Binding proteins of the human thrombin receptor PAR4
阅读说明：本技术 人凝血酶受体par4的结合蛋白 (Binding proteins of the human thrombin receptor PAR4 ) 是由 J·哈米尔顿 M·斯利曼 于 2018-09-11 设计创作，主要内容包括：本公开涉及人蛋白酶活化受体4(PAR4)结合蛋白(例如抗体)。特别地,作为人PAR4的拮抗剂的抗PAR4结合蛋白以及其方法和用途。(The present disclosure relates to human protease activated receptor 4(PAR4) binding proteins (e.g., antibodies). In particular, anti-PAR 4 binding proteins as antagonists of human PAR4 and methods and uses thereof.)
1. A protease activated receptor 4(PAR4) binding protein that is an anti-PAR 4 recombinant or synthetic or monoclonal antibody or antigen binding fragment thereof, wherein said binding protein inhibits cleavage of cell surface expressed human PAR4 by equal to or greater than 50% in the presence of thrombin.
2. The PAR4 binding protein of claim 1, wherein the protein inhibits cleavage by (i) 60% or more or (ii) 70% or more or (iii) 80% or more of PAR4 expressed on the surface of a cell in the presence of thrombin.
3. The PAR4 binding protein of claim 1 or 2, wherein the protein inhibits cleavage of cell surface expressed PAR4 by 90% or more in the presence of thrombin.
4. The PAR4 binding protein according to any one of claims 1 to 3, which specifically binds to an epitope spanning the thrombin cleavage site of PAR 4.
5. The PAR4 binding protein of claim 4, wherein the epitope comprises the sequence APRGY, and wherein the thrombin cleavage site corresponds to RG.
6. PAR4 binding protein according to claim 4 or 5 wherein the epitope comprises or consists of a sequence selected from ILPAPGRGY or APRGYPGQV.
7. The PAR4 binding protein according to any of claims 1 to 6, wherein the antibody binds to Ala120 and/or Thr120 variants of human PAR 4.
8. The PAR4 binding protein of any one of claims 1 to 7, wherein the protein does not bind or does not substantially bind to human PAR1, PAR2 or PAR 3.
9. The PAR4 binding protein according to any of claims 1 to 8 comprising a variable heavy chain (VH) sequence set forth in SEQ ID NO:
QX1QLVESGGGVVQPGRSLRLSCX2AS 3 4 5 6GFXXSXXGMHWVRQAPGKGLEWVX7V 8 9 10 11IWXDGXXXX12YX13DSVX14GRFX15ISRDX16SKNTX17X18LQMNX19LRAEDTAVYYC 20 21 22 23 24 25 26AREXXXXXXXPFDYWGQGTLVTVSS
wherein
X1Is V or I;
X2is A or V;
X3is T or A;
X4is L or F;
X5is N or S;
X6is Y or D;
X7is S or A;
X8is Y or F;
X9is S or R;
X10is N or S;
X11is K or R;
X12is H or Y;
X13is A, L or T;
X14is K or R;
X15is T or D;
X16is N or T;
X17is L or Q;
X18is Y or F;
X19is S or I;
X20is S or T;
X21is I, S or A;
X22is V, I, M or L;
X23is E, S, V or I;
X24is V, T, R or G;
X25is L, R or G; and is
X26Is P or V.
10. The PAR4 binding protein according to any of claims 1 to 9 comprising a variable light chain (VL) sequence listed in SEQ ID NO:
X1IVLTQSPGTLSLSPGERX2TLSCX3X4S 5 6 7 8QXXRXXYLAWX9QQKPGQAPRL
X10IYGASSRATGX11PDRFSGSGSGTDFX12X13TIX14RLEPEDF AX15YYC 16QQYGXSYTFGQGTKLEIK
wherein
X1Is K or E;
X2is V or A;
X3is R or G;
X4is A or T;
X5is R or S;
X6is V or I;
X7is N or S;
X8is N or S;
X9is F or Y;
X10is F or L;
X11is I or T;
X12is I or T;
X13is F or L;
X14is S or T;
X15is V or L; and is
X16Is N, R or S.
11. The PAR4 binding protein of 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(SEQ ID NO:75)。
12. the PAR4 binding protein of any one of claims 9 to 11, 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(SEQ ID NO:76)。
13. the PAR4 binding protein of any one of claims 9 to 12, 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(SEQ ID NO:77)。
14. the PAR4 binding protein of claim 10, wherein the VL comprises a CDR1 sequence selected from the group consisting of seq id no:
(i)QRVRNNY(SEQ ID NO:16)；
(ii) QSVRSSY (SEQ ID NO: 57); and
(iii)QSIRSNY(SEQ ID NO:78)。
15. the PAR4 binding protein of claim 10 or 14, wherein the VL comprises the CDR2 sequence GAS (SEQ ID NO: 28).
16. The PAR4 binding protein of claim 10, 14 or 15, wherein the VL comprises a CDR3 sequence selected from the group consisting of seq id no:
(i)QQYGNSYT(SEQ ID NO:18)；
(ii) QQYGRSYT (SEQ ID NO: 62); and
(iii)QQYGSSYT(SEQ ID NO:58)。
17. the PAR4 binding protein according to any of claims 1 to 8 comprising a variable heavy chain (VH) sequence set forth in SEQ ID NO:
QVQLQQWGAGLLKPSETLX1LX2CAX3X4 5 6 7XGSXSXYX8WX9WIX10QPPGKGLEWIGE 11 12 13IXHXGXTX14YNPSLKSRVTISVDTSKX15QX16SLX17LSSVTAADTAVYYC 18 19 20 21 22 23XXEXSXSXGXYYYGMDVWGQGTTVTVSS
wherein
X1Is A or S;
X2is T or A;
X3is V or I;
X4is Y or S;
X5is G or S;
X6is L or F;
X7is N, D or T;
X8is Y or F;
X9is S or R;
X10is R or H;
X11is N or I;
X12is S or T;
X13is T or S;
X14is N or T;
X15is K or N;
X16is F or L;
X17is K or N;
X18is A or K;
X19is I, F or V;
X20is Y or H;
X21is N or S;
X22is R, G or S; and is
X23Is V or H.
18. The PAR4 binding protein according to any of claims 1 to 9 or 17 comprising a variable light chain (VL) sequence as set forth in:
DIQMTQSPSSLSASX1GDRX2TITCRAS 3 4QXISXYLNWYQQX5PGKAPX6LLIYAASX7LX8SGVPSRFSGSGSGTDFTLTISSLQPEDFX9X10YYC 11 12 13XQXYXTPLTFGGGTKX14IK
wherein
X1Is V or A;
X2is V or I;
X3is S or T;
X4is S, Y or N;
X5is K or I;
X6is N or K;
X7is R or S;
X8is R or Q;
X9is T or A;
X10is T or S;
X11is Q or R;
X12is T, S or N;
X13is N or N;
X14is E or G.
19. The PAR4 binding protein of 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(SEQ ID NO:66)。
20. the PAR4 binding protein of 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 of 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 of any one of claims 18 to 21, 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. the PAR4 binding protein of any one of claims 18 to 22, wherein the VL comprises the CDR2 sequence AAS (SEQ ID NO: 51).
24. The PAR4 binding protein according to any of claims 18 to 23, 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(SEQ ID NO:67)。
25. The PAR 4-binding protein according to any preceding claim comprising a variable heavy chain (VH) having CDR1, CDR2 and CDR3 sequences, the CDR1, CDR2 and CDR3 sequences comprising or consisting of, respectively:
(i) 13, 14 and 15;
(ii) 47, 48 and 49;
(iii) 24, 25 and 26;
(iv) 55, 14 and 56;
(v) 59, 60 and 61;
(vi) 63, 64 and 65;
(vii) 68, 14 and 69;
(viii) 70, 71 and 72;
(ix) 55, 73 and 74;
(x) 75, 76 and 77;
(xi) 79, 80 and 81;
(xii) 82, 80 and 83;
(xiii) 55, 73 and 74; or
(xiv) 86, 87 and 88.
26. The PAR4 binding protein according to claim 25, said PAR4 binding protein further comprising a variable light chain (VL) having CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3 sequences comprising or consisting of, respectively:
(i) 16, 17 and 18;
(ii) 50, 51 and 52;
(iii) 27, 28 and 29;
(iv) 57, 28 and 58;
(v) 57, 28 and 62;
(vi) 66, 51 and 67;
(vii) 57, 28 and 58;
(viii) 57, 28 and 58;
(ix) 78, 28 and 62;
(x) 84, 51 and 85;
(xi) 57, 28 and 58;
(xii) 57, 51 and 58; or
(xiii) 109, 51 and 85 SEQ ID NO.
27. The PAR4 binding protein according to any preceding claim, comprising a VH sequence that is at least 95% identical to a sequence set forth in any 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 de-immunized version thereof.
28. The PAR4 binding protein according to claim 27, further comprising a VL sequence at least 95% identical to a sequence set forth in any 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, chimeric or de-immunized version thereof.
29. The PAR4 binding protein of any preceding claim, the PAR4 binding protein 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) the VH set forth in SEQ ID NO. 22 and the 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) the VH set forth in SEQ ID NO. 93 and the 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) VH set forth in SEQ ID NO:97 and VL set forth in SEQ ID NO: 98;
(x) VH set forth in SEQ ID NO 99 and 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 PAR4 binding protein of any preceding claim, wherein the antigen binding fragment is:
(i) single chain Fv fragment (scFv);
(ii) dimeric scFv (di-scFv);
(iii) at least one of (i) and/or (ii) linked to a heavy chain constant region or Fc or heavy chain constant domains (CH)2 and/or CH 3.
31. The PAR4 binding protein according to any one of claims 1 to 29 wherein the antigen binding fragment is:
(i) a diabody;
(ii) a trisomy antibody;
(iii) a tetrad antibody;
(iv)Fab；
(v)F(ab′)2；
(vi) fv; or
(vii) (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or heavy chain constant domains (CH)2 and/or CH 3.
32. The PAR4 binding protein according to any preceding claim conjugated to a moiety.
33. The PAR4 binding protein according to claim 32, 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 a PAR4 binding protein according to any preceding claim.
35. The PAR4 binding protein according to claim 34, comprising the VH nucleic acid sequence set forth in SEQ ID NO 20 and/or comprising the VL nucleic acid sequence set forth in SEQ ID NO 21.
36. The PAR4 binding protein according to claim 34, comprising the VH nucleic acid sequence set forth in SEQ ID NO 30 and/or comprising the VL nucleic acid sequence set forth in SEQ ID NO 31.
37. A composition comprising a PAR4 binding protein according to any of claims 1 to 33 and a suitable carrier.
38. A method for treating or preventing a thrombotic or thromboembolic disorder in a subject, the method comprising administering to the subject a PAR4 binding protein or antibody according to any one of claims 1 through 33 or a composition according to claim 37.
39. A method of treating, preventing or ameliorating a thrombotic or thromboembolic disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a PAR4 binding protein or antibody according to any one of claims 1-33 or a composition according to claim 37.
[ technical field ] A method for producing a semiconductor device
The present disclosure relates to human protease activated receptor 4(PAR4) binding proteins (e.g., antibodies). In particular, anti-PAR 4 binding proteins as antagonists of human PAR4 and methods and uses thereof.
[ BACKGROUND OF THE INVENTION ]
Activated platelets are a key cellular component of arterial thrombosis, the most common cause of Death and disability in the world (Rosendaal FR et al (2014)384:1653-4), accounting for nearly 40% of deaths in many countries (mozafferian D et al (2015) Circulation 131e29-322), including Australia (australian statistical office, cats of Death, Australia, 20113303, chapter 42011 (2013)). Arterial thrombosis results in heart attacks and ischemic strokes.
Platelets are cells that form an arterial thrombus. Platelets are activated by a combination of endogenous agonists that trigger platelet aggregation and promotion of coagulation, which together promote pathological thrombosis. Antiplatelet drugs therefore constitute the primary drug therapy for the prevention of arterial thrombosis. However, despite many such agents, limitations in safety and/or efficacy necessitate rationalization of new drug targets. There is an urgent need for improved antiplatelet drugs in a wide range of clinical settings and there is a great interest in developing new agents.
Thrombin is the most potent known activator of human platelets and is also a key effector protease of the coagulation cascade. Thrombin activates platelets primarily via protease-activated Cell surface receptors (PAR), namely PAR1 and PAR4, and is the most potent platelet activator in humans (Vu T-KH et al (1991) Cell 64: 1057-68; Coughlin SR (1992) J Clininvest 89: 351-5; Coughlin SR (2000) Nature 407: 258-64). Loss receptors belong to a unique family of seven transmembrane receptors, G protein-coupled receptors (GPCRs), which are activated by proteolysis of their N-terminus. Once cleaved, the newly exposed N-terminus serves as a tethered ligand for activation of the receptor by binding to extracellular loop 2 (Vu T-KH et al (1991) Cell 64: 1057-68). 4 members of the PAR family (PAR1-4) are widely expressed and activated by a variety of proteases.
Mice lacking all platelet PAR function (PAR4)-/-) Are protected from thrombosis and do not exhibit spontaneous bleeding (Hamilton J et al (2004) Thromb Haemost 2: 1429-35; hamilton J et al (2009) BloodRev 23:61-5), indicating the potential to target these receptors for antithrombotic therapy. Two major PAR species are present on human platelets, PAR1 and PAR 4. Among these, PAR1 is a higher affinity thrombin receptor and has been the focus of antiplatelet drug development using two PAR1 antagonists that have been evaluated in clinical trials, namely, atorvastatin (E5555) (Goto S et al (2010) Eur Heart J31:2601-13) and Wallaparsa (Tricoci P et al (2012) New Engl J Med 366: 20-33; Morrow DA et al (2012) New Engl J Med 366: 13). Walapasha received US FDA approval at the end of 2014 and was planned by TGA for prevention of myocardial infarction and peripheral arterial disease at mid-2016. However, the combination of vorapaxate with single or dual anti-platelet therapies is associated with significantly increased intracranial bleeding rates, particularly in patients with a history of stroke or other predisposing factors (Tricoci P et al (2012) New Engl J Med 366: 20-33; Morrow DA et al (2012) New Engl J Med 366: 1404-13).
Therefore, the development of PAR4 antagonists has attracted considerable interest. The functional role of PAR4 is mainly elucidated with respect to platelets. A key feature that distinguishes PAR4 is its ability to form hetero-oligomers with both PAR1 and the ADP receptor P2Y12, which allows PAR4 to influence thrombin-and ADP-triggered signaling (Li D et al (2011) J Biol Chem 286: 3805-14). One major difference between the PARs of two platelets relates to the kinetics of intracellular signaling (Holinstat M et al (2006) JBiol Chem 281: 26665-74; Voss B et al (2007) Mol Pharmacol 71: 1399-. Both PAR1 and PAR4 signal via Gq to mobilize intracellular calcium and drive platelet function, 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 J376: 733-40). The cellular consequences of this sustained platelet activation downstream of PAR4 have not been fully characterized, but in view of the dependence of these phenomena on sustained, elevated intracellular calcium levels, may involve sustained platelet secretion kinetics (Jonnalagadad 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) Biochemistry34: 11625-34).
No studies have examined the contribution of PAR4 to procoagulant activity in a human thrombotic setting, probably 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 pepducin P4pal-10 and P4pal-il (Leger AJ et al (2006) Circulation113: 1244-54; Covic L et al (2002) PNAS 99: 643-8; Stampfuss JJ et al (2003) Nat Med9: 1447). However, in studies using human platelets, these agents are not widely available (e.g., YD-3) or are reported to lack specificity (e.g., pepducin) and/or efficacy (e.g., tc-YPGKF-NH)2) (Stampfuss JJ et al (2003) Nat Med9: 1447; hollenberg MD et al (2004) Br J Pharmacol 143: 443-54; wu CC et al (2002) ThrombHaemost 87: 1026-33). PAR4 antagonist (BMS-986141) is currently being examined in early clinical trials for the treatment of thrombosis, however, the common PAR4 variant (present in 19% -82% of people, depending on the population) renders the receptor insensitive to small molecule inhibitors such as BMS-986141.
Based on the foregoing, it will be apparent to the skilled artisan that it would be useful to identify improved human PAR4 binding proteins for use in the pharmacological treatment of thrombosis. Furthermore, treatment of thrombosis with minimal adverse side effects is a significant unmet medical need. Thus, there is a need in the art for PAR4 antagonists that provide advantages over existing strategies and provide improved therapeutic profiles relative to targeting PAR1 in treating or preventing thrombosis.
[ summary of the invention ]
Current clinical programs are developing small molecule orthosteric PAR4 inhibitors. However, it has recently been revealed that this method is completely ineffective in most patients. Specifically, the single nucleotide polymorphism (SNP; rs773902) in PAR4 desensitizes the receptor to antagonism of the normal PAR4 (Edelstein LC et al (2014) Blood 124: 3450-3458). This Small Nucleotide Polymorphism (SNP) determines whether amino acid 120 is alanine (Ala120) or threonine (Thr 120). Pharmacological studies have shown that orthosteric PAR4 antagonism effectively inhibits platelet activation induced by PAR4 in patients genotyped as Ala120, but even at high concentrations has no effect at all on platelets from patients genotyped as Thr 120. Heterozygosity results only in partial inhibition (Edelstein LC et al (2014) Blood 124: 3450-. The frequency of PAR4 against the Thr120 form of the inhibitor was very high (> 80% in some populations), indicating a significant effect of this antagonism insensitivity. The Thr120 allele is racially diad, occurring in 63% of self-identified blacks in a cohort of 154 north americans, compared to 19% of whites. Data from the Human Genome Diversity Program (HGDP) show that SNP rs773902 is not regiospecific, with up to 80% of people in the region of sahara south africa and about two thirds of babu and melanisa having the Thr120 PAR4 variant.
