Antibodies to Ticagrelor and Methods of Use Thereof

Abstract

The present invention relates to antibodies to ticagrelor and methods of using the same. This disclosure generally provides antibodies and antigen-binding fragments of antibodies that bind ticagrelor and metabolites of ticagrelor. This disclosure also provides compositions comprising these antibodies, nucleic acid molecules encoding these antibodies, methods of treating patients by administering these antibodies, and methods of making and using these antibodies.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580053771.X, "Antibodies to Ticagrelor and Methods of Use Thereof", with a filing date of September 30, 2015.

[0002] Reference to Electronically Submitted Sequence Listing

[0003] This application incorporates by reference the sequence listing in computer-readable form that is submitted together with this application as a text file, the sequence listing being named "Ticagrelor_SeqList_ST25.TXT", created on October 1, 2014, and having a size of 33,900 bytes. Technical Field

[0004] This application relates to the field of antibodies, and more particularly to antibodies that bind to ticagrelor and metabolites of ticagrelor, and antigen-binding fragments of the antibodies. Background Art

[0005] Ticagrelor (BRILINTA TM (BRILINTA TM or BRILIQUE TM )) is an orally active cyclopentyltriazolopyrimidine, a selective and reversible binding adenosine diphosphate (ADP) receptor antagonist. In patients with acute coronary syndrome (ACS), ticagrelor at 90 mg twice daily in combination with low-dose aspirin is approved for reducing major cardiovascular (CV) events. Ticagrelor acts through a dual pathway of mediating antiplatelet effects (P2Y 12 ) and enhanced adenosine response (ENT-1) (Cattaneo M et al., 2014, Journal of the American College of Cardiology 63(23):2503-9). Although the indications have not been approved, ongoing and planned studies are evaluating ticagrelor for reducing major cardiovascular events in patients with old myocardial infarction, established peripheral arterial disease, and acute stroke, as well as in patients with diabetes and established coronary atherosclerosis.

[0006] Ticagrelor has two major metabolites: ticagrelor active metabolite (TAM) and ticagrelor inactive metabolite (TIM) (Teng et al., 2010, Drug Metabolism and Disposition 38:1514-1521). TAM, also known as AR-C124910XX, is the major circulating metabolite of ticagrelor and is involved in P2Y 12Equally effective in antagonist activity. TAM is typically present in patients taking BRILINTA / BRILIQUE at approximately 30%-40% of the parent ticagrelor concentration. The circulating half-lives of ticagrelor and TAM are 8 and 12 hours, respectively. TIM, also known as AR-C133913XX, is 12 inactive, constitutes less than 10% of the parent ticagrelor, is undetectable after 8 hours, and is the major metabolite excreted in the urine.

[0007] Irrespective of the treatment strategy (pharmacological or invasive), the Platelet Inhibition and Patient Outcomes (PLATO) trial confirmed greater efficacy of ticagrelor compared to clopidogrel in a broad patient population with acute coronary syndromes (unstable angina (UA), non-ST-segment elevation myocardial infarction (NSTEMI), ST-segment elevation myocardial infarction (STEMI)), without an increase in overall major bleeding. (Wallentin et al. 2009, The New England Journal of Medicine, 361(11): 1045-1057). However, as with all antiplatelet agents, there is a possibility of bleeding in patients using ticagrelor. If a patient on dual antiplatelet therapy (DAPT) experiences severe bleeding, there are limited treatment options. If a bleeding event occurs in a DAPT patient, platelet transfusion or administration of coagulation factors can be used in an attempt to enhance hemostasis. However, there is currently no clinical data available to evaluate the hemostatic benefit of platelet transfusion or the use of recombinant factor VIIa after or during a major bleeding event in subjects taking ticagrelor (Dalen M( M) et al., 2013, Journal of Cardiothoracic and Vascular Anesthesia 27(5): e55-7).

[0008] Thus, the availability of an antidote, such as a ticagrelor-specific neutralizing antibody, would allow for better clinical management of the balance between the desired antithrombotic effect and bleeding control. Since ticagrelor is the only commercially available reversible platelet-binding inhibitor, the antibody can provide reversal of platelet inhibition without the need for fresh platelet transfusion, thereby avoiding the risks associated with platelet transfusion. The availability of a reagent that overcomes the inhibition of ADP-induced platelet aggregation associated with ticagrelor and TAM would meet an important unmet clinical need, such as in patients who experience major bleeding or require emergency surgery. SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure relates to an antibody that specifically binds to a cyclopentyltriazolopyrimidine compound having the chemical formula (Ia):

[0010]

[0011] Wherein

[0012] R1 is selected from the group consisting of: C1-C6 alkoxy and C1-C6 alkylthio;

[0013] R2 is selected from the group consisting of: H, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; and

[0014] R3 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanol.

[0015] In some embodiments, the antibody binds to an epitope within a moiety of the compound labeled by the parentheses in Chemical Formula (IIa)

[0016]

[0017] wherein R1, R2, and R3 are as defined above.

[0018] In other embodiments, the antibody binds to an epitope within a moiety of the compound labeled by the parentheses in Chemical Formula (IIIa)

[0019]

[0020] wherein R2 and R3 are as defined above, and R'1 is selected from the group consisting of: C1-C4 alkyl.

[0021] In other embodiments, the antibody binds to a compound selected from the group consisting of:

[0022] Ticagrelor;

[0023] Ticagrelor active metabolite (TAM); and Ticagrelor inactive metabolite (TIM).

