Binding proteins for the human thrombin receptor PAR4

Abstract

The present disclosure is directed to human protease-activated receptor 4 (PAR4) binding proteins (e.g., antibodies). In particular, it is directed to anti-PAR4 binding proteins that are antagonists of human PAR4, and methods and uses thereof. [Selection diagram] None

Description

[Technical Field]

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

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

[0003] The entire contents of the electronic sequence listing submission are incorporated by reference in their entirety for all purposes.

[0004] Field of the Disclosure The present disclosure is directed to human protease-activated receptor 4 (PAR4) binding proteins (e.g., antibodies). In particular, it is directed to anti-PAR4 binding proteins that are antagonists of human PAR4, and methods and uses thereof. [Background technology]

[0005] Activated platelets are key cellular components in arterial thrombosis, 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 42011 (2013)). Arterial thrombosis can lead to heart attacks and ischemic strokes.

[0006] Platelets are the cells that form arterial thrombi. Platelets are activated by a combination of endogenous agonists that cause platelet aggregation and procoagulation, which together drive pathological thrombus formation. Therefore, antiplatelet drugs constitute the primary pharmacotherapy for the prevention of arterial thrombosis. However, despite the large number of such drugs, limitations in safety and / or efficacy necessitate the rationalization of novel drug targets. There is a significant need for improved antiplatelet drugs across a wide range of clinical settings, and there is considerable interest in the development of novel agents.

[0007] Thrombin is the most potent known human platelet activator and a key effector protease in the coagulation cascade. Thrombin activates platelets primarily through the protease-activated cell surface receptors (PAR1 and PAR4), making it the body's most potent platelet activator (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), that are activated by N-terminal proteolysis. Once cleaved, the newly exposed N-terminus acts as a tethered ligand, activating the receptor by binding extracellular loop 2 (Vu T-KH et al. (1991) Cell 64:1057-68). There are four members of the PAR family (PAR1-4) that are expressed and activated by multiple proteases.

[0008] All platelet PAR functions were lost (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), demonstrating the potential for targeting such receptors for antithrombotic therapy. There are two predominant PARs on human platelets: PAR1 and PAR4. Of these, PAR1 is a higher-affinity thrombin receptor and has been the target of antiplatelet drug development. Two PAR1 antagonists, atopaxar (E5555) (Goto S et al. (2010) Eur Heart J 31:2601-13) and vorapaxar (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. Vorapaxar was approved by the US FDA in late 2014 and scheduled by the TGA for the prevention of myocardial infarction and peripheral arterial disease in mid-2016. However, vorapaxar in combination with single or dual antiplatelet therapy was associated with a significant increase in the rate of intracerebral hemorrhage, 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 developing PAR4 antagonists. The functional role of PAR4 has been elucidated primarily in platelets. A key feature that characterizes PAR4 is its ability to form hetero-oligomers with both PAR1 and the ADP receptor P2Y12, allowing PAR4 to affect both thrombin- and ADP-initiated 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 through Gq to mobilize intracellular calcium, driving platelet functions including integrin activation, granule secretion, and phosphatidylserine (PS) exposure. However, PAR4 activation induces a slower and 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 due, in part, to the anionic sequence C-terminal to 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 events depend on sustained elevations in intracellular calcium levels, they may involve sustained platelet secretory dynamics (Jonnalagadda D et al. (2012) 120:5209-16) and platelet procoagulant function (Williamson P et al. (1995) 34:10448-55; Dachary-Prigent J et al. (1995) Biochemistry 34:11625-34).

[0010] There are no studies validating the contribution of PAR4 to procoagulant activity in human thrombosis settings, likely due to the limited availability of suitable PAR4 antagonists required for such studies. The most commonly used PAR4 antagonists are the small molecule YD-3 (Wu CC et al. (2000) Br J Pharmacol 130:1289-96), the peptidomimetic tc-YPGKF-NH2 (Hollenberg MD et al. (2001) 79:439-42), and the pepducins P4pal-10 and P4pal-11 (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 agents are not widely available (e.g., YD-3) or have been reported to lack specificity (e.g., pepducins) and / or efficacy (e.g., tc-YPGKF-NH2) in studies with human platelets (Stampfuss JJ et al. (2003) Nat Med 9:1447; Hollenberg MD et al. (2004) Br J Pharmacol 143:443-54; Wu CC et al. (2002) Thromb Haemost 87:1026-33). A PAR4 antagonist (BMS-986141) has been tested in early clinical trials for the treatment of thrombosis, but common PAR4 variants (present in 19–82% of the population, depending on the population) render the receptor insensitive to small molecule inhibitors (e.g., BMS-986141).

[0011] Based on the above, it will be clear to those skilled in the art that the identification of improved human PAR4 binding proteins for the medical treatment of thrombosis will be useful.In addition, the treatment of thrombosis with minimal harmful 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 provide improved therapeutic profiles in targeting PAR1 in the treatment or prevention of thrombosis. Summary of the Invention

[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 renders the receptor insensitive to orthosteric PAR4 antagonism (Edelstein LC et al. (2014) Blood 124:3450-3458). This SNP determines whether amino acid 120 is alanine (Ala120) or threonine (Thr120). Pharmacological studies have shown that orthosteric PAR4 antagonism potently inhibits PAR4-induced platelet activation in patients with the Ala120 genotype but has no effect on platelets from patients with the Thr120 genotype, even at high concentrations. Heterozygosity results in only partial inhibition (Edelstein LC et al (2014) Blood 124:3450-3458). The prevalence of inhibitor-resistant Thr120 forms of PAR4 is remarkably high (over 80% in some populations), indicating the significant impact of this antagonism. The Thr120 allele is racially dimorphic; in a cohort of 154 North Americans, it occurred in 63% of those who self-identified as black, compared with 19% of whites. Data from the Human Genome Diversity Project (HGDP) indicate 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 carrying the Thr120 PAR4 variant.

[0013] In addition to rendering PAR4 resistant to orthosteric inhibitors, SNP rs773902 increases the sensitivity of PAR4 to receptor activation, resulting in 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-of-care antiplatelet agents (aspirin and / or P2Y12 inhibitors).

[0014] The inventors have developed a monoclonal antibody that specifically binds to human PAR4 and inhibits PAR4 activation by thrombin. 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 have found that targeting PAR4 is less likely to cause bleeding complications than targeting PAR1 due to its different mechanism of action and an overall broader safety profile.

[0015] Specifically, the antibodies identified by the inventors are effective against both PAR4 receptor variants, i.e., Ala120 and Thr120, meaning that they are effective in treating thromboembolic disorders in all subjects, not just those with sensitive PAR4 variants. Furthermore, because the antibodies specifically bind to PAR4 with minimal cross-reactivity with PAR1, bleeding complications can be minimized or avoided. Thus, the antibodies of the present invention are distinguished from prior art PAR1 antagonists and PAR4 small molecule inhibitors.

[0016] Additionally, we found that the antibody significantly inhibited thrombin-induced PAR4 cleavage and aggregation of human platelets, and these effects could be reversed by decompeting the antibody with the immunizing peptide.

[0017] Thus, the present disclosure provides various reagents for diagnosing or predicting thrombosis in a subject. The present disclosure also provides methods for treating, preventing, or ameliorating thrombosis or a thromboembolic disorder in a subject.

[0018] The present 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 one example, the PAR4-binding protein is not a small molecule antagonist (e.g., an imidazothiadiazole derivative described in WO2013 / 163244 or a synthetic peptide analog described in US7879792).

[0020] The present 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 contacted 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, the PAR4-binding protein inhibits the externalization of phosphatidylserine (PS) on the cell surface of cells (e.g., platelets) that express PAR4. In one example, the PAR4-binding protein reduces thrombus volume as measured by a whole blood thrombosis assay.

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

[0023] Those skilled in the art can measure PAR4 cleavage using suitable in vitro assays.As a non-limiting example, PAR4 cleavage can be evaluated in a cell line that expresses fluorescently tagged PAR4 protein on the surface.In the presence of thrombin, the loss of FLAG from the cell surface caused by PAR4 cleavage can be quantified.In a specific example, PAR4 cleavage can be measured in transfected HEK293 cells that contain a nucleic acid encoding PAR4 that contains a fluorescently tagged FLAG tag.In the presence of thrombin (for example, 0.1U / ml), the loss of FLAG from the cell surface caused by PAR4 cleavage can be quantified by using flow cytometry.

[0024] In one example, the binding protein inhibits cleavage of cell surface-expressed human PAR4 by 50% or more 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 one example, the PAR4 binding protein specifically binds to an epitope including residues contained within the sequence set forth as GDDSTPSILPAPRGYPGQVC (SEQ ID NO: 2). In one example, the peptide consists of SEQ ID NO: 1 or SEQ ID NO: 2. For example, the peptide is displayed on the surface of a phage.

[0026] In another example, the epitope comprises the sequence APRGY (SEQ ID NO: 42), where the thrombin cleavage site corresponds to RG. In another example, the epitope comprises 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 comprising the sequence set forth 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 the human PAR4 sequence according to SEQ ID NO: 19. In one example, the PAR4 binding protein binds to a sequence matching residues 35-54 of the human PAR4 sequence set forth in SEQ ID NO: 19. In another example, the PAR4 binding protein binds to the sequence set forth as SEQ ID NO: 2, optionally additionally comprising keyhole limpet hemocyanin (KLH) or other immunostimulatory molecule. In a further example, the PAR4 binding protein binds to the sequence set forth in SEQ ID NO: 4.

[0029] In another example, the PAR4 binding protein binds to the sequence set forth as SEQ ID NO: 2, optionally additionally comprising a C-terminal GGGG and streptavidin-k / biotin (SKB). In one example, the PAR4 protein binds to the sequence set forth as SEQ ID NO: 7.

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

[0031] In a further example, the PAR4 binding protein does not bind or does not substantially bind to a PAR sequence 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 (eg, a peptide according to SEQ ID NO: 2 or SEQ ID NO: 7) and contacting the peptide with a PAR4 binding protein.

[0033] Exemplary PAR4 binding proteins having such binding characteristics described herein include the variable regions and / or CDRs of the antibody designated 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 is 5A.RC3, 5 I .RG1, 5F.R F 3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, 5H.RF2, or a peptide consisting of the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:7 or human PAR4 at a similar level or substantially the same level, or with similar or substantially the same affinity, as the antibodies designated 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, 5H.RF2.

[0035] In another example, the PAR4 binding protein is 5A.RC3, 5A.RC4, 5A.RC5, 5A.RC6, 5A.RC7, 5A.RC8, 5A.RC9, 5A.RC10, 5A.RC11, 5A.RC12, 5A.RC13, 5A.RC14, 5A.RC15, 5A.RC16, 5A.RC17, 5A I .RG1, 5F.R F In a further example, the protein competitively inhibits the binding of antibodies designated 5A.RC3, 5F.RE6, 5H.RF2, 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. I .RG1, 5F.R F 3, 5G.RA1, 5D.RH4, 5H.RH4, 5G.RF6, 5G.RD6, 5H.RA3, 5G.RG1, 5H.RG4, 5G.RC5, 5F.RE6, 5H.RF2 from binding to a peptide consisting of the sequence set forth 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 set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:7 in an amount that is within 75% of the amount bound by an antibody comprising a VH comprising the sequence set forth 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 a VL comprising the sequence set forth 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 a PAR4-binding protein with a peptide consisting of the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7, and measuring the amount of PAR4-binding protein contacted with the peptide (e.g., 10 μg / ml). The amount of PAR4-binding protein bound to the peptide is then measured and compared with the amount of an antibody comprising a VH comprising the sequence set forth 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 a VH comprising the sequence set forth 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, each of which is bound to the peptide. In one example, the amount of PAR4 binding protein bound to the peptide is within about 80%, or 70%, or 60%, or 40% of the amount of antibody bound.

[0038] The present disclosure also provides PAR4 binding proteins that competitively inhibit the binding of the following antibodies: (i) the aforementioned 5A.RC3 antibody, comprising a VH comprising the sequence set forth in SEQ ID NO: 11 and a VL comprising the sequence set forth in SEQ ID NO: 12; (ii) 5I.RG1 comprising a VH comprising the sequence set forth in SEQ ID NO: 45 and a VL comprising the sequence set forth in SEQ ID NO: 46; (iii) 5F.RF3 comprising a VH comprising the sequence set forth in SEQ ID NO: 22 and a VL comprising the sequence set forth in SEQ ID NO: 23; (iv) 5G.RA1 comprising a VH comprising the sequence set forth in SEQ ID NO: 53 and a VL comprising the sequence set forth in SEQ ID NO: 54; (v) 5D.RH4 comprising a VH comprising the sequence set forth in SEQ ID NO: 89 and a VL comprising the sequence set forth in SEQ ID NO: 90; (vi) 5H.RH4 comprising a VH comprising the sequence set forth in SEQ ID NO: 91 and a VL comprising the sequence set forth in SEQ ID NO: 92; (vii) 5G.RF6 comprising a VH comprising the sequence set forth in SEQ ID NO: 93 and a VL comprising the sequence set forth in SEQ ID NO: 94; (viii) 5G.RD6 comprising a VH comprising the sequence set forth in SEQ ID NO: 95 and a VL comprising the sequence set forth in SEQ ID NO: 96; (ix) 5H.RA3 comprising a VH comprising the sequence set forth in SEQ ID NO: 97 and a VL comprising the sequence set forth in SEQ ID NO: 98; (x) 5G.RG1 comprising a VH comprising the sequence set forth in SEQ ID NO: 99 and a VL comprising the sequence set forth in SEQ ID NO: 100; (xi) Sequence number 101 VH comprising the sequence shown in SEQ ID NO: 102 5H.RG4, which contains a VL containing the sequence shown in (xii) 5G.RC5 comprising a VH comprising the sequence set forth in SEQ ID NO: 103 and a VL comprising the sequence set forth in SEQ ID NO: 104; (xiii) 5F.RE6 comprising a VH comprising the sequence set forth in SEQ ID NO: 105 and a VL comprising the sequence set forth in SEQ ID NO: 106; or (xiv) 5H.RF2 comprising a VH comprising the sequence set forth in SEQ ID NO: 107 and a VL comprising the sequence set forth in SEQ ID NO: 108, a peptide comprising or consisting of the sequence set forth 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., has PAR4 antagonist activity). In one example, the PAR4-binding protein (e.g., at a concentration of 10 μg / ml) 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., has PAR4 antagonist activity). 4 5G.RC5, 5F.RE6, or 5H.RF2, or the variable regions or complementarity determining regions (CDRs) of antibodies containing the CDRs of such antibodies.

[0040] In one example, PAR4 binding protein binds to the peptide comprising 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 2nM or less (for example, 1.5nM or less, for example, 1nM or less).In one example, KD is between about 0.01nM and about 2nM, for example, between about 0.05nM and about 1nM, for example, between about 0.1nM and about 1nM, for example, between about 0.3nM and about 1nM.In one example, KD is between about 0.01nM and 1nM, for example, between about 0.05nM and 0.9nM, for example, between about 0.09nM and about 0.7nM, for example, between about 0.1nM and 0.6nM.

[0041] In one example, KD is assessed by utilizing a streptavidin chip, capturing a biotin-conjugated 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 across it.

[0042] In one example, KD is assessed by utilizing a streptavidin chip, capturing a biotin-conjugated 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 across it.

[0043] Exemplary PAR4 binding proteins of the present disclosure have a KD of about 0.01 to 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 one example, the PAR4 binding protein has a KD shown in Table 4 corresponding to any one of the PAR4 binding proteins herein.

[0044] In one example, a PAR4 binding protein of the present disclosure specifically binds to human PAR4. In one example, protein binding is assessed by ELISA and high-throughput antigen microarray.

[0045] In one example, the PAR4 binding protein binds to the same epitope in human PAR4 or to an epitope in human PAR4 that overlaps with the epitope bound by the following antibody: (i) the aforementioned 5A.RC3 antibody, comprising a VH comprising the sequence set forth in SEQ ID NO: 11 and a VL comprising the sequence set forth in SEQ ID NO: 12; (ii) 5I.RG1 comprising a VH comprising the sequence set forth in SEQ ID NO: 45 and a VL comprising the sequence set forth in SEQ ID NO: 46; (iii) 5F.RF3 comprising a VH comprising the sequence set forth in SEQ ID NO: 22 and a VL comprising the sequence set forth in SEQ ID NO: 23; (iv) 5G.RA1 comprising a VH comprising the sequence set forth in SEQ ID NO: 53 and a VL comprising the sequence set forth in SEQ ID NO: 54; (v) 5D.RH4 comprising a VH comprising the sequence set forth in SEQ ID NO: 89 and a VL comprising the sequence set forth in SEQ ID NO: 90; (vi) 5H.RH4 comprising a VH comprising the sequence set forth in SEQ ID NO: 91 and a VL comprising the sequence set forth in SEQ ID NO: 92; (vii) 5G.RF6 comprising a VH comprising the sequence set forth in SEQ ID NO: 93 and a VL comprising the sequence set forth in SEQ ID NO: 94; (viii) 5G.RD6 comprising a VH comprising the sequence set forth in SEQ ID NO: 95 and a VL comprising the sequence set forth in SEQ ID NO: 96; (ix) 5H.RA3 comprising a VH comprising the sequence set forth in SEQ ID NO: 97 and a VL comprising the sequence set forth in SEQ ID NO: 98; (x) 5G.RG1 comprising a VH comprising the sequence set forth in SEQ ID NO: 99 and a VL comprising the sequence set forth in SEQ ID NO: 100; (xi) Sequence number 101 VH comprising the sequence shown in SEQ ID NO: 102 5H.RG4, which contains a VL containing the sequence shown in (xii) 5G.RC5 comprising a VH comprising the sequence set forth in SEQ ID NO: 103 and a VL comprising the sequence set forth in SEQ ID NO: 104; (xiii) 5F.RE6 comprising a VH comprising the sequence set forth in SEQ ID NO: 105 and a VL comprising the sequence set forth in SEQ ID NO: 106; or (xiv) VH comprising the sequence shown in SEQ ID NO: 107 and a VL comprising the sequence shown in SEQ ID NO: 108 Includes 5H.RF2.

[0046] The present disclosure also provides a PAR4 binding protein that specifically binds to human PAR4 and is an anti-PAR4 recombinant, synthetic, or monoclonal antibody or antigen-binding fragment thereof.

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

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

[0049] In one example, the PAR4 binding protein is a chimeric antibody comprising human heavy and light chain constant region sequences, hi another example, the PAR4 binding protein is a humanized or fully human antibody.

[0050] In one example, the PAR4 binding protein inhibits cleavage of cell surface-expressed PAR4 by 60% or more in the presence of thrombin or a PAR1 antagonist. In a further example, the protein inhibits cleavage of PAR4 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, cleavage of PAR4 by thrombin is measured by loss of the Flag tag from HEK293 cells expressing flag-tagged PAR4. In another example, cleavage is measured by flow cytometry.