In addition to making PAR4 resistant to a constitutive inhibitor, SNP rs773902 also increases the sensitivity of PAR4 to receptor activation, leading to platelet hyperactivity in patients with the Thr120 variant (Edelstein LC et al (2013) NatMed 19: 1609-1616). This increased PAR4 function persists in patients treated with standard of care antiplatelet drugs (aspirin and/or P2Y12 inhibitors).
The inventors have developed monoclonal antibodies that specifically bind to human PAR4 and inhibit the activation of PAR4 by thrombin. Thus, these antibodies are antagonists of thrombin-induced PAR4 cleavage. The antibodies identified by the present inventors are capable of antagonizing or reducing PAR 4-mediated events (e.g., thrombosis). Furthermore, the inventors have found that targeting PAR4 is less likely to cause bleeding complications than targeting PAR1 (a common target of prior art antibodies) due to its unique mechanism of action and a broader safety profile overall.
In particular, the antibodies identified by the present inventors are effective against both PAR4 receptor variants, i.e. Ala120 and Thr120, which means that they are effective in the treatment of thromboembolic disorders in all subjects (not only those with sensitive PAR4 variants). In addition, because the antibody specifically binds to PAR4 with minimal cross-reactivity to PAR1, bleeding complications can be minimized or avoided. Thus, the antibodies of the invention are thus distinct from prior art PAR1 antagonists and PAR4 small molecule inhibitors.
In addition, the inventors found that the antibodies significantly inhibited thrombin-induced PAR4 cleavage and aggregation of human platelets. In addition, these effects can be reversed by competing the antibody with an immunopeptide.
Accordingly, the present disclosure provides various agents for diagnosing or prognosing thrombosis in a subject. The present disclosure also provides methods for treating, preventing, or ameliorating a thrombotic or thromboembolic disorder in a subject.
The present disclosure provides a PAR4 binding protein comprising an antigen binding domain, wherein the antigen binding domain specifically binds to human PAR4, and wherein the protein antagonizes at least one PAR 4-mediated event (e.g., thrombosis). In one example, the protein antagonizes at least one PAR 4-mediated event (e.g., thrombosis) in the presence of thrombin.
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, such as an imidazothiadiazole derivative described in WO2013/163244 or a synthetic peptide analog described in US 7879792.
The present disclosure also provides a human PAR4 binding protein comprising an antigen binding domain of an anti-PAR 4 antibody, wherein the antigen binding domain specifically binds to PAR4, and wherein the protein antagonizes at least one PAR4 mediated event (e.g., thrombosis) when contacted with a cell expressing PAR 4. In one example, the protein antagonizes at least one PAR 4-mediated event (e.g., thrombosis) in the presence of thrombin.
In one example, the PAR4 binding protein inhibits the externalization of Phosphatidylserine (PS) on the cell surface of a cell expressing PAR4 (e.g., a platelet). In one example, the PAR4 binding protein reduces thrombus volume when measured by a whole blood thrombosis assay.
The present disclosure provides a protease activated receptor 4(PAR4) binding protein that is an anti-PAR 4 recombinant or synthetic or monoclonal antibody or antigen binding fragment thereof, wherein the PAR4 binding protein substantially inhibits thrombin-induced cleavage of human PAR 4.
One skilled in the art would be able to measure PAR4 cleavage using an appropriate in vitro assay. As a non-limiting example, PAR4 cleavage can be assessed in a cell line expressing a fluorescently labeled PAR4 protein on its surface. Cleavage of PAR4 in the presence of thrombin resulted in loss of FLAG from the cell surface, which can be quantified. In one particular example, PAR4 cleavage can be determined in transfected HEK293 cells comprising a nucleic acid encoding PAR4 comprising a fluorescently labeled FLAG tag. Cleavage of PAR4 in the presence of thrombin (e.g., 0.1U/ml) resulted in loss of FLAG from the cell surface, which can be quantified by using flow cytometry.
In one example, the binding protein inhibits cleavage of cell surface expressed human PAR4 by equal to or greater than 50% in the presence of thrombin.
In one example, the PAR4 binding protein specifically binds to an epitope spanning the thrombin cleavage site of human PAR 4. In one example, the PAR4 binding protein specifically binds to an epitope comprising residues contained within a sequence as set forth in GDDSTPSILPAPRGYPGQVC (SEQ ID NO: 2). In one example, the peptide consists of
In another example, the epitope comprises the sequence APRGY (SEQ ID NO:42), wherein 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 another example, the PAR4 binding protein specifically binds to an epitope comprising a sequence as set forth in PRGYPG (SEQ ID NO: 1).
In one example, the PAR4 binding protein specifically binds to an Ala120 or Thr120 variant of human PAR 4. In another example, the PAR4 binding protein binds to both Ala120 and Thr120 variants of human PAR 4.
In one example, the PAR4 binding protein binds to the thrombin cleavage site in 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 a sequence as set forth in SEQ ID No. 2, optionally additionally comprising Keyhole Limpet Hemocyanin (KLH) or other immunostimulatory molecule. In another example, the PAR4 binding protein binds to the sequence set forth in SEQ ID NO. 4.
In another example, the PAR4 binding protein binds to a sequence as set forth in SEQ ID NO:2, optionally further comprising a C-terminal GGGG and streptavidin-k/biotin (SKB). In one example, the PAR4 protein binds to a sequence as set forth in SEQ ID NO. 7.
In one example, the PAR4 binding protein does not bind or does not substantially bind to human PAR3, PAR2, or
In another example, the PAR4 binding protein does not bind or does not substantially bind to a PAR sequence selected from the group consisting of: 3 (mouse PAR4), 8 (human PAR3), 9 (human PAR2) or 10 (human PAR 1).
In one example, the level of binding is assessed by immobilizing a peptide (e.g., a peptide according to SEQ ID NO:2 or SEQ ID NO: 7) and contacting the peptide with the PAR4 binding protein.
Exemplary PAR4 binding proteins having such binding characteristics described herein comprise the variable regions and/or CDRs of an antibody designated 5arc3.f10b.h4b (hereinafter 5a.rc3) or 5f.rf3.a7b.a1 (hereinafter 5 f.rf3).
In one example, the PAR4 binding protein binds to 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 to human PAR4 at a level or with a similar or substantially identical affinity to the antibodies designated 5a.rc3, 51.RG1, 5f.rg3, 5g.ra1, 5d.rh4, 5h.rh4, 5g.rf6, 5g.rd6, 5h.ra3, 5g.rg1, 5h.rg4, 5g.rc5, 5f.re6, 5 h.rf2.
In another example, the PAR4 binding protein competitively inhibits the binding of an antibody designated 5a.rc3, 51.RG1, 5f.rg3, 5g.ra1, 5d.rh4, 5h.rh4, 5g.rf6, 5g.rd6, 5h.ra3, 5g.rg1, 5h.rg4, 5g.rc5, 5f.re6, 5h.rf2 to human PAR 4. In another example, the protein competitively inhibits the binding of an antibody designated 5a.rc3, 51.RG1, 5f.rg3, 5g.ra1, 5d.rh4, 5h.rh4, 5g.rf6, 5g.rd6, 5h.ra3, 5g.rg1, 5h.rg4, 5g.rc5, 5f.re6, 5h.rf2 to a peptide consisting of the sequence set forth in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 7.
In one example, the PAR4 binding protein binds to a peptide consisting of the sequence set forth in
In one example, the amount of protein or antibody bound is assessed by contacting the PAR4 binding protein with a peptide consisting of the sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:7 and the amount of PAR4 binding protein contacted with the peptide (e.g., 10 μ g/ml). The amount of PAR4 binding protein bound to the peptide is then determined and compared with the amount of antibody bound to the peptide, which antibody comprises a VH comprising the sequence set forth in SEQ ID NO 11,
The present disclosure also provides a PAR4 binding protein that competitively inhibits binding of an antibody designated as:
(i) RC3, said 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) RG1, said 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) rf3, said 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, said 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) RH4, said 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) RH4, said 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) rd6, said 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) RA3, said 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) RG1, the 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) RG4, said 5H.RG4 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;
(xii) Rc5, said 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) RE6, said 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, said 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; binding to a peptide comprising or consisting of the sequence set forth in
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 μ g/ml) reduces thrombin-induced PAR4 expressing HEK293 cells (e.g., about 5 × 10)4Individual cells) at the same time (e.g., at 0.1U/ml). Exemplary antibodies having such activity comprise the variable regions or Complementarity Determining Regions (CDRs) of antibodies 5a.rc3, 5i.rg1, 5f.rf3, 5g.ra1, 5d.rh4, 5h.rh4, 5g.rf6, 5g.rd6, 5h.ra3, 5g.rg1, 5h.rg4, 5g.rc5, 5f.re6, or 5h.rf2 or antibodies comprising the CDRs thereof.
In one example, the PAR4 binding protein binds to a peptide comprising the thrombin cleavage site of human PAR4 as described herein or to the N-terminal extracellular region of human PAR4 with an affinity dissociation constant (KD) of 2nM or less, such as 1.5nM or less, for example 1nM or less. In one example, the KD is between about 0.01nM and about 2nM, such as between about 0.05nM and about 1nM, such as between about 0.1nM and about 1nM, such as between about 0.3nM and about 1 nM. In one example, the KD is between about 0.01nM and 1nM, such as between about 0.05nM and 0.9nM, for example between about 0.09nM and 0.7nM, for example between about 0.1nM and 0.6 nM.
In one example, KD is assessed by using a streptavidin chip and capturing a biotin-coupled human PAR4 peptide (e.g., a peptide according to SEQ ID NO: 7) on the surface of the chip and delivering the PAR4 binding protein thereon.
In one example, KD is assessed by using a streptavidin chip and capturing a biotin-coupled human PAR4 peptide (e.g., a peptide according to SEQ ID NO: 7) on the surface of the chip and delivering the PAR4 binding protein thereon.
Exemplary PAR4 binding proteins of the present disclosure have a KD between about 0.01 and 0.61 when evaluated by SA chip biotin peptide SPR. In one example, the PAR4 binding protein has a KD of about 0.4nM (e.g. +/-0.1 nM). In one example, the PAR4 binding protein has a KD as shown in table 4, corresponding to any of the PAR4 binding proteins therein.
In one example, a PAR4 binding protein of the disclosure specifically binds to human PAR 4. In one example, protein binding is assessed by ELISA and high-throughput antigen microarrays.
In one example, the PAR4 binding protein binds to the same epitope in human PAR4 or an epitope in human PAR4 that overlaps with the epitope bound by the following antibodies:
(i) RC3, said 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) RG1, said 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) rf3, said 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, said 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) RH4, said 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) RH4, said 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) rd6, said 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) RA3, said 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) RG1, the 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) RG4, said 5H.RG4 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;
(xii) Rc5, said 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) RE6, said 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) Rf2, said 5h.rf2 comprising a VH comprising the sequence set forth in SEQ ID NO: 107.
The present disclosure also provides a PAR4 binding protein that specifically binds to human PAR4 and is an anti-PAR 4 recombinant or synthetic or monoclonal antibody or antigen-binding fragment thereof.
In one example, the antibody substantially inhibits cleavage of PAR4 by thrombin.
In one example, PAR4 is expressed on human platelets.
In one example, the PAR4 binding protein is a chimeric antibody comprising human heavy and light chain constant region sequences. In another example, the PAR4 binding protein is a humanized or fully human antibody.
In one example, the PAR4 binding protein inhibits cleavage of cell surface expressed PAR4 by equal to or greater than 60% in the presence of thrombin or a PAR1 antagonist. In other examples, 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%.
In another example, cleavage of PAR4 by thrombin was measured by loss of Flag tag from HEK293 cells expressing Flag-tagged PAR 4. In another example, cleavage is measured by flow cytometry.
In one example, the PAR4 binding protein does not bind or does not substantially bind to human PAR1, PAR2, or PAR 3.
In one example, the PAR4 binding protein comprises a variable heavy chain (VH) sequence as set forth in:
QX1QLVESGGGVVQPGRSLRLSCX2AS 3 4 5 6GFXXSXXGMHWVRQAPGKGLEWVX7V 8 9 10 11IWXDGXXXX1 2YX13DSVX14GRFX15ISRDX16SKNTX17X18LQMNX19LRAEDTAVYYC 20 21 22 23 24 25 26AREXXXXXXXPFDYWGQGTLVTVSS
wherein
X1Is V or I;
X2is A or V;
X3is T or A;
X4is L or F;
X5is N or S;
X6is Y or D;
X7is S or A;
X8is Y or F;
X9is S or R;
X10is N or S;
X11is K or R;
X12is H or Y;
X13is A, L or T;
X14is K or R;
X15is T or D;
X16is N or T;
X17is L or Q;
X18is Y or F;
X19is S or I;
X20is S or T;
X21is I, S or A;
X22is V, I, M or L;
X23is E, S, V or I;
X24is V, T, R or G;
X25is L, R or G; and is
X26Is P or V.
In one example, the PAR4 binding protein further comprises a variable light chain (VL) sequence set forth in seq id no:
X1IVLTQSPGTLSLSPGERX2TLSCX3X4S 5 6 7 8QXXRXXYLAWX9QQKPGQAPRL
X10IYGASSRATGX11PDRFSGSGSGTDFX12X13TIX14RLEPEDFAX15YYC 16QQYGXSYTFGQGTKLEIK
wherein
X1Is K or E;
X2is V or A;
X3is R or G;
X4is A or T;
X5is R or S;
X6is V or I;
X7is N or S;
X8is N or S;
X9is F or Y;
X10is F or L;
X11is I or T;
X12is I or T;
X13is F or L;
X14is S or T;
X15is V or L; and is
X16Is N, R or S.
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(SEQ ID NO:75)。
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(SEQ ID NO:76)。
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(SEQ ID NO:77)。
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)。
in one example, the VL comprises the CDR2 sequence GAS (SEQ ID NO: 28).
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(SEQ ID NO:58)。
in another example, the PAR4 binding protein comprises a variable heavy chain (VH) sequence set forth in seq id no:
in another example, the PAR4 binding protein comprises a variable heavy chain (VH) sequence as set forth in seq id no:
QVQLQQWGAGLLKPSETLX1LX2CAX3X4 5 6 7XGSXSXYX8WX9WIX10QPPGKGLEWIGE 11 12 13IXHXGX TX14YNPSLKSRVTISVDTSKX15QX16SLX17LSSVTAADTAVYYC 18 19 20 21 22 23XXEXSXSXGXYYYGMDVWGQGTTVTVSS
wherein
X1Is A or S;
X2is T or A;
X3is V or I;
X4is Y or S;
X5is G or S;
X6is L or F;
X7is N, D or T;
X8is Y or F;
X9is S or R;
X10is R or H;
X11is N or I;
X12is S or T;
X13is T or S;
X14is N or T;
X15is K or N;
X16is F or L;
X17is K or N;
X18is A or K;
X19is I, F or V;
X20is Y or H;
X21is N or S;
X22is R, G or S; and is
X23Is V or H.
In one example, the PAR4 binding protein further comprises a variable light chain (VL) sequence set forth in:
DIQMTQSPSSLSASX1GDRX2TITCRAS 3 4QXISXYLNWYQQX5PGKAPX6LLIYAASX7LX8SGVPSRFSGSGSGTDFTLTISSLQPEDFX9X10YYC 11 12 13XQXYXTPLTFGGGTKX14IK
wherein
X1Is V or A;
X2is V or I;
X3is S or T;
X4is S, Y or N;
X5is K or I;
X6is N or K;
X7is R orS；
X8Is R or Q;
X9is T or A;
X10is T or S;
X11is Q or R;
X12is T, S or N;
X13is N or N;
X14is E or G.
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(SEQ ID NO:66)。
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)。
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)。
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)。
in one example, the VL comprises the CDR2 sequence AAS (SEQ ID NO: 51).
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(SEQ ID NO:67)。
The present disclosure also provides a PAR4 binding protein comprising a variable heavy chain (VH) having the sequences of CDR1, CDR2 and CDR3, the sequences of CDR1, CDR2 and CDR3 comprising or consisting of, respectively:
(i) 13, 14 and 15;
(ii) 47, 48 and 49;
(iii) 24, 25 and 26;
(iv) 55, 14 and 56;
(v) 59, 60 and 61;
(vi) 63, 64 and 65;
(vii) 68, 14 and 69;
(viii) 70, 71 and 72;
(ix) 55, 73 and 74;
(x) 75, 76 and 77;
(xi) 79, 80 and 81;
(xii) 82, 80 and 83;
(xiii) 55, 73 and 74; or
(xiv) 86, 87 and 88.