[0024] In other embodiments of the above aspects and embodiments, the antibody or fragment thereof comprises a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72; and a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, and SEQ ID NO: 77. In some embodiments, the antibody comprises a combination of VH and VL sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 7; SEQ ID NO: 12 and SEQ ID NO: 17; SEQ ID NO: 22 and SEQ ID NO: 27; SEQ ID NO: 32 and SEQ ID NO: 37; SEQ ID NO: 42 and SEQ ID NO: 47; SEQ ID NO: 52 and SEQ ID NO: 57; SEQ ID NO: 62 and SEQ ID NO: 67; and SEQ ID NO: 72 and SEQ ID NO: 77. In other embodiments, the antibody comprises a combination of VH and VL selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 57; SEQ ID NO: 62 and SEQ ID NO: 67; and SEQ ID NO: 72 and SEQ ID NO: 77.

[0025] In other embodiments of the above aspects and embodiments, the antibody or fragment thereof comprises framework regions (FRs) and complementarity determining regions (CDRs) 1, 2, and 3 of the heavy chain variable region and the light chain variable region, wherein the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region comprise SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3); SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3); SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3); SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3); SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3); SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3); SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3); or SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3); and wherein the CDR1, CDR2, and CDR3 sequences of the light chain variable region comprise SEQ ID NO: 8 (CDR1), SEQ ID NO: 9 (CDR2), and SEQ ID NO: 10 (CDR3); SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 20 (CDR3); SEQ ID NO: 28 (CDR1), SEQ ID NO: 29 (CDR2), and SEQ ID NO: 30 (CDR3); SEQ ID NO: 38 (CDR1), SEQ ID NO: 39 (CDR2), and SEQ ID NO: 40 (CDR3); SEQ ID NO: 48 (CDR1), SEQ ID NO: 49 (CDR2), and SEQ ID NO: 50 (CDR3); SEQ ID NO: 58 (CDR1), SEQ ID NO: 59 (CDR2), and SEQ ID NO: 60 (CDR3); SEQ ID NO: 68 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3);or SEQ ID NO: 78 (CDR1), SEQ ID NO: 79 (CDR2) and SEQ ID NO: 80 (CDR3). In other embodiments, the antibody comprises a combination of CDR regions selected from the group consisting of SEQ ID NO: 53 (VH CDR1), SEQ ID NO: 54 (VH CDR2), SEQ ID NO: 55 (VH CDR3), SEQ ID NO: 58 (VL CDR1), SEQ ID NO: 59 (VL CDR2), and SEQ ID NO: 60 (VL CDR3); SEQ ID NO: 63 (VH CDR1), SEQ ID NO: 64 (VH CDR2), SEQ ID NO: 65 (VH CDR3), SEQ ID NO: 68 (VL CDR1), SEQ ID NO: 69 (VL CDR2) and SEQ ID NO: 70 (VL CDR3); and SEQ ID NO: 73 (VH CDR1), SEQ ID NO: 74 (VH CDR2), SEQ ID NO: 75 (VH CDR3), SEQ ID NO: 78 (VL CDR1), SEQ ID NO: 79 (VL CDR2), and SEQ ID NO: 80 (VL CDR3).;

[0026] In the above aspects and embodiments, the antibody is selected from polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, single-chain Fv (scFv), single-domain antibodies, Fab, F(ab’)2, single-chain diabodies, antibody mimetics, and antibody variable domains. In some embodiments, the antibody comprises scFv. In some embodiments, the antibody comprises Fab.

[0027] In some embodiments, the above antibody binds to ticagrelor or a ticagrelor active metabolite (TAM).

[0028] In other embodiments, the antibody binds to ticagrelor or its metabolite or derivative with an IC 50 of about 200 nM or lower. In still other embodiments, the antibody binds to ticagrelor or its metabolite or derivative with an IC 50 in the range of about 100 nM to about 1 nM or 50 with an IC in the range of about 10 nM to about 1 nM.

[0029] In other embodiments, the antibody binds to ticagrelor or its metabolite or derivative with a KD of about 50 nM or lower. In other embodiments, the antibody binds to ticagrelor or its metabolite or derivative with a KD in the range of about 250 pM to about 1 pM, or in the range of about 100 pM to about 1 pM.

[0030] In other embodiments, the antibody binds to ticagrelor or its metabolites or derivatives and does not bind to compounds selected from the group consisting of fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate. In an embodiment, the antibody does not inhibit the activity of compounds selected from the group consisting of fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate. In other embodiments, the antibody exhibits an IC 50 of at least about 1000 μM for compounds selected from the group consisting of fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate.

[0031] In some embodiments, the antibody has an in vivo half-life of about 4 - 12 hours. In a specific embodiment, the antibody has an in vivo half-life of about 12 hours.

[0032] In some embodiments, the antibody neutralizes the antiplatelet effect of ticagrelor or the active metabolite of ticagrelor. In other embodiments, the antibody neutralizes the antiplatelet effect of ticagrelor or the active metabolite of ticagrelor within about 60 minutes of administration.

[0033] In some embodiments, the antibody has a dissociation rate for ticagrelor or the active metabolite of ticagrelor that permits resumption of treatment comprising ticagrelor.

[0034] In other aspects, the present disclosure provides a method of treating acute bleeding in a patient in need thereof, the method comprising administering to the patient an effective amount of the antibody disclosed herein. In some embodiments of the method, the patient has received or is receiving surgery and has been administered ticagrelor. In some embodiments of the method, the patient requires emergency care and / or emergency trauma management.