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

[0053] In one example, the PAR4 binding protein comprises the variable heavy (VH) sequence shown below. TIFF0007733443000001.tif23152 (in the sequence, X1 is V or I; X2 is A or V, X3 is T or A, X4 is L or F, X5 is N or S, X6 is Y or D, X7 is S or A; X8 is Y or F, X9 is 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 is L, R, or G, X 26 is P or V)

[0054] In one example, the PAR4 binding protein further comprises a variable light (VL) sequence shown below: TIFF0007733443000002.tif26152 (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 10is 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, the VH comprises a CDR1 sequence selected from the group consisting of: (i) GFTLSNYG (SEQ ID NO: 13); (ii) GFTFSSDG (SEQ ID NO: 59); (iii) GFTFSNYG (SEQ ID NO: 68); (iv) GFTFSSYG (SEQ ID NO: 55); (v) GFAFSSYG (SEQ ID NO: 70); and (vi) GFTLSSYG (sequence number 75).

[0056] In one example, the VH comprises a CDR2 sequence selected from the group consisting of: (i) IWYDGSNK (SEQ ID NO: 14); (ii) IWFDGRNK (SEQ ID NO: 60); (iii) IWYDGSNR (SEQ ID NO: 71); and (iv) IWYDGSSK (sequence number 76).

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

[0058] In one example, the VL comprises a CDR1 sequence selected from the group consisting of: (i) QRVRNNY (SEQ ID NO: 16); (ii) QSVRSSY (SEQ ID NO: 57); and (iii) QSIRSNY (SEQ ID NO: 78).

[0059] In one example, the VL comprises the CDR2 sequence GAS (SEQ ID NO: 28).

[0060] In one example, the VL comprises a CDR3 sequence selected from the group consisting of: (i) QQYGNSYT (SEQ ID NO: 18); (ii) QQYGRSYT (SEQ ID NO: 62); and (iii) QQYGSSYT (sequence number 58).

[0061] In another example, the PAR4 binding protein comprises the variable heavy (VH) sequence shown below:

[0062] In another example, the PAR4 binding protein comprises the variable heavy (VH) sequence shown below: TIFF0007733443000003.tif27151(in the sequence, X1 is A or S, X2 is T or A, X3 is V or I, X4 is Y or S, X5 is G or S, X6 is L or F, X7 is N, D, or T, X8 is Y or F, X9 is S or R, X 10 is R or H, X 11is N or I, X 12 is 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)

[0063] In one example, the PAR4 binding protein further comprises a variable light (VL) sequence shown below: TIFF0007733443000004.tif30155(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 14is E or G)

[0064] In one example, the VH comprises a CDR1 sequence selected from the group consisting of: (i) GGSLSDYY (SEQ ID NO: 86); (iii) SGSFSTYF (SEQ ID NO: 47); and (iv) GGSFSNYY (sequence number 66).

[0065] In one example, the VH comprises a CDR2 sequence selected from the group consisting of: (i) INHSGTT (SEQ ID NO: 87); (ii) IIHTGST (SEQ ID NO: 64); or (iii) INHSGST (SEQ ID NO: 48).

[0066] In one example, the VH comprises a CDR3 sequence selected from the group consisting of: (i) AIEYSNSRGYYYGMDV (SEQ ID NO: 88); (ii) AFEYSSSGGYYYGMDV (SEQ ID NO: 49); and (iii) KVEHSSSSGHYYYGMDV (SEQ ID NO: 65).

[0067] In one example, the VL comprises a CDR1 sequence selected from the group consisting of: (i) QTISNY (SEQ ID NO: 109); (ii) QSISSY (SEQ ID NO: 50); and (iii) QTISYY (SEQ ID NO: 66).

[0068] In one example, the VL comprises the CDR2 sequence AAS (SEQ ID NO: 51).

[0069] In one example, the VL comprises a CDR3 sequence selected from the group consisting of: (i) RQNYNTPLT (SEQ ID NO: 85); (iii) QQTYSTPLT (SEQ ID NO: 52); or (iv) QQSYSTPLT (sequence number 67).

[0070] The present disclosure also provides a PAR4 binding protein comprising a variable heavy chain (VH) having CDR1, CDR2, and CDR3 sequences that comprise or consist of, respectively, the following sequences: (i) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; (ii) SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49; (iii) SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; (iv) SEQ ID NO:55, SEQ ID NO:14, and SEQ ID NO:56; (v) SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61; (vi) SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65; (vii) SEQ ID NO: 68, SEQ ID NO: 14, and SEQ ID NO: 69; (viii) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; (ix) SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 74; (x) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77; (xi) SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; (xii) SEQ ID NO: 82, SEQ ID NO: 80, and SEQ ID NO: 83; (xiii) SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 74; or (xiv) SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88.

[0071] The present disclosure also provides a PAR4 binding protein comprising a variable light chain (VL) having CDR1, CDR2, and CDR3 sequences that each comprise or consist of the following sequences: (i) SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; (ii) SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52; (iii) SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29; (iv) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (v) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:62; (vi) SEQ ID NO: 66, SEQ ID NO: 51, and SEQ ID NO: 67; (vii) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (viii) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (ix) sequence number 57 , SEQ ID NO: 28, and SEQ ID NO: 58 ; (x) sequence number 78 , sequence number 28 , and SEQ ID NO: 62 ; (xi) SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 58; (xii) SEQ ID NO: 57, SEQ ID NO: 51, and SEQ ID NO: 58; (xiii) SEQ ID NO: 84, SEQ ID NO: 51, and SEQ ID NO: 85; or (xiv) SEQ ID NO: 109, SEQ ID NO: 51, and SEQ ID NO: 85.

[0072] In one example, the PAR-4 binding protein includes: (i) a VH comprising a sequence at least 50% identical to any one of 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, or a humanized, chimeric, or deimmunized version thereof; and / or (ii) A VL comprising a sequence that is at least 85% identical to the sequence set forth 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, the VH comprises a sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to any one of 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.

[0074] In one example, the VL comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to any one of 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, the VH comprises a sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 97%, 98%, 99%, or 99.5% identical to any one of 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 (excluding the CDR sequences).

[0076] In one example, the VL comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to 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 (excluding the CDR sequences).

[0077] In one example, the CDRs are defined by the IMGT numbering system.

[0078] The present disclosure also provides a PAR4 binding protein, including: (i) VH set forth in SEQ ID NO: 11 and VL set forth in SEQ ID NO: 12; (ii) a VH set forth in SEQ ID NO: 45 and a VL set forth in SEQ ID NO: 46; (iii) VH set forth in SEQ ID NO: 22 and VL set forth in SEQ ID NO: 23; (iv) VH set forth in SEQ ID NO: 53 and VL set forth in SEQ ID NO: 54; (v) a VH set forth in SEQ ID NO: 89 and a VL set forth in SEQ ID NO: 90; (vi) a VH set forth in SEQ ID NO: 91 and a VL set forth in SEQ ID NO: 92; (vii) a VH set forth in SEQ ID NO: 93 and a VL set forth in SEQ ID NO: 94; (viii) VH set forth in SEQ ID NO: 95 and VL set forth in SEQ ID NO: 96; (ix) a VH set forth in SEQ ID NO: 97 and a VL set forth in SEQ ID NO: 98; (x) a VH set forth in SEQ ID NO: 99 and a VL set forth in SEQ ID NO: 100; (xi) VH set forth in SEQ ID NO: 101 and VL set forth in SEQ ID NO: 102; (xii) VH set forth in SEQ ID NO: 103 and VL set forth in SEQ ID NO: 104; (xiii) VH set forth in SEQ ID NO: 105 and VL set forth in SEQ ID NO: 106; or (xiv) VH shown in SEQ ID NO: 107 and VL shown in SEQ ID NO: 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) linked to a 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) linked to a protein that binds to platelets (e.g., von Willebrand factor (vWF)).

[0080] In another example of the present disclosure, the VL and VH are separate polypeptide chains. For example, a PAR4 binding protein may comprise: (i) diabody; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab′)2; (vi) Fv; or (vii) at least one of (i)-(vi) linked to a 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) linked to a protein that binds to platelets (e.g., vWF).

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

[0082] Based on the present disclosure, it will be apparent to one of skill in the art that the PAR4 binding proteins of the present disclosure encompass human, humanized, synhumanized, chimeric, and primatized proteins.

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

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

[0085] In one example, a PAR4 binding protein or antibody of the present disclosure is conjugated to a moiety, such as a moiety selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the PAR4 binding protein in a subject, and mixtures thereof.

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

[0087] The present disclosure additionally provides an expression construct comprising a nucleic acid of the present disclosure operably linked to a promoter. Such an expression construct can be in a vector, for example, a plasmid.

[0088] In examples of the present disclosure directed to a single polypeptide PAR4 binding protein, the expression construct can include a promoter linked to a nucleic acid encoding that polypeptide chain.

[0089] In an example directed to multiple polypeptides that form a PAR4 binding protein, the expression construct of the present disclosure includes a nucleic acid encoding one of the polypeptides (e.g., including a VH) operably linked to a promoter and a nucleic acid encoding another of the polypeptides (e.g., including a VL) operably linked to a separate promoter.

[0090] In another example, the expression construct is a bicistronic expression construct, e.g., comprising the following components operably linked in 5' to 3' order: (i) promoters; (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide.

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

[0092] The present disclosure also contemplates separate expression constructs, one encoding a first polypeptide (e.g., comprising a VH and optionally a heavy chain constant region or portion thereof) and another encoding a second polypeptide (e.g., comprising a VL and optionally a light chain constant region). For example, the present disclosure contemplates separate expression constructs, one encoding a first polypeptide (e.g., comprising a VH and optionally a heavy chain constant region or portion thereof) and another encoding a second polypeptide (e.g., comprising a VL and optionally a light chain constant region). (i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH operably linked to a promoter); and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL operably linked to a promoter). Also provided is a composition comprising: wherein the first and second polypeptides associate to form a PAR4 binding protein of the disclosure.

[0093] The present disclosure additionally provides an isolated cell expressing the PAR4-binding protein or antibody of the present disclosure, or a recombinant cell genetically modified to express the PAR4-binding protein or antibody of the present disclosure. In one example, the cell is an isolated hybridoma. In another example, the cell contains a nucleic acid or expression construct of the present disclosure, or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH) operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL) operably linked to a promoter; The present invention relates to a PAR4-binding protein comprising 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] The present disclosure additionally provides a composition comprising a PAR4-binding protein or nucleic acid or expression construct or cell of the present disclosure and a suitable carrier. In one example, the composition comprises a PAR4-binding protein of the present disclosure.

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

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

[0097] The present disclosure also provides a method for treating or preventing thrombosis or thromboembolic disorders in a subject, comprising administering to the subject the PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure.In one example, the subject is at risk of PAR4-mediated events such as thrombosis.In one example, the subject has or has previously had PAR4-mediated events (for example, thrombosis).

[0098] In one example, the method comprises administering to a subject an antibody comprising a VH comprising the sequence set forth in any one of 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, or a humanized or deimmunized version thereof, and a VH comprising the sequence set forth in any one of 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 or deimmunized version thereof.

[0099] The present disclosure additionally provides a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure for use in medicine.

[0100] The present disclosure additionally provides a PAR4-binding protein or nucleic acid or expression construct or cell or composition of the present disclosure for use in treating or preventing a PAR4-mediated event (eg, thrombosis).

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

[0102] In some examples, the present disclosure provides a method for treating, preventing, or ameliorating thrombosis in a subject in need thereof, comprising administering to the subject in need thereof a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure.

[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 a PAR4-binding protein, nucleic acid, expression construct, cell, or composition of the present disclosure either before and / or after the surgical procedure. In one example, the surgical procedure is liver transplantation and the thrombosis is hepatic artery thrombosis.

[0104] In some examples, the present disclosure provides methods for determining whether a dose of a PAR4-binding protein or antibody according to the present disclosure is appropriate, including (i) obtaining a blood sample from a subject treated with a PAR4-binding protein or antibody of the present disclosure, (ii) treating platelets from the blood sample with a PAR4 agonist in vitro, (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 a blood sample obtained before treatment with the PAR4-binding protein or antibody.

[0105] Examples of suitable PAR4 agonists will be familiar to those skilled in the art, and non-limiting examples include agonist peptides such as AYPGKF-NH2 (Tocris).

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

[0107] In some embodiments, the present disclosure includes a method for inhibiting or preventing platelet aggregation, comprising administering to a subject (e.g., a human) in need thereof a therapeutically effective amount of a PAR4 binding protein according to the present disclosure.

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

[0109] Preferably, the subject is a human.

[0110] The present disclosure additionally provides uses of the PAR4 binding proteins or nucleic acids or expression constructs or cells or compositions of the present disclosure in the medical field.

[0111] The present disclosure additionally provides the use of a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure in the manufacture of a medicament for treating or preventing thrombosis or a thromboembolic disorder.

[0112] The present disclosure additionally 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 detecting the complex indicates PAR4 in the sample.

[0113] The present disclosure also provides a vaccine antigen comprising or consisting of a sequence according to SEQ ID NO: 4 together with a pharmaceutically acceptable carrier for generating antagonistic antibodies to human PAR4.

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

[0115] Thus, in one example, the present disclosure also provides a PAR4 binding protein comprising: (i) a VH comprising a sequence at least 50% identical to the sequence set forth in SEQ ID NO: 32, or a humanized, chimeric, or deimmunized version thereof; and / or (ii) A VL comprising a sequence that is at least 85% identical to the sequence set forth in SEQ ID NO: 33, or a humanized, chimeric, or deimmunized version thereof. [Brief explanation of the drawings]

[0116] [Figure 1] Schematic representation of the extracellular location of the thrombin cleavage and activation site of PAR4 and the anti-PAR4 target regions of the antibodies described herein. [Figure 2] Figure 1 shows the percentage of intact PAR4 present on the cell surface of HEK293 cells transfected with human PAR4 containing an N-terminal FLAG tag, as quantified by flow cytometry. Cells were pretreated with five different hybridoma supernatants (MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BBRH3, and MoB5CRC4) from the first hybridoma screen and then treated with thrombin (2 U / ml for 10 minutes). The positive control was a polyclonal anti-PAR4 antibody (French et al. (2016) Journal of Thrombosis and Haemostasis 14:1642-1654). Data are the mean + standard error of the mean for three individual data points. [Figure 3] Figure 1 shows the percentage of intact PAR4 (measured by % FLAG epitope) present on the cell surface of HEK293 cells transfected with PAR4 containing an N-terminal FLAG tag, as quantified by flow cytometry. Cells were pretreated with supernatants from two subclones of 5A.RC3 (designated B6b and H4b) and then treated with thrombin (2 U / ml for 10 minutes). Data are the mean + standard error of the mean for four individual data points. [Figure 4] Figure 1 shows the percentage of intact PAR4 (measured by % FLAG epitope) present on the cell surface of HEK293 cells transfected with either Thr120 or Ala120 of human PAR4 containing an N-terminal FLAG tag, as quantified by flow cytometry. Cells were pretreated with various concentrations of 5A.RC3 subclones as indicated and then treated with thrombin (2 U / ml). [Figure 5] (A) ELISA-based screening of the binding of hybridoma supernatants 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 of the binding affinity of PAR4 antibodies from 5A.RC3 subclones to human PAR4 peptides (measured at different concentrations). [Figure 6] 5A.RC3 inhibits thrombin cleavage of both Ala120 and Thr120 PAR4 variants. To assess thrombin cleavage of PAR4 variants, HEK293T cells were transiently transfected with either PAR4-120Ala or PAR4-120Thr variants, which contain a FLAG epitope upstream of the thrombin cleavage site. (A) Cells were stimulated with increasing doses (0.1–2 U / mL) of thrombin, and the amount of thrombin cleavage was measured by flow cytometry as loss of the FLAG epitope using a FITC-conjugated anti-FLAG antibody. (B) Preincubation of transfected cells with 5A.RC3 prior to thrombin stimulation resulted in similar and nearly complete inhibition of thrombin cleavage, regardless of the PAR4 variant. Doses of 1, 10, and 100 μg / mL of 5A.RC3 were compared. [Figure 7] The 5A.RC3 subclone inhibits the upregulated thrombin-induced platelet aggregation response in donors with the PAR4 Thr120 variant. The platelet aggregation response to a PAR4 agonist was assessed in human isolated platelets with different PAR4 variants. As expected, the presence of the T allele is associated with higher maximal aggregation in response to (A) PAR4-AP and (B) thrombin in the mid-dose range. A similar trend is observed in the case of thrombin stimulation in the presence of PAR1 blockade with (C) vorapaxal (90 nM). (D) The concentration response of 5A.RC3 inhibitory activity on thrombin-induced platelet aggregation in the presence of a PAR1 antagonist (i.e., PAR4-dependent) demonstrates efficacy for the Thr120 variant, indicating that antibody-mediated PAR4 inhibition is similarly effective across all genotypes. (E) IC50 of 5A.RC3 inhibitory activity on PAR4-dependent thrombin-induced platelet aggregation. [Figure 8] Flow cytometry shows that 5A.RC3 (10 ug / mL) binds to human PAR4 in isolated platelets as shown against an isotype control. [Figure 9]These results demonstrate that the PAR4 genotype is associated with an increased procoagulant platelet phenotype and can be targeted by antibody-mediated inhibition. The procoagulant activity of isolated human platelets was assessed by measuring phosphatidylserine exposure in response to (A) PAR4-activating peptide (AP) stimulation and (B) thrombin stimulation. Note that the Thr120 variant results in increased PS exposure in PAR4-AP-stimulated platelets. A similar trend is 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] These results demonstrate that the 5A.RC3 subclone inhibits 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 clotting conditions: (A) platelet deposition (PE-conjugated anti-CD9), thrombin activity (FRET-based thrombin probe), fibrin volume (Dylight650-conjugated anti-fibrin antibody), and fibrin per clot ratio (data shown is for the 10-minute endpoint). Note that the direct thrombin inhibitor hirudin (800 U / mL) abolished thrombin activity and fibrin volume despite continued platelet deposition. [Figure 10-2]These results demonstrate that the 5A.RC3 subclone inhibits 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 clotting conditions: (B-E) No significant differences in these parameters were observed across PAR4 genotypes. 5A.RC3 (open bars, 100 μg / mL) had no effect on (F) platelet deposition, but significantly inhibited (G) thrombin activity, (H) fibrin volume, and (I) the ratio of fibrin to clot volume compared to control (black bars). Data are mean ± SEM for N = 4-6 per genotype (n = 15 total; color indicates PAR4 genotype: black circles = AA, gray circles = AT, white circles = TT). For (G) and (H), data were normalized to hirudin baseline and expressed as a percentage of untreated controls. Statistical significance was determined by one-way ANOVA (B–E) or Student's t-test (F–G). * indicates P<0.05. [Figure 11] 1 shows the functional screening of initial hybridoma supernatants on platelet aggregation. [Figure 12] Figure 1 shows the amino acid sequences of VH (A) and VL (B) of MoB5A-RC3.F10b.H4b (5A.RC3) with CDRs identified according to IMGT numbering. VH = heavy chain variable region, VL = light chain variable region, CDR = complementarity-determining region, and FWR = framework region. This antibody demonstrated PAR4 inhibitory function. [Figure 13] Figure 1 shows the amino acid sequences of VH (A) and VL (B) of MoB5H-RD2.A7b (5H.RD2) with CDRs identified according to IMGT numbering. 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 function against PAR4. [Figure 14]Figure 1 shows the amino acid sequences of VH (A) and VL (B) of MoB5F-RF3.A7b.C9 (5F.RF3) with CDRs identified according to IMGT numbering. VH = heavy chain variable region, VL = light chain variable region, CDR = complementarity-determining region, and FWR = framework region. This antibody demonstrated PAR4 inhibitory function. [Figure 15] Figure 1 shows the reactivity of purified mAb with hPAR4 peptide by ELISA. Purified mAb bound to hPAR4 peptide in a dose-dependent manner as detected by anti-mouse Fc conjugated with alkaline phosphatase (Ap). No binding was observed with IC (isotype control) mAb raised against an unrelated non-PAR4 antigen. [Figure 16] ELISA shows that at 10 μg / ml, purified anti-HPAR4 mAb binds specifically to the hPAR4 biotinylated peptide and does not react nonspecifically with hPAR1, hPAR2, and hPAR3 (biotinylated peptides). [Figure 17] Flow cytometry shows concentration-dependent binding of purified anti-hPAR4 mAbs vs. isotype control to isolated human platelets. Raw data expressed as geometric mean fluorescence intensity are shown. Each mAb binds in a concentration-dependent manner. N=3-5. Data points are mean ± SEM. [Figure 18] Figure 1 shows the concentration-dependent inhibition of human platelet aggregation induced by 0.1 U / ml thrombin by three anti-hPAR4 mAb clones. Near-maximal inhibition was achieved by each clone at the highest concentration tested. The IC50 values ​​(μg / ml) determined from these concentration-inhibition curves are also shown in the figure. N=4-8. Data points are mean ± SEM. [Figure 19] Figure 1 shows inhibition of human clot formation by monoclonal antibodies 5A.RC3 and 5D.RH4. Pretreatment of blood with either 5D.RH4 or 5A.RC3 (both at 100 μg / ml) reduced total clot volume. Individual data points are shown. Bars are mean ± SEM. *P<0.05 (unpaired Student's t-test). [Figure 20] 1 shows the sequence of the variable heavy chain of the anti-PAR4 mAb showing the complementarity determining regions (CDRs) according to the IMGT numbering system. [Figure 21] 1 shows the sequence of the variable light chain of the anti-PAR4 mAb showing the complementarity determining regions (CDRs) according to the IMGT numbering system. [Figure 22] Synthetic peptides for epitope mapping of anti-hPAR4 mAb are shown. Overlapping peptides spanning the original human PAR4 antigen were synthesized. These consisted of amino acid residues 1–9, amino acid residues 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] Reactivity of purified hPAR4 mAb with peptides 1-9, 8-15, and 11-20 is shown. Absorbance values ​​were subtracted from background levels. A non-hPAR4 control mAb did not react with any of the three peptides.

