6B shows the results of PAR4 cleavage in HEK293 cells with 5A.RC3 (10 μg / ml) compared to either the vehicle or isotype control, co-incubated with thrombin (0.1 U / ml) for 10 minutes.

[0391] The monoclonal antibody 5A.RC3 significantly inhibited thrombin-induced PAR4 cleavage of both Ala120 and Thr120 variants of PAR4, as well as activation of human PAR4. The functionality of this anti-hPAR4 antibody 5A.RC3 was restored by the addition of an immunized peptide.

[0392] [Example 5: Inhibition of platelet aggregation] To determine whether the anti-hPAR4 antibody MoB5ARC3.H4b(5A.RC3) can inhibit the effect of thrombin on PAR4 observed by platelet aggregation, an ex vivo platelet aggregation assay was performed on human platelets. The response to PAR4 agonists was evaluated in isolated human platelets with different PAR4 variants. Three different genotypes (TT, AT, and AA) were tested, as shown in Figure 7.

[0393] As shown in Figures 7A and 7B, the presence of the T allele was associated with higher maximal aggregation in response to PAR4-activating peptide (AP) and thrombin in the medium dose range. PAR4-AP is a selective PAR4 agonist having the C-terminal amidated peptide sequence AYPGKF-NH2.

[0394] Figure 7C shows thrombin stimulation in the presence of PAR1 blockade by borapaxal (90 nm).

[0395] Figure 7D shows that platelet aggregation was inhibited in a dose-dependent manner by 5A.RC3. This dose-dependent inhibition was equally effective across all genotypes.

[0396] Figure 7E shows the inhibitory concentration (IC) in the 5A.RC3 subclone. 50 This indicates that...

[0397] [Example 6: MoB5ARC3.F10b.H4b (hereinafter referred to as 5A.RC3) binds to PAR4 on human platelets.] The binding of clone 5A.RC3 to isolated human platelets in vitro was tested. Binding was determined using flow cytometry. Isolated platelets were incubated with either mouse IgG1 (isotype control) or 5A.RC3, and binding to PAR4 was tested. CD41a, a marker expressed on platelets, was used as a positive control. As shown in Figure 8, 5A.RC3 (10 μg / ml) binds to human PAR4 in isolated platelets.

[0398] [Example 7: Inhibition of procoagulation activity in isolated platelets as demonstrated by MoB5ARC3.F10b.H4b (hereinafter referred to as 5A.RC3)] Platelet surface phosphatidylserine (PS) exposure was determined by measuring annexin V binding. (Human isolated platelets (5 × 10⁶) 7 The sample ( / ml) was pre-treated with the indicated concentration of 5A.RC3 for 5 minutes, incubated with Alexa Fluor 488-conjugated annexin V (1:100), and then stimulated with thrombin. After stimulation, the sample was resuspended in modified Tyrode's buffer for flow cytometry analysis (FACSCalibur, BD Biosciences).

[0399] The procoagulation activity of isolated human platelets was validated by measuring phosphatidylserine (PS) exposure in response to stimulation with PAR4-AP or thrombin (1 U / ml). The percentage of annexin V-positive cells was measured. Platelets were pre-incubated with 5A.RC3 for 5 minutes. Blood was flowed for 10 minutes, and data were collected in real time. 10 minutes (final data point) is shown for simplicity.

[0400] Figure 9 shows the percentage of annexin V-positive cells in response to either PAR4-AP stimulation (A) or thrombin stimulation (B). The thr120 variant resulted in increased PS exposure in PAR4-AP stimulated platelets. A similar trend was observed in thrombin-stimulated platelets.

[0401] Figure 9C shows that pretreatment with 5A.RC3 (5 minutes) inhibited thrombin-induced phosphatidylserine exposure in a dose-dependent manner, regardless of donor genotype.

[0402] [Example 8: Antithrombotic effect of MoB5ARC3.F10b.H4b (hereinafter 5A. RC3)] Thrombosis parameters, including platelet deposition, thrombin activity, fibrin volume, and the ratio of fibrin per thrombus, were measured in real time over a 10-minute period in a total thrombosis assay as described herein under coagulation conditions.

[0403] The platelet deposition (PE-conjugated anti-CD9), thrombin activity (FRET-based thrombin probe), fibrin volume (Dylight650-conjugated anti-fibrin antibody), and fibrin-to-thrombin ratio at the 10-minute endpoint are shown in Figure 10A, obtained using a Nikon A1r equipped with a 25× lens and 2× digital magnification (to enable volume measurement) and confocal microscopy.