[0035] Other aspects of the present disclosure provide a composition comprising a combination of the antibody of any of the foregoing aspects and embodiments with a pharmaceutically acceptable carrier.

[0036] Some additional aspects provide nucleic acid molecules that comprise a nucleotide sequence encoding an antibody according to any one of the foregoing schemes. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71, and SEQ ID NO: 76.

[0037] Other aspects of the present disclosure provide compositions, vectors, and host cells that can comprise at least one nucleic acid molecule disclosed herein. In some embodiments, the compositions, vectors, and host cells comprise a first nucleic acid molecule and a second nucleic acid molecule encoding one or more proteins disclosed herein.

[0038] The present invention specifically relates to the following schemes:

[0039] 1. An antibody that specifically binds to a cyclopentyltriazolopyrimidine compound having the chemical formula (Ia):

[0040]

[0041] wherein

[0042] R1 is selected from the group consisting of: C1-C6 alkoxy and C1-C6 alkylthio;

[0043] R2 is selected from the group consisting of: H, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; and

[0044] R3 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanol.

[0045] 2. The antibody according to Scheme 1, wherein the antibody binds to the cyclopentyltriazolopyrimidine compound having the chemical formula (Ia) at an epitope within the moiety of the compound marked by the chemical formula (IIa)

[0046]

[0047] 3. The antibody according to Scheme 1, wherein the antibody binds to the cyclopentyltriazolopyrimidine compound having the chemical formula (Ia) at an epitope within the moiety of the compound marked by the chemical formula (IIIa)

[0048]

[0049] Wherein R'1 is selected from the group consisting of: C1-C4 alkyl.

[0050] 4. An antibody that specifically binds to a cyclopentyltriazolopyrimidine compound having the chemical formula (Ib):

[0051]

[0052] 5. The antibody according to aspect 4, wherein R1 is C1-C6 alkylthio.

[0053] 6. The antibody according to aspect 4, wherein the compound having the chemical formula (Ib) is ticagrelor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol) or its metabolite or derivative.

[0054] 7. The antibody according to aspect 4, wherein the compound having the chemical formula (Ib) is selected from the group consisting of:

[0055] Ticagrelor;

[0056] Ticagrelor active metabolite (TAM); and

[0057] Ticagrelor inactive metabolite (TIM).

[0058] 8. The antibody according to any one of aspects 1-7, wherein the antibody is selected from polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, single-chain Fv (scFv), single-domain antibodies, Fab, F(ab′)2, single-chain diabodies, antibody mimetics, and antibody variable domains.

[0059] 9. The antibody according to aspect 8, wherein the antibody comprises scFv.

[0060] 10. The antibody according to aspect 8, wherein the antibody comprises Fab.

[0061] 11. The antibody according to any one of aspects 1-10, wherein the antibody binds to ticagrelor or its metabolite or derivative with an IC 50 of about 200 nM or lower.

[0062] 12. The antibody according to any one of Aspects 1-10, wherein the antibody has an IC in the range of about 100 nM to about 1 nM 50 that binds to ticagrelor or its metabolite or derivative.

[0063] 13. The antibody according to any one of Aspects 1-10, wherein the antibody has an IC in the range of about 10 nM to about 1 nM 50 that binds to ticagrelor or the active metabolite of ticagrelor.

[0064] 14. The antibody according to any one of Aspects 1-10, wherein the antibody has a K of about 50 nM or lower D that binds to ticagrelor or its metabolite or derivative.

[0065] 15. The antibody according to any one of Aspects 1-10, wherein the antibody has a K in the range of about 250 pM to about 1 pM D that binds to ticagrelor or its metabolite or derivative.

[0066] 16. The antibody according to any one of Aspects 1-10, wherein the antibody has a K in the range of about 100 pM to about 1 pM D that binds to ticagrelor or the active metabolite of ticagrelor.

[0067] 17. The antibody according to any one of Aspects 1-10, wherein the antibody shows an IC of no more than about 50 μM for a compound selected from the group consisting of: fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate. 50 , the group consisting of the following: fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate.

[0068] 18. The antibody according to any one of Aspects 1-10, wherein the in vivo half-life of the antibody is about 12 hours.

[0069] 19. The antibody according to any one of Aspects 1-10, wherein the antibody neutralizes the antiplatelet effect of ticagrelor or the active metabolite of ticagrelor.

[0070] 20. The antibody according to any one of Aspects 1-10, wherein the antibody neutralizes the antiplatelet effect of ticagrelor or the active metabolite of ticagrelor within about 30 minutes of administration.

[0071] 21. The antibody according to any one of Aspects 1-10, wherein the antibody restores ADP-induced platelet aggregation in the presence of ticagrelor or the active metabolite of ticagrelor.

[0072] 22. An antibody or fragment thereof that specifically binds to a cyclopentyltriazolopyrimidine compound having the chemical formula (Ia):

[0073]

[0074] wherein

[0075] R1 is selected from the group consisting of: C1-C6 alkoxy and C1-C6 alkylthio;

[0076] R2 is selected from the group consisting of: H, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; and

[0077] R3 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanol;

[0078] the antibody or fragment thereof comprises

[0079] a heavy chain variable region (VH) sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72; and

[0080] a light chain variable region (VL) sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, and SEQ ID NO: 77.