[0117] Explanation of the sequence listing SEQ ID NO: 1: Epitope sequence of PAR4 SEQ ID NO: 2: Sequence of hPAR4 (naked) SEQ ID NO: 3: Sequence of mPAR4 (naked) SEQ ID NO: 4: Sequence of hPAR4 (KLH) used as immunogen SEQ ID NO: 5: Sequence of mPAR4 (KLH) used as immunogen SEQ ID NO: 6: Sequence of mPAR4 (biotin) SEQ ID NO: 7: Sequence of hPAR4 (biotin) SEQ ID NO: 8: Sequence of hPAR3 (biotin) SEQ ID NO: 9: Sequence of hPAR2 (biotin) SEQ ID NO: 10: Sequence of hPAR1 (biotin) SEQ ID NO: 11: Amino acid sequence of 5A.RC3 VH SEQ ID NO: 12: Amino acid sequence of 5A.RC3 VL SEQ ID NO: 13: Sequence of 5A.RC3 VH CDR1 SEQ ID NO: 14:5A.RC3 VH CDR2 sequence SEQ ID NO: 15:5A.RC3 VH CDR3 sequence SEQ ID NO: 16:5A.RC3 VL CDR1 sequence SEQ ID NO: 17:5A.RC3 VL CDR2 sequence SEQ ID NO: 18:5A.RC3 VL CDR3 sequence SEQ ID NO: 19: Sequence of human PAR4 SEQ ID NO: 20:5A.RC3 VH nucleic acid sequence SEQ ID NO: 21:5A.RC3 VL nucleic acid sequence SEQ ID NO: 22:5A. Amino acid sequence of RC3 VH SEQ ID NO: 23: Amino acid sequence of 5F.RF3 VL (5F.RF3) SEQ ID NO: 24: Sequence of 5F.RF3 VH CDR1 SEQ ID NO: 25: Sequence of 5F.RF3 VH CDR2 SEQ ID NO: 26: Sequence of 5F.RF3 VH CDR3 SEQ ID NO: 27: Sequence of 5F.RF3 VL CDR1 SEQ ID NO: 28: Sequence of 5F.RF3 VL CDR2 SEQ ID NO: 29: Sequence of 5F.RF3 VL CDR3 SEQ ID NO: 30: Nucleic acid sequence of 5F.RF3 VH SEQ ID NO: 31: Nucleic acid sequence of 5F.RF3 VL SEQ ID NO: 32: Amino acid sequence of 5H.RD2 VH SEQ ID NO: 33: Amino acid sequence of 5H.RD2 VL SEQ ID NO: 34: Sequence of 5H.RD2 VH CDR1 SEQ ID NO: 35: Sequence of 5H.RD2 VH CDR2 SEQ ID NO: 36: Sequence of 5H.RD2 VH CDR3 SEQ ID NO: 37: Sequence of 5H.RD2 VL CDR1 SEQ ID NO: 38: Sequence of 5H.RD2 VL CDR2 SEQ ID NO: 39: Sequence of 5H.RD2 VL CDR3 SEQ ID NO: 40: Nucleic acid sequence of 5H.RD2 VH SEQ ID NO: 41: Nucleic acid sequence of 5H.RD2 VL SEQ ID NO: 42: Epitope sequence SEQ ID NO: 43: Epitope sequence SEQ ID NO: 44: Epitope sequence SEQ ID NO: 45: Amino acid sequence of 5I.RG1 VH SEQ ID NO: 46: Amino acid sequence of 5I.RG1 VL SEQ ID NO: 47: Sequence of 5I.RG1 VH CDR1 SEQ ID NO: 48: Sequence of 5I.RG1 VH CDR2 SEQ ID NO: 49: Sequence of 5I.RG1 VH CDR3 SEQ ID NO: 50: Sequence of 5I.RG1 VL CDR1 SEQ ID NO: 51: Sequence of 5I.RG1 VL CDR2 SEQ ID NO: 52: Sequence of 5I.RG1 VL CDR3 SEQ ID NO: 53: Amino acid sequence of 5G.RA1 VH SEQ ID NO: 54: Amino acid sequence of 5G.RA1 VL SEQ ID NO: 55: Sequence of 5G.RA1 VH CDR1 SEQ ID NO: 14: Sequence of 5G.RA1 VH CDR2 SEQ ID NO: 56: Sequence of 5G.RA1 VH CDR3 SEQ ID NO: 57: Sequence of 5G.RA1 VL CDR1 SEQ ID NO: 28: Sequence of 5G.RA1 VL CDR2 SEQ ID NO: 58: Sequence of 5G.RA1 VL CDR3 SEQ ID NO: 89: Amino acid sequence of 5D.RH4 VH SEQ ID NO: 90: Amino acid sequence of 5D.RH4 VL SEQ ID NO: 59: Sequence of 5D.RH4 VH CDR1 SEQ ID NO: 60: Sequence of 5D.RH4 VH CDR2 SEQ ID NO: 61: Sequence of 5D.RH4 VH CDR3 SEQ ID NO: 57: Sequence of 5D.RH4 VL CDR1 SEQ ID NO: 28: Sequence of 5D.RH4 VL CDR2 SEQ ID NO: 62: Sequence of 5D.RH4 VL CDR3 SEQ ID NO: 91: Amino acid sequence of 5H.RH4 VH SEQ ID NO: 92: Amino acid sequence of 5H.RH4 VL SEQ ID NO: 63: Sequence of 5H.RH4 VH CDR1 SEQ ID NO: 64: Sequence of 5H.RH4 VH CDR2 SEQ ID NO: 65: Sequence of 5H.RH4 VH CDR3 SEQ ID NO: 66: Sequence of 5H.RH4 VL CDR1 SEQ ID NO: 51: Sequence of 5H.RH4 VL CDR2 SEQ ID NO: 67: Sequence of 5H.RH4 VL CDR3 SEQ ID NO: 93: Amino acid sequence of 5G.RF6 VH SEQ ID NO: 94: Amino acid sequence of 5G.RF6 VL SEQ ID NO: 68: Sequence of 5G.RF6 VH CDR1 SEQ ID NO: 14: Sequence of 5G.RF6 VH CDR2 SEQ ID NO: 69: Sequence of 5G.RF6 VH CDR3 SEQ ID NO: 57: Sequence of 5G.RF6 VL CDR1 SEQ ID NO: 28: Sequence of 5G.RF6 VL CDR2 SEQ ID NO: 58: Sequence of 5G.RF6 VL CDR3 SEQ ID NO: 95: Amino acid sequence of 5G.RD6 VH SEQ ID NO: 95: Amino acid sequence of 5G.RD6 VL SEQ ID NO: 70: Sequence of 5G.RD6 VH CDR1 SEQ ID NO: 71: Sequence of 5G.RD6 VH CDR2 SEQ ID NO: 72: Sequence of 5G.RD6 VH CDR3 SEQ ID NO: 57: Sequence of 5G.RD6 VL CDR1 SEQ ID NO: 28: Sequence of 5G.RD6 VL CDR2 SEQ ID NO: 58: Sequence of 5G.RD6 VL CDR3 SEQ ID NO: 97: Amino acid sequence of 5H.RA3 VH SEQ ID NO: 98: Amino acid sequence of 5H.RA3 VL SEQ ID NO: 55: Sequence of 5H.RA3 VH CDR1 SEQ ID NO: 73: Sequence of 5H.RA3 VH CDR2 SEQ ID NO: 74: 5H.RA3 VH CDR3 sequence SEQ ID NO: 57: Sequence of 5H.RA3 VL CDR1 SEQ ID NO: 28: Sequence of 5H.RA3 VL CDR2 SEQ ID NO: 58: Sequence of 5H.RA3 VL CDR3 SEQ ID NO: 99: Amino acid sequence of 5G.RG1 VH SEQ ID NO: 100: Amino acid sequence of 5G.RG1 VL SEQ ID NO: 75: Sequence of 5G.RG1 VH CDR1 SEQ ID NO: 76: Sequence of 5G.RG1 VH CDR2 SEQ ID NO: 77: Sequence of 5G.RG1 VH CDR3 SEQ ID NO: 78: Sequence of 5G.RG1 VL CDR1 SEQ ID NO: 28: Sequence of 5G.RG1 VL CDR2 SEQ ID NO: 62: Sequence of 5G.RG1 VL CDR3 SEQ ID NO: 101: Amino acid sequence of 5H.RG4 VH SEQ ID NO: 102: Amino acid sequence of 5H.RG4 VL SEQ ID NO: 79: Sequence of 5H.RG4 VH CDR1 SEQ ID NO: 80: Sequence of 5H.RG4 VH CDR2 SEQ ID NO: 81: Sequence of 5H.RG4 VH CDR3 SEQ ID NO: 57: Sequence of 5H.RG4 VL CDR1 SEQ ID NO: 28: Sequence of 5H.RG4 VL CDR2 SEQ ID NO: 58: Sequence of 5H.RG4 VL CDR3 SEQ ID NO: 103: Amino acid sequence of 5G.RC5 VH SEQ ID NO: 104: Amino acid sequence of 5G.RC5 VL SEQ ID NO: 82: Sequence of 5G.RC5 VH CDR1 SEQ ID NO: 80: Sequence of 5G.RC5 VH CDR2 SEQ ID NO: 83: Sequence of 5G.RC5 VH CDR3 SEQ ID NO: 57: Sequence of 5G.RC5 VL CDR1 SEQ ID NO: 51: Sequence of 5G.RC5 VL CDR2 SEQ ID NO: 58: Sequence of 5G.RC5 VL CDR3 SEQ ID NO: 105: Amino acid sequence of 5F.RE6 VH SEQ ID NO: 106: Amino acid sequence of 5F.RE6 VL SEQ ID NO: 55: Sequence of 5F.RE6 VH CDR1 SEQ ID NO: 73: Sequence of 5F.RE6 VH CDR2 SEQ ID NO: 74: Sequence of 5F.RE6 VH CDR3 SEQ ID NO: 84: Sequence of 5F.RE6 VL CDR1 SEQ ID NO: 51: Sequence of 5F.RE6 VL CDR2 SEQ ID NO: 85: Sequence of 5F.RE6 VL CDR3 SEQ ID NO: 107: Amino acid sequence of 5H.RF2 VH SEQ ID NO: 108: Amino acid sequence of 5H.RF2 VL SEQ ID NO: 86: Sequence of 5H.RF2 VH CDR1 SEQ ID NO: 87: Sequence of 5H.RF2 VH CDR2 SEQ ID NO: 88: Sequence of 5H.RF2 VH CDR3 SEQ ID NO: 109: Sequence of 5H.RF2 VL CDR1 SEQ ID NO: 51: Sequence of 5H.RF2 VL CDR2 SEQ ID NO: 85: Sequence of 5H.RF2 VL CDR3. DETAILED DESCRIPTION OF THE INVENTION

[0118] overview Throughout this specification, unless expressly stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0119] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, as well as any combination or any two or more of such steps or features.

[0120] The present disclosure is not intended to be 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 the present disclosure.

[0121] Any example of the present disclosure shall be construed as applying mutatis mutandis to any other example of the present disclosure, unless otherwise specified.

[0122] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0123] Unless otherwise indicated, the recombinant protein, cell culture, 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 in such sources as Perbal (1984), Sambrook et al. (1989), Brown (1991), Glover and Hames (1995 and 1996), Ausubel et al. (1988, including all current revisions), Harlow and Lane (1988), Coligan et al. (including all current revisions), 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 a discussion of the IMGT 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.

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

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

[0127] The present invention employs conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook et al., "Molecular Cloning," A Laboratory Manual (1989).

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

[0129] Furthermore, as used herein, "and / or" shall be construed as specifically disclosing the two specified features or components, with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in phrases 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); and 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 stated value. In general, the term "about" is used herein to modify a numerical value above and below by a variance of 10 percent (%) above or below (high or low) the stated value.

[0131] It is understood that the PAR4-binding proteins and antibodies, nucleic acids, cells, and vectors described herein are in isolated form. "Isolated" refers to a polypeptide, antibody, polynucleotide, vector, or cell in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, or cells include those that have been purified to the extent that they are no longer in a form 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 4 (PAR4)" refers to all or a portion 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). The polypeptide is characterized by having the ligand activation properties (including agonist activation and antagonist inhibition properties) and tissue distribution described herein. The term includes those PAR4 portions capable of binding to thrombin or the PAR4 receptor portion set forth in SEQ ID NO:2.

[0133] The term "PAR4 antagonist" refers to an inhibitor of platelet aggregation that binds to PAR4 and inhibits PAR4 cleavage and / or signaling. Typically, PAR4 activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in a dose-dependent manner 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 a fully human antibody. For naming and non-limiting 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 comprising murine constant region sequences and human variable region sequences.

[0136] The term "antibody" describes a natural, partially or wholly synthetically produced, or recombinantly produced immunoglobulin. The term also encompasses any polypeptide or protein having a binding domain that is, or is homologous to, an antibody binding domain. CDR-grafted antibodies are also contemplated by the term. An "antibody" is any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. The term encompasses polyclonal, monoclonal, multivalent, multispecific, chimeric, humanized, and human antibodies. The term "antibody" also refers to a protein comprising at least two immunoglobulin heavy (H) chains and two immunoglobulin light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). A CH is typically composed of three domains, CH1, CH2, and CH3 (e.g., IgM has an additional domain, CH4). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The CL is composed of one domain and can be of lambda or kappa type. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWRs). Each VH and VL is composed of three CDRs and four FWRs, arranged from the amino terminus to the carboxy terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. In certain embodiments, both the VH and VL comprise a binding domain that interacts with an antigen. In other embodiments, a single VH domain or a single VL domain can specifically interact with an 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 of the classical complement system (C1q).Antibody molecules can 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" also includes "chimeric" antibodies in which portions of the heavy and / or light chains are identical to 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(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Basic antibody structure in vertebrate systems is well understood. See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press). Any "antigen-binding fragment" is also included within 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) a Fab fragment, i.e., a monovalent fragment consisting of the VL and CL, VH and CH1 domains; (ii) an F(ab)2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and CL domains of a single antibody arm; (v) a single-domain antibody fragment or dAb (Ward et al., Nature 341:544-546 (1989)), consisting of only the VH or VL domain; and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VH and VL, are encoded by separate genes, they can be joined using recombinant or synthetic methods, for example, by a synthetic linker that enables them to be produced as a single protein chain, where the VH and VL regions pair to form a monovalent molecule (known as a single-chain 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). scFvs are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility 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 framework regions (FRs). 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 the present 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 antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced from any animal, e.g., mouse, rat, rabbit, pig, etc., or can be synthetically produced and can have partially or fully 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 generate antibodies against that antigen. Polyclonal antibodies can be generated from any mammal, e.g., mouse, rat, rabbit, pig, human, etc., or can be generated synthetically, e.g., as a phage display library of VH and VL genes.

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

[0142] The term "humanized antibody" shall be understood to refer to chimeric molecules, generally prepared using recombinant techniques, having an epitope-binding site derived from an immunoglobulin from a non-human species, while the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site preferably comprises complementarity-determining regions (CDRs) from the non-human antibody grafted into appropriate framework regions within the variable domains of a human antibody, with the remaining regions from the human antibody.

[0143] The term "human antibody," as used herein in connection with antibody molecules and binding proteins, refers to variable antibody regions (e.g., VH, VL, CDR, and FR regions) and constant antibody regions that are derived from or correspond to sequences found in a human (e.g., in a human germline or somatic cell).