[0404] The direct thrombin inhibitor hirudin (800 U / mL) eliminated thrombin activity and fibrin volume despite continued platelet deposition. Figures 10B-E show that no significant differences in the above parameters were observed across all PAR4 genotypes. Pretreatment with 5A.RC3 (100 μg / ml), shown as a white bar, did not affect platelet deposition (F) compared to the control (black bar), but significantly inhibited thrombin activity (G), thrombin activity (H), fibrin volume (H), and the ratio of fibrin to thrombin volume (I).

[0405] [Example 9: Further screening of additional clones] Clones identified through binding screening as possessing reasonable affinity and specificity for human PAR4 were further validated using a functional platelet aggregation assay.

[0406] Thrombin-induced platelet aggregation (i.e., PAR4-dependent aggregation) in the presence of a PAR1 antagonist has been demonstrated for the supernatants of 30 monoclonal antibodies that bind to human platelets. 8 The cells were mixed in a 1:1 ratio. Figure 11 shows the maximum aggregation achieved at 50 minutes, expressed as a percentage of the control (individual donor n=3).

[0407] [Example 10: Sequence of two inhibitor clones and one non-inhibitor clone] Sequencing of monoclonal antibodies, all of which are IgG1 kappa isotypes, was performed at the Monash Antibody Technologies Facility at Monash University.

[0408] (i) 5A.RC3 subclone The nucleotide and amino acid sequences of the heavy chain variable region and the light chain variable region were determined. These are shown in the sequence listing, which forms part of this disclosure, and also in Figure 12. This antibody is an antagonist and belongs to the IgG2a isotype.

[0409] The sequence of the complementarity determination region (CDR) is shown below. 5A.RC3 Double-chain CDR: CDR1:GFTLSNYG (Sequence ID 13) CDR2:IWYDGSNK(Sequence ID 14) CDR3:ARESIVEVLPPFDY(Sequence ID 15) 5A.RC3 Light Chain CDR: CDR1:QRVRNNY(Sequence ID 16) CDR2:GAS (Sequence ID 17) CDR3:QQYGNSYT (Sequence ID 18)

[0410] (ii) 5F.RF3 subclone The nucleotide and amino acid sequences of the heavy chain variable region and the light chain variable region were determined. These are shown in the sequence listing, which forms part of this disclosure, and also in Figure 14. This antibody is an antagonist. The antibody isotype is IgG2b kappa.

[0411] The sequence of the complementarity determination region (CDR) is shown below. 5F.RF3 heavy-chain CDR: CDR1:AYTFTNYG (Sequence ID 24) CDR2:ISPYNGNT (Sequence ID 25) CDR3:AREYNRSSRGRYYYYGMDV (Sequence ID 26) 5F.RF3 Light Chain CDR: CDR1:QSVSSNY (Sequence ID 27) CDR2:GAS (Sequence ID 28) CDR3:QQYGSSPWT (Sequence ID 29)

[0412] (iii) 5H.RD2 subclone The nucleotide and amino acid sequences of the heavy chain variable region and the light chain variable region were determined. These are shown in the sequence listing, which forms part of this disclosure, and also in Figure 13. This antibody binds to PAR4 but does not function as an antagonist. The isotype of this antibody is IgG1 kappa.

[0413] The sequence of the complementarity determination region (CDR) is shown below. 5H.RD2 double-chain CDR: CDR1: GFTFFNTW (Sequence ID 34) CDR2:VKSKNDGGTK(Sequence ID 35) CDR3:TTDPHYDFWSAY (Sequence ID 36) 5H.RD2 Light Chain CDR: CDR1:QSLVHSDGNT (Sequence ID 37) CDR2:VKSKNDGGTK(Sequence ID 38) CDR3:LQATQFMYT (Sequence ID 39)

[0414] The CDR and framework area were determined using the IMGT / V-Quest program.

[0415] [Example 11: Measurement of binding kinetics of seven purified monoclonal antibody clones by two different surface plasmon resonance assays (SPR)] The binding kinetics of the seven purified anti-hPAR4 mAbs listed in Table 4 below were measured using two different methods.