The present disclosure also provides a PAR4 binding protein comprising a variable light chain (VL) having CDR1, CDR2, and CDR3 sequences, the CDR1, CDR2, and CDR3 sequences comprising or consisting of, respectively:
(i) 16, 17 and 18;
(ii) 50, 51 and 52;
(iii) 27, 28 and 29;
(iv) 57, 28 and 58;
(v) 57, 28 and 62;
(vi) 66, 51 and 67;
(vii) 57, 28 and 58;
(viii) 57, 28 and 58;
(ix) 78, 28 and 62;
(x) 84, 51 and 85;
(xi) 57, 28 and 58;
(xii) 57, 51 and 58; or
(xiii) 109, 51 and 85 SEQ ID NO.
In one example, the PAR-4 binding protein comprises:
(i) a VH comprising a sequence that is at least 50% identical to a sequence set forth in any one of SEQ ID NO 11,
(ii) A VL comprising a sequence that is at least 85% identical to a sequence set forth in
In one example, the VH comprises a sequence at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.5% identical to any of
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 of
In one example, the VH comprises a sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99.5% identical to
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
In one example, the CDRs are defined by the IMGT numbering system.
The disclosure also provides a PAR4 binding protein comprising a PAR4 binding protein
(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) the VH set forth in SEQ ID NO. 22 and the 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) the VH set forth in SEQ ID NO. 93 and the 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) VH set forth in SEQ ID NO:97 and VL set forth in SEQ ID NO: 98;
(x) VH set forth in SEQ ID NO 99 and VL set forth in
(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;
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 CH 3; or
(iv) At least one of (i) and/or (ii) linked to a protein that binds to platelets, such as von Willebrand factor (vWF).
In another example of the disclosure, the VL and VH are in separate polypeptide chains. For example, the PAR4 binding protein is:
(i) a diabody;
(ii) a trisomy antibody;
(iii) a tetrad antibody;
(iv)Fab；
(v)F(ab′)2；
(vi) fv; or
(vii) (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or heavy chain constant domains (CH)2 and/or CH 3; or
(viii) (vii) to at least one of (i) to (vi) attached to a protein (e.g., vWF) that binds to platelets.
The present disclosure also provides a chimeric antibody comprising a VH and a VL as 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.
Based on the disclosure herein, it will be apparent to those skilled in the art that the PAR4 binding proteins of the present disclosure encompass human, humanized, homohumanized, chimeric, and primatized proteins.
Antibodies of the present disclosure may belong to any class, including IgM, IgG, IgE, IgA, IgD, or subclass. Exemplary subclasses of IgG are IgG1, IgG2, IgG3, and IgG 4.
In one example, the PAR4 binding protein is recombinant. In one example, the PAR4 binding protein is synthetic.
In one example, a PAR4 binding protein or antibody of the present disclosure is conjugated to a moiety. For example, 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.
The present disclosure also provides an isolated nucleic acid encoding a PAR4 binding protein or antibody of the 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.
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 may be in a vector, for example in a plasmid.
In examples of the disclosure involving a single polypeptide PAR4 binding protein, the expression construct may comprise a promoter linked to a nucleic acid encoding the polypeptide chain.
In examples involving the formation of a plurality of polypeptides of a PAR 4-binding protein, an expression construct of the disclosure comprises a nucleic acid encoding one of the polypeptides (e.g., comprising a VH) operably linked to a promoter and a nucleic acid encoding the other of the polypeptides (e.g., comprising a VL) operably linked to another promoter.
In another example, the expression construct is a bicistronic expression construct, e.g., comprising in 5 'to 3' order the following operably linked components:
(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.
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.
The present disclosure also contemplates isolated expression constructs, one of which encodes a first polypeptide (e.g., comprising a VH and optionally a heavy chain constant region or a portion thereof) and the other of which encodes a second polypeptide (e.g., comprising a VL and optionally a light chain constant region). For example, the present disclosure also provides a composition comprising:
(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),
wherein the first polypeptide associates with the second polypeptide to form a PAR4 binding protein of the disclosure.
The present disclosure additionally provides an isolated cell expressing a PAR4 binding protein or antibody of the present disclosure; or a recombinant cell genetically modified to express a PAR4 binding protein or antibody of the disclosure. In one example, the cell is an isolated hybridoma. In another example, the cell comprises a nucleic acid or expression construct of the 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,
wherein the first polypeptide associates with the second polypeptide to form a PAR4 binding protein or antibody of the disclosure.
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 vector. In one example, the composition comprises a PAR4 binding protein of the disclosure.
In one example, the carrier is pharmaceutically acceptable.
The compositions of the present disclosure may be administered alone or in combination with other treatments, therapeutic agents or agents, either simultaneously or sequentially.
The present disclosure additionally provides a method for treating or preventing a thrombotic or thromboembolic disorder in a subject, the method comprising administering to the subject a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure. In one example, the subject is a subject at risk for a PAR 4-mediated event, such as thrombosis. In one example, the subject is a subject who has or has previously had a PAR 4-mediated event (e.g., thrombosis).
In one example, the method comprises administering to the subject an antibody comprising a VH comprising a 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 VL comprising a sequence set forth in any one of SEQ ID NO 12,
The present disclosure additionally provides PAR4 binding proteins or nucleic acids or expression constructs or cells or compositions of the present disclosure for use in medicine.
The present disclosure additionally provides PAR4 binding proteins or nucleic acids or expression constructs or cells or compositions of the present disclosure for treating or preventing PAR4 mediated events (e.g., thrombosis).
In one example, the present disclosure provides a method of treating, preventing, or ameliorating a thrombotic or thromboembolic disorder, the method comprising administering to a subject in need thereof a PAR4 binding protein or nucleic acid or expression construct or composition of cells of the present disclosure.
In some examples, the disclosure provides a method of treating, preventing, or ameliorating thrombosis in a subject in need thereof, the method comprising administering to a subject in need thereof a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the disclosure.
In one example, the present disclosure provides a method of reducing the risk of thrombosis associated with a surgical procedure, the method comprising administering to the subject a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure before and/or after a surgical procedure. In one example, the surgical procedure is a liver transplant and the thrombosis is hepatic artery thrombosis.
In some examples, the disclosure provides a method for determining whether a dosage of a PAR4 binding protein or antibody according to the disclosure is appropriate. Such methods comprise (i) obtaining a blood sample from a subject that has been 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 platelet activation in a blood sample after treatment with the PAR4 binding protein or antibody to platelet activation in a blood sample obtained prior to treatment with the PAR4 binding protein or antibody.
Examples of suitable PAR4 agonists are familiar to those skilled in the art. Non-limiting examples include agonist peptides, such as AYPGKF-NH2 (Tocris).
In one example, platelet activation is measured according to the methods exemplified in the examples herein.
In some embodiments, the disclosure includes a method of inhibiting or preventing platelet aggregation comprising the step of administering a therapeutically effective amount of a PAR4 binding egg according to the disclosure to a subject (e.g., a human) in need thereof.
In some examples, the disclosure provides a method of treating or preventing a thrombotic or thromboembolic disorder, the method involving administering to a subject (e.g., a human) in need thereof a therapeutically effective amount of a PAR 4-binding protein according to the disclosure and inhibiting PAR4 cleavage and/or signal transduction, wherein the subject has a dual PAR1/PAR4 platelet receptor repertoire.
Preferably, the subject is a human.
The present disclosure further provides for the use of a PAR4 binding protein or nucleic acid or expression construct or cell or composition of the present disclosure in medicine.
The present disclosure further provides for the use of a PAR4 binding protein or nucleic acid or expression construct or cell of the present disclosure in the manufacture of a medicament for treating or preventing a thrombotic or thromboembolic disorder.
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 such that an antigen-protein complex forms, and detecting the complex, wherein detection of the complex is indicative of PAR4 in the sample.
The present disclosure also provides a vaccine antigen comprising or consisting of a sequence according to SEQ ID No. 4 and a pharmaceutically acceptable carrier for the production of antagonist antibodies to human PAR 4.
The present disclosure also provides PAR-4 binding proteins that do not or only partially inhibit the cleavage of PAR-4 in the presence of thrombin.
Thus, in one example, the disclosure also provides a PAR4 binding protein, the 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 form thereof; and/or
(ii) A VL comprising a sequence at least 85% identical to the sequence set forth in SEQ ID NO. 33, or a humanized, chimeric or deimmunized form thereof.
[ description of the drawings ]
Figure 1 shows a schematic of the extracellular location of the thrombin cleavage and activation site of PAR4 and the anti-PAR 4 target region of the antibodies described herein.
Figure 2 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 obtained from the primary hybridoma screen (MoB5ARC3, MoB5BRB4, MoB5BRC6, MoB5BBRH3, and MoB5CRC4) prior to treatment with thrombin (2U/ml for 10 minutes). The positive control was a polyclonal anti-PAR 4 antibody (French et al (2016) Journal of Thrombosis and Haemostasis 14: 1642-1654). Data are mean ± standard error of mean of three individual data points.
Figure 3 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 (as measured by FLAG epitope%. Cells were pre-treated with supernatants from two subclones of 5a. rc3 (designated B6B and H4B) before treatment with thrombin (2U/ml for 10 min). Data are mean + standard error of mean for four individual data points.
Figure 4 shows the percentage of intact PAR4 present on the cell surface of HEK293 cells transfected with Thr120 or Ala120 variants of human PAR4 containing an N-terminal FLAG tag (measured by FLAG epitope%) as quantified by flow cytometry. The cells were pre-treated with 5a. rc3 subclones at various concentrations as indicated, followed by treatment with thrombin (2U/ml).
Figure 5 shows an ELISA-based screen of (a) binding of 5a. rc3 (dark) and 5b. rb4 (light) hybridoma supernatants on immobilized proteins corresponding to the immunogen PAR4 and corresponding regions of PAR1, 2 and 3. (B) Biacore analysis of the binding affinity of the 5a. rc3 subcloned PAR4 antibody to human PAR4 peptide measured at different concentrations.
Figure 65 a. rc3 inhibits thrombin cleavage in both Ala120 and Thr120 PAR4 variants. To assess thrombin cleavage of PAR4 variants, HEK293T cells were transiently transfected with PAR4-120Ala or PAR4-120Thr variants comprising a FLAG epitope upstream of the thrombin cleavage site. (A) Cells were stimulated with increasing doses of thrombin (0.1-2U/mL) and the amount of thrombin cleavage as loss of FLAG epitope was measured by flow cytometry using FITC-conjugated anti-FLAG antibody. (B) Pre-incubation of transfected cells with 5a. rc3 prior to thrombin stimulation provided almost complete inhibition of thrombin cleavage, to the same extent regardless of the PAR4 variant. Dosages of 5a. rc3 of 1, 10 and 100 μ g/ml were compared.
Figure 75 a. rc3 subclone inhibited up-regulated thrombin-induced platelet aggregation response in donors with PAR4 Thr120 variant. Human isolated platelets of different PAR4 variants were evaluated for their response to platelet aggregation response to PAR4 agonists. As predicted, the presence of the T allele was associated with higher maximal aggregation in response to moderate doses of (a) PAR4-AP and (B) thrombin, with a similar trend observed for (C) thrombin stimulation in the presence of PAR1 blocked with walaparsat (90 nM). (D) The concentration response of 5a.rc3 showing efficacy against the Thr120 variant, indicative of antibody-mediated inhibition of PAR4, against the inhibitory activity of thrombin-induced platelet aggregation in the presence of a PAR1 antagonist (i.e., PAR 4-dependent) was effective on all genotypes. (E) IC50 of 5a. rc3 inhibitory activity in PAR 4-dependent thrombin-induced platelet aggregation.
Fig. 8 shows that 5a. rc3(10ug/mL) binds human PAR4 in isolated platelets as shown by flow cytometry against isotype control.
Figure 9 shows that the PAR4 genotype is associated with an increase in the procoagulant platelet phenotype and can be targeted by antibody-mediated inhibition. Procoagulant activity of human isolated platelets was assessed via measurement of phosphatidylserine exposure in response to (a) PAR4 Activating Peptide (AP) stimulation and (B) thrombin stimulation. Note that the Thr120 variant resulted in an increase in PS exposure in PAR4-AP stimulated platelets, with a similar trend observed in thrombin stimulated platelets. (C) Rc3 subcloning was used to dose-dependently inhibit thrombin-induced phosphatidylserine exposure regardless of donor genotype.
Fig. 10-1 and 10-2 show that 5a. rc3 subclone inhibited thrombus formation regardless of donor genotype. Under clotting conditions, in a human whole blood thrombosis assay, the following thrombosis parameters were measured in real time over a 10 minute period: (A) platelet deposition (PE-conjugated anti-CD 9), thrombin activity (FRET-based thrombin probe), fibrin volume (Dylight 650-conjugated anti-fibrin antibody) and fibrin ratio per thrombus (data shown at 10 min end point). Note that the direct thrombin inhibitor hirudin (800U/mL) eliminates thrombin activity and fibrin volume despite sustained platelet deposition. (B-E) no significant difference in these parameters across the PAR4 genotype was observed. Pretreatment with 5a. rc3 (open bar, 100 μ G/mL) had no effect on (F) platelet deposition, but significantly inhibited (G) thrombin activity (H) fibrin volume and (I) fibrin ratio per thrombus volume compared to control (black bar). Data are mean ± SEM of each genotype N-4-6 (total N-15; color indicates PAR4 genotype: black circle AA, gray circle AT, white circle TT). For (G) and (H), the data were normalized to the hirudin baseline and expressed as a percentage of the untreated control. Statistical significance was determined by one-way ANOVA (B-E) or student T-test (F-G), meaning P <0.05
Figure 11 shows a functional screen of initial hybridoma supernatants for platelet aggregation.
FIG. 12 shows the VH (A) and VL (B) amino acid sequences 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 Ab exhibited PAR4 inhibitory function.
Figure 13 shows vh (a) and vl (b) amino acid sequences of MoB5H-rd2.a7b (5h.rd2) having CDRs identified according to IMGT numbers. VH ═ heavy chain variable region, VL ═ light chain variable region, CDR ═ complementarity determining region, and FWR ═ framework region. This Ab binds to PAR4, but does not exhibit inhibitory function against PAR 4.
Figure 14 shows vh (a) and vl (b) amino acid sequences 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 Ab exhibited PAR4 inhibitory function.
Figure 15 shows the reactivity of mabs purified by ELISA with hPAR4 peptide. Purified mAb detected with anti-mouse Fc conjugated to alkaline phosphatase (Ap) bound to the hPAR4 peptide in a dose-dependent manner. No binding was observed for IC (isotype control) mabs against an irrelevant non-PAR 4 antigen.
Figure 16 shows that purified anti-HPAR 4mAb specifically binds to HPAR4 biotinylated peptide by ELISA and does not react non-specifically with HPAR1, HPAR2 and HPAR3 (biotinylated peptide) at 10 μ g/ml.
Figure 17 shows concentration-dependent binding of purified anti-hPAR 4mAb versus isotype control to human isolated platelets by flow cytometry. Shown are raw data, expressed as geometric mean fluorescence intensity. Each mAb binds in a concentration-dependent manner. N-3-5. Data points are mean ± SEM.
FIG. 18 shows the concentration-dependent inhibition of human platelet aggregation induced by 0.1U/ml thrombin by three anti-hPAR 4mAb clones. Near-maximal inhibition was achieved for each clone at the highest concentration tested. IC determined from these concentration inhibition curves50The values (. mu.g/ml) are also shown in the figure. And N is 4-8. Data points are mean ± SEM.
Figure 19 shows the inhibition of human thrombosis by monoclonal antibodies 5a. rc3 and 5 d.rh4. Pretreatment of blood with 5d.rh4 or 5a.rc3 (both 100 μ g/ml) reduced overall thrombus volume. Individual data points are shown. Bars are mean ± SEM. P <0.05 (unpaired student t-test).
Figure 20 shows the sequence of the variable heavy chain of the anti-PAR 4mAb showing the Complementarity Determining Regions (CDRs) according to the IMGT numbering system.
FIG. 21 shows the sequence of the variable light chain of the anti-PAR 4mAb showing the Complementarity Determining Regions (CDRs) according to the IMGT numbering system
FIG. 22 shows synthetic peptides for epitope mapping of anti-hPAR 4 mAb. Overlapping peptides spanning the original human PAR4 antigen were synthesized consisting of amino acid residues 1-9, amino acid residues 8-15, and amino acid residues 11-20. The sequence of the thrombin cleavage site is underlined. The C-terminal cysteine residue has been removed to prevent multimer formation.
FIG. 23 shows the reactivity of purified hPAR4mAb with peptides 1-9, 8-15 and 11-20. The absorbance values were subtracted from the background levels and the non-hPAR 4 control mAb did not react with any of the three peptides.