[0144] As used herein, "IMGT numbering" refers to a numbering system used to identify CDR and FWR sequences of antibody variable regions. IMGT unique numbering is defined to compare 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 the 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 ndThe amino acid sequence of each CDR-IMGT fragment is a sequence of amino acids 118-118 (CYS), phenylalanine, or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides standardized boundaries for the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, and FR4-IMGT: positions 118-128) and the complementarity determining regions (CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, and CDR3-IMGT: positions 105-117). Gaps represent unoccupied positions, so the CDR-IMGT lengths are crucial information. The IMGT unique numbering is used in the 2D diagrammatic representations named 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 in the 3D structures 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" is intended to mean that a protein of the disclosure reacts with or associates with a particular cell or substance more frequently, rapidly, for longer duration, and / or with greater affinity than with alternative cells or substances. Reading this definition also indicates that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. Thus, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another antigen, as is intended by the term "selective binding."

[0146] "Transfected" and "transfected cell," and the like, refer to a cell that has been introduced into it (or into an ancestor of it) by means of genetic engineering a DNA molecule encoding PAR4 (or DNA encoding a biologically active fragment or analog thereof). Such a DNA molecule is "positioned for expression," meaning that the DNA molecule is positioned adjacent to DNA sequences that direct the transcription and translation of the sequences (i.e., drive the production of the PAR4 protein or fragment or analog thereof).

[0147] The term "identity" and its grammatical variations mean that two or more referenced entities are the same. Thus, 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 relative to a defined area (region or domain) of the sequence. The percent identity of polynucleotides 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 specified, the query sequence is at least 45 nucleotides in length, 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 in length, and the GAP analysis aligns the two sequences over a region of at least 100 nucleotides. Most preferably, the two sequences are aligned over their entire length.

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

[0149] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, and / or other problem or complication, within the bounds of sound medical evaluation, and commensurate with a reasonable benefit / risk ratio.

[0150] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder (e.g., the progression of a thromboembolic condition (e.g., acute coronary syndrome)). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), both detectable and undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0151] As used herein, "prophylaxis" or "prevention" refers to preventative treatment of an asymptomatic disease state in a mammal, particularly a human, with the goal of reducing the likelihood of a clinical disease state occurring. Patients are selected for preventative therapy based on factors known to increase their risk of suffering from a clinical disease state compared to the general population. "Prophylactic" therapy is classified as (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in subjects who have not yet exhibited a clinical disease state, while secondary prevention is defined as preventing the secondary occurrence of the same or similar clinical disease state.

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

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

[0154] "Subject" refers to any subject, particularly a 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, such as non-human primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles, etc. As used herein, phrases such as "subjects with a PAR4-mediated condition or disorder" include subjects, e.g., mammalian subjects, that would benefit from the administration of a PAR4 antagonist.

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

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

[0157] The term "competitively inhibit" is intended to mean that the protein of the present disclosure reduces or prevents the binding of the recited produced antibody (e.g., 5A.RC3) to the thrombin cleavage site of PAR4 or a fragment thereof. From the above, it will be apparent that the protein need not completely inhibit antibody binding; rather, it is sufficient if it reduces binding by a statistically significant amount, e.g., 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 a fragment thereof 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, the protein is considered to competitively inhibit antibody binding. In one example, the protein and antibody are exposed to PAR4 substantially simultaneously. Additional methods for measuring competitive inhibition of binding will be apparent to those skilled in the art and / or are described herein. In one example, the antigen-binding domain of the protein competitively inhibits antibody binding.

[0158] "Overlapping," in the context of two epitopes, shall be interpreted to mean that the two epitopes share a sufficient number of amino acid residues so that an antibody that binds to one epitope can competitively inhibit the binding of an antibody that binds to the other epitope. For example, the epitopes share at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids.

[0159] As used herein, the term "does not detectably bind" shall be understood to mean that a protein (e.g., an antibody) binds to a candidate antigen at a level that is less than 10%, or less than 8%, or less than 6%, or less than 5% above background. 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 using a biosensor assay (e.g., Biacore) where the protein is immobilized and contacted with the antigen.

[0160] antibody For the avoidance of doubt, the monoclonal antibody mAb ARC3 is synonymous with other names for this antibody, such as MoB5A-RC3, as shown in the Examples. This antibody was further subcloned to form the derivative monoclonal antibody MoB5-ARC3.F10b.H4b, which is also referred to as mAb ARC3.H4b. The sequence corresponding to this antibody is found in SEQ ID NOs: 11-18.

[0161] Functionally equivalent antibodies The present disclosure also contemplates anti-PAR4 antibodies or antigen-binding fragments thereof that contain one or more amino acid additions, deletions, or substitutions in the heavy and light chain variable region sequences of antibody mAb ARC3.H4b, but still retain the function of mAb ARC3.H4b. In some examples, the PAR4-binding protein contains 10 or fewer conservative amino acid substitutions, for example, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitution. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydrophobicity and / or hydrophilicity. The hydropathic index is described, for example, in Kyte and Doolittle (1982), and the hydrophilicity index is described, for example, in US4554101.

[0162] Such modifications may be deliberate, for example through site-directed mutagenesis, or may be accidental, such as modifications acquired through mutations in hosts expressing the antibody.

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

[0164] In designing amino acid sequence variants, the location of the mutation site and the nature of the mutation will depend on the feature(s) to be modified. The sites for mutation can be modified individually or sequentially, for example, by (1) substituting initially with conservative amino acid choices, followed by more radical choices depending on the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the positioned site.

[0165] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues, and typically about 1 to 5 contiguous residues.

[0166] Substitutional variants involve removing at least one amino acid residue in an antibody and / or immunoglobulin chain molecule, including the variable regions, and inserting a different residue in its place. Sites of greatest interest for 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 human antibodies and / or immunoglobulin chains, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading "Exemplary Substitutions."

[0167] Conservative amino acid substitutions are also contemplated by the present invention and are intended to mean the amino acid substitutions shown in the table below.

[0168] [Table 1]

[0169] The amino acids described herein are preferably in the "L" isomeric form. However, residues in the D isomeric form may be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin binding is retained by the polypeptide. Modifications also include structural and functional analogs, e.g., peptidomimetics having synthetic or unnatural amino acids or amino acid analogs and derivatized forms.

[0170] The present disclosure also contemplates non-conservative amino acid changes.For example, of particular interest is the substitution of a charged amino acid with another charged amino acid and a neutral or positively charged amino acid.In some examples, the PAR4 binding protein comprises 10 or less non-conservative amino acid substitutions, for example, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0171] Mutant forms of the PAR4-binding protein described herein according to any of the examples 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 contacted with immobilized PAR4 or a peptide containing the thrombin cleavage site of PAR4 (e.g., as shown in SEQ ID NO: 2). After washing, the bound label is detected. The labeled PAR4-binding protein is also contacted 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 (e.g., PAR1, PAR2, or PAR3) or mutant proteins or fragments of PAR4, 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 the present disclosure. In another example, the mutation(s) occur within the CDR of the PAR4 binding protein of the present 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, in such methods, an Fnl4 protein or an immunogenic fragment or epitope-containing, or expressing and displaying, cell (i.e., immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen 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 the immunized animal at various time points after immunization. One or more further immunizations can be given if necessary to achieve the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When the desired level of immunogenicity is obtained, the immunized animal is bled and the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0175] Monoclonal antibodies are exemplary antibodies contemplated by the present disclosure. The term "monoclonal antibody" or "mAb" or "MAb" refers to a homogeneous antibody population capable of binding to the same antigen(s), e.g., the same epitope within the antigen. The term is not intended to be limited by the source of the antibody or the manner in which it is made.

[0176] For the production of mAbs, any one of several known techniques can be used, for example, the procedures exemplified in U.S. Pat. No. 4,196,265, 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 the protein or its immunogenic fragment or epitope, or cells expressing 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 but not murine immunoglobulin proteins can also be used to generate antibodies of the present disclosure (e.g., 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 the immunized animal are then fused with cells of immortalized myeloma cells. The myeloma cells are generally 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 by Kohler and Milstein, (1975); and Kohler and Milstein, (1976). Methods using polyethylene glycol (PEG) (e.g., 37% (v / v) PEG) are described by Gefter et al., (1977) Somatic Cell Genet. 3(2):231-6. Electrically induced fusion methods are also suitable.

[0179] The hybrids are amplified by culturing in selective medium containing drugs that block de novo synthesis of nucleotides in tissue culture medium. Exemplary drugs are aminopterin, methotrexate, and azaserine.

[0180] The expanded hybridomas are subjected to functional selection for antibody specificity and / or titer, e.g., by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.). The present disclosure also contemplates subcloning of antibody-producing cells, e.g., as exemplified herein.

[0181] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate 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 by display libraries, eg, phage display libraries, such as those described in US Pat. No. 6,300,064, EP 0,368,684, and / or US Pat. No. 5,885,793.

[0183] Chimeric antibodies and proteins One example of an antibody or PAR4-binding protein of the present disclosure is a chimeric antibody. That is, the 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 murine animal such as a mouse or a rat) or in a protein belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in a protein derived from another species (e.g., a primate animal 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 comprising a VH and a VL from a non-human antibody (e.g., a murine antibody), and the remaining regions of the antibody are 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 accomplished by standard means (e.g., those described in 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 Nos. 5,807,715; 4,816,567; and 4,816,397). Furthermore, it is contemplated that the human constant region of the chimeric antibody of the invention may be selected from an IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19 constant region.

[0184] Humanized and Human Antibodies / Proteins The PAR4 binding proteins of the present disclosure can be humanized or human.

[0185] The term "humanized protein" shall be understood to refer to a protein comprising human-like variable regions (including CDRs) from an antibody from a non-human species grafted or inserted into FRs from a human antibody (this type of antibody is also called a "CDR-grafted antibody"). Humanized proteins also include proteins in which one or more residues of the human protein have been modified by one or more amino acid substitutions and / or one or more FR residues of the human protein have been replaced by corresponding non-human residues. Humanized proteins may also contain residues that are not found in either human or non-human antibodies. Any additional regions of the protein (e.g., the Fc region) are human. Humanization can be performed using methods known in the art, for example, US Pat. No. 5,225,539, US Pat. No. 6,054,297, US Pat. No. 7,566,771, or US Pat. No. 5,585,089. The term "humanized protein" also encompasses superhumanized proteins, for example, as described in US Pat. No. 7,732,578.

[0186] In one example, the humanized protein comprises the 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 sequences disclosed herein (numbering according to the IMGT numbering system).

[0187] As used herein, the term "human protein" refers to a protein having variable antibody regions and, optionally, constant antibody regions derived from or corresponding to sequences found in humans (e.g., in human germline or somatic cells). A "human" antibody can include amino acid residues not encoded by human sequences, e.g., mutations introduced in vitro by random or site-specific mutagenesis (particularly mutations involving conservative substitutions or mutations of a small number of residues in the protein, e.g., 1, 2, 3, 4, or 5 residues in the protein). Such "human antibodies" need not actually be produced as the result of a human immune response; rather, they 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 U.S. Pat. No. 5,565,332). The term also encompasses affinity-matured forms of such antibodies. Human proteins are also contemplated to include proteins comprising FRs from human antibodies or FRs comprising sequences from consensus sequences of human FRs, wherein one or more CDRs are random or semi-random as described in US6300064 and / or US6248516.

[0188] Human proteins or antibodies that recognize a selected epitope can also be generated using a technique called "directed selection," in which a selected non-human monoclonal antibody (e.g., a murine antibody) is used to direct 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 proteins of the present disclosure comprise the variable region of a human antibody.

[0190] Synthetic Humanized and Primatized Proteins The PAR4-binding protein of the present disclosure may be a synthetic humanized protein. The term "synthetic humanized protein" refers to a protein prepared by the method described in WO2007 / 019620. The synthetic humanized PAR4-binding protein comprises an antibody variable region whose variable region comprises FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region. For example, the synthetic humanized PAR4-binding protein comprises an antibody variable region whose variable region comprises FRs from a New World primate antibody variable region and CDRs from a mouse antibody (e.g., those described herein). In one example, the synthetic humanized PAR4-binding protein is a PAR4-binding antibody in which one or both variable regions are synthetically humanized.

[0191] The PAR4 binding protein of the present disclosure may be a primatized protein. A "primatized protein" comprises a variable region(s) from an antibody generated after immunization of a non-human primate (e.g., a cynomolgus macaque). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to produce a primatized antibody. An exemplary method for producing a primatized antibody is described in US6113898.

[0192] Deimmunized antibodies and proteins The present disclosure also contemplates deimmunized antibodies or PAR4-binding proteins. Deimmunized antibodies remove (i.e., mutate) one or more epitopes, such as B-cell epitopes or T-cell epitopes, thereby reducing the likelihood that a subject will develop an immune response to the antibody or protein. Methods for producing deimmunized antibodies and proteins are known in the art and are described, for example, in WO00 / 34317, WO2004 / 108158, and WO2004 / 064724.

[0193] Methods for introducing suitable mutations and expressing and assaying the resulting proteins will be apparent to those of skill in the art based on the disclosure herein.

[0194] Antibody variable region containing proteins. Single Domain Antibodies In some instances, the PAR4 binding proteins of the present disclosure are single domain antibodies (this term is used interchangeably with the terms "domain antibody" or "dAb"). Single domain antibodies are single polypeptide chains that contain all or part of the heavy chain variable region of an antibody. In certain instances, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., US6248516; WO90 / 05144; and / or WO2004 / 058820).

[0195] Diabodies, triabodies, and tetrabodies Exemplary PAR4 binding proteins that comprise an antibody antigen-binding domain 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 comprising the structure VL-X-VH or VH-X-VL (where VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, and X is absent or a linker comprising incomplete residues to allow the VH and VL within a single polypeptide chain to associate (or form an Fv), and the VH of one polypeptide chain binds to the VL of the other polypeptide chain to form an antigen-binding site, i.e., an Fv molecule capable of specifically binding to one or more antigens). The VL and VH can be the same in each polypeptide chain, or the VL and VH can be different in each polypeptide chain to form a bispecific diabody (i.e., comprising two Fvs with different specificities).

[0197] Single chain Fv (scFv) fragment Those skilled in the art will recognize that an scFv comprises a VH domain and a VL domain within a single polypeptide chain. The polypeptide chain further comprises a polypeptide linker between the VH and VL that enables the scFv to form the desired structure for antigen binding (i.e., the desired association of the VH and VL of the single polypeptide chain to form an Fv). For example, the linker comprises more than 12 amino acid residues, with (Gly4Ser)3 being one of the more preferred linkers for scFvs.

[0198] The present disclosure also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs) in which a single cysteine ​​residue is introduced into the VH FR or VL FR, and the cysteine ​​residues are linked by a disulfide bond to yield a stable Fv (see, e.g., Brinkmann et al. (1993) Proc Natl Acad Sci USA 90:547-551).

[0199] Alternatively or additionally, the present disclosure provides a dimeric scFv, i.e., a protein comprising two scFv molecules linked non-covalently or covalently, for example, by a leucine zipper domain (e.g., 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 both scFvs to form and bind to antigen, as described, for example, in US20060263367.

[0200] For a review of scFvs, 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 a minibody comprises the VH and VL domains of an antibody fused to the CH2 and / or CH3 domains of the antibody. Optionally, the minibody includes a hinge region between the VH and VL; this configuration is sometimes referred to as a Flex minibody. Minibodies do not contain a CH1 or CL. In one example, the VH and VL domains are fused to the hinge region and CH3 domain of an 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 their production are described, for example, in WO 94 / 09817.

[0202] Other antibody variable region containing proteins The present disclosure also contemplates other variable regions comprising PAR4 binding proteins, including, for example: (i) "key and hole" bispecific proteins as described in US 5,731,168; (ii) heteroconjugate proteins (e.g., those described in US 4,676,980); (iii) heteroconjugate proteins produced using chemical cross-linkers (e.g., those described in U.S. Pat. No. 4,676); (iv) Fab′-SH fragments (e.g., those described in Shalaby (1992) j Exp Med 1;175(1):217-25); (v) a single-chain Fab; or (vi) Fab3 (e.g., as described in EP19930302894).

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

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

[0205] Heavy chain immunoglobulins Heavy chain immunoglobulins are structurally distinct from many other immunoglobulin forms (e.g., antibodies) insofar as they contain heavy chains but no light chains. Thus, such immunoglobulins are also referred to as "heavy chain-only antibodies." Heavy chain immunoglobulins are found, for example, in camelids and cartilaginous fish (also called IgNARs).

[0206] The variable regions present in naturally occurring heavy chain immunoglobulins are generally referred to as "VHH domains" in camelid Igs and V-NARs in IgNARs to distinguish them from the heavy chain variable regions (called "VH domains") present in traditional four-chain antibodies and the light chain variable regions (called "VL domains") present in traditional four-chain antibodies.

[0207] Heavy chain immunoglobulins do not require the presence of light chains to bind relevant antigens with high affinity and specificity. This means that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easily expressed and generally stable and soluble. A general description of heavy chain immunoglobulins and their variable regions from camelids and methods for their production and / or isolation and / or use can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0208] A general description of heavy chain immunoglobulins and their variable regions from Chondrichthyes and methods for their production and / or isolation and / or use can be found, inter alia, in WO2005 / 118629.

[0209] V-like protein One example of a PAR4-binding protein of the present disclosure is a T cell receptor. T cell receptors have two V domains that combine to form a structure similar to the Fv module of an antibody. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991, describe how the two V domains (designated alpha and beta) of a T cell receptor can be fused and expressed as a single polypeptide chain, and how surface residues can be modified to reduce hydrophobicity, similar to antibody scFvs. Other publications describing the production of single-chain or multimeric T cell receptors containing two V-alpha and V-beta domains include WO1999 / 045110 or 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 disclosure of such V-like domain-containing proteins is contained in WO1999 / 045110.

[0211] Adnectins In one example, the PAR4 binding protein of the present disclosure is an adnectin.

[0212] Adnectins are based on the tenth fibronectin type III (10Fn3) domain of human fibronectin, in which the loop regions have been modified to confer antigen binding. For example, the three loops at one end of the 10Fn3 beta sandwich can be engineered to enable Adnectins to specifically recognize antigens. For further details, see US20080139791 or WO2005 / 056764.

[0213] Anticalin In a further example, the PAR4-binding protein of the present disclosure is an anticalin. Anticalins are derived from lipocalins, a family of extracellular proteins that transport hydrophobic small molecules (e.g., steroids, bilins, retinoids, and lipids). Lipocalins have a rigid beta-sheet secondary structure with multiple loops at the open end of a cone-shaped structure, which can be engineered to bind to antigens. Such engineered lipocalins are known as anticalins. For further description of anticalins, see US7250297B1 or US20070224633.