[0416] For the mAb capture method, low levels of ligand mAbs and the analyte hPAR4 were used as an attempt to induce a 1:1 binding interaction. This method was successfully used for kinetic and affinity KD measurements (Kamat V and Rafique A (2017) Analytical Biochemistry 530:75-86) despite the use of total IgG. For the streptavidin capture method, interactions greater than 1:1 can occur by using total antibody in this analysis, even with very low levels of the analyte (mAb).

[0417] Binding of purified mAbs to hPAR4 was analyzed by ELISA (Figure 15), and the data were expressed as an ELISA positive:negative ratio. The dynamics of the binding interaction between mAbs and hPAR4 were measured using two different methods with surface plasmon resonance (SPR; Biacore): 1. an anti-mouse Fc mAb capture method, and 2. a streptavidin (SA) tip for capturing biotinylated peptides. Using Biacore evaluation software, the ka (on) velocity, kd (off) velocity, and KD were measured and fitted to a Langmuir model.

[0418] [Table 7]

[0419] [Example 12: Binding of anti-hPAR4 monoclonal antibody to hPAR4 peptide] The reactivity of purified mAbs binding to the hPAR4 peptide was identified using a method similar to the ELISA screening method described in Example 11. The experiment was completed using purified mAbs diluted in PBS instead of hybridoma supernatant.

[0420] The reactivity of seven purified hPAR4 monoclonal antibodies was analyzed by ELISA (see Table 4). Dilutions of the mAbs were reacted with hPAR4 peptide-coated wells. The binding curves are shown in Figure 15. The strongest binding was observed with mAb 5F RF3.A7b.C9 (5F.RF3), followed by three mAbs with very similar binding curves: 5H RA3.D3b.A2b (5H.RA3), 5A RC3.F10b.H4b (5A.RC3), and 5D RH4.G7.E6.C7b.G7 (5D.RH4).

[0421] The reactivity of 5G RA1.E10.G3 (5G.RA1) mAb was slightly lower, and among the mAbs, 5H RF2.A5b.D3.C2 (5H.RF2) and 5I RG1.D6.C1b (5I.RG1) showed the lowest reactivity with the hPAR4 peptide. This data can also be expressed as the ELISA positive:negative ratio shown in Table 4.

[0422] [Example 13: Specificity of anti-hPAR4 monoclonal antibody against human PAR4 peptide] The wells of a streptavidin-coated plate (Superblock-blocked Thermo Scientific Pierce streptavidin high-binding capacity coated plate, code #15500) were washed three times with washing buffer (PBS containing 0.05% TWEEN® 20 and 0.1% bovine serum albumin). Biotinylated peptides (hPAR1-4) were diluted in washing buffer and bound to the blocked streptavidin-coated wells at 10 ug / ml (100 ul per well) at room temperature for 1 hour with gentle mixing. The wells were washed three times as described above, and purified anti-hPAR4 mAb prepared in 10 ug / ml of washing buffer was bound to them at room temperature for 1 hour with gentle mixing (100 ul / well). The plate was washed three times as described above, and anti-mouse Fc conjugates conjugated with alkaline phosphatase (AP) at 0.3 ug / ml (100 ul) were added to the wells over 1 hour as described above. The wells were washed three times as described above, and the supernatant screening ELISA was colored with alkaline phosphatase substrate as described.

[0423] To further characterize the purified anti-hPAR4 mAbs, specificity of the mAbs for hPAR1, hPAR2, hPAR3, and hPAR4 biotinylated peptides was investigated. The binding data clearly demonstrate that all seven purified monoclonal antibodies are highly specific, binding only to hPAR4 and not reacting with hPAR1, hPAR2, and hPAR3 peptides.

[0424] [Example 14: Binding and Inhibition Characteristics of Five Purified Anti-hPAR4 Monoclonal Antibodies] The binding of five anti-PAR4 monoclonal antibodies to human platelets was examined by flow cytometry. Table 5 shows 1) binding to human platelets by flow cytometry (expressed as geometric mean fluorescence intensity [GMFI] observed at 10 μg / ml compared to the same concentration of isotype control) and 2) IC257 inhibition of human platelet aggregation in response to 0.1 U / ml thrombin. 50 The results for each clone regarding the value are shown.

[0425] [Table 8]

[0426] Figure 17 shows the concentration-dependent binding of purified anti-hPAR4 mAbs to isolated human platelets. Each antibody showed concentration-dependent binding compared to the relevant isotype control.