[ Key points of sequence Listing ]
1, SEQ ID NO: epitope sequence of PAR4
2, SEQ ID NO: sequence of hPAR4 (naked)
3, SEQ ID NO: sequence of mPAR4 (naked)
4, SEQ ID NO: sequences of hPAR4(KLH) for use as immunogens
5, SEQ ID NO: sequences of mPAR4(KLH) for use as immunogens
6 of SEQ ID NO: sequence of mPAR4 (Biotin)
7, SEQ ID NO: sequence of hPAR4 (Biotin)
8, SEQ ID NO: sequence of hPAR3 (Biotin)
9 of SEQ ID NO: sequence of hPAR2 (Biotin)
10, SEQ ID NO: sequence of hPAR1 (Biotin)
11, SEQ ID NO: amino acid sequence of RC3 VH
12, SEQ ID NO: amino acid sequence of RC3 VL
13 in SEQ ID NO: sequences of RC3 VH CDR1
14, SEQ ID NO: sequences of RC3 VH CDR2
15, SEQ ID NO: sequences of RC3 VH CDR3
16 in SEQ ID NO: sequence of RC3 VL CDR1
17 in SEQ ID NO: sequence of RC3 VL CDR2
18, SEQ ID NO: sequence of RC3 VL CDR3
19, SEQ ID NO: human PAR4 sequence
20, SEQ ID NO: nucleic acid sequence of RC3 VH
21, SEQ ID NO: nucleic acid sequence of RC3 VL
22, SEQ ID NO: amino acid sequence of RC3 VH
23, SEQ ID NO: amino acid sequence of 5F.RF3 VL (5F.RF3)
24, SEQ ID NO: sequence of RF3 VH CDR1
25 in SEQ ID NO: sequence of RF3 VH CDR2
26, SEQ ID NO: sequence of RF3 VH CDR3
27 of SEQ ID NO: sequence of RF3 VL CDR1
28, SEQ ID NO: sequence of RF3 VL CDR2
29 in SEQ ID NO: sequence of RF3 VL CDR3
30 of SEQ ID NO: nucleic acid sequence of RF3 VH
31, SEQ ID NO: nucleic acid sequence of RF3 VL
32 in SEQ ID NO: amino acid sequence of RD2 VH
33, SEQ ID NO: amino acid sequence of RD2 VL
34 of SEQ ID NO: sequence of rd2 VH CDR1
35 in SEQ ID NO: sequence of rd2 VH CDR2
36, SEQ ID NO: sequence of rd2 VH CDR3
37, SEQ ID NO: sequence of rd2 VL CDR1
38, SEQ ID NO: sequence of rd2 VL CDR2
39, SEQ ID NO: sequence of rd2 VL CDR3
40 of SEQ ID NO: nucleic acid sequence of RD2 VH
41 in SEQ ID NO: nucleic acid sequence of RD2 VL
42 of SEQ ID NO: epitope sequences
43 of SEQ ID NO: epitope sequences
44 of SEQ ID NO: epitope sequences
45 in SEQ ID NO: amino acid sequence of RG1 VH
46 of SEQ ID NO: amino acid sequence of RG1 VL
47 of SEQ ID NO: sequences of RG1 VH CDR1
48 of SEQ ID NO: sequences of RG1 VH CDR2
49 of SEQ ID NO: sequences of RG1 VH CDR3
50 of SEQ ID NO: sequences of RG1 VL CDR1
51 of SEQ ID NO: sequences of RG1 VL CDR2
52, SEQ ID NO: sequences of RG1 VL CDR3
53, SEQ ID NO: amino acid sequence of RA1 VH
54, SEQ ID NO: amino acid sequence of RA1 VL
55 in SEQ ID NO: sequence of RA1 VH CDR1
14, SEQ ID NO: sequence of RA1 VH CDR2
56 in SEQ ID NO: sequence of RA1 VH CDR3
57 in SEQ ID NO: sequence of 5G.RA1 VL CDR1
28, SEQ ID NO: sequence of 5G.RA1 VL CDR2
58 in SEQ ID NO: sequence of 5G.RA1 VL CDR3
89 of SEQ ID NO: amino acid sequence of RH4 VH
90 in SEQ ID NO: amino acid sequence of RH4 VL
59 of SEQ ID NO: sequence of RH4 VH CDR1
60 of SEQ ID NO: sequence of RH4 VH CDR2
61: sequence of RH4 VH CDR3
57 in SEQ ID NO: sequence of RH4 VL CDR1
28, SEQ ID NO: sequence of RH4 VL CDR2
62 of SEQ ID NO: sequence of RH4 VL CDR3
91 of SEQ ID NO: amino acid sequence of RH4 VH
92, SEQ ID NO: amino acid sequence of RH4 VL
63, SEQ ID NO: sequence of RH4 VH CDR1
64 in SEQ ID NO: sequence of RH4 VH CDR2
65 of SEQ ID NO: sequence of RH4 VH CDR3
66 of SEQ ID NO: sequence of RH4 VL CDR1
51 of SEQ ID NO: sequence of RH4 VL CDR2
67, SEQ ID NO: sequence of RH4 VL CDR3
93 in SEQ ID NO: amino acid sequence of RF6 VH
94, SEQ ID NO: amino acid sequence of 5G.RF6 VL
68 of SEQ ID NO: sequence of RF6 VH CDR1
14, SEQ ID NO: sequence of RF6 VH CDR2
69 of SEQ ID NO: sequence of RF6 VH CDR3
57 in SEQ ID NO: sequence of the 5G.RF6 VL CDR1
28, SEQ ID NO: sequence of the 5G.RF6 VL CDR2
58 in SEQ ID NO: sequence of the 5G.RF6 VL CDR3
95 in SEQ ID NO: amino acid sequence of RD6 VH
95 in SEQ ID NO: amino acid sequence of RD6 VL
70 of SEQ ID NO: sequence of RD6 VH CDR1
71 of SEQ ID NO: sequence of RD6 VH CDR2
72 of SEQ ID NO: sequence of RD6 VH CDR3
57 in SEQ ID NO: sequence of RD6 VL CDR1
28, SEQ ID NO: sequence of RD6 VL CDR2
58 in SEQ ID NO: sequence of RD6 VL CDR3
97 of SEQ ID NO: amino acid sequence of RA3 VH
98 of SEQ ID NO: amino acid sequence of RA3 VL
55 in SEQ ID NO: sequence of RA3 VH CDR1
73 in SEQ ID NO: sequence of RA3 VH CDR2
74 of SEQ ID NO: sequence of RA3 VH CDR3
57 in SEQ ID NO: sequence of RA3 VL CDR1
28, SEQ ID NO: sequence of RA3 VL CDR2
58 in SEQ ID NO: sequence of RA3 VL CDR3
99 in SEQ ID NO: amino acid sequence of RG1 VH
100, SEQ ID NO: amino acid sequence of RG1 VL
75 of SEQ ID NO: sequences of RG1 VH CDR1
76 of SEQ ID NO: sequences of RG1 VH CDR2
77 of SEQ ID NO: sequences of RG1 VH CDR3
78, SEQ ID NO: sequence of RG1 VL CDR1
28, SEQ ID NO: sequence of RG1 VL CDR2
62 of SEQ ID NO: sequence of RG1 VL CDR3
101, SEQ ID NO: amino acid sequence of RG4 VH
102, SEQ ID NO: amino acid sequence of RG4 VL
79 in SEQ ID NO: sequences of RG4 VH CDR1
80, SEQ ID NO: sequences of RG4 VH CDR2
81 of SEQ ID NO: sequences of RG4 VH CDR3
57 in SEQ ID NO: sequence of RG4 VL CDR1
28, SEQ ID NO: sequence of RG4 VL CDR2
58 in SEQ ID NO: sequence of RG4 VL CDR3
103, SEQ ID NO: amino acid sequence of RC5 VH
104 of SEQ ID NO: amino acid sequence of RC5 VL
82 in SEQ ID NO: RC5 VH CDR1 sequences
80, SEQ ID NO: RC5 VH CDR2 sequences
83 of SEQ ID NO: RC5 VH CDR3 sequences
57 in SEQ ID NO: RC5 VL CDR1 sequences
51 of SEQ ID NO: RC5 VL CDR2 sequences
58 in SEQ ID NO: RC5 VL CDR3 sequences
105 of SEQ ID NO: amino acid sequence of RE6 VH
106 of SEQ ID NO: amino acid sequence of 5F.RE6 VL
55 in SEQ ID NO: sequence of RE6 VH CDR1
73 in SEQ ID NO: sequence of RE6 VH CDR2
74 of SEQ ID NO: sequence of RE6 VH CDR3
84, SEQ ID NO: sequence of the 5 f.6 VL CDR1
51 of SEQ ID NO: sequence of the 5 f.6 VL CDR2
85 of SEQ ID NO: sequence of the 5 f.6 VL CDR3
107 of SEQ ID NO: amino acid sequence of RF2 VH
108 in SEQ ID NO: amino acid sequence of RF2 VL
86 of SEQ ID NO: sequence of RF2 VH CDR1
87, SEQ ID NO: sequence of RF2 VH CDR2
88 of SEQ ID NO: sequence of RF2 VH CDR3
109, SEQ ID NO: sequence of 5H.RF2 VL CDR1
51 of SEQ ID NO: sequence of 5H.RF2 VL CDR2
85 of SEQ ID NO: sequence of rf2 VL CDR 3.
[ detailed description ] embodiments
General rule
In this specification, a reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be taken to encompass one or more (i.e., one or more) of those steps, compositions of matter, group of steps, or group of compositions of matter, unless otherwise specifically indicated or the context requires otherwise.
It will be appreciated by persons skilled in the art that the present invention 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 disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.
Any embodiment of the disclosure herein should be understood to apply mutatis mutandis to any other embodiment of the disclosure unless explicitly stated otherwise.
Unless otherwise specifically defined, all technical and scientific terms used herein are to be understood as having 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).
Unless otherwise indicated, recombinant proteins, cell culture and immunological techniques used in the present disclosure are standard procedures that are numerical to those of skill in the art. Throughout the literature, in such cases as Perbal (1984); sambrook et al, (1989); brown (1991); glover and Hames (1995 and 1996) and Ausubel et al, (1988, including all updates to date); harlow and Lane, (1988); coligan et al, (including all updates so far); and Zola (1987) have described and explained such techniques.
The variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof, described and defined herein can be determined by Kabat,1987 and/or 1991; the discussion in Bork et Al, 1994 and/or Chothia and Lesk,1987 and/or 1989 or Al-Lazikani et Al, 1997 or the IMGT number of Lefranc M. -P., (1997) Immunology 5Today 18,509 is further elucidated.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the term "derived from" shall be meant to indicate that the specified integer is obtainable from a particular source, even if not necessarily directly from that source.
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).
Selected definition
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The term "a/an" and the terms "one or more" and "at least one" are used interchangeably herein.
Further, "and/or" as used herein is considered a specific disclosure of the presence or absence of the other for each of the two specified features or components. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B," "a or B," "a" (alone) and "B" (alone). Also, the term "and/or" as 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).
The term "about" is used herein to mean approximately, about, or around … …. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values set forth. In general, the term "about" is used herein to modify a numerical value by a variation of 10% (%) above and below the stated value (up or down).
It is understood that the PAR4 binding proteins and antibodies, nucleic acids, cells, and vectors described herein are in isolated form. By "isolated" is meant 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 the form that they exist in nature. In some aspects, the isolated antibody, polynucleotide, vector or cell is substantially pure. In some aspects, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.
As used herein, the term "protease activated receptor (PAR 4)" 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 polypeptides are characterized by having the ligand activation properties (including agonist activation properties and antagonist inhibition properties) and tissue distribution described herein. The term includes those portions of PAR4 that are capable of binding thrombin or the PAR4 receptor portion as set forth in SEQ ID NO: 2.
The term "PAR 4 antagonist" refers to an inhibitor of platelet aggregation that binds to PAR4 and inhibits PAR4 cleavage and/or signaling. Typically, the PAR4 activity is reduced by at least 10% >, 20% >, 30% >, 40% >, 50% >, 60% >, 70% >, 80% >, 90% >, or 100% in a dose-dependent manner as compared to such activity in control cells. Control cells are cells that have not been treated with a compound. PAR4 activity is determined by any standard method in the art, including those methods described herein (e.g., calcium mobilization, platelet aggregation in cells expressing PAR4, platelet activation assays and hemostasis models measuring, e.g., calcium mobilization, p-selectin or CD40L release or thrombosis).
The term "hPAR 4" or "human PAR 4" refers to fully human antibodies. For purposes of nomenclature, but not limitation, the amino acid sequence of hPAR4 is set forth in SEQ ID NO. 19.
The term "mAb" as used herein is intended to refer to a monoclonal antibody comprising mouse constant region sequences and human variable region sequences.
The term "antibody" describes an immunoglobulin, either naturally or partially or wholly synthetically produced or recombinantly produced. 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 this term. An "antibody" is any immunoglobulin that binds 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 or antigen-binding portion thereof comprising at least two heavy (H) and two light (L) immunoglobulin chains that are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). CH typically consists of three domains, CH1, CH2 and CH3 (IgM, e.g. with an additional constant region domain CH 4). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). CL consists of one domain and can be of the λ or κ type. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FWRs). Each VH and VL consists of three CDRs and four FWRs, arranged from amino-terminus to carboxy-terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR 4. In certain embodiments, the VH and VL together comprise a binding domain that interacts with an antigen. In other embodiments, a single VH or a single VL domain may specifically interact with an antigen. The CH domain of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells), cells lining the walls of blood vessels, other cells expressing receptors for the CH domain of the immunoglobulin, and the first component of the classical complement system (C1 q). Immunoglobulin 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 IgA 2). The term "antibody" as used herein also includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain(s) is identical or homologous to corresponding sequences in antibodies derived 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)). The basic antibody structure in vertebrate systems is relatively well understood. (see, e.g., Harlow et al (1988) Antibodies: A Laboratory Manual (2 nd edition; Coldspring Harbor Laboratory Press.) the term "antibody" also includes within its meaning any "antigen-binding fragment".
The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Fragments of full-length antibodies 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, which is a monovalent fragment consisting of the VL domain and the CL, VH and CH1 domains; (ii) a f (ab)2 fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH domain and the CH1 domain; (iv) fv fragments consisting of the VH domain and the CL domain of a single arm of an antibody; (v) single domain antibody fragments or dAbs (Ward et al, (1989) Nature 341:544-546) consisting of only VH domains or vl domains; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant or synthetic methods, e.g., by a synthetic linker that enables them to be made in the form of a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scfv)); see, e.g., Bird et al (1988) Science242: 423-; and Huston et al (1988) Proc.Natl.Acad.Sci.USA 85: 5879-. scFv are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as intact antibodies.
As used herein, "antibody variable region" refers to the portions of the light and heavy chains of an antibody molecule that include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR3) and the framework region (FWR). VH refers to the heavy chain variable region. VL refers to the light chain variable region. According to the methods used in the present invention, the amino acid positions assigned to the CDRs and FRs may 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.
The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibodies exhibit 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 produced synthetically and are part or all of human sequences.
The term "polyclonal antibody" as used herein refers to a mixture of antibodies purified from the serum of a mammal into which an antigen has been injected to produce antibodies against the antigen. Polyclonal antibodies can be produced from any mammal, e.g., mouse, rat, rabbit, pig, human, etc., or can be produced synthetically, e.g., as a VH and VL gene phage display library.
The term "chimeric antibody" refers to an antibody that: wherein a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine) or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical or homologous to corresponding sequences in antibodies derived from another species (e.g., primate) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
The term "humanized antibody" is understood to mean a chimeric molecule, typically prepared using recombinant techniques, which has an epitope binding site derived from an immunoglobulin from a non-human species and 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 onto an appropriate framework region in the variable domain of the human antibody and the remaining region from the human antibody.
The term "human antibody" as used herein in connection with antibody molecules and binding proteins refers to antibodies having variable (e.g., VH, VL, CDR and FR regions) and constant antibody regions derived from or corresponding to sequences found in humans, e.g., in the human germline or somatic cells.
As used herein, "IMGT numbering" refers to the numbering system used to identify the CDR and FWR sequences of an antibody variable region. IMGT unique numbering has been defined to compare variable domains regardless of antigen receptor, chain type or species (lefrancm. -p., Immunology 5Today 18,509(1997)/Lefranc m. -p., The Immunologist,7, 132-. In the unique numbering of IMGT, conserved amino acids have the same positions throughout, such as cysteine 23 (1 st CYS), tryptophan 41 (conserved TRP), hydrophobic amino acid 89, cysteine 104 (2 nd CYS), phenylalanine or tryptophan 118(J-PHE or J-TRP). The unique numbering of IMGT provides the framework regions (FR 1-IMGT:
As used herein, the term "specifically binds" will be used to refer to a protein of the present disclosure reacting or associating with a particular cell or substance more frequently, more rapidly, with a longer duration and/or with greater affinity than with a replacement cell or substance. It is also understood by reading the definition that, for example, a protein that specifically binds a first antigen may or may not specifically bind a second antigen. As such, "specific binding" does not necessarily require an exclusive or undetectable binding of another antigen, which is to be interpreted by the term "selective binding".
"transfected", "transfected cell" and the like refer to a cell into which (or into an ancestor of which) a DNA molecule encoding PAR4 (or a DNA encoding a biologically active fragment or analog thereof) has been introduced by genetic engineering. Such a DNA molecule is "positioned for expression" means that the DNA molecule is positioned adjacent to a DNA sequence that directs the transcription and translation of the sequence (i.e., promotes the production of the PAR4 protein or fragment or analog thereof).