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

[0215] Abima In a further example, the PAR-binding protein of the present disclosure is an avimer. An avimer is a multi-domain protein derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by shuffling the natural variants represented by the A-domain family. For further details, see WO2002088171.

[0216] DARPins In a further example, the PAR4-binding protein of the present disclosure is a designed ankyrin repeat protein (DARPin). DARPins are 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 a-helices and one β-turn. DARPins can be engineered to bind to different target antigens by randomizing residues within the first a-helix and β-turn of each repeat. The binding interface of a DARPin can be increased by increasing the number of modules (affinity maturation). For further details, see US20040132028.

[0217] Other non-antibody polypeptides Other non-antibody proteins that contain binding domains include those based on human gamma-crystallin and human ubiquitin (affilin), the Kunitz-type domains of human protease inhibitors, the PDZ domain of the Ras-binding protein AF-6, scorpion toxin (charybdotoxin), and C-type lectin domains (tetranectin).

[0218] constant region The present disclosure encompasses PAR4 binding proteins comprising a variable region and a constant region or domain(s) thereof (e.g., CH2 and / or CH3). Those skilled in the art will be aware of 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 producing the PAR4-binding proteins of the present disclosure can be obtained from several different sources. In some examples, the constant region of the PAR4-binding protein or a portion thereof is derived from a human antibody. Furthermore, the constant domain or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, human isotype IgG1 is used.

[0220] A variety of constant region gene sequences are available in the form of publicly accessible deposits, or the sequences are available from publicly available databases. Constant regions can be selected with particular effector functions (or lacking particular effector functions) or with particular modifications to reduce immunogenicity.

[0221] In one example, a protein of the present disclosure has or exhibits an effector function that promotes or enables at least partial depletion, substantial depletion, or elimination of cells expressing PAR4. Such effector function can be enhanced binding affinity to an Fc receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0222] In one example, a PAR4 binding protein can induce enhanced levels of effector function.

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

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

[0225] Those skilled in the art will appreciate that greater effector function may be manifested in multiple ways, for example, as a greater level of effect, a more sustained effect, or a more rapid effect. Exemplary constant region modifications include amino acid substitutions, such as S239D / I332E (numbering according to the EU index of Kabat), or S239D / A330L / I332E (numbering according to the EU index of Kabat).

[0226] Further amino acid substitutions that increase the ability of an Fc region to induce effector function are known in the art and / or are described, for example, in US Pat. No. 6,737,056 or US Pat. No. 7,317,091.

[0227] In one example, glycosylation of the constant region is modified to increase the ability to induce enhanced effector function. In some examples, the Fc region according to the present disclosure comprises a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region, i.e., the Fc region is "non-fucosylated." Such variants may have improved ADCC induction ability. Methods for producing non-fucosylated antibodies include expressing Fnl4-binding proteins in cell lines that cannot express α1,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., 2004). Other methods include using cell lines that inherently produce antibodies capable of inducing enhanced effector function (e.g., duck embryonic stem cells for viral vaccine production (WO2008 / 129058); production of recombinant proteins in avian EBX® cells (WO2008 / 142124)).

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

[0229] Methods for determining the ability of an antibody or antigen-binding fragment thereof to induce effector function are known in the art and / or described herein.

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

[0231] Exemplary 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 protein of the present disclosure can comprise an IgG4 constant region or a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" should be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange, or the tendency to form Fab arm exchange or half antibodies, or to form half antibodies. "Fab arm exchange" refers to a type of protein modification in human IgG4 in which one IgG4 heavy chain and attached light chain (half molecule) are exchanged with a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro with purified blood cells or reducing agents such as reduced glutathione. A "half antibody" form is formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0232] In one example, a stabilized IgG4 constant region contains a 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 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 the Fc and Fab regions, conferring mobility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in disulfide bonding between the heavy chains. The hinge region is defined as stretching from Glu226 to Pro243 of human IgG11 according to the Kabat numbering system. Hinge regions of other IgG isotypes can be aligned with the IgGl1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions (see, for example, WO2010 / 080538).

[0233] modified proteins The present disclosure provides PAR4 binding proteins that have at least 80% identity to the sequences of the present disclosure and the same functional characteristics as described or claimed herein.

[0234] In one example, a PAR4 binding protein of the present 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 to a VL sequence disclosed herein (e.g., SEQ ID NO: 11).

[0235] In another example, a PAR4 binding protein of the present 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 to a VH disclosed herein (e.g., SEQ ID NO: 12).

[0236] The present disclosure also provides nucleic acids encoding the aforementioned proteins or nucleic acids that hybridize to the nucleic acids under medium to high stringency conditions.

[0237] The present disclosure also encompasses nucleic acids that encode proteins comprising the sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, which differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0238] Percent 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 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 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 lengths.

[0239] The glycosylation pattern of an antibody may be altered from the original glycosylation pattern of a reference antibody. Alteration refers to the deletion of one or more carbohydrate moieties found in the antibody and / or the addition of one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used). Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains the above-described tripeptide sequence (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0240] Modified glycoforms of the antibodies of the invention may be useful for various purposes, including, but not limited to, enhancing or reducing effector function and / or modifying the half-life of the antibody (see, e.g., WO / 2007 / 010401). Such modifications may result in decreased or increased C1q binding and CDC, or decreased or increased FcγR binding and ADCC. Substitutions may be made, for example, at one or more amino acid residues in the heavy chain constant region, resulting in altered effector function while retaining the ability to bind to antigen compared to the modified antibody (see, e.g., U.S. Pat. Nos. 5,624,821 and 5,648,260). Engineered glycoforms can be produced by any method known to one of skill in the art, for example, by use of engineered or variant expression strains, co-expression with one or more enzymes (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTII 1)), expression of the antibody or fragment thereof in a different organism or in a cell line from a different organism, or modification of the carbohydrate(s) after expression of the antibody or fragment. Methods for purifying engineered glycoforms are known in the art and include, but are not limited to, 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).See, e.g., WO00061739; EA01229125; US20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0241] Modifications of the effector function of the antibodies herein may be desirable, for example, to enhance 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 ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing or antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1-191-1-195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers such as those described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody may be engineered which contains dual Fc regions and may have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

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

[0243] affinity maturation In a further example, an existing PAR4-binding protein of the present disclosure is affinity matured to produce an antibody capable of binding to PAR4 with increased affinity. For example, the VL and / or VH-encoding sequence is isolated, and the CDR-encoding region (e.g., the region encoding the CDR3 of the VL and / or VH) is mutated to introduce one or more amino acid substitutions. The resulting mutated PAR4-binding protein is then screened for binding to PAR4, e.g., in a competitive assay.

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

[0245] Protein production In one example, a PAR4 binding protein of the present disclosure is produced by culturing a cell line (e.g., a hybridoma under conditions sufficient to produce the protein, e.g., 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 into one or more expression constructs (e.g., expression vector(s)) and then transfected into host cells (e.g., cells capable of providing disulfide bridges or bonds, such as 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 to achieve this end are known in the art and are described, for example, in 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] Once isolated, the nucleic acid encoding the protein of the present disclosure is inserted into an expression construct or replicable vector for further cloning (DNA amplification) or for expression in a cell-free system or within a cell. For example, the nucleic acid is operably linked to a promoter. As used herein, the term "promoter" is intended to be interpreted in its broadest context and includes the transcriptional control sequences of a genomic gene (including the TATA box or initiation element required for proper transcription initiation), with or without additional control elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that modify expression of the nucleic acid, for example, in response to developmental and / or external stimuli or in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe recombinant, synthetic, or fusion nucleic acids or derivatives that confer, activate, or enhance expression of an operably linked nucleic acid. Exemplary promoters can include additional copies of one or more specific control elements to further enhance expression of the nucleic acid and / or modify spatial and / or temporal expression.

[0248] As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0249] Cell-free expression systems are also contemplated by the present disclosure. For example, a nucleic acid encoding an Fnl4-binding protein of the present disclosure is operably linked to a suitable promoter (e.g., a 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 not limited to, the TNT T7 and T3 TNT systems (Promega), pEXP1-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 the present 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 secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

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

[0252] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-oc promoter (EF1), the small nuclear RNA promoters (Ula and Ulb), the oc-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter, and hybrid control elements containing the CMV enhancer / β-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, AUSTRALIAN CELL BANK CRL 1651); human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, AUSTRALIAN CELL BANK CCL 10); or Chinese hamster ovarian (CHO) cells.

[0253] Exemplary 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, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0254] Means for introducing isolated nucleic acid molecules or genetic 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 the known technique that has been used successfully. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection 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 microparticle bombardment (e.g., using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA), among others).

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

[0256] Protein isolation The PAR4 binding proteins of the present disclosure can be isolated or purified.

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

[0258] When using recombinant techniques, the PAR4 binding protein of the present disclosure can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.When producing the protein intracellularly, as a first step, particulate debris, host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration.When secreting the protein into the medium, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter (for example, Amicon or Millipore Pellicon ultrafiltration unit).A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious 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 of the foregoing. These methods are known in the art and are described, for example, in WO99 / 57134 or Zola (1997).

[0260] Those skilled in the art will also recognize that the PAR4-binding proteins of the present disclosure can be modified to include a tag to facilitate purification or detection, such as a polyhistidine tag, e.g., a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus type 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. For example, the tag is a hexa-his tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-his tag can be purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which 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 protein. Alternatively or additionally, a ligand or antibody that binds to the tag can be used in affinity purification methods.

[0261] combination The present disclosure also provides conjugates of the PAR4-binding proteins described herein according to any of the examples. 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, antiangiogenic agents, anti-angiogenic and / or other angiogenic agents, antiproliferative agents, pro-apoptotic agents, chemotherapeutic agents, or therapeutic nucleic acids. Toxins include any agent that is detrimental to cells (e.g., kills cells). For a description of classes of drugs known in the art and their mechanisms of action, see Goodman et al. (1990). Further techniques suitable for preparing immunoglobulin-immunotoxin conjugates are provided, for example, in US Pat. No. 5,194,594. Exemplary toxins include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diansin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, e.g., WO93 / 21232.

[0262] In one example, the PAR4 binding protein described herein according to any example is bound or linked to another protein, for example, an immunomodulator, or a half-life extending protein or peptide, or other protein that specifically binds to serum albumin.Exemplary serum albumin binding peptides or proteins are described in US20060228364 or US20080260757.

[0263] In another example, a protein may be conjugated to a "receptor" (e.g., streptavidin) for use in cell pre-targeting, in which case the conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a therapeutic agent (e.g., a radionucleotide).

[0264] The PAR4-binding protein of the present disclosure can be modified to include additional non-protein moieties that are known in the art and readily available. For example, moieties suitable 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 protein of the present 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, the PAR4 binding protein described herein according to any example includes one or more detectable markers to facilitate detection and / or isolation. For example, the compound includes fluorescent labels such as fluorescein (FITC), 5,6-carboxymethylfluorescein, Texas Red, nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2-phenylindole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), and rhodamine (5,6-tetramethylrhodamine). The absorption and emission maxima of 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 of the examples are labeled, for example, with fluorescent semiconductor nanocrystals (e.g., as described in U.S. Pat. No. 6,306,610).

[0266] Alternatively or additionally, the PAR4 binding protein is labeled with a magnetic or paramagnetic compound such as, for example, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt-platinum, or strontium ferrite.

[0267] Protein immobilization In one example, 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 proteins on a specific matrix, such as those described in WO99 / 56126 or WO02 / 26292.For example, immobilization can help stabilize proteins to prevent their activity from being reduced or adversely modified by biological, chemical, or physical exposure, especially during storage or single-batch use.Various methods for immobilizing proteins on a matrix are known in the art, including cross-linking, binding with a carrier, and retention in a semi-permeable matrix.Exemplary matrices include porous gel, aluminum oxide, bentonite, agarose, starch, nylon, or polyacrylamide.

[0268] Assaying the Activity of Binding Proteins of the Disclosure Binding assay One form of such an assay is an antigen-binding assay, such as that described in Scopes (1994) Protein Purification: Principles and Practice (Springer-Verlag). Such methods generally involve labeling a PAR4-binding protein and contacting it with an immobilized antigen or a fragment thereof, e.g., a protein containing the extracellular portion of PAR4 fused to biotin (e.g., as set forth in SEQ ID NO: 6). After washing to remove nonspecifically bound proteins, the amount of label, and consequently, bound protein, is detected. Of course, the PAR4-binding protein can be immobilized and the antigen labeled. Panning-type assays can also be used. The examples herein describe a binding assay based on flag-tagged PAR4, which can be expressed on the surface of HEK cells. Inhibition of PAR4 cleavage by the PAR4-binding protein in the presence of thrombin can be measured by flow cytometry.

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

[0270] Competitive binding assay Assays for determining PAR4-binding proteins that competitively inhibit the binding of an antibody of the present disclosure (e.g., mAb ARC3.H4b) will be clear to those skilled in the art. For example, an antibody of the present 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 contacted with PAR4 or its extracellular domain fused to the Fc region of the antibody or a peptide containing its epitope. The level of the labeled antibody is then measured and compared to the level measured when the labeled antibody is contacted 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 that in the absence of the PAR4-binding protein, the PAR4-binding protein competitively inhibits the binding of the antibody.

[0271] Optionally, the test PAR4-binding protein is conjugated to a label that is different from the antibody, which allows for the level of binding of the test PAR4-binding protein to the protein or epitope to be detected.

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

[0273] Epitope mapping assay In another example, the epitope to which the PAR4-binding protein described herein binds has been mapped. Methods for epitope mapping will be apparent to those skilled in the art. For example, a series of overlapping peptides, e.g., peptides containing 10-15 amino acids, spanning the PAR4 sequence or a region thereof containing the epitope of interest are generated. The PAR4-binding protein is then contacted with each peptide or a combination thereof to determine the peptide(s) to which the protein binds. This allows the determination of the peptide(s) containing the epitope to which the PAR4-binding protein binds. If the PAR4-binding protein binds to multiple non-contiguous peptides, it is possible that the PAR4-binding protein binds to a conformational epitope.

[0274] In one example, random fragments of PAR4 are expressed on the surface of phage, and the phage are contacted with a PAR4-binding protein. Phages bound by antibodies are then isolated, and the amino acid sequence of the expressed peptide can be deduced using the encoding nucleic acid contained within the phage. By isolating a series of phages with overlapping peptides, peptides containing regions of PAR4 that contain residues contained 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 binding of the PAR4-binding protein. Any mutation that reduces or prevents binding of the PAR4-binding protein may be present within the epitope to which the PAR4-binding protein binds.

[0276] A further method involves binding PAR4 or a region thereof to an immobilized PAR4-binding protein of the present disclosure and digesting the resulting complex with a protease. The peptides that remain bound to the immobilized PAR4-binding protein are then isolated and analyzed, for example, using mass spectrometry, to determine the sequence of the peptides.

[0277] Another method is to convert the hydrogen in PAR4 or its region into deuterium nucleus (deutron), and then bind the resulting protein to the immobilized PAR4 binding protein of the present disclosure.Then, convert the deuterium nucleus back to hydrogen, isolate PAR4 or its region, digest it with enzyme, and analyze it by, for example, mass spectrometry, to identify the region that contains deuterium nucleus.It is believed that this region is protected from being converted to hydrogen by the binding of the PAR4 binding protein described herein.

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

[0279] Affinity assay Optionally, the dissociation constant (Kd) or association constant (Ka) or binding constant (KD, i.e., Ka / Kd) of the PAR4-binding protein for PAR4 or its epitope-containing peptide is measured. For example, these constants for the PAR4-binding protein are measured by a radiolabeled or fluorescently labeled PAR4 binding assay. In this assay, the PAR4-binding protein is equilibrated with a minimum concentration of labeled PAR4 in the presence of a titration series of unlabeled PAR4. After washing to remove unbound PAR4, the amount of label is measured. According to another example, the coefficient is measured 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 assays One example of the present disclosure is detecting the presence of PAR4 or cells (e.g., platelets) expressing PAR4. The amount, level, or presence of proteins or cells is measured using any of a variety of techniques known to those skilled in the art, for example, a technique selected from the group consisting of flow cytometry, immunohistochemistry, immunofluorescence, immunoblot, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay, 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 technology, evanescent fiber optic technology, or protein chip technology.

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

[0282] For example, the protein is detected by immunoassay, e.g., using an assay selected from the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), Western blotting, radioimmunoassay (RIA), biosensor assay, protein chip assay, immunostaining assay (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 the present disclosure or a protein that binds to a different epitope of PAR4 on a solid matrix, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick, or a glass support.The sample is then brought into physical contact with the immobilized protein, and PAR4 is bound or "captured."The bound PAR4 is then detected using a second labeled compound that binds to a different epitope of PAR4.Alternatively, a third labeled antibody that binds to the second (detection) antibody may be used.It will be clear to those skilled in the art that the assay format described herein is applicable to high-throughput formats, such as automated screening processes or microarray formats.In addition, variations of the above-mentioned assays, such as competitive ELISA, will be clear to those skilled in the art.

[0285] In another example, polypeptides are detected in 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 generally use an electrode surface combined with a current- or impedance-measuring element to integrate an assay substrate (e.g., as described in US5567301) into the device. The PAR4-binding proteins of the present disclosure are incorporated onto the surface of the biosensor device, and a biological sample is contacted with the device. A change in current or impedance detected by 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 reflectance at the surface plasmon resonance surface indicates protein binding 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, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This allows for the simultaneous detection of several proteins or peptides in small volumes of body fluids.

[0288] 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 antibodies useful herein include in vitro exposure of lymphocytes to PAR4 protein or PAR4 peptides (e.g., as described herein) and selection of antibody display libraries in phage or similar vectors (e.g., by using immobilized or labeled PAR4 protein or peptide). Genes encoding polypeptides with promising PAR4 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage (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 for peptides that interact with known targets, which can 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 art (Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484, and Ladner et al., U.S. Pat. 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 to tag cells, isolate homologous polypeptides by affinity purification, or be directly or indirectly conjugated to drugs, toxins, radionucleotides, and the like. Such binding proteins can also be used, for example, in expression libraries and analytical methods for screening neutralizing activity. Binding proteins can also be used in diagnostic assays to measure circulating levels of polypeptides or to detect or quantify soluble polypeptides as markers of underlying pathologies 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 believed to be useful for inhibiting cellular responses to protease-activated PAR4.

[0290] Various assays known to those skilled in the art can be used to detect antibodies that specifically bind to PAR4 protein or peptide.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 assay, inhibition or competition assay, and sandwich assay.In addition, antibodies can be screened for binding to wild-type vs. mutant PAR4 protein, polypeptide, or fragment.