[0427] Figure 18 shows the concentration-dependent binding of 0.1 U / ml thrombin-induced human platelet aggregation for three anti-hPAR4 mAb clones (5A.RC3, 5D.RH4, and 5G.RA1). Near maximum inhibition was observed for each clone at the highest concentration tested.

[0428] [Example 15: Antithrombin effect of two anti-PAR4 clones (5D.RH4 and 5A.RC3)] The inhibition of human thrombus formation by mAb 5A.RC3 and mAb 5D.RH4 was verified using confocal microscopy. In an ex vivo human whole blood thrombosis assay, the volume of human thrombi formed after 3 minutes was quantified using confocal microscopy. When blood was pretreated with either mAb at 100 μg / ml, the total thrombus volume was reduced, as shown in Figure 19.

[0429] [Example 16: Sequence of purified anti-hPAR4 mAb clone] Sequencing of monoclonal antibodies, all of which are IgG1 kappa isotypes, was performed at the Monash Antibody Technologies Facility at Monash University. The designation of the CDR and framework region was determined using the IMGT / V-Quest program.

[0430] Figure 20 shows the variable heavy chain sequences of purified mAbs relative to human PAR4. The complementarity-determining regions (CDRs) are shown in the figure according to the IMGT numbering system.

[0431] Figure 21 shows the variable light chain sequences of purified mAbs relative to human PAR4. The complementarity-determining regions (CDRs) are shown in the figure according to the IMGT numbering system.

[0432] Table 6 shows the complementarity-determining region sequences of the antibodies.

[0433] [Table 9] JPEG0007895907000018.jpg24657

[0434] [Table 10] JPEG0007895907000020.jpg247146JPEG0007895907000021.jpg245155JPEG0007895907000022.jpg24533

[0435] [Example 17: Epitope Mapping] To determine the minimum epitope to which the anti-hPAR4 monoclonal antibody binds, three duplicate peptides corresponding to the hPAR4 peptide were synthesized as shown below and in Figure 22. JPEG0007895907000023.jpg31148

[0436] The sequence RG of the thrombin cleavage site is underlined. The C-terminal cysteine ​​was removed to prevent multimer formation. See Table 2 for the original hPAR4 peptide (KLH and naked peptide).

[0437] Screening using an ELISA assay as described in Example 11 was performed to identify whether the mAbs showed greater reactivity to any of the shorter duplicate peptides spanning the original peptide antigen. Briefly, peptides were coated overnight in coating buffer (0.1 M sodium carbonate pH 8.0) at 10 ug / ml on a Nunc maxisorp ELISA plate. Purified mAbs diluted in PBS were used instead of the supernatant.

[0438] As shown in Figure 23, purified mAb 5A.RC3, 5G.RA1, 5D.RH4, and 5F.RF3 preferentially reacted with core peptide amino acid residues 8-15, which contain the thrombin cleavage site. This indicates that the epitope is located within this region of the original hPAR4 peptide.

[0439] As shown in Figure 23, purified mAb 5G.RA3 reacted with peptide amino acid residues 11-20. These residues also contain thrombin cleavage sites, indicating that 5G.RA3 recognizes a slightly different hPAR4 epitope compared to other mAbs.