The term "identity" and grammatical variations thereof refers to the identity of two or more referenced entities. Thus, where two antibody sequences are identical, they have identical amino acid sequences, at least within the reference region or portion. When two nucleic acid sequences are identical, they have the same polynucleotide sequence at least within the reference region or portion. The identity may be over a defined region (region or domain) of the sequence. The% identity of a polynucleotide is determined by GAP (Needleman and Wunsch, j.mol biol.48:444-453.1970) analysis (GCG program) at a GAP creation penalty of 5 and a GAP extension penalty of 0.3. Unless otherwise stated, the query sequence is at least 45 nucleotides 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. More preferably, the two sequences are aligned over their entire length.
The term "pharmaceutical composition" as used herein refers to any composition containing at least one therapeutic or bioactive agent and suitable for administration to a patient. Any of these formulations can be prepared by methods well known and recognized in the art. See, e.g., Gennaro, A.R., eds., Remington: The Science and Practice of Pharmacy, 20th edition, Mack Publishing Co., Easton, Pa. (2000).
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and/or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the terms "treatment" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the progression of an undesirable physiological change or disorder, such as thromboembolism, e.g., acute coronary syndrome. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean prolonging survival compared to that expected in the absence of 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 for which prevention of the condition or disorder is intended.
As used herein, "prophylaxis" or "prevention" refers to prophylactic treatment of a sub-clinical disease state in a mammal, particularly a human, that aims to reduce the likelihood of the occurrence of a clinical disease state. Patients are selected for prophylactic treatment based on known factors that increase the risk of developing clinical disease compared to the general population. "prevention" therapy can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of a subject who has not yet exhibited a clinical disease state, while secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.
The term "therapeutically effective amount" is understood to mean an amount of a PAR4 binding protein or antibody sufficient to reduce or inhibit one or more symptoms of PAR4 activation to a level below that observed and accepted as a clinical feature of the disorder. One skilled in the art will appreciate that such amounts will vary depending on the type or severity or level of the specific antibody, fragment, and/or particular subject and/or disorder. Accordingly, the terms should not be construed to limit the invention to specific amounts.
As used herein, the term "PAR 4 antagonist therapy" refers to the treatment of a subject with a PAR4 antagonist.
By "subject" is meant any subject, particularly a mammalian subject, in need of diagnosis, prognosis or treatment. 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, and the like. As used herein, phrases such as "a subject having a PAR4 mediated disorder or condition" include subjects, such as mammalian subjects, that would benefit from administration of a PAR4 antagonist.
As used herein, reference to a level of binding that is "similar" is understood to refer to an antibody that binds to an antigen at a level within about 30% or 25% or 20% of the level at which it binds to another antigen. The term can also mean that one antibody binds to an antigen at a level within about 30% or 25% or 20% of the level at which another antibody binds to the same antigen.
As used herein, reference to a level of binding that is "substantially the same" is understood to refer to an antibody that binds to an antigen at a level that is within about 15% or 10% or 5% of the level at which it binds to another antigen. The term can also mean that one antibody binds to an antigen at a level within about 5% or 4% or 3% of the level at which another antibody binds to the same antigen.
The term "competitively inhibits" is understood to mean that a protein of the disclosure reduces or prevents the binding of the antibody produced (e.g., 5a. rc3) to the thrombin cleavage site of PAR4 or a fragment thereof. It will be apparent from the above that the protein need not completely inhibit binding of the antibody, but it need only reduce binding by a statistically significant amount, for example by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determining 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 in the presence or absence of a protein. A protein is considered to competitively inhibit binding of the antibody if less antibody binds in the presence of the protein than in the absence of the protein. In one example, the protein and antibody are exposed to PAR4 substantially simultaneously. Additional methods for determining competitive inhibition of binding will be apparent to those skilled in the art and/or described herein. In one example, the antigen binding domain of the protein competitively inhibits binding of the antibody.
"overlap" in the context of two epitopes will be used to refer to two epitopes sharing a sufficient number of amino acid residues to allow an antibody binding to one epitope to competitively inhibit binding of an antibody binding to the other epitope. For example, the epitopes share at least one or two or three or four or five or six or seven or eight or nine or ten amino acids.
As used herein, the term "non-detectably binds" is understood to mean that the protein (e.g., antibody) binds to the candidate antigen at a level less than 10%, 8%, or 6% or 5% above background. The background can be the level of binding signal detected in the absence of 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) in which the protein is immobilized and contacted with the antigen.
Antibodies
For the avoidance of doubt, the monoclonal antibody mAb ARC3 is synonymous with the other names for such an antibody, as shown in the examples, such as MoB5A-RC3. This antibody has been further subcloned into the derivative monoclonal antibody MoB5-ARC3.F10b. H4b. This seed clone is also known as mAb ARC3.H4 b. The sequences corresponding to this antibody are shown in SEQ ID NO 11-18.
Functionally equivalent antibodies
The present disclosure also contemplates anti-PAR 4 antibodies or antigen-binding fragments thereof that have one or more amino acid additions, deletions, or substitutions in the heavy and light chain variable region sequences of the antibody mAb arc3.h4b, but still retain the function of mAb arc3.h 4b. In some examples, the PAR4 binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. 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. Hydrophobic (hydropathic) indices are described, for example, in Kyte and Doolittle (1982), and hydrophilic (hydropathic) indices are described, for example, in US 4554101.
These modifications may be deliberate, such as, for example, by site-directed mutagenesis; or may be incidental such as those obtained by mutations in the host in which the antibody is expressed.
The mutated (altered) polypeptide may be prepared using any technique known in the art. For example, polynucleotides of the present disclosure can be subjected to in vitro mutagenesis. This in vitro mutagenesis technique involves subcloning the polynucleotide into a suitable vector, converting the vector into a "mutant" strain, such as E.coli XL-1 Red (Stratagene), and propagating 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.
In designing amino acid sequence mutants, the position of the mutation site and the nature of the mutation will depend on the feature or features to be altered. The site of mutation may be altered individually or sequentially, for example, by (1) selective substitution with conserved amino acids first and then selective substitution with more groups (depending on the result achieved), (2) deletion of the target residue, or (3) insertion of other residues adjacent to the site located.
Amino acid sequence deletions typically range from about 1 to 15 residues, more preferably from about 1 to 10 residues and typically from about 1 to 5 contiguous residues.
Substitution mutants remove at least one amino acid residue from the antibody and/or immunoglobulin chain molecule (including in the variable region) and insert a different residue in its place. Sites of most interest for substitution mutagenesis include sites identified as important for antigen binding. These sites, particularly those that fall within the sequence of human antibodies and/or at least three other equally conserved sites of immunoglobulin chains, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in table 1 under the heading of "exemplary substitutions".
Conservative amino acid substitutions are also contemplated by the present invention. These are considered to refer to the amino acid substitutions listed in the following table.
Table 1 exemplary substitutions
The amino acids described herein are preferably in the "L" isomeric form. However, residues in the form of the D isomer may be substituted for any L-amino acid residue as long as the polypeptide retains the desired functional properties of immunoglobulin binding. Modifications also include structural and functional analogs, such as peptidomimetics having synthetic or unnatural amino acids or amino acid analogs and derivatized forms.
Non-conservative amino acid changes are also encompassed by the present disclosure. For example, it is particularly noteworthy that a charged amino acid is substituted with another charged amino acid and with a neutral or positively charged amino acid. In some examples, the PAR4 binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1, non-conservative amino acid substitutions.
A mutant form of the PAR4 binding protein described herein according to any example retains the ability to specifically bind to PAR 4. Methods for determining specific binding to PAR4 are described herein. For example, a labeled PAR4 binding protein is contacted with immobilized PAR4 or a peptide comprising the thrombin cleavage site of PAR4 (e.g., as set forth in SEQ ID NO: 2). After washing, bound label is detected. The labeled PAR4 binding protein was also contacted with immobilized PAR4 and related proteins or mutant forms of PAR4 or fragments of PAR4 as described above and the bound label was detected after washing. A marker that detects binding to PAR4 but not to a protein of interest (e.g., PAR1, PAR2, or PAR3) or mutant protein or fragment of PAR4 indicates that the mutant PAR4 binding protein retains the ability to specifically bind to PAR 4.
In one example, the mutation occurs within the FWR of the PAR4 binding protein of the present disclosure. In another example, the mutation occurs within a CDR of a PAR4 binding protein of the disclosure.
Antibody production
Methods for producing antibodies are known in the art and/or described in Harlow and Lane (1988) or Zola (1987). Generally, in such methods, the Fn14 protein or immunogenic fragment thereof or cell containing an epitope or expressing and displaying it (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal subject, e.g., a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.
The production of polyclonal antibodies can be monitored by sampling blood from the immunized animal at various points after immunization. If desired, one or more further immunizations may be given to achieve the desired antibody titer. The process of boosting and titration is repeated until a suitable titer is obtained. 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 produce monoclonal antibodies (mabs).
Monoclonal antibodies are exemplary antibodies encompassed by the present disclosure. The term "monoclonal antibody" or "mAb" refers to a homogeneous population of antibodies capable of binding to the same antigen and, for example, to the same epitope within the antigen. This term is not intended to be limiting with respect to the source or manner of preparation of the antibody.
To generate the mAb, any of a variety of known techniques may be used, such as, 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 Zola (1987) Monoclonal Antibodies: A manual of techniques.
For example, a suitable animal is immunized with an effective amount of a protein or immunogenic fragment or epitope thereof or a cell expressing the protein or immunogenic fragment or epitope thereof under conditions sufficient to stimulate antibody-producing cells. Rodents such as rabbits, mice, and rats are exemplary animals, with mice being the most common. Mice genetically engineered to express human immunoglobulin proteins but not murine immunoglobulin proteins can also be used to produce antibodies of the disclosure (e.g., as described in WO 2002/066630).
After immunization, somatic cells, particularly B lymphocytes (B cells), that have the potential to produce antibodies are selected for use in the mAb generation protocol. These cells may be obtained from spleen, tonsils or lymph node biopsies or from peripheral blood samples. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, which is generally derived from the same species as the animal immunized with the immunogen. The B cells and immortalized cells are fused by incubating a mixture of cell types in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. The fusion method using Sendai virus has been 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) social Cell Gene.3 (2): 231-6). The use of an electrically induced fusion method is also suitable.
The hybrids are amplified by culturing in a selective medium comprising an agent that blocks de novo synthesis of nucleotides in the tissue culture medium. Exemplary agents are aminopterin, methotrexate and azapurines.
The amplified hybridomas are functionally selected for antibody specificity and/or titer, such as, for example, 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.
Alternatively, ABL-MYC technology (NeoClone, Madison Wi 53713, USA) was used to generate mAb-secreting cell lines (e.g., as described in Kumar et al, (1999) Immunol Lett.65(3): 153-9).
Antibodies can also be produced or isolated by screening display libraries, e.g., phage display libraries, e.g., as described in US6300064EP0368684 and/or US 5885793.
Chimeric antibodies and proteins
In one example, an antibody or PAR4 binding protein of the disclosure is a chimeric antibody, or a PAR4 binding protein is a chimeric protein. The term "chimeric protein" refers to the following proteins: wherein the antigen-binding domain VH or VL is identical or homologous to corresponding sequences in proteins derived from a particular species (e.g. murine, such as mouse or rat) or belonging to a particular antibody class or subclass, and the remainder of the chain protein is identical or homologous to corresponding sequences in proteins derived from another species (e.g. primate, such as human) or belonging to another antibody class or subclass. In one embodiment, 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 methods (as described, for example, in US 6331415; US 5807715; US4816567 and US 4816397). The production of such chimeric antibodies is known in the art and can be achieved by standard means (as described, for example, 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. Pat. Nos. 5,807,715; 4,816,567 and 4,816,397). It is further contemplated that the human constant region of the chimeric antibody of the invention may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19 constant regions.
Humanized and human antibodies/proteins
The PAR4 binding proteins of the present disclosure may be humanized or human.
The term "humanized protein" is understood to mean a protein comprising human-like variable regions, including the grafting of CDRs of an antibody from a non-human species onto or insertion into FRs from a human antibody (this type of antibody is also referred to as "CDR-grafted antibody"). Humanized proteins also include proteins in which one or more residues of the human protein are modified by one or more amino acid substitutions and/or one or more FR residues of the human protein are replaced with corresponding non-human residues. Humanized proteins may also comprise residues that are not found in human antibodies or in non-human antibodies. Any additional region of the protein (e.g., the Fc region) is typically human. Humanization may be performed using methods known in the art, for example US5225539, US6054297, US7566771 or US 5585089. The term "humanized protein" also encompasses super-humanized proteins, e.g., as described in US 7732578.
In one example, the humanized protein comprises regions between 26 and 33, between 51 and 58, and between 97 and 110, and between 27 and 33, between 51 and 53, and between 90 and 97 (numbered according to the IMGT numbering system) in the heavy chain sequences disclosed herein.
The term "human protein" as used herein refers to a protein having a variable and optionally constant antibody region derived from or corresponding to a sequence found in humans, e.g., in human germline or somatic cells. A "human" antibody may comprise amino acid residues not encoded by human sequences, for example mutations introduced by random or site-directed mutagenesis in vitro (especially mutations comprising conservative substitutions or mutations in a small number of residues of the protein, for example mutations in 1, 2, 3,4 or 5 residues of the protein). These "human antibodies" need not in fact be produced by a human immune response, rather they may be produced using recombinant methods (e.g., screening phage display libraries) and/or by transgenic animals (e.g., mice) comprising nucleic acids encoding human antibody constant and/or variable regions and/or using guided selection (e.g., as described in or US 5565332). This term also encompasses affinity matured forms of such antibodies. Human proteins will also be considered to include proteins comprising FRs from human antibodies or FRs comprising sequences from the consensus sequence of human FRs, and wherein one or more of the CDRs is random or semi-random, for example as described in US6300064 and/or US 6248516.
Human proteins or antibodies that recognize selected epitopes can also be produced using a technique known as "guided selection". In this method, selected non-human monoclonal antibodies, e.g., mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers LS et al, (1988) Biotechnology 12(9): 899-.
The human PAR4 binding proteins of the present disclosure comprise the variable regions of human antibodies.
Homohumanised and primatized proteins
The PAR4 binding proteins of the present disclosure can be homohumanized proteins. The term "homohumanized protein" refers to a protein prepared by the method described in WO 2007/019620. Humanized PAR4 binding proteins include the variable regions of an antibody, wherein the variable regions comprise FRs from the variable region of a New world primate antibody and CDRs from the variable region of a non New world primate antibody. For example, a humanized PAR4 binding protein includes the variable region of an antibody, wherein the variable region comprises FWRs from the variable region of a new world primate antibody and CDRs from a mouse antibody, e.g., as described herein. In one example, the humanized PAR4 binding protein is a PAR4 binding antibody wherein one or both of the variable regions are homohumanized.
The PAR4 binding proteins of the present disclosure can be primatized proteins. A "primatized protein" comprises the variable regions from antibodies produced after immunization of a non-human primate (e.g., cynomolgus monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to make a primatized antibody. An exemplary method for producing primatized antibodies is described in US 6113898.
Deimmunized antibodies and proteins
Deimmunized antibodies or PAR4 binding proteins are also contemplated by the present disclosure. Deimmunized antibodies have one or more epitopes removed (i.e., mutated), such as B cell epitopes or T cell epitopes, thereby reducing the likelihood that a subject will mount 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 WOOO/34317, WO2004/108158, and WO 2004/064724.
Methods for introducing suitable mutations and expressing and assaying the resulting proteins will be apparent to those skilled in the art based on the description herein.
A protein comprising an antibody variable region.
Single domain antibodies
In some examples, the PAR4 binding proteins of the present disclosure are single domain antibodies (which are used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable region of the antibody. In certain examples, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6248516; WO90/05144 and/or WO 2004/058820).
Diabody, triabody, and tetrabody antibodies
Exemplary PAR4 binding proteins comprising an antibody antigen binding domain are diabodies, triabodies, tetrabodies and higher order protein complexes such as those described in WO98/044001 and WO 94/007921.
For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to allow association of VH and VL in a single polypeptide chain (or formation of an Fv) or is absent, and wherein the VH of one polypeptide chain binds to the VL of another polypeptide chain to form an antigen binding site, i.e. to form an Fv molecule capable of specifically binding to one or more antigens. The VL and VH in each polypeptide chain can be the same, or the VL and VH in each polypeptide chain can be different so as to form a bispecific diabody (i.e., comprising two fvs with different specificities).
Single chain fv (scFv) fragments
The skilled person will appreciate that the scFv comprises VH and VL regions in a single polypeptide chain. The polypeptide chains also comprise a polypeptide linker between the VH and VL that enables the scFv to form the desired structure for antigen binding (i.e., enables the VH and VL of a single polypeptide chain to associate with each other to form the Fv). For example, the linker comprises a super linker12 amino acid residues in which (Gly)4Ser)3Is one of the more advantageous linkers for scFv.
The present disclosure also encompasses disulfide-bond stabilized fvs (or divvs or dsfvs) in which a single cysteine residue is introduced in the FR of VH and the FR of VL and the cysteine residues are linked by a disulfide bond to produce a stable Fv (see, e.g., Brinkmann et al, (1993) Proc Natl Acad Sci USA 90: 547-.