[0081] 23. The antibody according to embodiment 22, wherein the antibody comprises a combination of VH and VL sequences selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 7; SEQ ID NO: 12 and SEQ ID NO: 17; SEQ ID NO: 22 and SEQ ID NO: 27; SEQ ID NO: 32 and SEQ ID NO: 37; SEQ ID NO: 42 and SEQ ID NO: 47; SEQ ID NO: 52 and SEQ ID NO: 57; SEQ ID NO: 62 and SEQ ID NO: 67; and SEQ ID NO: 72 and SEQ ID NO: 77.

[0082] 24. The antibody according to embodiment 23, wherein the antibody comprises a combination of VH and VL, said VH and VL being selected from the group consisting of: SEQ ID NO: 52 and SEQ ID NO: 57; SEQ ID NO: 62 and SEQ ID NO: 67; and SEQ ID NO: 72 and SEQ ID NO: 77.

[0083] 25. An antibody or fragment thereof that specifically binds to a cyclopentyltriazolopyrimidine compound of formula (Ia):

[0084]

[0085] wherein

[0086] R1 is selected from the group consisting of: C1-C6 alkoxy and C1-C6 alkylthio;

[0087] R2 is selected from the group consisting of: H, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; and

[0088] R3 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanol;

[0089] The antibody or fragment thereof comprises framework regions (FR) and complementarity determining regions (CDR) 1, 2, and 3 of a heavy chain variable region and a light chain variable region, wherein the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region comprise,

[0090] SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3);

[0091] SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3);

[0092] SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3);

[0093] SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3);

[0094] SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3);

[0095] SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3);

[0096] SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3); or

[0097] SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3); and

[0098] wherein the CDR1, CDR2, and CDR3 sequences of the light chain variable region comprise

[0099] SEQ ID NO: 8 (CDR1), SEQ ID NO: 9 (CDR2), and SEQ ID NO: 10 (CDR3);

[0100] SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 20 (CDR3);

[0101] SEQ ID NO: 28 (CDR1), SEQ ID NO: 29 (CDR2), and SEQ ID NO: 30 (CDR3);

[0102] SEQ ID NO: 38 (CDR1), SEQ ID NO: 39 (CDR2), and SEQ ID NO: 40 (CDR3);

[0103] SEQ ID NO: 48 (CDR1), SEQ ID NO: 49 (CDR2), and SEQ ID NO: 50 (CDR3);

[0104] SEQ ID NO: 58 (CDR1), SEQ ID NO: 59 (CDR2), and SEQ ID NO: 60 (CDR3);

[0105] SEQ ID NO: 68 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3); or

[0106] SEQ ID NO: 78 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 80 (CDR3).

[0107] 26. The antibody according to embodiment 25, wherein the antibody comprises a combination of CDR regions selected from the group consisting of:

[0108] SEQ ID NO: 53 (VH CDR1), SEQ ID NO: 54 (VH CDR2), SEQ ID NO: 55 (VH CDR3), SEQ ID NO: 58 (VL CDR1), SEQ ID NO: 59 (VL CDR2), and SEQ ID NO: 60 (VL CDR3);

[0109] SEQ ID NO: 63 (VH CDR1), SEQ ID NO: 64 (VH CDR2), SEQ ID NO: 65 (VH CDR3), SEQ ID NO: 68 (VL CDR1), SEQ ID NO: 69 (VL CDR2), and SEQ ID NO: 70 (VL CDR3); and

[0110] SEQ ID NO: 73 (VH CDR1), SEQ ID NO: 74 (VH CDR2), SEQ ID NO: 75 (VH CDR3), SEQ ID NO: 78 (VL CDR1), SEQ ID NO: 79 (VL CDR2), and SEQ ID NO: 80 (VL CDR3).

[0111] 27. The antibody according to any one of embodiments 22 - 26, wherein the antibody is selected from polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, single-chain Fv (scFv), single-domain antibodies, Fab, F(ab′)2, single-chain diabodies, antibody mimetics, and antibody variable domains.

[0112] 28. The antibody according to embodiment 27, wherein the antibody comprises scFv.

[0113] 29. The antibody according to embodiment 27, wherein the antibody comprises Fab.

[0114] 30. The antibody according to any one of embodiments 22 - 29, wherein the antibody binds ticagrelor or its metabolite or derivative with an IC 50 of about 100 nM or lower.

[0115] 31. The antibody according to any one of embodiments 22 - 29, wherein the antibody binds ticagrelor or its metabolite or derivative with an IC 50 in the range of about 100 nM to about 1 nM.

[0116] 32. The antibody according to any one of aspects 22 - 29, wherein the antibody has an IC in the range of about 100 nM to about 1 nM 50 and binds to ticagrelor or an active metabolite of ticagrelor.

[0117] 33. The antibody according to any one of aspects 22 - 29, wherein the antibody has a K of about 50 nM or lower D and binds to ticagrelor or its metabolite or derivative.

[0118] 34. The antibody according to any one of aspects 22 - 29, wherein the antibody has a K in the range of about 250 pM to about 1 pM D and binds to ticagrelor or its metabolite or derivative.

[0119] 35. The antibody according to any one of aspects 22 - 29, wherein the antibody has a K in the range of about 100 pM to about 1 pM D and binds to ticagrelor or an active metabolite of ticagrelor.

[0120] 36. The antibody according to any one of aspects 22 - 29, wherein the antibody exhibits an IC of no more than about 50 μM for a compound selected from the group consisting of: fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2 - MeS adenosine diphosphate, and 2 - MeS adenosine triphosphate. 50 , the group consisting of the following: fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2 - MeS adenosine diphosphate, and 2 - MeS adenosine triphosphate.

[0121] 37. The antibody according to any one of aspects 22 - 29, wherein the in - vivo half - life of the antibody is about 12 hours.