[0440] Those skilled in the art will understand that numerous variations and / or modifications can be applied to the present invention as shown in the embodiments described above without departing from the broadly described scope of the invention. Therefore, the embodiments of the present invention should be considered in all respects as illustrative and not restrictive. The present invention provides the following: 1. A protease-activated receptor 4 (PAR4) binding protein which is an anti-PAR4 recombinant antibody, synthetic antibody, monoclonal antibody, or antigen-binding fragment thereof, wherein the protease-activated receptor 4 (PAR4) binding protein inhibits the cleavage of human PAR4 expressed on the cell surface by 50% or more in the presence of thrombin. 2. The PAR4-binding protein described in 1 above, which inhibits (i) cleavage of 60% or more, or (ii) cleavage of 70% or more, or (iii) cleavage of 80% or more of cell surface-expressed PAR4 in the presence of thrombin. 3. The PAR4-binding protein described in 1 or 2 above, which inhibits the cleavage of more than 90% of cell surface-expressed PAR4 in the presence of thrombin. 4. A PAR4-binding protein according to any of items 1-3 above, which specifically binds to an epitope spanning the thrombin cleavage site of PAR4. 5. The PAR4-binding protein described in 4 above, wherein the epitope contains the sequence APRGY and the thrombin cleavage site corresponds to RG. 6. The PAR4-binding protein according to 4 or 5 above, wherein the epitope comprises or consists of a sequence selected from ILPAPRGY or APRGYPGQV. 7. A PAR4-binding protein according to any one of items 1 to 6 above, wherein the antibody binds to the Ala120 and / or Thr120 variant of human PAR4. 8. A PAR4-binding protein according to any one of items 1 to 7 above, wherein the protein does not bind to or substantially binds to human PAR1, PAR2, or PAR3. 9. A PAR4-binding protein according to any of items 1 to 8 above, comprising the variable heavy chain (VH) sequence shown below. JPEG0007895907000024.jpg26150 (in the array, X1 is V or I, X2 is either A or V, X3 is either T or A. X4 is L or F. X5 is N or S. X6 is either Y or D. X7 is S or A. X8 is Y or F, X9 is either S or R. X 10 is N or S, X 11 is K or R, X 12 is H or Y, X 13 is A, L, or T, X 14 is K or R, X 15 is T or D, X 16 is N or T, X 17 is L or Q, X 18 is Y or F, X 19 is S or I, X 20 is S or T, X 21 is I, S, or A; X 22 is V, I, M, or L, X 23 is E, S, V, or I, X 24 is V, T, R, or G, X 25 It is L, R, or G. X 26 (is P or V) 10. A PAR4-binding protein according to any of items 1 to 9 above, comprising the variable light chain (VL) sequence shown below. JPEG0007895907000025.jpg28150(in the array, X1 is either K or E. X2 is either V or A. X3 is R or G. X4 is A or T, X5 is R or S, X6 is V or I, X7 is N or S. X8 is N or S. X9 is either F or Y. X 10 is F or L, X 11 is I or T, X 12 is I or T, X 13 is F or L, X 14 is S or T, X 15 is V or L, X 16 (is N, R, or S) 11. The PAR4 binding protein described in 9 or 10 above, wherein the VH comprises a CDR1 sequence selected from the group consisting of the following: (i) GFTLSNYG(sequence number 13); (ii) GFTFSSDG (Sequence ID 59); (iii) GFTFSNYG (Sequence No. 68); (iv) GFTFSSYG (Sequence ID 55); (v) GFAFSSYG (Sequence No. 70); and (vi)GFTLSSYG (sequence number 75). 12. The PAR4 binding protein according to any of 9 to 11 above, wherein the VH contains a CDR2 sequence selected from the group consisting of the following: (i)IWYDGSNK(sequence number 14); (ii) IWFDGRNK (Sequence ID 60); (iii) IWYDGSNR(sequence number 71); and (iv) IWYDGSSK (Sequence ID 76). 13. The PAR4 binding protein according to any one of 9 to 12 above, wherein the VH contains a CDR3 sequence selected from the group consisting of the following: (i) ARESIVEVLPPFDY(Sequence ID 15); (ii) ARESSISTRPPFDY (Sequence No. 61); (iii)ARETIMVRGVPFD(sequence number 69); (iv)ARETALVRGVPFDY(Sequence ID 56); (v)ARETAMVRGVPFDY(Sequence ID 72); and (vi)ARETILIGGVPFDY(Sequence ID 77). 14. The PAR4 binding protein described in 10 above, wherein the VL contains a CDR1 sequence selected from the group consisting of the following: (i) QRVRNNY (sequence number 16); (ii) QSVRSSY (sequence number 57); and (iii) QSIRSNY (Sequence ID 78). 15. The PAR4-binding protein according to 10 or 14 above, wherein the VL contains the CDR2 sequence GAS (SEQ ID NO: 28). 16. The PAR4 binding protein according to 10, 14, or 15 above, wherein the VL contains a CDR3 sequence selected from the group consisting of the following: (i) QQYGNSYT (sequence number 18); (ii) QQYGRSYT (sequence number 62); and (iii) QQYGSSYT (Sequence ID 58). 17. A PAR4-binding protein according to any of items 1 to 8 above, comprising the variable heavy chain (VH) sequence shown below. JPEG0007895907000026.jpg25154(in the array, X1 is either A or S. X2 is either T or A. X3 is V or I, X4 is either Y or S. X5 is either G or S. X6 is L or F. X7 is N, D, or T. X8 is Y or F, X9 is either S or R. X 10 is R or H, X 11 is N or I, X 12is S or T, X 13 is T or S, X 14 is N or T, X 15 is K or N, X 16 is F or L, X 17 is K or N, X 18 is A or K, X 19 is I, F, or V, X 20 is Y or H, X 21 is N or S, X 22 is R, G, or S, X 23 is V or H) 18. The PAR4 binding protein according to any one of 1 to 9 or 17 above, comprising the variable light chain (VL) sequence shown below. JPEG0007895907000027.jpg27153 (In the sequence, X1 is V or A, X2 is V or I, X3 is S or T, X4 is S, Y, or N, X5 is K or I, X6 is N or K, X7 is R or S, X8 is R or Q, X9 is T or A, X 10 is T or S, X 11 is Q or R, X 12 is T, S, or N, X 13 is N or N, X 14 is E or G) 19. The PAR4 binding protein described in 18 above, wherein the VH comprises a CDR1 sequence selected from the group consisting of the following: (i)GGSLSDYY(array number 86); (iii) SGSFSTYF (sequence number 47); and (iv) GGSFSNYY (Sequence No. 66). 20. The PAR4 binding protein described in 18 or 19 above, wherein the VH comprises a CDR2 sequence selected from the group consisting of the following: (i) INHSGTT (sequence number 87); (ii) IIHTGST (Sequence No. 64); or (iii) INHSGST (Sequence No. 48). 