Alternatively or additionally, the present disclosure provides dimeric scfvs, i.e., proteins comprising two scFv molecules linked by a non-covalent or covalent linkage (e.g., via a leucine zipper domain (e.g., derived from Fos or Jun)) (see, e.g., Kruif and Logtenberg, 1996). Alternatively, the two scfvs are linked by a peptide linker of sufficient length to allow the two scfvs to form and bind to an antigen, e.g. as described in US 20060263367.
For a review of scFv, see Ahmad ZA et al, (2012) Clinical and developmental immunology doi: 10.1155/2012/980250.
Minibody
The skilled artisan will appreciate that minibodies comprise the VH and VL domains of the antibody fused to (CH2 and/or (CH3 domain, optionally, minibodies comprise a hinge region between the VH and VL, this conformation sometimes being referred to as Flex minibodies the minibodies do not comprise CH1 or CL. in one example, the VH and VL domains are fused to the hinge region and CH3 domain of the antibody at least one of the variable regions of the minibodies binds to par4 in the manner disclosed herein exemplary minibodies and methods of production thereof are described, for example, in WO 94/09817.
Other proteins containing antibody variable regions
Other PAR4 binding proteins containing variable regions are also contemplated by the present disclosure, such as:
(i) a "key and well" bispecific protein as described in US5,731,168; (ii) heteroconjugate proteins, e.g., as described in US4,676,980;
(iii) heteroconjugate proteins produced using chemical cross-linking agents, for example, as described in US4, 676;
(iv) fab' -SH fragments, e.g., as described by Shalaby (1992)
(v) a single chain Fab; or
(vi) Fab3 (e.g. as described in EP 19930302894).
Non-antibody based antigen binding domain containing proteins
Immunoglobulins and immunoglobulin fragments
Examples of compounds of the present disclosure are proteins comprising the variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., IgNA, camelid antibodies).
The term "immunoglobulin" is understood to include any antigen binding protein comprising an immunoglobulin domain. Exemplary immunoglobulins are antibodies. Additional proteins encompassed by the term "immunoglobulin" include domain antibodies, camelized antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin neoantigen receptor (IgNAR)). Generally, camelid antibodies and ignars comprise a VH, however, lack a VL and are commonly referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T cell receptors.
Heavy chain immunoglobulins
Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulins (e.g., antibodies) so long as they comprise a heavy chain but not a light chain. Thus, these immunoglobulins are also referred to as "heavy chain-only antibodies". Heavy chain immunoglobulins are present in e.g. camelids and cartilaginous fish (also known as IgNAR).
The variable regions found in naturally occurring heavy chain immunoglobulins are commonly referred to as the "VHH domain" in camelid igs and the V-NAR in ignars in order to distinguish them from the heavy chain variable regions found in conventional 4 chain antibodies (which are referred to as "VH domains") and the light chain variable regions found in conventional 4 chain antibodies (which are referred to as "VL domains").
Heavy chain immunoglobulins do not require the presence of a light chain to bind to the relevant antigen with high affinity and high specificity. This means that the single domain binding fragment may be derived from a heavy chain immunoglobulin which is readily expressed and is generally stable and soluble. General descriptions of heavy chain immunoglobulins derived from camelids and their variable regions and methods for their production and/or isolation and/or use are found, inter alia, in the following references WO94/04678, WO97/49805 and WO 97/49805.
A general description of heavy chain immunoglobulins from cartilaginous fish and their variable regions and methods for their production and/or isolation and/or use are found in particular in WO 2005/118629.
V-like proteins
An example of a PAR4 binding protein of the present disclosure is the T cell receptor. The T cell receptor has 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-. Other publications describing the generation of single-chain T-cell receptors or multimeric T-cell receptors comprising two V-alpha and V-beta domains include WO1999/045110 or WO 2011/107595.
Other non-antibody proteins that comprise an antigen-binding domain include proteins with a V-like domain, which are typically monomeric. Examples of proteins comprising such V-like domains include CTLA-4, CD28 and ICOS. Further disclosures of proteins comprising such V-like domains are included in WO 1999/045110.
Adnectin
In one example, a PAR4 binding protein of the present disclosure is an adnectin.
Adnectins are based on the tenth fibronectin type III (10Fn3) domain of human fibronectin, in which the loop region is altered to confer antigen binding. For example, three loops at one end of the β -sandwich of the 10Fn3 domain can be engineered to enable adnectins to specifically recognize antigens. For further details, see US20080139791 or WO 2005/056764.
Anti-transporter proteins
In another example, a PAR4 binding protein of the present disclosure is an anti-transporter protein. Antiporters are derived from lipocalins, a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile pigments (bilins), retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with multiple loops at the open end of a conical structure, which can be engineered to bind to an antigen. Such engineered lipocalins are referred to as antiporters. For further description of the anti-transporter proteins see US7250297B1 or US 20070224633.
Affibody
In another example, the PAR4 binding proteins of the present disclosure are affibodies. Affibodies are the Z domain (antigen binding domain) of protein a derived from staphylococcus aureus, which can be engineered to bind to an antigen. The Z domain consists of a triple helix bundle of about 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see EP 1641818.
Avimer
In another example, the PAR binding protein of the disclosure is Avimer. Avimer is a multidomain protein derived from the A-domain scaffold family. The natural domain of about 35 amino acids assumes a defined disulfide-bonded structure. Diversity is generated by shuffling of natural variations displayed by the a-domain family. For further details, see WO 2002088171.
DARPin
In another 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 integral membrane proteins to the cytoskeleton. The single ankyrin repeat is a33 residue motif consisting of two alpha helices and beta turns. They can be engineered by randomizing residues in the first alpha-helix and beta-turn of each repeat sequence to bind different target antigens. The binding interface can be increased by increasing the number of modules (method of affinity maturation). For further details, see US 20040132028.
Other non-antibody polypeptides
Other non-antibody proteins containing binding domains include those based on human gamma-crystallin and human ubiquitin (affilins), kunitz-type domain of human protease inhibitor, PDZ domain of Ras binding protein AF-6, scorpion toxin (charcot-ton), C-type lectin domain (tetranectin).
Constant region
The present disclosure encompasses PAR4 binding proteins comprising a variable region and a constant region or one or more domains thereof (e.g., Fc, CH2, and/or CH3 domains). Based on the disclosure herein and the references discussed herein, the skilled person will know the meaning of the terms constant region and constant domain.
The constant region sequences useful for producing the PAR4 binding proteins of the present disclosure may be obtained from a number of different sources. In some examples, the constant region of the PAR4 binding protein, or portion thereof, is derived from a human antibody. Furthermore, the constant domains or portions thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE; and any antibody isotype, including IgGl, IgG2, IgG3, and IgG 4. In one example, the human isotype IgGl is used.
The various constant region gene sequences may be obtained as publicly available deposits, or their sequences may be obtained from publicly available databases. Constant regions with specific effector functions (or lack specific effector functions) or with specific modifications can be selected to reduce immunogenicity.
In one example, a protein of the disclosure has or displays effector function that contributes to or enables at least partial depletion, substantial depletion, or elimination of cells expressing PAR 4. Such effector functions may be enhanced binding affinity to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).
In one example, the PAR4 binding protein is capable of inducing enhanced levels of effector function.
In one example, the level of effector function induced by the constant region is enhanced relative to the Fc region of a wild-type IgG1 antibody or the Fc region of a wild-type IgG3 antibody.
In another example, the constant region is modified to enhance the level of effector function that it is capable of inducing compared to a constant region without the modification. Such modifications may be at the amino acid level and/or secondary structure level and/or tertiary structure level and/or glycosylation of the Fc region.
One skilled in the art will appreciate that higher effector function may be manifested in any of a variety of ways, such as by a higher level of action, more sustained action, or a faster rate of action. Exemplary constant region modifications include amino acid substitutions, such as S239D/I332E, numbered according to the EU index of Kabat, or S239D/a330L/I332E, numbered according to the EU index of Kabat.
Additional amino acid substitutions that enhance the ability of the Fc region to induce effector function are known in the art and/or described in, for example, US6737056 or US 7317091.
In one example, the glycosylation of the constant region is altered to enhance its ability to induce enhanced effector function. In some examples, an Fc region according to the present disclosure comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region, i.e., the Fc region is "afucosylated". Such variants may have an increased ability to induce ADCC. Methods for producing afucosylated antibodies include expressing the Fnl4 binding protein in a cell line incapable of expressing a-l, 6-fucosyltransferase (FUT8) (e.g., as described by yunane-ohniki et al, 2004). Other methods include the use of cell lines that inherently produce antibodies capable of inducing enhanced effector function (e.g., duck embryo-derived stem cells for the production of viral vaccines, WO 2008/129058; in aviansProduction of recombinant proteins in cells, WO 2008/142124).
PAR4 binding proteins may also comprise an Fc region capable of inducing enhanced CDC levels. For example, a hybrid of IgG1 and IgG3 produces antibodies with enhanced CDC activity (Natsume et al, 2008).
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.
In another example, the protein comprises one or more amino acid substitutions that increase the half-life of the PAR4 binding protein. For example, the PAR4 binding protein comprises a constant region comprising one or more amino acid substitutions that increase the affinity of the constant region for a neonatal Fc region (FcRn). For example, the constant region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in endosomes. In one example, the constant region has an increased affinity for FcRn at about pH 6 compared to the affinity at about pH7.4, which facilitates the re-release of Fc into the blood after cell circulation. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.
Exemplary amino acid substitutions include T250Q and/or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F, according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US 7083784. The neutralizing PAR4 binding proteins of the present disclosure may comprise an IgG4 constant region or a stabilized IgG4 constant region. The term "stable IgG4 constant region" will be understood to refer to an IgG4 constant region that has been modified to reduce Fab arm exchange or to undergo Fab arm exchange or the propensity to form or form half antibodies. "Fab arm exchange" refers to a class of protein modifications to human IgG4 in which the IgG4 heavy chain and attached light chain (half molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Thus, the IgG4 molecule can obtain two different Fab arms that discriminate between two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A "half antibody" is formed when an IgG4 antibody is broken down to form two molecules each containing a single heavy chain and a single light chain.
In one example, the stable IgG4 constant region comprises a proline at position 241 of the hinge region according to the system of Kabat. This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, the residue is typically serine. After serine substitution for proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, the skilled person will know that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that links the Fc region and the Fab region that confers mobility to the two Fab arms of the antibody. The hinge region includes cysteine residues, which are involved in the disulfide bonds between the heavy chains. Numbering system according to Kabat, which is generally defined as the stretch from Glu226 to Pro243 of
Modified proteins
The present disclosure provides PAR4 binding proteins having at least 80% identity to the sequences of the present disclosure and having the same functional characteristics as described or claimed herein.
In one example, a PAR4 binding protein of the 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.
In another example, a PAR4 binding protein of the 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 disclosed VH described herein, e.g., seq id No. 12.
The present disclosure also provides nucleic acids encoding the aforementioned proteins or nucleic acids that hybridize thereto under medium to high stringency conditions.
The present disclosure also encompasses nucleic acids encoding proteins comprising the sequences set forth in SEQ ID NO:11 and SEQ ID NO:12 that differ from the sequences exemplified herein due to the degeneracy of the genetic code.
The% identity of a nucleic acid or polypeptide is determined by GAP (Needleman and wunsch.1970) analysis (GCG program) at a GAP creation penalty of 5 and a GAP extension penalty of 0.3. The query sequence is at least 50 residues in length, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length, and the GAP analysis aligns the two sequences over a region of at least 100 residues. In one embodiment, the two sequences are aligned over their entire length.
The glycosylation pattern of an antibody can be altered from the original glycosylation pattern of a reference antibody. Alteration refers to deletion of one or more carbohydrate moieties found in the antibody, and/or addition of one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a 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 the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any 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 may also be used. The addition of a glycation site to the antibody is preferably achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for an N-linked glycation site). Alterations may also be caused by the addition or substitution of one or more serine or threonine residues to the sequence of the original protein (for O-linked glycosylation sites).
Modified glycoforms of the antibodies of the invention may be used for a variety of purposes, including, but not limited to, enhancing or reducing effector function and/or altering the half-life of the antibody (see, e.g., WO/2007/010401). such alterations may result in a reduction or increase in C1q binding and CDC or fcyr binding and ADCC. for example, substitutions may be made in one or more amino acid residues of the heavy chain constant region, thereby causing a change in effector function while retaining the ability to bind to the antigen as compared to the modified antibody, see US5,624,821 and US5,648,260. the engineered glycoforms may be produced by any method known to those skilled in the art, e.g., by using an engineered or variant expressing strain, by contacting with one or more enzymes (e.g., β (l,4) -N-acetylglucosamine transferase III (GnTIl 1))Co-expression, by expressing the antibody or fragment thereof in various organisms or cell lines derived from various organisms, or by modifying one or more carbohydrates after the antibody or fragment thereof has been expressed. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, those described in: umana et al, 1999, nat. Biotechnol 17: 176-180; davies et al, 2007Biotechnol Bioeng 74: 288-294; shield et al, 2002, J Biol Chem 277: 26733-26740; shinkawa et al, 2003, J Biol Chem278: 3466-; U.S. Pat. nos. 6,602,684; U.S. serial No. 10/277,370; U.S. serial No. 10/113,929; PCT WO 00/
For effector function, it may be desirable to modify an antibody of the invention, e.g., in order 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 in the Fc region of the antibody. Alternatively or in addition, cysteine residues may be introduced into the Fc region, allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J.ExpMed.176: 1191-1195 (1992) and shop, B.J.Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al Cancer Research 53:2560 + 2565 (1993). Alternatively, antibodies with dual Fc regions can be engineered and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al Anti-Cancer Drug Design 3:219-230 (1989).
To increase the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), for example, as described in U.S. patent No. 5,739,277. The term "salvage receptor binding epitope" as used herein refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for extending the in vivo serum half-life of the IgG molecule. D. Alternatively, the half-life of the antibody can be increased by pegylation.
Affinity maturation
In another example, an existing PAR4 binding protein of the present disclosure is affinity matured to produce an antibody capable of binding PAR4 with increased affinity. For example, sequences encoding VL and/or VH are isolated and CDR encoding regions (e.g., regions encoding CDR3 of VL and/or VH) are mutated such that one or more amino acid substitutions are introduced. The resulting mutant PAR 4-binding proteins are then screened for binding to PAR4, for example in a competitive assay.
PAR4 binding proteins according to the present disclosure can be soluble secreted proteins and can be presented as fusion proteins on the surface of a cell, or as particles (e.g., phage or other virus, ribosome, or spore). Exemplary phage display methods are described in, for example, US 5821047; US6248516 and US 6190908. Phage display particles produced using these methods are then screened to identify displayed PAR 4-binding proteins with a conformation sufficient to bind to a target antigen, such as PAR 4.
Protein production
In one example, a PAR4 binding protein of the 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 as known in the art).
Recombinant expression
In the case of recombinant proteins, the nucleic acid encoding the recombinant protein is placed in one or more expression constructs (e.g., expression vectors), which are then transfected into a host cell, such as a cell that produces disulfide bridges or bonds, e.g., an E.coli cell, a yeast cell, an insect cell, or a mammalian cell. Exemplary mammalian cells include simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. Molecular cloning techniques to achieve these goals are known in the art and are described, for example, in Ausubel or Sambrook. A variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art. See US 4816567; US7923221 and US 7022500.
After isolation, the nucleic acid encoding the protein of the disclosure is inserted into an expression construct or replicable vector for further cloning (amplification of the DNA) or expression in a cell-free system or cell. For example, the nucleic acid is operably linked to a promoter, and as used herein, the term "promoter" is to be understood in its broadest sense and includes transcriptional regulatory sequences of genomic genes, including the TATA box or initiator element required for precise transcription initiation, with or without other regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter nucleic acid expression (e.g., in response to developmental and/or external stimuli, or in a tissue-specific manner). In the context of the present disclosure, the term "promoter" is also used to describe a recombinant, synthetic or fused nucleic acid, or derivative that confers, activates or enhances expression of the nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more particular regulatory elements to further enhance expression and/or alter spatial expression and/or temporal expression of the nucleic acid.
As used herein, the term "operably linked" means that a promoter is positioned relative to a nucleic acid such that expression of the nucleic acid is under the control of the promoter.
The present disclosure also contemplates cell-free expression systems. For example, a nucleic acid encoding the Fn1-4 binding proteins of the present disclosure is operably linked to a suitable promoter, such as the T7 promoter, and the resulting expression construct is exposed to conditions sufficient for transcription and translation. Typical expression vectors for in vitro expression or cell-free expression have been described, including but not limited to TNTT7 and TNT 3 system (Promega), pEXP1-DEST and pEXP2-DEST vector (Invitrogen).
Many vectors are available for expression in cells. Carrier components typically include, but are not limited to, one or more of the following: signal sequences, sequences encoding the Fnl4 binding proteins of the disclosure (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. One skilled in the art will know the sequences suitable for expression of a protein. 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 a leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signals).
Exemplary promoters include promoters active in prokaryotes (e.g., phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters, such as the tac promoter).
Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-oc promoter (EF1), micronucleus RNA promoter (Ula and Ulb), oc-myosin heavy chain promoter,
Typical promoters suitable for expression in a yeast cell, such as, for example, a yeast selected from the group consisting of Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Schizosaccharomyces pombe (S.pombe), include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUPl promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.
The means for introducing an isolated nucleic acid molecule or a genetic construct comprising said nucleic acid into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transfection, liposome (e.g., using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA)) mediated transfection, PEG-mediated DNA uptake, electroporation, viral transduction (e.g., using lentiviruses), and microprojectile bombardment (e.g., using DNA-coated tungsten or gold particles (Agracetus inc., WI, USA)), among others.
Host cells for producing the PAR4 binding proteins of the present disclosure can be cultured in a variety of media depending on the cell type used. Commercially available media (e.g., Ham's F10(Sigma), minimal essential medium ((MEM), (Sigma)), RPMl-1640(Sigma), and Dulbecco's modified eagle's medium ((DMEM), Sigma)) are suitable for culturing mammalian cells.
Isolation of proteins
The PAR4 binding proteins of the present disclosure can be isolated or purified.
Methods for purifying PAR4 binding proteins of the present disclosure are known in the art and/or described herein.
When using recombinant techniques, the PAR4 binding proteins of the present disclosure may be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the antibody is produced intracellularly, either the host cells or the particulate debris of the lysed fragments are removed as a first step, for example, by centrifugation or ultrafiltration. In the case of secretion of the protein into the culture medium, the supernatant from such expression systems is first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). A protease inhibitor (e.g., PMSF) may be included in any of the foregoing steps to inhibit proteolysis and an antibiotic may be included to prevent the growth of adventitious contaminants.
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 methods. Such methods are known in the art and are described, for example, in W099/57134 or Zola (1997).
The skilled artisan will also appreciate that the PAR4 binding proteins of the present disclosure may be modified to include tags to facilitate purification or detection, such as a polyhistidine tag, e.g., a hexa-polyhistidine tag, or an influenza virus Hemagglutinin (HA) tag, or a simian virus 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 comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on a solid or semi-solid support that specifically binds the hexa-his tag, washing the sample to remove unbound protein, and then eluting bound protein. Alternatively or additionally, ligands or antibodies bound to a tag are used in the affinity purification method.
Conjugates
The present disclosure also provides conjugates of the PAR4 binding proteins described herein according to any example. Examples of compounds to which a protein may 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 a protein in a subject, and mixtures thereof. Exemplary therapeutic agents include, but are not limited to, anti-angiogenic agents, anti-neovascularization and/or other angiogenic agents anti-proliferative agents, pro-apoptotic agents, chemotherapeutic agents or therapeutic nucleic acids. Toxins include any agent that is harmful (e.g., killing) to a cell. For a description of these classes of drugs and their mechanisms of action known in the art, see Goodman et al, (1990). Additional techniques related to the preparation of immunoglobulin-immunotoxin conjugates are provided, for example, in US 5194594. Exemplary toxins include diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, cantonese a chain, α -sarcin, Aleurites fordii protein, dianthin protein, pokeweed (phytopaca americana) protein (PAPI, PAPII and PAP-S), momordica charantia (momordica charrantia) inhibitor, curcin, crotin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitogellin (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (neomycin) and trichothecene (tricothecene). See, e.g., W093/21232.
In one example, a PAR4 binding protein as described herein according to any example is conjugated or linked to another protein, such as an immunomodulator or a half-life extending protein or a peptide or other protein that binds to serum albumin, and the like. Exemplary serum albumin binding peptides or proteins are described in US20060228364 or US 20080260757.
In another example, the protein is conjugated to a "receptor" (such as streptavidin) for use in cell pre-targeting, where the conjugate is administered to a patient, followed by removal of unbound conjugate from circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) conjugated to a therapeutic agent (e.g., a radionucleotide).
PAR4 binding proteins of the present disclosure can be modified to contain additional non-protein moieties known in the art and readily available. For example, moieties suitable for derivatization of proteins are physiologically acceptable polymers, such as water soluble polymers. Such polymers may be used to increase stability and/or decrease clearance (e.g., through the kidney) and/or decrease immunogenicity of Fn14 binding proteins of the present disclosure. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propylene glycol (PPG).
In one example, a PAR4 binding protein as described herein according to any example comprises one or more detectable labels to facilitate detection and/or isolation. For example, the compounds comprise fluorescent labels, such as, for example, Fluorescein (FITC), 5, 6-carboxymethylfluorescein, Texas Red, nitrobenzene-2-oxa-1, 3-oxadiazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4' -6-diamidino-2-phenylindole (DAPI) and cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5, 6-tetramethylrhodamine). The absorption and emission maxima of these phosphors are respectively: fn1-4 binding proteins as described herein according to any example, alternatively or additionally, are labelled with, for example, fluorescent semiconductor nanocrystals (e.g. as described in US6,306,610), FITC (490 nm; 520nm), Cy3(554 nm; 568nm), Cy3.5(581 nm; 588nm), Cy5(652nm:672nm, Cy5.5(682 nm; 703nm) and Cy7(755 nm; 778 nm).
Alternatively or additionally, PAR4 binding proteins are labeled with, for example, magnetic or paramagnetic compounds, such as iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt-platinum, or strontium ferrite.
Immobilized proteins
In one example, the PAR4 binding protein is immobilized on a solid or semi-solid matrix. The term "immobilization" is to be understood as referring to various methods and techniques for immobilizing proteins on a specific substrate, e.g.as described in W099/56126 or WO 02/26292. For example, immobilization can serve to stabilize the protein such that its activity is not reduced or adversely altered by biological, chemical, or physical exposure, particularly during storage or single batch use. Various methods for immobilizing proteins on a matrix are known in the art, including cross-linking, binding to a carrier, retention within a semi-permeable matrix. Exemplary matrices include porous gel, alumina, bentonite, agarose, starch, nylon, or polyacrylamide.
Determining the Activity of binding proteins of the disclosure
Binding assays
One form of such an assay is an antigen binding assay, for example, as described in Scopes (1994) protein purification: primers and pathogen Springer-Verlag. Such methods generally involve labeling and contacting a PAR4 binding protein with an immobilized antigen or fragment thereof, e.g., a protein comprising the extracellular portion of PAR4 fused to biotin (e.g., as set forth in SEQ ID NO: 6). After washing to remove non-specifically bound proteins, the amount of label, and thus the amount of bound protein, is detected. Of course, the PAR4 binding protein may be immobilized and the antigen may be labeled. Panning-type assays may also be used. The examples herein describe binding assays for PAR4 based on flag markers, which can be expressed on the surface of HEK cells, PAR 4. Inhibition of PAR4 cleavage by PAR4 binding protein in the presence of thrombin can be measured by flow cytometry.
Conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA), can be used to screen and identify PAR4 binding proteins that competitively inhibit binding of the PAR4 antibodies of the invention to an epitope.
Competitive binding assays
Assays for assaying PAR4 binding proteins that competitively inhibit binding of an antibody of the present disclosure (e.g., mAb arc3.h4b) will be apparent to the skilled artisan. For example, an antibody of the present disclosure is conjugated to a detectable label, e.g., a fluorescent label or a radioactive label. The labeled antibody and test PAR4 binding proteins are then mixed and contacted with PAR4 or its extracellular domain fused to the Fc region of the antibody or peptide comprising an epitope thereof. The level of labeled antibody is then determined and compared to the level determined when the labeled antibody is contacted with PAR4 or a PAR4-Fc fusion or a peptide comprising an epitope thereof in the absence of a PAR4 binding protein. The PAR4 binding protein competitively inhibits the binding of a labeled antibody if the level of the antibody is reduced in the presence of the PAR4 binding protein as compared to the level in the absence of the test PAR4 binding protein.
Optionally, the test PAR4 binding protein is conjugated to a different label than the antibody. This allows the detection of the level of binding of the test PAR4 binding protein to the protein or epitope.
In another example, a test PAR4 binding protein is bound to PAR4 or PAR4-Fc fusion or a peptide comprising an epitope thereof prior to contacting PAR4 or a PAR4-Fc fusion or a peptide comprising an epitope thereof with an antibody described herein. A decrease in the amount of antibody bound in the presence of the PAR4 binding protein compared to the absence of the PAR4 binding protein indicates that the PAR4 binding protein competitively inhibits the binding of the antibody to PAR 4. A mutual assay may also be performed using a labeled PAR4 binding protein and first allowing the antibody to bind to PAR4 or a PAR4-Fc fusion or peptide comprising an epitope thereof. In this case, a decrease in the amount of labeled PAR4 binding protein that binds to PAR4 or a PAR4-Fc fusion or a peptide comprising an epitope thereof in the presence of the antibody as compared to the absence of the antibody indicates that the PAR4 binding protein competitively inhibits the binding of the antibody to PAR 4.
Epitope mapping assay
In another example, the epitope bound by the PAR4 binding proteins described herein is mapped. Methods of epitope mapping will be apparent to those skilled in the art. For example, a series of overlapping peptides spanning the PAR4 sequence comprising the epitope of interest or a region thereof, e.g., peptides comprising 10-15 amino acids, are generated. The PAR4 binding protein is then contacted with various peptides or combinations thereof and the peptides to which it binds are determined. This allows the determination of peptides comprising the epitope to which the PAR4 binding protein binds. A PAR4 binding protein may bind a conformational epitope if multiple non-adjacent peptides are bound by the PAR4 binding protein.
In one example, random fragments of PAR4 are expressed on the surface of a bacteriophage and the bacteriophage is contacted with a PAR4 binding protein. The phage bound by the antibody can then be isolated and the amino acid sequence of the expressed peptide deduced from the coding nucleic acid contained in the phage. By isolating a series of phages with overlapping peptides, peptides comprising the PAR4 region of residues contained in the epitope were identified.
Alternatively or additionally, amino acid residues within PAR4 are mutated, for example by alanine scanning mutagenesis, and mutations are determined which reduce or prevent binding of the PAR4 binding protein. Any mutation that reduces or prevents binding of the PAR4 binding protein is likely to be within the epitope bound by the PAR4 binding protein.
Another method comprises binding PAR4 or a region thereof to an immobilized PAR4 binding protein of the disclosure and digesting the resulting complex with a protease. The peptides that remain bound to the immobilized PAR4 binding protein are then isolated and their sequence determined, for example, using mass spectrometry.
Yet another method involves converting hydrogen in PAR4 or a region thereof to a deuterium atom and allowing the resulting protein to bind to an immobilized PAR4 binding protein of the present disclosure. The deuterons are then converted back to hydrogen, PAR4 or regions thereof are isolated, digested with enzymes and analyzed, e.g., using mass spectrometry, to identify those regions containing deuterons that may have been protected from conversion to hydrogen by binding of the PAR4 binding protein described herein.
In the preceding paragraph, reference to PAR4 encompasses recombinant PAR4, including the extracellular domain thereof.
Affinity assay
Optionally, the dissociation constant (Kd) or association constant (Ka) or binding constant (Kd, i.e. Ka/Kd) of a PAR4 binding protein is determined for PAR4 or an epitope-containing peptide thereof. In one example, these constants for the PAR4 binding protein are measured by a radiolabeled or fluorescently labeled PAR4 binding assay. This assay allowed the PAR4 binding protein to be in equilibrium with the lowest concentration of labeled PAR4 in the presence of a titration series of unlabeled PAR 4. After washing to remove unbound PAR4, the amount of label was determined. According to another example, the constant is measured by using a surface plasmon resonance assay, for example using BIAcore surface plasmon resonance (BIAcore, inc., Piscataway, NJ) with immobilized PAR4 or a region thereof.
Protein detection assay
One example of the disclosure detects the presence of PAR4 or a cell (e.g., a platelet) expressing PAR 4. The amount, level or presence of a protein or cell is determined using any of a variety of techniques known to the skilled artisan, such as, for example, a technique selected from the group consisting of: flow cytometry, immunohistochemistry, immunofluorescence, immunoblotting, western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), enzyme immunoassay, Fluorescence Resonance Energy Transfer (FRET), matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g., LC MS/MS), biosensor technology, evanescent fiber technology, or protein chip technology.
In one example, the assay used to determine the amount or level of protein is a semi-quantitative assay. In another example, the assay used to determine the amount or level of protein is a quantitative assay.
For example, proteins are detected with an 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 blot, Radioimmunoassay (RIA), biosensor assay, protein chip assay, and immunostaining assay (e.g., immunofluorescence).
Standard solid phase ELISA or FLISA formats are particularly useful for determining the concentration of proteins from various samples.
In one form, the ELISA or FLISA comprises immobilizing a PAR4 binding protein of the disclosure or a protein that binds to a different epitope of PAR4 on a solid substrate (such as, for example, a membrane, polystyrene or polycarbonate microwell, polystyrene or polycarbonate paper, or glass support). The sample is then physically associated with the immobilized protein and PAR4 is bound or "captured". Bound PAR4 was then detected using a second labeled compound that bound to a different epitope of PAR 4. Alternatively, a third labeled antibody that binds to the second (detection) antibody may be used. It will be apparent to the skilled person that the assay formats described herein are suitable for high throughput formats, such as for example automated or microarray formats of screening processes. Furthermore, variations of the above assays will be apparent to those skilled in the art, such as, for example, competitive ELISA.
In an alternative example, the polypeptide is detected in or on the cell using methods known in the art, such as, for example, immunohistochemistry or immunofluorescence. Methods using immunofluorescence are exemplary in that 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.
Biosensor devices typically employ an electrode surface in combination with a current or impedance measuring element to be integrated into the device in combination with an assay substrate (such as described in US 5567301). The PAR4 binding proteins of the present disclosure are incorporated onto the surface of a biosensor device and a biological sample is contacted with the device. A change in the detected current or impedance of the biosensor device indicates binding of the protein to the PAR4 binding protein. Some forms of biosensors known in the art also rely on Surface Plasmon Resonance (SPR) to detect protein interactions, whereby changes in the reflected surface plasmon resonance surface indicate binding of the protein to a ligand or antibody (US5485277 and US 5492840).
Biosensors are particularly useful in high-throughput analysis due to the ease of adapting such systems to micro-or nano-scale. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing multiplexing of diagnostic reagents in a single biosensor unit. This allows the simultaneous detection of several proteins or peptides in small amounts of body fluid.
Flow cytometry can also be used to detect binding of a protein to PAR4 as described in the examples herein.
anti-PAR 4 antibody production and selection
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to PAR4 protein or PAR4 peptide (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 having a potential PAR4 polypeptide binding domain can be obtained by screening random peptide libraries displayed on phage (phage display) or bacteria (e.g., e.coli). The nucleotide sequence encoding the polypeptide can be obtained in a variety of ways, such as by random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides that interact with known targets, which may be proteins or polypeptides, such as ligands or receptors, biological or synthetic macromolecules, or organic or inorganic substances. Techniques for generating 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, for example, from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Pirataway, N.J.). Random peptide display libraries can be screened using the PAR4 sequences disclosed herein to identify proteins that bind to PAR 4. These "binding proteins" that interact with the PAR4 polypeptide can be used to label cells; isolating the homologous polypeptide by affinity purification; they may be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like. These binding proteins may also be used in assays, for example for screening expression libraries and for neutralizing activity. Binding proteins can also be used in diagnostic assays to determine circulating levels of polypeptides; for detecting or quantifying soluble polypeptides as markers of underlying pathology or disease. These binding proteins may also act as PAR4 "antagonists" to block PAR4 binding and signaling in vitro and in vivo. These anti-PAR 4 binding proteins can be used to inhibit cellular responses to protease-activated PAR 4.
Antibodies that specifically bind to PAR4 protein or peptide can be detected using a variety of assays known to those skilled in the art. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: parallel immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant PAR4 proteins, polypeptides, or fragments.
Determination of the functional characteristics of PAR4 binding proteins
The antithrombotic activity of the PAR-4 binding proteins across all PAR4 variants can be examined in an ex vivo platelet aggregation assay using blood from humans genotyped as homozygous Ala120 or Thr120 or heterozygous to confirm the efficacy of the full variant.
Can be prepared by the absence and presence of existing antiplatelet drug pathway inhibitors (aspirin (50 μ M), P2Y12Inhibitor 2-MeSAMP (50 μ M or 100 μ M)) to examine the usefulness of the PAR4 binding protein as an antithrombotic agent, and the PAR1 inhibitor valaparsat (100mM) was also used in an ex vivo platelet aggregation assay for comparative studies of antithrombotic effect together with the PAR4 binding protein. When the inventors have demonstrated that thrombosis occurs independently of these mechanisms, high shear conditions will be included (3000 s)-1) (Neeves KB et al 2008) J ThrombHaemost 6: 2193-2201).