[0122] 38. The antibody according to any one of aspects 22 - 29, wherein the antibody neutralizes the anti - platelet effect of ticagrelor or an active metabolite of ticagrelor.

[0123] 39. The antibody according to any one of aspects 22 - 29, wherein the antibody neutralizes the anti - platelet effect of ticagrelor or an active metabolite of ticagrelor within about 30 minutes of administration.

[0124] 40. The antibody according to any one of aspects 22 - 29, wherein the antibody restores ADP - induced platelet aggregation in the presence of ticagrelor or an active metabolite of ticagrelor.

[0125] 41. A method of treating acute bleeding in a patient who has been administered ticagrelor, the method comprising administering to the patient an effective amount of the antibody according to any one of aspects 1 - 40.

[0126] 42. A method for treating severe bleeding in a patient who has received or is receiving surgery and has been administered ticagrelor, the method comprising administering to the patient an effective amount of an antibody as described in any one of Protocols 1-40.

[0127] 43. A method for preventing bleeding in a patient who has been administered ticagrelor, the method comprising administering to the patient an effective amount of an antibody as described in any one of Protocols 1-40.

[0128] 44. A method for inhibiting the action of ticagrelor or its active metabolite on the (P2Y 12 ) receptor of a patient, the method comprising administering to the patient an effective amount of an antibody as described in any one of Protocols 1-40.

[0129] 45. A method for inhibiting the binding of ticagrelor or its active metabolite to the P2Y 12 receptor in a patient, the method comprising administering to the patient an effective amount of an antibody as described in any one of Protocols 1-40.

[0130] 46. A method for activating ADP-induced platelet aggregation in a patient who has been administered ticagrelor, the method comprising administering to the patient an effective amount of an antibody as described in any one of Protocols 1-40.

[0131] 47. The method according to any one of Protocols 43-46, wherein the patient is scheduled for surgery.

[0132] 48. A composition comprising a nucleic acid molecule, the nucleic acid molecule comprising a sequence encoding an antibody as described in any one of Protocols 22-26.

[0133] 49. The composition according to Protocol 48, wherein the nucleic acid molecule comprises SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71 or SEQ ID NO: 76.

[0134] 50. A vector comprising a nucleic acid molecule, the nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71 or SEQ ID NO: 76.

[0135] 51. A host cell comprising the vector as described in embodiment 50.

[0136] 52. The antibody according to any one of embodiments 1-10 or 22-29, wherein the antibody does not bind to an epitope comprising a hydroxyethyl group on the cyclopentyl ring of ticagrelor.

[0137] 53. The antibody according to any one of embodiments 1-10 or 22-29, wherein the antibody binds to an epitope comprising a cyclopropyl-difluorophenyl group of ticagrelor.

[0138] 54. The antibody according to any one of embodiments 1-10 or 22-29, wherein the antibody binds to an epitope comprising the cyclopropyl-difluorophenyl of ticagrelor, but does not bind to an epitope comprising a hydroxyethyl group on the cyclopentyl ring of ticagrelor.

[0139] Other aspects will be apparent to those skilled in the art upon reading the following description and the description of the examples. Description of the Drawings

[0140] For the purpose of illustrating the present disclosure, certain aspects of the present disclosure are depicted in the drawings. However, the present disclosure is not limited to the exact arrangements and means of the aspects depicted in the drawings.

[0141] Figure 1 PK / PD modeling of neutralizing Fab of ticagrelor based on Phase III PLATO data is described. Neutralizing Fab was added at time zero after a 180 mg loading dose and 90 mg ticagrelor twice daily. Patients were rechallenged with ticagrelor on day 1. It is predicted that Fab rapidly neutralizes ticagrelor and TAM, thereby restoring platelet aggregation in 99% of patients. The "peak" between day 0 and day 1 represents the redistribution of ticagrelor from other tissues in 1% of patients with slower clearance of ticagrelor.

[0142] Figure 2A 、 2B and 2C - hapten and Fab specificity. Figure 2AProvide the chemical structures of ticagrelor, ticagrelor active metabolite (TAM), ticagrelor inactive metabolite (TIM), and adenosine. The unique R groups (difluorophenyl-cyclopropyl and thiopropyl substituents) are highlighted with a dashed line. Figure 2B Is the specific spectrum of TICA0072. Figure 2C Is the specific spectrum of TICA0212 (MEDI2452). The specific spectrum includes ticagrelor, TAM, TIM, adenosine, ADP, ATP, and Figure 4 Three of the twelve related compounds (for clarity; at concentrations up to 0.1 mM, no binding of any of the twelve compounds was detected). Data are the mean and SEM of three replicates.

[0143] Figure 3 Shows the correlation of the scFv that binds to biotinylated linker ticagrelor (x-axis) with the scFv that binds to biotinylated linker ticagrelor in 50-fold excess of unmodified ticagrelor (y-axis). The inhibition of scFv binding in the presence of excess unmodified ticagrelor is shown by lines of 0%, 50%, 80%, and 90% inhibition.

[0144] Figure 4 Shows compounds that have a certain degree of 2D, 3D, or electrostatic similarity to ticagrelor.

[0145] Figure 5 Provides selectivity studies of TlCA0049Fab and TICA0072Fab. a-c Competition of TICA0049Fab for binding to biotinylated ticagrelor by the listed compounds. d-f Competition of TICA0072Fab for binding to biotinylated ticagrelor by the listed compounds. Data are DMSO-normalized.