[0291] Assay of functional characteristics of PAR4-binding proteins The antithrombotic activity of PAR4-binding proteins against all PAR4 variants can be verified in 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 drugs was confirmed by the combination of inhibitors of existing antiplatelet pathways (aspirin (50 μM), P2Y 12 This can be verified by measuring the antithrombotic effect in the absence and presence of the inhibitor 2-MeSAMP (50 μM or 100 μM). The PAR1 inhibitor vorapaxal (100 mM) is also used in ex vivo platelet aggregation assays for comparative studies of the antithrombotic effect with PAR4-binding proteins. If we show that thrombosis occurs independently of these mechanisms, high shear conditions (3000 s -1 ) (Neeves KB et al. (2008) J Thromb Haemost 6:2193-2201).

[0293] Additionally or alternatively, in vivo thrombosis experiments in mice (Lee H et al. (2012) Brit J Pharmacol 166:2188-2197; Mountford JK et al. (2015) Nat Commun 6:6535) may be used to verify the functionality of PAR4-binding proteins. To ensure the inclusion of a positive control for anti-PAR4 activity in such mouse experiments, antigens corresponding to either the mouse or human receptor sequences (Table 2) can be used for screening as outlined above to verify the antithrombotic effects of the generated antibodies. Note that primates are the only species known to have platelets that express only the combination of PAR1 and PAR4. Mouse platelets express PAR3 and PAR4, and only PAR4 is functional. Therefore, while such studies are limited to in vivo mechanistic validation, they represent the most relevant in vivo validation of the antithrombotic activity of PAR4-binding proteins outside of human trials and preclinical studies in nonhuman primates. Electrolytic injury of the carotid artery in anesthetized mice can be used to examine the effects of PAR4-binding proteins 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 assessed for thrombus formation (time to arterial occlusion) and stability (number and extent of recanalization events after occlusion), as well as total blood flow through the injured artery and thorough examination of thrombus histology by Carstair staining of paraffin-embedded arterial cross 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 (e.g., HEK293T cells) transfected with PAR4 constructs or PAR4 polymorphic variants are useful systems for studying PAR4 antagonists. PAR4-transfected cells are used to screen for ligands for the receptor and also for antagonists of the natural ligand. Briefly, this approach involves combining the cDNA or receptor-encoding gene with other genetic elements (e.g., transcription promoter) required for its expression, and then inserting the resulting expression vector into host cells. Cells that express the DNA and produce functional receptors 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 biological responses in target cells. An increase in metabolism above control values ​​indicates a test compound that modulates PAR4 activity or response. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected, for example, by measuring the incorporation of tritiated thymidine or by a colorimetric assay based on the metabolic decomposition of 3-(4,5-dimethylthiazol-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 containing a receptor of interest on the cell surface and receptor (-) cells that do not express the receptor of interest. Such cells can be engineered to express a reporter gene. The reporter gene is linked to a promoter or response element responsive to a receptor-linked pathway, and the assay detects transcriptional activity of the reporter gene. Suitable response elements include cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response elements (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-77, 1990), and serum response elements (SRE) (Shaw et al., Cell 56:563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263(19):9063-66; 1988; and Habener, Molec. Endocrinol. 4(8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44, 1989. A preferred promoter element in this regard is a serum response element, or SRE (see, e.g., Shaw et al., Cell 56:563-72, 1989).A preferred reporter gene is the luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). The expression of the luciferase gene 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-conditioned medium, fungal broth, soil samples, water samples, etc. This type of assay detects compounds that directly block PAR4 ligand binding and compounds that further block the processes of cellular pathways subsequent to receptor-ligand binding. Alternatively, compounds or other samples can be tested for direct blocking of PAR4 binding using moieties tagged with detectable labels (e.g., 125 I, biotin, horseradish peroxidase, FITC, etc.). In 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 using ligand-binding receptors or antibodies or their binding fragments and commercially available biosensor devices (BIAcore, Pharmacia Biosensor, Piscataway, NJ) can 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 using amine or sulfhydryl chemistry to dextran fibers attached to a gold film within a flow cell. A test sample is passed through the cell. If a ligand or epitope is present in the sample, it 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 allows for the measurement of on- and off-rates (from which binding affinity can be calculated) and assessment of the stoichiometry of binding.

[0298] Ligand-binding receptor polypeptides can also be used in other assay systems known in the art, including the Statchard assay (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) for measuring binding affinity.

[0299] The FLIPR assay is an exemplary in vitro assay for measuring the activity of the PAR4 antagonists of the present invention, in which a PAR4 agonist induces intracellular calcium mobilization in PAR4-expressing cells and monitors calcium mobilization.

[0300] The PAR4-binding proteins of the present disclosure can be tested in vitro for their ability to inhibit platelet aggregation induced by gamma-thrombin. Gamma-thrombin is a proteolytic product of alpha-thrombin 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 1b," J. Biol. Chem., 276:21173-21183 (2001)). Platelet aggregation can be monitored in a 96-well microplate aggregation assay format or using a standard aggregometer. Aggregation assays 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 ability of selective PAR4 antagonists to inhibit platelet aggregation can be measured using a standard optical aggregometer.

[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 adding tissue factor and CaCl2. Tissue factor, the initiator of the extrinsic coagulation cascade, is highly elevated in human atherosclerotic plaques. Exposing blood to tissue factor at the atherosclerotic site activates robust thrombin generation, inducing the formation of an occlusive thrombus.

[0303] The effectiveness 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 effectiveness of the PAR4 antagonist of the present invention as an antithrombotic agent include, but are not limited to, guinea pigs and primates.Relevant efficacy models include, but are not limited to, electrolyte injury carotid artery thrombosis, FeCl3-induced carotid artery thrombosis, and arteriovenous shunt thrombosis.Kidney bleeding time, renal bleeding time, and other bleeding time measurement models 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 for their ability to inhibit thrombus formation induced by electrolytic injury to the carotid artery.

[0305] Platelet aggregation assay Microplate-based platelet light transmission aggregometry can be used to measure platelet aggregation (French et al (2016) Journal of Thrombosis and Haemostasis 14:1642-1654).

[0306] This test (Born GV (1962) Nature 194:927-929) assesses in vitro platelet clump formation, i.e., aggregation, a key function of platelets, in a glycoprotein (GP) IIb / IIIa-dependent manner. The assay is based on measuring the increase in light transmittance through an optically dense sample 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 settling of platelet aggregates. This determines the increase in light transmittance through the plasma sample. The device photometrically records the rate and maximum percentage of this increase 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 to a graphical curve corresponding to the increase in light transmittance during platelet aggregation. Available aggregometers are easy-to-use devices with automatic settings (100% and 0%), software for storing results, and disposable cuvettes with a stir bar. The slope of the curve, the maximum degree of aggregation (%), and the latency time (induction period) are automatically measured parameters, allowing the change in shape and primary and secondary aggregation to be viewed graphically. Different agonists are added to samples of PRP or washed platelets to stimulate different platelet activation pathways, obtaining information about several features of platelet function. The Born aggregometry method 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 proteins can be measured using bleeding time (BT) (Duke WW et al (1910) JAMA 55:1185-1192). BT assesses platelet ability to generate a hemostatic plug by recording the time it takes platelets to close an in vivo skin wound to stop bleeding.

[0308] Impedance whole blood aggregometry (WBA) allows the evaluation of platelet function without any sample processing by using anticoagulated whole blood (WB) as the environment (Mackie IJ, et al. (1984) J Clin Pathol. 37:874-878). The 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 in a WB sample. Platelet aggregation is assessed by detecting the increase in electrical impedance generated by the aggregation of other platelets on the platelets immobilized on the electrodes. Therefore, reducing the current intensity increases the electrical impedance. The magnitude of the increase in impedance is recorded in ohms.

[0309] Luminal aggregometry allows for 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 activated platelets by different agonists using luminescence techniques in PRP, washed platelets (WP), or WB. The assay is based on the conversion of ADP released from platelet dense granules to ATP, which reacts with a luciferin-luciferase reagent. The emitted light is proportional to the ATP concentration and is quantified by the aggregometer.

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

[0311] Calcium signaling assay Calcium flux 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 can be utilized by those skilled in the art to further or additionally screen, evaluate, and / or confirm the antibodies or fragments thereof of the present disclosure, including further evaluating PAR4 activation or antithrombotic effects in vivo. Such animal models include, but are not limited to, models that subject the carotid artery to electrolytic injury to examine thrombus formation (time to arterial occlusion), stability (number and extent of recanalization events after occlusion), and total blood flow through the injured artery.

[0313] An exemplary or suitable 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 active ingredient) of the present disclosure is useful for formulation into pharmaceutical compositions for parenteral, topical, oral, or localized administration, aerosol administration, or transdermal administration for preventive or therapeutic treatment.Pharmaceutical compositions can be administered in various unit dosage forms depending on the administration method.For example, suitable unit dosage forms for oral administration include powder, tablet, pill, capsule, and lozenge.

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

[0316] Compositions for administration will generally comprise a solution of the PAR4-binding protein of the present disclosure dissolved in a pharmaceutically acceptable carrier, e.g., 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 the present disclosure in such formulations can vary widely and is selected primarily based on liquid volume, viscosity, body weight, and the like, depending on the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose 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 minor amounts of additives, such as buffers and preservatives, that enhance isotonicity and chemical stability.

[0317] The PAR4 binding proteins of the present disclosure can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, transdermal, or other such routes, including peristaltic administration and direct instillation (intracavitary administration) into tumor or disease sites. The preparation of an aqueous composition containing a compound of the present disclosure as an active ingredient will be known to those skilled in the art.

[0318] Suitable pharmaceutical compositions according to the present disclosure generally comprise an amount of a PAR4-binding protein of the present disclosure mixed with an acceptable pharmaceutical carrier (e.g., a sterile aqueous solution) to provide a range of final concentrations depending on the intended use. Preparation techniques are well known in the art, as exemplified by Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company, 1980.

[0319] Upon formulation, the compounds of the present disclosure are administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically / prophylactically effective. Suitable dosages of the compounds of the present disclosure will vary depending on the particular compound, the condition being treated, and / or the subject being treated. It is within the skill of one of ordinary skill in the art to determine suitable dosages, for example, by starting with a suboptimal dosage and gradually increasing the dosage to determine an optimal or useful dosage.

[0320] Exemplary dosages and administration timing will be clear to those skilled in the art based on the disclosure herein. A preferred dose of a PAR4 antagonist is a biologically active dose. A biologically active dose is a dose that inhibits PAR4 cleavage and / or signaling and has an antithrombotic effect. Desirably, a 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% compared to the level of an untreated control. The level of PAR4 in platelets can be measured by any method known in the art, including, for example, receptor binding assay, platelet aggregation, platelet activation assay (e.g., p-selectin expression by FACS), Western blot, or ELISA analysis. Alternatively, the biological activity of PAR4 can be measured by evaluating intracellular signaling induced by PAR4 (e.g., calcium mobilization or other second messenger assay).

[0321] In some examples, the therapeutically effective amount 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 more preferred embodiments, the therapeutically effective amount of the PAR4 compound is less than 5 mg / kg. In the most preferred embodiment, the therapeutically effective amount of the PAR4 compound is less than 1 mg / kg. As will be appreciated by those skilled in the art, the effective dose will vary depending on the route of administration and the use of excipients.

[0322] In some instances, liposomes and / or nanoparticles may also be used with PAR4-binding proteins. The formation and use of liposomes is well known to those skilled in the art. Liposomes can be formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 angstroms and containing aqueous solution at their cores. When dispersed in water, phospholipids can form various structures other than liposomes, depending on the lipid to water molar ratio. Liposomes at low ratios are the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but at elevated temperatures, they undergo a phase transition that significantly changes their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the fluid state.

[0323] The compositions may be administered alone or in combination with other treatments, therapies, or agents, either simultaneously or sequentially, including, but not limited to: (i) an anticoagulant, for example, an Fxa inhibitor, a FXIa inhibitor such as apixaban or rivaroxaban, or a thrombin inhibitor such as dabigatran; (ii) an antiplatelet agent, e.g., aspirin or a P2Y12 antagonist (e.g., clopidogrel, ticagrelor, or prasugrel); (iii) Angiogenic agents, for example, angiogenesis inhibitors.

[0324] Treatment method As discussed herein, the PAR4 binding proteins of the present disclosure can be used to treat, prevent, or ameliorate a thrombosis or thromboembolic disorder in a subject.

[0325] Thrombosis refers to the formation or presence of a blood clot (thrombus: plural thrombi) within a blood vessel, which can cause ischemia or infarction of the tissue supplied by the blood vessel.

[0326] Thromboembolic disorders are characterized by the sudden blockage of an artery by a blood clot (e.g., an embolism) or foreign body carried by the bloodstream to a site of deposition. "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 (defined above).

[0327] Thromboembolic disorders include arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the ventricles or peripheral circulation. As used herein, the term "thromboembolic disorders" also includes specific disorders selected from, but not limited to, 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 artery 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 artificial surfaces that promote thrombosis. Medical implants or devices include, but are not limited to, prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, 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 embolic stroke or atherothrombotic stroke resulting from occlusive thrombosis in the carotid communis, carotid interna, or intracerebral arteries.

[0329] kit The present disclosure also provides therapeutic / prophylactic / diagnostic kits containing the compounds of the present disclosure for use in the detection / diagnosis / prognosis / treatment / prevention methods of the present disclosure. Such kits generally contain the PAR4-binding proteins of the present disclosure in suitable packaging. The kits can also contain other compounds, for example, for detection / isolation / diagnosis / imaging or combination therapy. For example, such kits can contain any one or more of a series of anticoagulants or antiplatelet agents.

[0330] In one example, the kit is for treating or preventing a condition.In such kit, PAR4 binding protein can be provided in solution or lyophilized form, optionally with a solution for resuspension.PAR4 binding protein can be conjugated with a therapeutic compound, or the kit can include a therapeutic compound for conjugation.

[0331] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0332] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent.

[0333] The present invention is further illustrated in the following non-limiting examples. [Example]

[0334] method Antibody generation Anti-PAR4 monoclonal antibodies were produced by immunizing HumAb mice (Regeneron Pharmaceuticals) with the C-terminal KLH (keyhole limpet hemocyanin)-conjugated peptides listed in Table 2 below. The immunizing peptides correspond to the N-terminal thrombin cleavage and activation site of hPAR4 (SEQ ID NO: 2). The cleavage site is indicated by RG and is underlined in the human PAR4 sequence below. Screening was performed against the naked peptide (SEQ ID NO: 3) TIFF0007733443000006.tif1475.

[0335] Mice were intraperitoneally immunized three times at two-week intervals with a combination of 16 μg of antigen and immune adjuvant (Sigma Aldrich Catalog No. S6322) combined with methylated CpG. Serum samples were collected from immunized mice, and reactivity to the antigen was tested by ELISA at dilutions of 1:250 and 1:1250 and compared with pre-immunization samples. A difference of more than three-fold increase was required between pre- and post-immunization serum titers at 1:250 and 1:1250.

[0336] Mice with the highest titers were selected for fusions.

[0337] Immunizing peptide The immunizing peptide and SKB-labeled peptide (shown in Table 2) were synthesized by Auspep (Melbourne, Australia) using solid phase synthesis.

[0338] Biotin was attached to the peptide at the C-terminus using a serine (S) and lysine (K) linker (SK) (see Table 2). Biotin was added to the C-terminal lysine residue by chemical conjugation to generate SKB, as shown in Table 2.

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

[0340] Hybridoma growth To generate hybridoma cells, mouse spleens were removed, dissociated into single-cell suspensions, and 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, at which point the number of hybridoma colonies (expressed as fusion efficiency) was determined, and after an additional 3 days of incubation, aliquots of antibody supernatants were taken for screening. Reactivity of the supernatants to antigen and any screening samples was assayed first by microarray and then by ELISA for any IgG-positive clones.

[0342] The most responsive ELISA-positive clones were then grown in 24-well tissue culture plates for 3-4 days, at which point they were expanded into 6-well tissue culture plates. Cells were seeded at 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. The supernatants from the supernatant wells were pooled and frozen at -20°C.

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

[0344] Supernatants from the subclonal cell lines were tested with a commercially available assay kit to determine the isotype of the monoclonal antibodies being produced.

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

[0346] ELISA screening of monoclonal antibodies 96-well ELISA plates were 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)). Plates were incubated overnight at 4°C.

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

[0348] Fifty microliters of undiluted hybridoma cell culture supernatant was added to the appropriate wells, incubated for 1 hour at room temperature, and 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 1x PBS (8% sodium chloride (NaCl, Merck #1.06404.5000), 0.2% potassium chloride (KCl, Merck #1.04936.0500), 1.44% disodium hydrogen phosphate (NaHPO, Merck #1.06586.0500), 0.24% potassium dihydrogen orthophosphate (KHPO, Merck #1.04873.0500)). 50 μl of diluted antibody was added to each well and incubated for 1 hour at room temperature. Wells were washed as described above. A set of substrate tablets (1x silver, 1x gold) was placed in 20 ml of ddH2O by vigorously shaking on a thermomixer for 6 minutes at room temperature. 50 μl of substrate (SIGMAFAST™ p-nitrophenyl phosphate (Sigma-Aldrich, N2770-50SET) tablets) was added to each well and incubated for 20–25 minutes at room temperature. 50 μl of stop solution (2 M sodium hydroxide, solid (NaOH) (Merck, Cas#1310-73-2)) was added to each well to terminate the reaction. Absorbance was read at 405 nm using an ELISA reader (RdrOle4) immediately after adding the stop solution.

[0349] human blood samples After obtaining informed consent, blood was collected from healthy adults (male and female, aged 21–50 years) who had not taken antiplatelet medications in the past 10 years. Blood was collected from the antecubital vein using a 19-gauge butterfly needle into a syringe containing one-seventh volume of acid citrate dextrose (ACD) (7:1 v / v final concentration) for platelet isolation or one-tenth volume of trisodium citrate (0.32% w / v final concentration) for whole blood flow experiments as previously described (Mountford JK et al. (2015) Nat Commun 6:6535).

[0350] mouse HumAb mice (Murphy AJ et al. (2014) Proc Natl Acad Sci USA 111:5153-5158) were obtained from Regeneron Pharmaceuticals. HumAb mice have been genetically engineered to enable the generation of human antibody responses by replacing 3 Mb segments of the mouse heavy variable Ig locus and kappa 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 exhibit robust humoral responses that result in large monoclonal antibody diversity. They are used by Regeneron as a platform for producing fully human monoclonal antibodies against a range of targets.