Additionally or alternatively, in vivo thrombosis experiments in mice (Lee H et al (2012) Brit J Pharmacol166: 2188-2197; Mountford JK et al (2015) Nat Commun6:6535) can be used to examine the functionality of PAR4 binding proteins. To ensure that a positive control for anti-PAR 4 activity was included in these mouse experiments, the anti-thrombotic effect of antibodies generated using antigens corresponding to mouse and human receptor sequences (table 2) was examined and screened as described above. It should be noted that primates are the only species known to have platelets that express only the combination of PAR1 and PAR 4. Mouse platelets express PAR3 and PAR4, and only PAR4 is functional. Therefore, these studies are limited to in vivo mechanistic validation, but outside human trials and preclinical studies in non-human primates, are the most appropriate in vivo examination for the antithrombotic activity of PAR4 binding proteins. Electrolytic injury of carotid arteries in anesthetized mice can be used to examine the effect of PAR4 binding protein on thrombus formation and stability in vivo (Lee H et al (2012) Brit J Pharmacol166: 2188-. Blood flow was recorded using a doppler flow probe. Endpoints can assess thrombus formation (time to arterial occlusion) and stability (number and extent of recanalization events following occlusion) as well as a thorough examination of thrombus histology by total blood flow to the injured artery and by cardair staining of paraffin-embedded cross sections of the artery.
PAR4 activation can be studied by determining phosphoinositide hydrolysis following protease stimulation. Epitope-tagged PAR4 assays as described herein can also be used to examine the cleavage and activation of PAR4 by PAR4 binding proteins.
Mammalian cells (e.g., HEK293T cells) transfected with the PAR4 construct or the PAR4 polymorphic variant are useful systems for studying antagonists of PAR 4. PAR4 transfected cells were used to screen for ligands for receptors as well as antagonists of the natural ligand. To summarize this approach, a cDNA or gene encoding the receptor is combined with other genetic elements required for its expression (e.g., a transcription promoter), and the resulting expression vector is inserted into a host cell. Cells expressing DNA and producing functional receptors are selected and used in a variety of screening systems.
Cells expressing functional PAR4 were used in the screening assay. A variety of suitable assays are known in the art. These assays are based on the detection of biological responses in target cells. An increase in metabolism above the control value is indicative of 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 incorporation of tritiated thymidine or by colorimetric assays based on the metabolic breakdown of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) (Mosman, j.immunol.meth.65:55-63,1983). Another assay involves measuring the effect of a test compound on receptor (+) cells containing a receptor of interest on their cell surface and receptor (-) cells that do not express the receptor of interest. These cells can be engineered to express a reporter gene. The reporter gene is linked to a promoter element or response element responsive to the receptor linkage pathway, and the assay detects activation of transcription of the reporter gene. Suitable response elements include cyclic AMP response element (CRE), Hormone Response Element (HRE), Insulin Response Element (IRE) (Nasrin et al, Proc. Natl. Acad. Sci. USA87: 5273-. 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, in the same manner as described above. Hormone response elements are reviewed by Beato, Cell56: 335-44; 1989. In this regard, preferred promoter elements are serum response elements or SREs (see, e.g., Shaw et al, Cell56:563-72, 1989). A preferred such reporter gene is the luciferase gene (de Wet et al, mol.cell.biol.7:725,1987). Luciferase gene expression is detected by luminescence using methods known in the art (e.g., Baumgartner et al, J.biol. chem.269:29094-101, 1994; Schenborn and Goiffin, Promega notes 41:11,1993). Luciferase activity assay kits are commercially available from, for example, Promega corp. This type of target cell line can be used to screen libraries of chemicals, cell conditioned media, fungal broths, soil samples, water samples, and the like. This type of assay will detect compounds that directly block PAR4 ligand binding, as well as compounds that block processes in the cellular pathway following receptor-ligand binding. Alternatively, a moiety labeled with a detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, etc.) can be used to test whether a compound or other sample directly blocks PAR4 binding. In this type of assay, the ability of the test sample to inhibit PAR4 activation is indicative of inhibitory activity, which can be confirmed by a secondary assay. The ability of the test sample to stimulate PAR4 activity can also be determined and confirmed by a secondary assay.
Assay systems using ligand-bound receptors or antibodies or binding fragments thereof, as well as commercially available Biosensor instruments (BIAcore, Pharmacia Biosensor, Piscataway, n.j.) may be advantageously employed. Such receptors, antibodies or fragments are immobilized on the surface of the receptor chip. The use of this instrument is described by Karlsson, J.Immunol.methods 145: 229-; and Cunningham and Wells, J.mol.biol.234:554 one 63, 1993. The receptor, antibody or fragment is covalently linked using amine or sulfhydryl chemicals to dextran fibers that are attached to gold membranes in the flow cell. Passing a test sample through the cell. If a ligand or epitope is present in the sample, it will bind 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 gold film surface plasmon resonance. Such a system allows determination of association and dissociation rates, from which binding affinities can be calculated and the stoichiometry of binding assessed.
Ligand binding receptor polypeptides may also be used in other assay systems known in the art. Such systems include Scatchard analysis (see Scatchard, Ann. NY Acad. Sci.51:660-72,1949) and calorimetric assays (Cunningham et al, Science 253:545-48, 1991; Cunningham et al, Science 245:821-25,1991) for determining binding affinity.
The FLIPR assay is an exemplary in vitro assay for measuring the activity of the PAR4 antagonists of the present invention. In this assay, intracellular calcium mobilization was induced in cells expressing PAR4 by a PAR4 agonist and monitored.
The ability of the PAR4 binding proteins of the present disclosure to inhibit platelet aggregation induced by gamma-thrombin can be tested in vitro. Gamma-thrombin, a proteolytic product of alpha-thrombin which no longer interacts with PAR1, selectively cleaves and activates PAR4(Soslau, G., et al, "Unique pathway of thrombin-induced platelet aggregated by glycoprotin lb", J.biol.chem.,276:21173-21183 (2001)). Platelet aggregation can be monitored in a 96-well microplate aggregation assay format or using a standard platelet aggregometer. The aggregation assay may also be used to test the selectivity of a compound for inhibiting platelet aggregation induced by the PAR4 agonist peptide, ADP, or the thromboxane analog U46619.
Another example is the alpha-thrombin induced platelet aggregation assay as shown in the examples herein. Alpha-thrombin activates both PAR1 and PAR 4. The ability of a selective PAR4 antagonist to inhibit platelet aggregation can be measured using a standard optical agglutination meter.
Another example is a tissue factor-induced platelet aggregation assay. The conditions in such an assay mimic physiological events during thrombus formation. In this assay, platelet aggregation in human PRP is determined by the addition of tissue factor and CaCl2And (4) initiating. Tissue factor (the initiator of the extrinsic coagulation cascade) is highly elevated in human atherosclerotic plaques. Exposure of blood to tissue factors at the site of atherosclerosis triggers the massive production of thrombin and induces the formation of an obstructive thrombus.
The efficacy of the PAR4 binding proteins of the invention in preventing thrombosis can also be measured in a variety of in vivo assays. Thrombogenic and hemostatic models can be provided to test the PAR of the inventionExemplary mammals for the effectiveness of 4 antagonists as antithrombotic agents include, but are not limited to, guinea pigs and primates. Relevant efficacy models include, but are not limited to, electrolytic injury induced carotid thrombosis, FeCl3Induced carotid thrombosis and arteriovenous shunt thrombosis. Renal bleeding time, and other bleeding time measurement models can be used to assess bleeding risk.
PAR4 binding proteins can be tested in an in vivo model of aortic thrombosis in cynomolgus monkeys. The ability of PAR4 binding proteins to inhibit electrolytic injury-induced thrombosis of carotid arteries can be tested in this model.
Platelet aggregation assay
Microplate-based light-transmission platelet aggregation methods can be used to measure platelet aggregation (French et al (2016) Journal of Thrombosis and Haemostasis 14: 1642-.
This test (Born GV (1962) Nature 194:927-929) assesses in vitro the clot formation, i.e.aggregation, of the most important function of platelets between platelets in a Glycoprotein (GP) IIb/IIIa-dependent manner. The assay is based on measurement of the increase in light transmission by Platelet Rich Plasma (PRP) or washed platelet optical density samples after addition of exogenous platelet agonist. During the assay, PRP or washed platelet preparations become clearer after addition of agonist due to the precipitation of platelet aggregates. This determines the increase in light transmission through the plasma sample. The device records the rate and maximum percentage of this increase by photometer from 0% (maximum optical density of PRP or washed platelets) to 100% (optical density of autologous platelet-free plasma or Tyrodes buffer). This signal is automatically converted into a graphical curve that parallels the increase in light transmission during platelet aggregation. Available agglutination meters are easy-to-use devices equipped with automated settings (100% and 0%), software for storing results, and disposable cuvettes with stir bars. The slope of the curve, maximum aggregation (%) and latency (lag phase) are parameters that are automatically measured, and shape changes as well as primary and secondary aggregation can be viewed graphically. Different agonists are added to PRP or washed platelet samples to stimulate different platelet activation pathways, thereby obtaining information about several characteristics of platelet function. The platelet aggregation method of Born is the most widely used method for detecting platelet dysfunction and monitoring anti-platelet therapy.
In vivo analysis of platelet function following administration of PAR4 binding protein can be determined using Bleeding Time (BT) (DukeWW et al (1910) JAMA 55: 1185-1192). BT evaluates the ability of platelets to form hemostatic plugs by recording the time required for platelets to occlude skin wounds in vivo to stop bleeding.
Impedance Whole Blood Agglutination (WBA) allows assessment of platelet function by using anticoagulated Whole Blood (WB) as an environment without any sample processing (Mackie IJ, et al (1984) J Clin Pathol.37: 874-878). It is based on the following principle: activated platelets adhere via their surface receptors to the artificial surfaces of the two electrodes within the WB sample positioned at a determined distance between the two electrodes. Platelet aggregation is assessed by detecting an increase in electrical impedance resulting from aggregation of other platelets fixed to the electrodes. Thus, by reducing the current intensity, the electrical impedance increases. The degree of increase in impedance is recorded in ohms.
The Lumi aggregation method can simultaneously measure the release of adenine nucleotides from platelet particles and platelet aggregation (Holmsen H, et al (1966) Anal biochem.17: 456-47). The method is based on the evaluation of Adenosine Triphosphate (ATP) released from activated platelets by different agonists by 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 that reacts with luciferin-luciferase reagent. The emitted light, which is proportional to the ATP concentration, is quantified by a lumi-agglomerator.
Other platelet function tests are reviewed in Panicica R et al (2015) Vasc Health RiskManag.11: 133-148.
Calcium signalling assay
Calcium flux can be measured in isolated platelets by microscopic imaging assays using two-dye ratiometric (Nesbitt WS et al (2012) Methods Mol Biol 788: 73-89)).
Animal model
The skilled artisan can utilize in vivo animal models of thrombosis to further or additionally screen, assess and/or validate the antibodies or fragments thereof of the present disclosure, including further assessment of PAR4 activation or anti-thrombotic effects in vivo. Such animal models include, but are not limited to, models that experience electrolytic injury to the carotid artery, and thus thrombosis (arterial occlusion time), and examine stability (number and extent of recanalization events after occlusion) and total blood flow through the injured artery.
An exemplary or suitable mouse model is the PAR 4-/-mouse (Sambrano GR et al (2001) Nature 2000407: 258-64; Mao Y et al (2010) J Cereb Blood Flow Metab.30(5): 1044-1052).
Pharmaceutical composition
The PAR4 binding proteins (synonym: active ingredient) of the present disclosure may be used to formulate pharmaceutical compositions for parenteral, topical, oral or topical administration, aerosol administration or transdermal administration, for prophylactic or therapeutic treatment. The pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include powders, tablets, pills, capsules, and lozenges.
The pharmaceutical compositions of the present disclosure may be used for parenteral administration, such as intravenous administration or subcutaneous administration.
Compositions for administration will typically comprise a solution of the PAR4 binding protein of the present disclosure dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable carriers required to mimic physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the PAR4 binding proteins of the present disclosure in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.
The PAR4 binding proteins of the present disclosure can be formulated for parenteral administration, e.g., for injection via intravenous, intramuscular, subcutaneous, transdermal or other such routes, including peristaltic administration and direct instillation into the tumor or disease site (intraluminal administration). The preparation of aqueous compositions containing the compounds of the present disclosure as active ingredients is known to those skilled in the art.
Suitable pharmaceutical compositions according to the present disclosure will generally comprise an amount of the PAR4 binding protein of the present disclosure blended with an acceptable pharmaceutical carrier (e.g., a sterile aqueous solution) to give a final concentration range (depending on the intended use). Preparation techniques are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences, 16 th edition Mack publishing Company, 1980.
Upon formulation, the compounds of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amounts as are therapeutically/prophylactically effective. Suitable dosages for 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 ability of the skilled practitioner to determine the appropriate dose, for example by starting with a sub-optimal dose and gradually changing the dose to determine the optimal or useful dose.
Exemplary dosages and administration times will be apparent to those skilled in the art based on the disclosure herein. Preferred doses of the PAR4 antagonist are biologically active doses. The biologically active dose is a dose which will inhibit the cleavage and/or signaling of PAR4 and have an antithrombotic effect. Desirably, the PAR4 antagonist has the ability to reduce the activity of PAR4 by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% below untreated control levels. The level of PAR4 in platelets is measured by any method known in the art including, for example, receptor binding assays, platelet aggregation, platelet activation assays (e.g., p-selectin expression by FACS), western blot or ELISA analysis. Alternatively, the biological activity of PAR4 is measured by assessing cell signaling (e.g., calcium mobilization or other second messenger assay) elicited by PAR 4.
In some examples, a therapeutically effective amount of a PAR4 compound is preferably about less than 100mg/kg, 50mg/kg, 10mg/kg, 5mg/kg, 1mg/kg, or less than 1 mg/kg. In a more preferred embodiment, the therapeutically effective amount of the PAR4 compound is less than 5 mg/kg. In a most preferred embodiment, the therapeutically effective amount of the PAR4 compound is less than 1 mg/kg. As recognized by one skilled in the art, effective dosages vary depending upon the route of administration and excipient usage.
In some examples, liposomes and/or nanoparticles may also be used with PAR4 binding proteins. The formation and use of liposomes is generally known to those skilled in the art. Liposomes can be formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs may typically have a diameter of 25nm to 4 μm. Sonication of MLVs results in the formation of Small Unilamellar Vesicles (SUVs) in the range of 200 to 500 angstroms in diameter, with an aqueous solution in the core. When phospholipids are dispersed in water, they can form various structures other than liposomes depending on the molar ratio of lipid to water. In low ratios, liposomes are the preferred structure. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but undergo phase transitions at high temperatures that significantly change their permeability. Phase transition involves a change from a tightly packed ordered structure (known as the gel state) to a loosely packed disordered structure (known as the fluid state).
The compositions may be administered alone or in combination with other therapies, therapeutic agents or agents, simultaneously or sequentially, including but not limited to:
(i) anticoagulants, for example FXa inhibitors, FXIa inhibitors (such as apixaban or rivaroxaban) or thrombin inhibitors (such as dabigatran);
(ii) antiplatelet agents, such as aspirin or a P2Y12 antagonist, such as clopidogrel, ticagrelor, or prasugrel;
(iii) an angiogenic agent, such as an angiogenesis inhibitor.
Method of treatment
As discussed herein, PAR4 binding proteins of the present disclosure are useful for treating, preventing, or ameliorating a thrombotic or thromboembolic disorder in a subject.
Thrombosis refers to the formation or presence of thrombi (thrombi) within a blood vessel that can lead to ischemia or infarction of the tissues supplying the blood vessel.
Thromboembolic disorders are characterized by a clot (e.g., an embolism) or foreign body that has been brought to its site of residence by the blood stream suddenly occluding an artery. "thromboembolism" refers to the occlusion of a blood vessel by thrombotic material carried by the blood stream from the site of origin to another vessel. The term "thromboembolic disorder" includes both "thrombotic" disorders and "embolic" disorders (defined above).
Thromboembolic disorders include arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the ventricles of the heart or in the peripheral circulation. The term "thromboembolic disorder" as used herein also includes specific disorders selected from, but not limited to: unstable angina or other acute coronary syndrome, atrial fibrillation, primary or recurrent myocardial infarction, sudden ischemic death, transient ischemic attack, stroke, atherosclerosis, peripheral arterial occlusive disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary thrombosis, cerebral arterial thrombosis, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. Medical implants or devices include, but are not limited to: artificial valves, prosthetic valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices, and artificial hearts or heart chambers, as well as vascular grafts. Programs 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.
As used herein, the term "stroke" refers to an embolic or atherosclerotic stroke due to occlusive thrombosis in the common carotid artery, the internal carotid artery, or the internal cerebral artery.
Medicine box
The present disclosure also provides therapeutic/prophylactic/diagnostic kits comprising a compound of the present disclosure for use in the detection/diagnosis/prognosis/therapy/prevention methods of the invention. Such kits will typically comprise a PAR4 binding protein of the disclosure in a suitable container. The kit may also contain other compounds, e.g. for detection/isolation/diagnosis/imaging or combination therapy. For example, such kits may contain any one or more of a range of anticoagulant or antiplatelet agents.
In one example, the kit is for treating or preventing a condition. In such kits, the PAR4 binding protein may be provided in solution or in lyophilized form, optionally together with a solution for resuspension. The PAR4 binding protein can be conjugated to a therapeutic compound or the kit can include a therapeutic compound for conjugation thereto.
It will be appreciated by persons skilled in the art 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. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
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 description herein, utilize the present invention to its fullest extent.
The invention is further described in the following non-limiting examples.
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