[0146] Figure 6 Provides the competition curves of the parent TICA0072 and the optimized variants TICA0152Fab, TICA0162 Fab, and TICA0212Fab in a second-generation epitope competition assay.

[0147] Figure 7 Shows the results of the selectivity study of TICA0162 Fab and TICA0212 Fab. a-c Competition of TICA0162 Fab for binding to biotinylated ticagrelor by the listed compounds. d-f Competition of TICA0212 Fab for binding to biotinylated ticagrelor by the listed compounds. Data are DMSO-normalized.

[0148] Figure 8A 、 8BAnd 8C show the results of concentration-dependent reversal of ticagrelor by TICA0212 / MEDI2452 or TICA0072. Figure 8A : Inhibition of 20 μM ADP-induced aggregation mediated by 1 μM ticagrelor (▲) or 1 μM TAM (●) of TICA0212 / MEDI2452. Figure 8B : TICA0212 / MEDI2452 shows a decrease in the plasma free ticagrelor concentration in the presence of 1 μM ticagrelor. Mean (n = 5) ± standard error of the mean. Figure 8C : P2Y 12 Reversal of ticagrelor and TAM inhibition of P2Y signaling by TICA0072.

[0149] Figure 9 Show the results of reversal of ADP-induced whole blood aggregation ex vivo mediated by TICA0212 (250 mg / kg) after administration to ticagrelor-treated mice.

[0150] Figure 10 Show partial views of the crystal structures of the complexes of TICA0072 (A) and TICA0212 / MEDI2452 (B) with ticagrelor. Fab is shown as a ribbon, with the amino acid residues within ticagrelor shown as sticks. Some backbone atoms are omitted for clarity. The light chain is shown in beige and the heavy chain in light blue. CDR3 from both chains is colored green. V H CDR3 could not be modeled in the TICA0072 structure and is drawn as a dashed line in the provisional position. The orange arrow indicates the displacement of V L CDR3 observed in TICA0212 / MEDI2452 compared to TICA0072. Residues follow the Kabat numbering and are prefixed with L or H to indicate the light or heavy chain.

[0151] Figure 11 Show reversal of ADP-induced whole blood aggregation ex vivo. (A) Individual data for each treatment group after cessation of ticagrelor infusion. Vehicle control (■), ticagrelor alone (●), ticagrelor + TICA0212 / MEDI2452 (○), and ticagrelor + isotype control (Δ). Bars represent mean data (n = 4). AU = aggregation unit. At 15 minutes, data were collected only for the ticagrelor + TICA0212 / MEDI2452 group. (B) Percentage of reversal induced by TICA0212 / MEDI2452, as mean data (n = 4) ± SEM

[0152] Figure 12Shows reversal of ticagrelor-induced bleeding. (A) Individual data for total blood loss and (B) total bleeding time. Vehicle control (■), ticagrelor alone (●), and ticagrelor + TICA0212 / MEDI2452 (○). Bars represent mean data (n = 12). Detailed Description

[0153] Before proceeding further with the detailed description of the disclosure, it is to be understood that the disclosure is not limited to the specific compositions or method steps, as these may vary. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0154] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide one of ordinary skill in the art with a general dictionary of many of the terms used in this invention.

[0155] Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0156] Unless otherwise specified, the numbering of amino acids in the variable domains, complementarity-determining regions (CDRs), and framework regions (FRs) of an antibody follows the Kabat definition, which is listed in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids that correspond to a shortening or insertion in the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and insertion residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the “standard” Kabat-numbered sequence in the homologous regions. The maximum alignment of framework residues often requires the insertion of “spacer” residues in the numbering system to be used for the Fv region. In addition, due to interspecies or allelic differences, the identity of certain individual residues at any given Kabat site numbering may differ between antibody chains.

[0157] As used herein, the terms “antibody” (and “antibodies”) also referred to as immunoglobulins, encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments (e.g., multispecific antibodies such as those described in PCT Publication WO 2009018386, PCT Application No. PCT / US 2012 / 045229, which are incorporated herein by reference in their entirety), bispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments, antibody fragments that exhibit the desired biological activity (e.g., antigen-binding portions), disulfide-linked Fv (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies that target the antibodies of the present invention), intracellular antibodies, and epitope-binding fragments of any of the foregoing. In the specific embodiments provided herein, the antibody refers to an active binding fragment of an antibody, i.e., a molecule that contains at least one antigen-binding site, such as scFv and Fab. Antibodies also include peptide fusions with an antibody or a portion thereof, such as a protein fused to an Fc domain. Immunoglobulin molecules can have any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, such as Glm(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)). Antibodies can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g., chickens).

[0158] As used herein, C1-C6 alkyl refers to straight-chain and branched-chain alkyl groups having one to six carbon atoms and includes methyl, ethyl, propyl, n-butyl, isobutyl, pentyl, isopentyl, neopentyl, and hexyl.

[0159] As used herein, C1-C6 alkoxy refers to an alkyl group as described above having an oxygen in the group. In some embodiments, the oxygen atom is located at the position where the substituent is attached to the core structure (i.e., the ring structure).

[0160] As used herein, C1-C6 alkylthio refers to an alkyl group as described above having a sulfur in the group. In some embodiments, the sulfur atom is located at the position where the substituent is attached to the core structure (i.e., the ring structure).

[0161] As used herein, C1-C6 alkanol refers to an alkyl group as described above having a hydroxyl group at the end of the substituent structure.