[0351] Detection of PAR4 by flow cytometry Washed human or mouse platelets (5 × 10 7 Platelets (12 mM NaHCO3, 10 mM HEPES pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5.5 mM D-glucose, 1 mM CaCl2) were 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 samples were 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's medium + 10% fetal bovine serum) were seeded in 12-well plates 24 hours prior to transfection. Once confluent, they were transfected with 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) plus 4 μL of Lipofectamine 2000 according to the manufacturer's instructions. 48 hours after transfection, cells were harvested, washed twice with PBS, and resuspended at 1 × 10 6 The cells (50 μL assay / 0.5 x 10 5 Cells (cells / condition) were pretreated 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) for 15 minutes at 37°C. Cells were then stimulated with 2 U / mL thrombin for 10 minutes. The reaction was stopped by adding 4 U / mL hirudin. Cells were then washed once, resuspended in PBS containing a 1:200 dilution of FITC anti-FLAG antibody (Sigma, clone M2), and incubated for 1 hour at room temperature in the dark. Cells were then fixed with 1% paraformaldehyde (final concentration) and read on a FACSCalibur flow cytometer to measure the percentage of FC1 / FITC-positive events. Data were normalized to the quiescent sample (100% without thrombin treatment).

[0353] Surface plasmon resonance (SPR) assay Surface plasmon resonance (SPR) is a biosensor technology that allows for label-free, real-time measurement of protein-protein interactions. SPR binding analysis of proteins and antibodies was performed using a Bio-Rad ProteOn XPR36 array system or a Biacore T200 (GE system) using standard techniques.

[0354] 5A. Methods for measuring RC3 kinetics ProteOn is an SPR biosensor with a multichannel module and interaction array sensor chip for analyzing up to 36 protein interactions in a single injection step. Analyses were performed using a ProteOn NLC biosensor chip, which contains a surface composed of NeutrAvidin bound to an alginate polymer for capture of biotinylated proteins and peptides.

[0355] The NLC chip was conditioned with 50 mM NaOH and then 1 M NaCl at a flow rate of 30 μl / min. Conditioning was performed in both horizontal and vertical channels. A biotinylated peptide (ligand sample) at a concentration of 25 μg / ml was captured on the chip in the vertical channel at a flow rate of 30 μl / min (see Table 1 for the ligand sample setup on the biosensor chip). To ensure stable capture of the peptide ligand before analyte injection, running buffer (1x PBS, 0.005% Tween®, pH 7.4) was injected across the vertical channel. The chip was then rotated horizontally, and a dilution series of mAb 5ARC3.F10b.H4b (analyte) was injected across channels A1–A6 at a flow rate of 100 μl / min (see Table 1 for the analyte concentrations tested). Note: The 6.25 nM mAb concentration was excluded from the final analysis due to its higher than normal reading.

[0356] [Table 2]

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

[0358] Antibody capture method A series S CM5 sensor chip was activated using standard EDC / NHS amine coupling chemistry with goat anti-mouse IgG Fc using the manufacturer's recommended protocol. Anti-hPAR4 mAb was captured at 1-2 μg / ml for 2 minutes on flow cell 2 (FC2) at 10 μl / min, and flow cell 1 (FC1) served 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), typically at 1000, 500, 250, 125, and 62.5 nM, and flowed through both FC1 and 2 at 30 μl / min for 60-100 seconds, with dissociation monitored for 80-120 seconds. A blank injection of buffer alone was also performed. All injections were performed in duplicate and in random order. After each capture / injection / dissociation cycle, the chip was regenerated with a 30 s injection of 0.1 M glycine pH 2.0.

[0359] Streptavidin Capture Method A series S sensor chip SA was prepared using the manufacturer's recommended protocol and immobilized with 1 μg / ml hPAR4-biotinylated peptide on FC2 and hPAR1-biotinylated peptide (control) on FC1. Anti-hPAR4 dilutions were prepared in running buffer, typically at 10, 5, 2.5, 1.25, 6.25, and 0 nM, and flowed through both FC1 and 2 at a flow rate of 100 μl / min for 60 seconds. Dissociation was monitored for 200 seconds. A blank injection of buffer alone was also performed. All injections were performed in duplicate and in random order. After each capture / injection / dissociation cycle, the chip was regenerated with a 30-second injection of 0.1 M glycine, pH 2.0.

[0360] Platelet aggregation assay Platelet aggregation was measured by light transmission aggregometry in a 96-well plate format. Human isolated platelets (2 × 10 8Platelets (1000 μg / ml) were pretreated for 10 min at 37°C with dimethyl sulfoxide (DMSO) (1% v / v), the PAR1 antagonist vorapaxal (90 nM), the anti-PAR4 antibody 5RC3 (20–100 μg / ml), or a combination of vorapaxal and 5RC3. Platelets were treated with thrombin (0.1 U / ml), and aggregation was analyzed at 37°C for 50 min using a 595 nm excitation filter in a FLUOstar OPTIMA plate reader (BMG Labtech) (10 read cycles with a 5 min double orbital shaking period between each reading). Optical density was normalized to blank (maximum) and unstimulated platelets (minimum) and expressed as % of maximum.

[0361] Whole blood thrombosis assay Human whole blood collected in 3.2% citrate was preincubated for 15 min at 37°C with PE-conjugated anti-CD9 antibody (4 μg / mL) and anti-fibrin antibody (5 μg / mL), and 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. The whole blood was recalcified with 5–7.5 mM CaCl2 (final concentration) to initiate clotting, 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 Wall shear rates of 1000 s were obtained. Dual-color confocal fluorescence images were recorded with excitation at 488 and 561 nm and collected through a 40x water-immersion objective. Confocal z-stacks were recorded continuously for 2 min before correction. Calcium-free Tyrode's buffer was then flowed through the thrombus, and a z-stack encompassing the entire thrombus field was recorded over a 10-min period. Anti-CD9-PE was used to define the platelet thrombus, and the mean fluorescence of the thrombus field was used to quantify fibrin volume. 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. Genotyping of rs773902 was performed on DNA samples using 10 ng of DNA with the Taqman SNP genotyping assay (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. PCR was performed in a Roche 96-well plate lightcycler using the following thermal cycling conditions: 95°C for 15 seconds, 60°C for 60 seconds, repeated for 40 cycles. Endpoint genotyping analysis using Mygo Pro software was used to distinguish between alleles. Ratiometric analysis of the relative fluorescence signal 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 as determined by either an unpaired two-tailed Student's t test or one-way ANOVA with Fisher's LSD test for multiple comparisons.

[0364] Antibody nomenclature Unless indicated to the contrary, the following nomenclature will be used to refer to the antibodies referred to herein, as shown in Table 2 below: For example, a shorthand reference to 5A.RC3 refers to the monoclonal antibody subclone 5A.RC3.F10b.H4b, unless indicated to the contrary.

[0365] [Table 3]

[0366] Example 1: Development of antagonistic monoclonal antibodies against human PAR4 The inventors sought to develop monoclonal antibodies targeting human PAR4. At the Monash Antibody Technology Facility (MATF), Prof. Mark Sleeman, a supervisor at the facility, performed antibody production and screening using Regeneron Pharmaceuticals' proprietary HuMab mice (VelocImmune®) to generate high affinity antibodies against PAR4, as described in further detail below.

[0367] (i) Immunization A KLH-conjugated peptide of the N-terminal thrombin cleavage and activation site of human PAR4 (hPAR4) was generated and used to immunize HumAb mice, followed by three boosts according to standard protocols. Serum titers were measured by ELISA using the naked hPAR4 peptide (Table 3).

[0368] A total of 12 HumAb mice were subjected to three separate immunization programs using the peptides. The sequences of the peptides used for immunization are shown in Table 3. These sequences contained an additional C-terminal cysteine ​​in some cases (as underlined). The peptides were conjugated at the C-terminus to keyhole limpet hemocyanin (KLH) or to biotin (SKB) via a cysteine-lysine bond.

[0369] [Table 4]

[0370] (ii) Generation of hybridomas Fusions were performed using standard fusion protocols 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 plated into twenty 96-well plates. Cells were grown in 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 lines. The monoclonal antibodies comprise human Ig variable regions linked to mouse constant regions (hence the term "mAb" used herein to refer to such chimeric antibodies).

[0373] Hybridoma supernatants were first screened for binding to the immunizing PAR4 peptide sequence (GDDSTPSILPAPRGYPGQVC-KLH) by antigen microarray. Binding was measured by fluorescence intensity above background (signal from medium alone). The top 46 clones per spleen fusion were selected for further ELISA-based screening of binding to the PAR4 antigen (both native and KLH-linked; fold binding of native vs. KLH-linked PAR4 peptide). Clones that showed greater than three-fold greater binding to the native PAR4 peptide than 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; fold binding of the PAR4 peptide vs. PAR1, 2, or 3 peptides). Clones that showed more than three-fold greater binding to the PAR4 peptide than to other PAR peptides were expanded and tested in functional bioassays (inhibition of PAR4 cleavage and inhibition of PAR4-induced platelet activation / aggregation) (Figs. 2 and 12).

[0374] Clones that demonstrated both binding and functionality were selected for subcloning and retesting. After each round of subcloning, clones were again tested by ELISA for binding to the native PAR4 peptide (compared to medium alone) to ensure maintenance of binding antibodies within the clones.

[0375] [Table 5] TIFF0007733443000011.tif237151TIFF0007733443000012.tif25249

[0376] [Table 6] TIFF0007733443000014.tif221120

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

[0378] Transfected HEK293 cells were incubated with either the anti-PAR4 polyclonal antibody (French SL et al. (2016) J Thromb Haemost 14, 1642-1654, used as a positive control) or the monoclonal antibody clones MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BRH3, or MoB5CRC4, or an untreated negative control, in the presence of thrombin (0.1 U / ml) for 10 minutes at room temperature. Thrombin-mediated cleavage of PAR4 was measured by loss of the FLAG tag from PAR4-expressing HEK293T cells using flow cytometry.

[0379] Treatment of cells with thrombin (2 U / ml for 10 min) resulted in the cleavage of approximately 50% of total PAR4 (negative control). Pretreatment with a polyclonal anti-PAR4 antibody was found to almost completely block thrombin-induced cleavage (positive control) (Figure 2). Initial screening of five hybridoma supernatants 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 thrombin-induced cleavage of PAR4 by at least 90%, clone B5.BRB4 blocked thrombin-induced cleavage by approximately 60%, B5BRC6 blocked thrombin-induced cleavage by approximately 50%, B5BRH3 blocked thrombin-induced cleavage by approximately 65%, and B5CRC4 blocked thrombin-induced cleavage by approximately 50%.

[0381] Clone 5A.RC3 was further subcloned by limiting dilution according to standard protocols and tested for its ability to block thrombin-induced cleavage of PAR4 as measured by flow cytometry, as shown in Figure 3. Hybridoma supernatants from both 5A.RC3 (subclone H4b) and 5A.RC3 (subclone B6b) in 100 μl supernatant significantly blocked thrombin-induced cleavage of PAR4 on HEK293T cells (100 μl cell suspension) by more than 90%.

[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 the 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 determine the specificity of the anti-hPAR clones for PAR4, ELISA screening was performed as described above. Figure 5A shows 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 the human PAR4 peptide over PAR1, PAR2, and PAR3. mAb 5B.RB4 bound similarly to all four human PAR peptides, with lower affinity for PAR4 compared to 5A.RC3.

[0385] We performed surface plasmon resonance (SPR) analysis using a Bio-Rad Proteon XPR36 to gain insight into the binding affinity (association and dissociation rates) and specificity of 5A.RC3.F10b.H4b. Using a streptavidin chip, we captured all biotin-conjugated human PAR peptides (Table 1) onto the surface and passed different concentrations of purified 5A.RC3 over them. Clone 5A.RC3.F10b.H4b was observed to have a dissociation constant (KD) of approximately 0.4 nM by SA chip SPR (Figure 5B).

[0386] Example 4: 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 for particular variants of PAR4 and therefore can only successfully inhibit platelet aggregation in individuals who have the relevant PAR4 receptor variant.

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

[0388] Cells were stimulated with 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, inhibition was dose-dependent.

[0389] Figure 6B shows that preincubation of transfected cells with 5A.RC3 (100 μg / ml) before thrombin stimulation resulted in a similar and nearly complete inhibition of thrombin cleavage, regardless of PAR4 variant.

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

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

[0392] Example 5: Inhibition of platelet aggregation To determine whether the anti-hPAR4 antibody MoB5ARC3.H4b (5A.RC3) could inhibit the effects of thrombin on PAR4, as 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 of 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 mid-dose range PAR4-activating peptide (AP) and thrombin. PAR4-AP is a selective PAR4 agonist with a C-terminal amidated peptide sequence, AYPGKF-NH2.

[0394] FIG. 7C shows thrombin stimulation in the presence of PAR1 blockade with vorapaxal (90 nM).

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

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

[0397] Example 6: MoB5ARC3.F10b.H4b (hereinafter, 5A.RC3) binds to PAR4 on human platelets. Clone 5A.RC3 was tested for in vitro binding to isolated human platelets. Binding analysis was determined using flow cytometry. Isolated platelets were incubated with either mouse IgG1 (isotype control) or 5A.RC3 and tested for binding to PAR4. 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 procoagulant activity in isolated platelets exhibited by MoB5ARC3.F10b.H4b (hereafter 5A.RC3) Platelet surface phosphatidylserine (PS) exposure was determined by measuring Annexin V binding. Human isolated platelets (5 × 10 7 Cells (1000 x g, ...

[0399] The procoagulant activity of isolated human platelets was examined 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 preincubated with 5A.RC3 for 5 minutes. Blood was allowed to flow for 10 minutes, and data were collected in real time. The 10-minute time point (the 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] FIG. 9C shows that pretreatment with 5A.RC3 (5 min) 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 fibrin per thrombus ratio were measured in real time over a 10-minute period in a whole blood thrombosis assay as described herein under clotting conditions.

[0403] Platelet deposition (PE-conjugated anti-CD9), thrombin activity (FRET-based thrombin probe), fibrin volume (Dylight650-conjugated anti-fibrin antibody), and fibrin per thrombin ratio at the 10-minute endpoint are shown in Figure 10A by confocal microscopy using a Nikon A1r equipped with a 25x lens and 2x digital magnification (to allow volumetric measurements).

[0404] The direct thrombin inhibitor hirudin (800 U / mL) abolished thrombin activity and fibrin volume despite continued platelet deposition. Figures 10B–E show that no significant differences in these parameters were observed across PAR4 genotypes. Pretreatment with 5A.RC3 (100 μg / mL), shown by open bars, had no effect on platelet deposition (F) compared to controls (black bars), but significantly inhibited thrombin activity (G), thrombin activity (H), fibrin volume (H), and the ratio of fibrin per thrombin volume (I).

[0405] Example 9: Further screening of additional clones Clones identified from the binding screen as having reasonable affinity and specificity for human PAR4 were further validated by 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 30 monoclonal antibody supernatants that bind to human platelets. Antibody supernatants were incubated with human platelets (2 × 10 8 Cells) at a 1:1 ratio. Figure 11 shows the maximum aggregation achieved at 50 minutes, expressed as a percentage of the control (n=3 individual donors).

[0407] Example 10: Sequences of two inhibitory clones and one non-inhibitory clone Sequencing of the monoclonal antibodies, all of the IgG1 kappa isotype, was carried out at the Monash Antibody Technologies Facility at Monash University.

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

[0409] The sequences of the complementarity determining regions (CDRs) are shown below. 5A.RC3 Heavy Chain CDRs: CDR1: GFTLSNYG (SEQ ID NO: 13) CDR2: IWYDGSNK (SEQ ID NO: 14) CDR3: ARESIVEVLPPFDY (SEQ ID NO: 15) 5A.RC3 Light Chain CDRs: CDR1: QRVRNNY (SEQ ID NO: 16) CDR2: GAS (SEQ ID NO: 17) CDR3: QQYGNSYT (SEQ ID NO: 18)

[0410] (ii) 5F.RF3 subclone The nucleotide and amino acid sequences of the heavy and light chain variable regions were determined and are shown in the Sequence Listing that forms part of this disclosure and in Figure 14. The antibody is an antagonist. The antibody isotype is IgG2b kappa.

[0411] The sequences of the complementarity determining regions (CDRs) are shown below. 5F.RF3 heavy chain CDRs: CDR1: AYTFTNYG (SEQ ID NO: 24) CDR2: ISPYNGNT (SEQ ID NO: 25) CDR3: AREYNRSSRGRYYYYGMDV (SEQ ID NO: 26) 5F.RF3 Light Chain CDRs: CDR1: QSVSSNY (SEQ ID NO: 27) CDR2: GAS (SEQ ID NO: 28) CDR3: QQYGSSPWT (SEQ ID NO: 29)

[0412] (iii) 5H.RD2 subclone The nucleotide and amino acid sequences of the heavy and light chain variable regions were determined and 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 antibody isotype is IgG1 kappa.

[0413] The sequences of the complementarity determining regions (CDRs) are shown below. 5H.RD2 heavy chain CDR: CDR1: GFTFFNTW (SEQ ID NO: 34) CDR2: VKSKNDGGTK (SEQ ID NO: 35) CDR3: TTDPHYDFWSAY (SEQ ID NO: 36) 5H.RD2 light chain CDR: CDR1: QSLVHSDGNT (SEQ ID NO: 37) CDR2: VKSKNDGGTK (SEQ ID NO: 38) CDR3: LQATQFMYT (SEQ ID NO: 39)

[0414] The IMGT / V-Quest program was used to determine the assignment of CDR and framework regions.

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

[0416] For the mAb capture method, low levels of the ligand mAb and analyte hPAR4 were used in an attempt to achieve a 1:1 binding interaction. This method was successfully used to measure kinetics and affinity KD (Kamat V and Rafique A (2017) Analytical Biochemistry 530:75-86), despite using whole IgG. For the streptavidin capture method, despite using very low levels of analyte (mAb), the use of whole antibody in this assay can result in interactions greater than 1:1.

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

[0418] [Table 7]

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

[0420] The reactivity of seven purified hPAR4 monoclonal antibodies was analyzed by ELISA (see Table 4). Dilutions of mAb were reacted with hPAR4 peptide-coated wells. Binding curves are shown in Figure 15. The strongest binding was observed with mAb 5F RF3.A7b.C9 (5F.RF3), followed by the binding curves of 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 5G RA1.E10.G3 (5G.RA1) mAb was slightly less reactive, and the mAbs with the least reactivity with the hPAR4 peptide were 5H RF2.A5b.D3.C2 (5H.RF2) and 5I RG1.D6.C1b (5I.RG1). The data are also expressed as ELISA positive:negative ratios, shown in Table 4.

[0422] Example 13: Specificity of anti-hPAR4 monoclonal antibodies to human PAR4 peptides Wells of a streptavidin-coated plate (Thermo Scientific Pierce Streptavidin High Binding Coated Plate, code #15500, blocked with Superblock) were washed three times with wash buffer (PBS containing 0.05% TWEEN® 20 and 0.1% bovine serum albumin). Biotinylated peptides (hPAR1-4) were diluted in wash buffer and bound to the blocked streptavidin-coated wells at 10 μg / ml (100 μl per well) for 1 hour at room temperature with gentle mixing. The wells were washed three times as above, and purified anti-hPAR4 mAb prepared at 10 μg / ml in wash buffer was bound (100 μl / well) for 1 hour at room temperature with gentle mixing. The plate was washed three times as above, and anti-mouse Fc conjugate conjugated to alkaline phosphatase (AP) at 0.3 μg / ml (100 μl) was added to the wells for 1 hour as above. Wells were washed three times as above and developed with alkaline phosphatase substrate as described in the supernatant screening ELISA.