21. The PAR4 binding protein according to 18, 19, or 20 above, wherein the VH contains a CDR3 sequence selected from the group consisting of the following: (i)AIEYSNSRGYYYGMDV(Sequence ID 88); (ii)AFEYSSSGGYYYGMDV(Sequence No. 49); and (iii) KVEHSSSSGHYYYGMDV (Sequence ID 65). 22. The PAR4 binding protein according to any one of items 18 to 21 above, wherein the VL contains a CDR1 sequence selected from the group consisting of the following: (i) QTISNY (sequence number 109); (ii) QSISSY (sequence number 50); and (iii) QTISYY (Sequence ID 66). 23. The PAR4 binding protein according to any one of items 18 to 22 above, wherein the VL contains the CDR2 sequence AAS (SEQ ID NO: 51). 24. The PAR4 binding protein according to any one of items 18 to 23 above, wherein the VL contains a CDR3 sequence selected from the group consisting of the following: (i)RQNYNTPLT(sequence number 85); (iii) QQTYSTPLT(sequence number 52); or (iv) QQSYSTPLT (Sequence ID 67). 25. Any of the above-mentioned PAR4-binding proteins, comprising a variable heavy chain (VH) having the following sequences, or having the following sequences, respectively: (i) Sequence IDs 13, 14, and 15; (ii) Sequence IDs 47, 48, and 49; (iii) Sequence IDs 24, 25, and 26; (iv) Sequence IDs 55, 14, and 56; (v) Sequence IDs 59, 60, and 61; (vi) Sequence IDs 63, 64, and 65; (vii) Sequence ID 68, Sequence ID 14, and Sequence ID 69; (viii) Sequence IDs 70, 71, and 72; (ix) Sequence IDs 55, 73, and 74; (x) Sequence IDs 75, 76, and 77; (xi) Sequence IDs 79, 80, and 81; (xii) Sequence IDs 82, 80, and 83; (xiii) Sequence ID 55, Sequence ID 73, and Sequence ID 74; or (xiv) Sequence IDs 86, 87, and 88. 26. The PAR4-binding protein described in 25 above, further comprising variable light chains (VLs) having CDR1, CDR2, and CDR3 sequences, each containing or comprising the following sequences: (i) Sequence IDs 16, 17, and 18; (ii) Sequence IDs 50, 51, and 52; (iii) Sequence IDs 27, 28, and 29; (iv) Sequence IDs 57, 28, and 58; (v) Sequence IDs 57, 28, and 62; (vi) Sequence ID 66, Sequence ID 51, and Sequence ID 67; (vii) Sequence IDs 57, 28, and 58; (viii) Sequence ID 57, Sequence ID 28, and Sequence ID 58; (ix) Sequence ID 78, Sequence ID 28, and Sequence ID 62; (x) Sequence ID 84, Sequence ID 51, and Sequence ID 85; (xi) Sequence ID 57, Sequence ID 28, and Sequence ID 58; (xii) Sequence ID 57, Sequence ID 51, and Sequence ID 58; or (xiii) Sequence ID 109, Sequence ID 51, and Sequence ID 85. 27. Any of the PAR4-binding proteins described above, comprising a VH sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 11, 22, 45, 53, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107, or a humanized, chimeric, or deimmunized version thereof. 28. The PAR4-binding protein described in 27, further comprising a VL sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NOs: 12, 23, 46, 54, 90, 92, 94, 96, 98, 100, 102, 104, 106, or 108, or a humanized, chimeric, or deimmunized version thereof. 29. Any of the PAR4-binding proteins listed above, including: (i) VH shown in Sequence ID 11 and VL shown in Sequence ID 12; (ii) VH shown in Sequence ID No. 45 and VL shown in Sequence ID No. 46; (iii) VH as shown in Sequence ID No. 22 and VL as shown in Sequence ID No. 23; (iv) VH as shown in Sequence ID 53 and VL as shown in Sequence ID 54; (v) VH as shown in Sequence ID 89 and VL as shown in Sequence ID 90; (vi) VH shown in Sequence ID 91 and VL shown in Sequence ID 92; (vii) VH as shown in sequence number 93 and VL as shown in sequence number 94; (viii) VH as shown in Sequence ID 95 and VL as shown in Sequence ID 96; (ix) VH as shown in Sequence ID 97 and VL as shown in Sequence ID 98; (x) VH shown in sequence number 99 and VL shown in sequence number 100; (xi) VH as shown in Sequence ID 101 and VL as shown in Sequence ID 102; (xii) VH shown in Sequence ID 103 and VL shown in Sequence ID 104; (xiii) VH shown in sequence number 105 and VL shown in sequence number 106; or (xiv) VH shown in sequence number 107 and VL shown in sequence number 108. 30. The antigen-binding fragment is (i) Single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) At least one of (i) and / or (ii) that is linked to the heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3 The PAR4-binding protein described above is one of the above-mentioned PAR4-binding proteins. 31. The antigen-binding fragment is (i) Diabody; (ii) Triabody; (iii) Tetrabody; (iv)Fab; (v)F(ab′)2; (vi)Fv; or (vii) At least one of (i) to (vi) that is linked to the heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3 The PAR4-binding protein described in any of items 1 to 29 above. 32. Any of the above-mentioned PAR4-binding proteins that are bound to a certain portion. 33. The PAR4-binding protein according to 32, wherein the portion is selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, compounds that increase the half-life of PAR4-binding proteins in a subject, and mixtures thereof. 34. A nucleic acid encoding any of the PAR4-binding proteins described above. 35. The PAR4-binding protein according to 34, comprising the VH nucleic acid sequence shown in SEQ ID NO: 20 and / or the VL nucleic acid sequence shown in SEQ ID NO: 21. 36. The PAR4-binding protein according to 34 above, comprising the VH nucleic acid sequence shown in SEQ ID NO: 30 and / or the VL nucleic acid sequence shown in SEQ ID NO: 31. 37. A composition comprising a PAR4-binding protein described in any of items 1 to 33 above and a suitable carrier. 38. A method for treating or preventing thrombosis or thromboembolic disorder in a subject, comprising administering to the subject a PAR4-binding protein or antibody described in any of items 1 to 33 above, or a composition described in item 37 above. 39. A method for treating, preventing, or improving thrombosis or thromboembolic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a PAR4-binding protein or antibody described in any of items 1 to 33 above, or a therapeutically effective amount of the composition described in item 37 above.