[0162] As used herein, C3-C6 cycloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0163] As used herein, "substituted" C3-C6 cycloalkyl and C1-C6 alkyl refer to the above alkyl and cycloalkyl substituted on at least one carbon atom by an aryl group (also substituted by 1-3 halogen groups).

[0164] As used herein, "ticagrelor" refers to the reversible P2Y 12 inhibitor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol), and has the following chemical structure:

[0165]

[0166] As used herein, "ticagrelor active metabolite" or "TAM" refers to the main active metabolite of ticagrelor, also known as AR-C124910XX, which is a reversible P2Y 12 inhibitor and has the following chemical structure:

[0167]

[0168] As used herein, "ticagrelor inactive metabolite" or "TIM" refers to the inactive metabolite of ticagrelor, also known as AR-C133913XX, and has the following chemical structure:

[0169]

[0170] antibody

[0171] In general, the present disclosure provides novel antibodies that bind to cyclopentyltriazolopyrimidine compounds having the chemical formula (Ia):

[0172]

[0173] wherein

[0174] R1 is selected from the group consisting of: C1-C6 alkoxy and C1-C6 alkylthio;

[0175] R2 is selected from the group consisting of: H, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; and

[0176] R3 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanol.

[0177] In a specific embodiment, the antibody specifically binds to a compound selected from the group consisting of:

[0178] Ticagrelor;

[0179] Ticagrelor active metabolite (TAM); and

[0180] Ticagrelor inactive metabolite (TIM).

[0181] In a specific aspect, the present disclosure provides an antibody that binds to ticagrelor and TAM and has any one or more of the following characteristics, including high binding specificity, high binding affinity, rapid onset time, and rapid offset time (e.g., allowing selective continuation or co-administration of a treatment comprising ticagrelor).

[0182] In some embodiments, the antibody binds to ticagrelor and neutralizes the antiplatelet aggregation activity of ticagrelor and TAM, thereby restoring ADP-induced platelet aggregation in the presence of ticagrelor and TAM.

[0183] In some embodiments, the half-life of the antibody in a subject is approximately the same as the half-lives of ticagrelor and TAM. In some embodiments, the half-life of the antibody is about 4 - 24 hours (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In some embodiments, the half-life of the antibody is about 4 - 12 hours (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours).

[0184] In some embodiments, the antibody provides rapid onset of activity. For example, in an embodiment, the onset time of the antibody or the time to neutralize ticagrelor- and TAM-mediated platelet inhibition is about 15 - 120 minutes, or about 15 - 60 minutes. In some embodiments, the onset time is less than 60 minutes.

[0185] In some embodiments, the antibody has a PK / PD profile that provides rapid offset of activity such that, for example, a subject who has been administered the antibody can resume a prescribed ticagrelor treatment. In some embodiments, a subject who has received the antibody disclosed herein (e.g., by intravenous infusion) can receive or resume ticagrelor treatment within twenty-four hours after administration of the antibody.

[0186] As discussed and illustrated in certain embodiments herein, the antibody binds to ticagrelor or its metabolite and does not bind to other structurally related compounds or compounds that may be administered in combination with ticagrelor. For example, suitably, the antibody does not inhibit the activity of compounds selected from the group consisting of fenofibrate, nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate.

[0187] The antibodies described herein may include antigen-binding fragments that contain only selected portions of antibody molecules such as Fab, F(ab′)2, Fab’, scFv, di-scFv, sdAb fragments, and may be used as diagnostic or therapeutic agents. Additionally, specific residues in the variable domains may be altered to improve the binding specificity and / or stability of the antibody and antibody fragments. Other residues that do not directly participate in antigen binding have been replaced in order to “humanize” regions of non-human antibodies and reduce the immunogenicity of the antibody.

[0188] In certain aspects, the antibody is a Fab fragment, such as a Fab fragment of an antibody or a recombinantly produced antigen-binding fragment that includes a variable light chain (VL), a constant light chain (CL), a variable heavy chain (VH), and a constant heavy chain portion (CH1). Optionally, the light and heavy chains of the Fab may be interconnected by one or more disulfide bonds, such as through a suitable antibody hinge region. As described herein, the Fab binds to an epitope of a cyclopentyltriazolopyrimidine compound of an orally active agent. In some embodiments, the Fab binds to ticagrelor or its metabolite.

[0189] In certain aspects, the Fab may be derived from or based on the sequence of an antibody, such as a conventional murine, humanized, or human antibody. In certain aspects, the Fab may be derived from or based on one or more scFvs, such as scFvs screened and derived from a library. In such embodiments, the Fab derived from or based on the sequence of a conventional antibody or scFv retains one or more functional activities of the conventional antibody (e.g., retains at least 80% or more (80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) of the functional activity). For example, in certain aspects, the Fab retains one or more of the affinity for an antigen (such as ticagrelor), inhibitory activity, and / or selectivity of the antibody or scFv.

[0190] Although the Fab fragment may contain a sequence that binds to an epitope of cyclopentyltriazolopyrimidine, in certain embodiments, the Fab binds to ticagrelor. In some aspects, the Fab binds to the active metabolite of ticagrelor. In certain aspects, the Fab may bind to both ticagrelor and the active metabolite of ticagrelor.

[0191] In some embodiments, the Fab may comprise a combination of CDR regions from different antibodies that bind ticagrelor or its active metabolite.