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

[0424] Example 14: Binding and Inhibitory 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 relative to the same concentration of an isotype control) and 2) IC value for inhibition of human platelet aggregation in response to 0.1 U / ml thrombin. 50 The results for each clone are shown for values ​​and .

[0425] [Table 8]

[0426] Concentration-dependent binding of purified anti-hPAR4 mAbs to isolated human platelets is shown in Figure 17. Each antibody showed concentration-dependent binding relative to the relevant isotype control.

[0427] 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) is shown in Figure 18. Near-maximal 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) Inhibition of human thrombus formation by mAb 5A.RC3 and mAb 5D.RH4 was verified by confocal microscopy. In an ex vivo human whole blood thrombosis assay, the volume of human thrombi formed after 3 minutes was quantified by confocal microscopy. Pretreatment of blood with 100 μg / ml of either mAb reduced total thrombus volume, as shown in Figure 19.

[0429] Example 16: Sequence of purified anti-hPAR4 mAb clones The monoclonal antibodies, all of which were of the IgG1 kappa isotype, were sequenced at the Monash Antibody Technologies Facility at Monash University. The IMGT / V-Quest program was used to determine the assignment of CDR and framework regions.

[0430] The sequence of the variable heavy chain of the purified mAb against human PAR4 is shown in Figure 20. The complementarity determining regions (CDRs) are indicated in the figure according to the IMGT numbering system.

[0431] The sequence of the variable light chain of the purified mAb against human PAR4 is shown in Figure 21. The complementarity determining regions (CDRs) are indicated in the figure according to the IMGT numbering system.

[0432] The antibody complementarity determining region sequences are shown in Table 6.

[0433] [Table 9] JPEG0007733443000018.jpg216153

[0434] [Table 10] TIFF0007733443000020.tif247146TIFF0007733443000021.tif245155TIFF0007733443000022.tif24533

[0435] [Example 17 Epitope Mapping] To determine the minimal epitope bound by anti-hPAR4 monoclonal antibodies, three overlapping peptides corresponding to the hPAR4 peptide were synthesized as shown below and in FIG. TIFF0007733443000023.tif31148

[0436] The thrombin cleavage site sequence RG 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 mAbs showing greater reactivity with shorter overlapping peptides spanning the original peptide antigen. Briefly, peptides were coated overnight on Nunc maxisorp ELISA plates at 10 μg / ml in coating buffer (0.1 M sodium carbonate pH 8.0). Purified mAbs diluted in PBS were used instead of supernatant.

[0438] As shown in Figure 23, purified mAbs 5A.RC3, 5G.RA1, 5D.RH4, and 5F.RF3 reacted preferentially with core peptide amino acid residues 8-15, which contains the thrombin cleavage site, thus indicating that the epitope resides within this region of the native hPAR4 peptide.

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

[0440] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the above-described embodiments without departing from the scope of the invention as broadly described, and the present embodiments are, therefore, to 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 that is an anti-PAR4 recombinant antibody, synthetic antibody, or monoclonal antibody or an antigen-binding fragment thereof, which inhibits the cleavage of cell surface-expressed human PAR4 by 50% or more in the presence of thrombin. 2. A 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. A PAR4-binding protein according to 1 or 2 above, which inhibits cleavage of 90% or more of cell surface-expressed PAR4 in the presence of thrombin. 4. A PAR4-binding protein according to any one of 1 to 3 above, which specifically binds to an epitope spanning the thrombin cleavage site of PAR4. 5. A PAR4 binding protein according to claim 4, wherein the epitope comprises the sequence APRGY and the thrombin cleavage site corresponds to RG. 6. A 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 described in any one of 1 to 6 above, wherein the antibody binds to the Ala120 and / or Thr120 variant of human PAR4. 8. A PAR4-binding protein described in any one of 1 to 7 above, wherein the protein does not bind or does not substantially bind to human PAR1, PAR2, or PAR3. 9. A PAR4-binding protein according to any one of 1 to 8 above, comprising the variable heavy chain (VH) sequence shown below. JPEG0007733443000024.jpg26150(in the array, X1 is V or I; X2 is A or V, X3 is T or A, X4 is L or F, X5 is N or S, X6 is Y or D, X7 is S or A, X8 is Y or F, X9 is 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 is L, R, or G, X 26 is P or V) 10. A PAR4 binding protein described in any one of 1 to 9 above, comprising the variable light chain (VL) sequence shown below. JPEG0007733443000025.jpg28150(in the array, 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) 11. The PAR4-binding protein according to claim 9 or 10, wherein the VH comprises a CDR1 sequence selected from the group consisting of: (i) GFTLSNYG (SEQ ID NO: 13); (ii) GFTFSSDG (SEQ ID NO: 59); (iii) GFTFSNYG (SEQ ID NO: 68); (iv) GFTFSSYG (SEQ ID NO: 55); (v) GFAFSSYG (SEQ ID NO: 70); and (vi) GFTLSSYG (sequence number 75). 12. The PAR4-binding protein according to any one of 9 to 11 above, wherein the VH comprises a CDR2 sequence selected from the group consisting of: (i) IWYDGSNK (SEQ ID NO: 14); (ii) IWFDGRNK (SEQ ID NO: 60); (iii) IWYDGSNR (SEQ ID NO: 71); and (iv) IWYDGSSK (sequence number 76). 13. The PAR4-binding protein according to any one of 9 to 12 above, wherein the VH comprises a CDR3 sequence selected from the group consisting of: (i) ARESIVEVLPPFDY (SEQ ID NO: 15); (ii) ARESSISTRPPFDY (SEQ ID NO: 61); (iii) ARETIMVRGVPFD (SEQ ID NO: 69); (iv) ARETALVRGVPFDY (SEQ ID NO: 56); (v) ARETAMVRGVPFDY (SEQ ID NO: 72); and (vi) ARETILIGGVPFDY (sequence number 77). 14. The PAR4-binding protein according to claim 10, wherein the VL comprises a CDR1 sequence selected from the group consisting of: (i) QRVRNNY (SEQ ID NO: 16); (ii) QSVRSSY (SEQ ID NO: 57); and (iii) QSIRSNY (SEQ ID NO: 78). 15. A PAR4 binding protein according to claim 10 or 14, wherein the VL comprises the CDR2 sequence GAS (sequence number 28). 16. The PAR4-binding protein according to claim 10, 14, or 15, wherein the VL comprises a CDR3 sequence selected from the group consisting of: (i) QQYGNSYT (SEQ ID NO: 18); (ii) QQYGRSYT (SEQ ID NO: 62); and (iii) QQYGSSYT (sequence number 58). 17. A PAR4-binding protein according to any one of 1 to 8 above, comprising the variable heavy chain (VH) sequence shown below. JPEG0007733443000026.jpg25154 (in the array, X1 is A or S, X2 is T or A, X3 is V or I, X4 is Y or S, X5 is G or S, X6 is L or F, X7 is N, D, or T, X8 is Y or F, X9 is 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. A PAR4 binding protein described in any one of 1 to 9 or 17 above, comprising the variable light chain (VL) sequence shown below. JPEG0007733443000027.jpg27153(in the array, 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 according to claim 18, wherein the VH comprises a CDR1 sequence selected from the group consisting of: (i) GGSLSDYY (SEQ ID NO: 86); (iii) SGSFSTYF (SEQ ID NO: 47); and (iv) GGSFSNYY (sequence number 66). 20. The PAR4-binding protein according to claim 18 or 19, wherein the VH comprises a CDR2 sequence selected from the group consisting of: (i) INHSGTT (SEQ ID NO: 87); (ii) IIHTGST (SEQ ID NO: 64); or (iii) INHSGST (SEQ ID NO: 48). 21. The PAR4-binding protein according to claim 18, 19, or 20, wherein the VH comprises a CDR3 sequence selected from the group consisting of: (i) AIEYSNSRGYYYGMDV (SEQ ID NO: 88); (ii) AFEYSSSGGYYYGMDV (SEQ ID NO: 49); and (iii) KVEHSSSSGHYYYGMDV (SEQ ID NO: 65). 22. The PAR4-binding protein according to any one of 18 to 21 above, wherein the VL comprises a CDR1 sequence selected from the group consisting of: (i) QTISNY (SEQ ID NO: 109); (ii) QSISSY (SEQ ID NO: 50); and (iii) QTISYY (SEQ ID NO: 66). 23. A PAR4-binding protein described in any one of 18 to 22 above, wherein the VL comprises the CDR2 sequence AAS (sequence number 51). 24. The PAR4-binding protein according to any one of 18 to 23 above, wherein the VL comprises a CDR3 sequence selected from the group consisting of: (i) RQNYNTPLT (SEQ ID NO: 85); (iii) QQTYSTPLT (SEQ ID NO: 52); or (iv) QQSYSTPLT (sequence number 67). 25. Any of the above-described PAR4 binding proteins, comprising a variable heavy chain (VH) having CDR1, CDR2, and CDR3 sequences comprising or consisting of the following sequences, respectively: (i) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; (ii) SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49; (iii) SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; (iv) SEQ ID NO:55, SEQ ID NO:14, and SEQ ID NO:56; (v) SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61; (vi) SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65; (vii) SEQ ID NO: 68, SEQ ID NO: 14, and SEQ ID NO: 69; (viii) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; (ix) SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 74; (x) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77; (xi) SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; (xii) SEQ ID NO: 82, SEQ ID NO: 80, and SEQ ID NO: 83; (xiii) SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 74; or (xiv) SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88. 26. The PAR4-binding protein according to claim 25, further comprising a variable light chain (VL) having CDR1, CDR2, and CDR3 sequences comprising or consisting of the following sequences, respectively: (i) SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; (ii) SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52; (iii) SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29; (iv) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (v) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:62; (vi) SEQ ID NO: 66, SEQ ID NO: 51, and SEQ ID NO: 67; (vii) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (viii) SEQ ID NO:57, SEQ ID NO:28, and SEQ ID NO:58; (ix) SEQ ID NO: 78, SEQ ID NO: 28, and SEQ ID NO: 62; (x) SEQ ID NO: 84, SEQ ID NO: 51, and SEQ ID NO: 85; (xi) SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 58; (xii) SEQ ID NO: 57, SEQ ID NO: 51, and SEQ ID NO: 58; or (xiii) SEQ ID NO: 109, SEQ ID NO: 51, and SEQ ID NO: 85. 27. Any of the above-described PAR4 binding proteins, comprising a VH sequence that is at least 95% identical to the sequence set forth in any one of 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, or a humanized, chimeric, or deimmunized version thereof. 28. The PAR4 binding protein described in 27 above, further comprising a VL sequence that is at least 95% identical to any one of the sequences set forth 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. 29. Any of the above-described PAR4 binding proteins, including: (i) VH set forth in SEQ ID NO: 11 and VL set forth in SEQ ID NO: 12; (ii) a VH set forth in SEQ ID NO: 45 and a VL set forth in SEQ ID NO: 46; (iii) VH set forth in SEQ ID NO: 22 and VL set forth in SEQ ID NO: 23; (iv) VH set forth in SEQ ID NO: 53 and VL set forth in SEQ ID NO: 54; (v) a VH set forth in SEQ ID NO: 89 and a VL set forth in SEQ ID NO: 90; (vi) a VH set forth in SEQ ID NO: 91 and a VL set forth in SEQ ID NO: 92; (vii) a VH set forth in SEQ ID NO: 93 and a VL set forth in SEQ ID NO: 94; (viii) VH set forth in SEQ ID NO: 95 and VL set forth in SEQ ID NO: 96; (ix) a VH set forth in SEQ ID NO: 97 and a VL set forth in SEQ ID NO: 98; (x) a VH set forth in SEQ ID NO: 99 and a VL set forth in SEQ ID NO: 100; (xi) VH set forth in SEQ ID NO: 101 and VL set forth in SEQ ID NO: 102; (xii) VH set forth in SEQ ID NO: 103 and VL set forth in SEQ ID NO: 104; (xiii) VH set forth in SEQ ID NO: 105 and VL set forth in SEQ ID NO: 106; or (xiv) VH set forth in SEQ ID NO: 107 and VL set forth in SEQ ID NO: 108. 30. The antigen-binding fragment: (i) Single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) at least one of (i) and / or (ii) linked to a heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3 Any of the above-described PAR4-binding proteins, 31. The antigen-binding fragment: (i) diabody; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab′)2; (vi) Fv; or (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3 30. The PAR4-binding protein according to any one of 1 to 29 above, 32. Any of the above PAR4-binding proteins, which is linked to a moiety. 33. The PAR4 binding protein described in 32 above, wherein the moiety is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the PAR4 binding protein in a subject, and mixtures thereof. 34. A nucleic acid encoding any of the above-described PAR4-binding proteins. 35. A PAR4-binding protein according to claim 34, comprising a VH nucleic acid sequence shown in SEQ ID NO: 20 and / or comprising a VL nucleic acid sequence shown in SEQ ID NO: 21. 36. A PAR4-binding protein according to claim 34, comprising a VH nucleic acid sequence shown in SEQ ID NO: 30 and / or comprising a VL nucleic acid sequence shown in SEQ ID NO: 31. 37. A composition comprising a PAR4-binding protein described in any one of 1 to 33 above and a suitable carrier. 38. A method for treating or preventing thrombosis or a thromboembolic disorder in a subject, the method comprising administering to the subject a PAR4 binding protein or antibody described in any of 1 to 33 above, or a composition described in 37 above. 39. A method for treating, preventing, or ameliorating thrombosis or a thromboembolic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a PAR4-binding protein or antibody described in any one of 1 to 33 above, or a therapeutically effective amount of a composition described in 37 above.

Claims

1. A protease-activated receptor 4 (PAR4) binding protein that is an anti-PAR4 recombinant or synthetic antibody or monoclonal antibody or antigen-binding fragment thereof, which specifically binds to an epitope spanning the thrombin cleavage site of PAR4: (i) a variable heavy chain (VH) having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and a variable light chain (VL) having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; (ii) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively; (iii) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively; (iv) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 55, SEQ ID NO: 14, and SEQ ID NO: 56, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 58, respectively; (v) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 62, respectively; (vi) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 66, SEQ ID NO: 51, and SEQ ID NO: 67, respectively; (vii) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 68, SEQ ID NO: 14, and SEQ ID NO: 69, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 58, respectively; (viii) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 55, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 57, SEQ ID NO: 28, and SEQ ID NO: 58, respectively; (ix) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 78, SEQ ID NO: 28, and SEQ ID NO: 62, respectively; (x) a VH having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 82, SEQ ID NO: 80, and SEQ ID NO: 83, respectively, and a VL having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO: 57, SEQ ID NO: 51, and SEQ ID NO: 58, respectively; or (xi) a VH having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO: 55, SEQ ID NO: 73 and SEQ ID NO: 74, respectively, and a VL having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO: 84, SEQ ID NO: 51 and SEQ ID NO: 85, respectively; The protease-activated receptor 4 (PAR4) binding protein, comprising:

2. The PAR4 binding protein of claim 1, which inhibits 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. 3. The PAR4 binding protein of claim 1 or 2, comprising a VH sequence at least 95% identical to the sequence set forth in any one of 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: 97, SEQ ID NO: 99, SEQ ID NO: 103, or SEQ ID NO: 105, or a humanized, chimeric, or deimmunized version thereof, and a VL sequence at least 95% identical to the sequence set forth in any one of 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: 98, SEQ ID NO: 100, SEQ ID NO: 104, or SEQ ID NO: 106, or a humanized, chimeric, or deimmunized version thereof.

4. 4. The PAR4 binding protein of any one of claims 1 to 3, comprising: (i) VH set forth in SEQ ID NO: 11 and VL set forth in SEQ ID NO: 12; (ii) VH set forth in SEQ ID NO: 45 and VL set forth in SEQ ID NO: 46; (iii) VH set forth in SEQ ID NO: 22 and VL set forth in SEQ ID NO: 23; (iv) VH set forth in SEQ ID NO: 53 and VL set forth in SEQ ID NO: 54; (v) VH set forth in SEQ ID NO: 89 and VL set forth in SEQ ID NO: 90; (vi) VH set forth in SEQ ID NO: 91 and VL set forth in SEQ ID NO: 92; (vii) VH set forth in SEQ ID NO: 93 and VL set forth in SEQ ID NO: 94; (viii) VH set forth in SEQ ID NO: 97 and VL set forth in SEQ ID NO: 98; (ix) a VH set forth in SEQ ID NO: 99 and a VL set forth in SEQ ID NO: 100; (x) a VH set forth in SEQ ID NO: 103 and a VL set forth in SEQ ID NO: 104; or (xi) VH shown in SEQ ID NO: 105 and VL shown in SEQ ID NO:

106.

5. A PAR4 binding protein according to any one of claims 1 to 4, comprising a VH having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, and a VL having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

6. A PAR4 binding protein according to any one of claims 1 to 4, comprising a VH having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, respectively, and a VL having CDR1, CDR2 and CDR3 sequences comprising or consisting of SEQ ID NO:57, SEQ ID NO:28 and SEQ ID NO:62, respectively.

7. the antigen-binding fragment: (i) single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) at least one of (i) and / or (ii) linked to a heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3; The PAR4 binding protein according to any one of claims 1 to 6,

8. 8. The PAR4 binding protein of any one of claims 1 to 7, which is linked to a moiety.

9. The PAR4 binding protein of claim 8, wherein the moiety is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the PAR4 binding protein in a subject, and mixtures thereof.

10. The PAR4 binding protein according to any one of claims 1 to 9, which binds to Ala120 and Thr120 variants of human PAR4.

11. 11. The PAR4 binding protein of any one of claims 1 to 10, which does not bind or does not substantially bind to human PAR1, PAR2, or PAR3.

12. A composition comprising a PAR4 binding protein according to any one of claims 1 to 11 and a suitable carrier.

13. 13. A PAR4 binding protein or antibody according to any one of claims 1 to 11, or a composition according to claim 12, for use in the treatment, prevention, or amelioration of thrombosis or a thromboembolic disorder.

14. A nucleic acid encoding the PAR4 binding protein according to any one of claims 1 to 13.

15. 15. The nucleic acid of claim 14, comprising the VH sequence shown in SEQ ID NO: 20 and the VL sequence shown in SEQ ID NO:

21.

16. 13. Use of a PAR4 binding protein or antibody according to any one of claims 1 to 11, or a composition according to claim 12, in the manufacture of a medicament for the treatment, prevention, or amelioration of thrombosis or a thromboembolic disorder.

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