Claims

1. A nucleic acid encoding both the variable heavy chain (VH) and variable light chain (VL) of a protease-activated receptor 4 (PAR4) binding protein, which is an anti-PAR4 recombinant antibody, synthetic antibody, or monoclonal antibody or its antigen-binding fragment, or a pair of nucleic acids in which one member encodes the VH of the PAR4 binding protein and the other member encodes the VL of the PAR4 binding protein, wherein the protein specifically binds to an epitope spanning the thrombin cleavage site of PAR4. (i) VH contains three complementarity-determining region (CDR) sequences: CDR1, CDR2, and CDR3 sequences, VL contains three CDR sequences: CDR1, CDR2, and CDR3 sequences, VH contains the amino acid sequence of SEQ ID NO: 89, and VL contains the amino acid sequence of SEQ ID NO: 90; or (ii) VH contains three CDR sequences: CDR1, CDR2, and CDR3 sequences, VL contains three CDR sequences: CDR1, CDR2, and CDR3 sequences, VH contains the amino acid sequence of SEQ ID NO: 11, and VL contains the amino acid sequence of SEQ ID NO:

12. The nucleic acid wherein the PAR4-binding protein binds to both the PAR4 receptor variants Ala120 and Thr120.

2. The nucleic acid according to claim 1, which inhibits the cleavage of cell surface-expressed human PAR4 by 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more in the presence of thrombin.

3. The nucleic acid according to claim 1 or 2, wherein the PAR4 binding protein does not react with human PAR1, PAR2, or PAR3 peptide.

4. The nucleic acid according to any one of claims 1 to 3, wherein pretreatment of human platelets with a protein inhibits thrombin-induced platelet aggregation.

5. The nucleic acid according to any one of claims 1 to 4, wherein the total thrombus volume is reduced by pretreatment of the blood with protein.

6. (i) VH contains the amino acid sequence of SEQ ID NO: 89, and the CDR1, CDR2, and CDR3 amino acid sequences within the VH sequence contain or consist of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively; VL contains the amino acid sequence of SEQ ID NO: 90, and the CDR1, CDR2, and CDR3 amino acid sequences within the VL sequence contain or consist of SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 62, respectively; or (ii) VH contains the amino acid sequence of SEQ ID NO: 11, and the CDR1, CDR2, and CDR3 amino acid sequences within the VH sequence contain or consist of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; VL contains the amino acid sequence of SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 amino acid sequences within the VL sequence contain or consist of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The nucleic acid according to any one of claims 1 to 5.

7. The nucleic acid according to any one of claims 1 to 6, wherein VH comprises the amino acid sequence of SEQ ID NO: 11 and VL comprises the amino acid sequence of SEQ ID NO:

12.

8. The nucleic acid according to any one of claims 1 to 6, wherein VH comprises the amino acid sequence of SEQ ID NO: 89 and VL comprises the amino acid sequence of SEQ ID NO:

90.

9. The nucleic acid according to any one of claims 1 to 8, which does not bind to or substantially binds to human PAR1, PAR2, or PAR3.

10. A composition comprising a nucleic acid according to any one of claims 1 to 9 and a suitable carrier.

11. An expression vector comprising the nucleic acid according to any one of claims 1 to 9.

12. A host cell comprising the expression vector described in claim 11.

13. A set of expression vectors comprising a first expression vector and a second expression vector, (i) The first expression vector comprises a nucleic acid encoding VH of a PAR4-binding protein containing the amino acid sequence of SEQ ID NO: 89, and the second expression vector comprises a nucleic acid encoding VL of a PAR4-binding protein containing the amino acid sequence of SEQ ID NO: 90; or (ii) The first expression vector comprises a nucleic acid encoding VH of a PAR4-binding protein containing the amino acid sequence of SEQ ID NO: 11, and the second expression vector comprises a nucleic acid encoding VL of a PAR4-binding protein containing the amino acid sequence of SEQ ID NO:

12. The set of the aforementioned expression vectors.

14. A composition comprising a first nucleic acid and a second nucleic acid, (i) a first nucleic acid encoding a first polypeptide comprising VH of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 89, and a second nucleic acid encoding a second polypeptide comprising VL of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 90; or (ii) The first nucleic acid encodes a first polypeptide comprising VH of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 11, and the second nucleic acid encodes a second polypeptide comprising VL of a PAR4-binding protein having the amino acid sequence of SEQ ID NO:

12. The aforementioned composition.

15. An isolated cell comprising a first nucleic acid and a second nucleic acid, (i) a first nucleic acid encoding a first polypeptide comprising VH of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 89, and a second nucleic acid encoding a second polypeptide comprising VL of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 90; or (ii) The first nucleic acid encodes a first polypeptide comprising VH of a PAR4-binding protein having the amino acid sequence of SEQ ID NO: 11, and the second nucleic acid encodes a second polypeptide comprising VL of a PAR4-binding protein having the amino acid sequence of SEQ ID NO:

12. The aforementioned cells.

16. A composition comprising the set of expression vectors according to claim 13, the composition according to claim 14, or isolated cells according to claim 15, and a suitable carrier.

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