[0192] In certain aspects, the Fab comprises a light chain portion (VL) that comprises an amino acid sequence listed in any one of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, and SEQ ID NO: 77. In additional embodiments, the Fab comprises a light chain portion that comprises an amino acid sequence listed in any one of SEQ ID NO: 57, SEQ ID NO: 67, and SEQ ID NO: 77. In certain aspects, the Fab comprises a heavy chain portion (VH) that comprises an amino acid sequence listed in any one of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72. In other embodiments, the Fab heavy chain portion comprises an amino acid sequence listed in any one of SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72. In certain aspects, the Fab is encoded by a nucleotide sequence encoding the light chain portion (VL) and a nucleotide sequence encoding the heavy chain portion (VH), such as a nucleotide sequence comprising a nucleic acid sequence listed in any one of SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, or SEQ ID NO: 71; and a nucleotide sequence comprising a nucleic acid sequence listed in any one of SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 16, SEQ ID NO: 16, SEQ ID NO: 16, SEQ ID NO: 16, SEQ ID NO: 16, or SEQ ID NO: 76.

[0193] In some aspects, the antibody can be a scFv. It should be understood that the scFv comprises a polypeptide chain that contains a variable heavy chain domain (VH) linked to a variable light chain domain (VL) by a flexible polypeptide linker. In some aspects, the polypeptide linker between VH and VL contains a protease cleavage site. The VH and VL domains of the scFv can be from the same or different antibodies. In some aspects, the VH or VL of the scFv can comprise one or more CDRs that bind to a target of interest, while the remainder of the VH or VL domain is derived from a different antibody or is synthetic. In some aspects, the scFv comprises at least one CDR of an antibody, such as an antibody that has binding activity to ticagrelor or its metabolite. In some aspects, the scFv comprises at least two CDRs of a given antibody. In some aspects, the scFv comprises at least three CDRs of a given antibody. In some aspects, the scFv comprises at least four CDRs of a given antibody. In some aspects, the scFv comprises at least five CDRs of a given antibody. In some aspects, the scFv comprises at least six CDRs of a given antibody.

[0194] Several methods can be used alone or in combination to improve the stability of the scFv molecule. One method that can be used alone or in combination with one or more other methods is to design the length and / or composition of the linker that connects the scFv domains to stabilize the scFv moiety.

[0195] Another possible method that can be used alone or in combination with one or more other methods described herein is to introduce at least two amino acid substitutions (also referred to as modifications or mutations) into the VH and / or VL domains of the scFv to facilitate disulfide bond formation (see, for example, Brinkmann et al., 1993, Proceedings of the National Academy of Sciences of the United States of America (PNAS), 90:7538-42; Zhu et al., 1997, Protein Science (Prot. Sci.) 6:781-8; Reiter et al., 1994, Biochemistry 33:5451-9; Reiter et al., 1996, Nature 14:1239-45; Luo et al., 1995, Journal of Biochemistry (J. Biochem.) 118:825-31; Young et al., 1995, Federation of European Biochemical Societies Letters (FEBS Let.) 377:135-9; Grenoble et al., 1990, Biochemistry 29:1362-7).

[0196] In some aspects, a mutation is introduced into each of the VH and VL domains of the scFv to facilitate the formation of inter-chain disulfide bonds between the VH and VL domains upon scFv expression. In another aspect, two mutations are introduced into the same domain of the chain. In some aspects, two mutations are introduced into different chains. In some aspects, multiple pairs of these two mutations are introduced to facilitate the formation of multiple disulfide bonds. In some aspects, cysteine is introduced to facilitate disulfide bond formation. Exemplary amino acids that can be mutated to cysteine include amino acids 43, 44, 45, 46, 47, 103, 104, 105, and 106 of VH2, and amino acids 42, 43, 44, 45, 46, 98, 99, 100, and 101 of VL2. The above numbering is based on Kabat numbering, which identifies positions only relative to VH2 and VL2 of the scFv (and not relative to the positions of amino acids in the full-length antibody sequence). Exemplary combinations of amino acid positions that can be mutated to cysteine residues include: VH 44-VL 100, VH 105-VL 43, VH 105-VL 42, VH 44-VL 101, VH 106-VL43, VH104-VL 43, VH 44-VL99, VH 45-VL98, VH 46-VL98, VH103-VL43, VH103-VL44, and VH103-VL45. In some aspects, amino acid 44 of VH and amino acid 100 of VL are mutated to cysteine.

[0197] Another potential approach that can be used alone or in combination with one or more of the other methods described herein is to select the order of the domains of the scFv. In some aspects, the orientation of the VH domain relative to the VL domain is optimized for stability. In some aspects, the scFv is in the VH-linker-VL orientation. In some aspects, the scFv is in the VL-linker-VH orientation.

[0198] Another method that can be used alone or in combination with one or more of the methods described herein is to introduce one or more stability mutations by mutating one or more surface residues of the scFv. In some aspects, one, two, three, four, five, six, or more than six residues are mutated in one or both of the VH and / or VL domains of the scFv. In certain aspects, changes are made only in the VH domain of the scFv. In certain aspects, changes are made only in the VL domain of the scFv. In certain aspects, changes are made in both the VH and VL domains of the scFv. The same number of changes can be made in each domain, or different numbers of changes can be made in each domain. In certain aspects, one or more of the changes are conservative amino acid substitutions from residues present in the unmodified parental scFv. In other aspects, one or more of the changes are non-conservative amino acid substitutions from residues present in the unmodified parental scFv. When multiple substitutions are made, in one or both of the VH or VL domains of the scFv, each substitution is independently a conservative or non-conservative substitution. In certain aspects, all substitutions are conservative substitutions. In certain aspects, all substitutions are non-conservative. In certain aspects, at least one substitution is conservative. In certain aspects, at least one substitution is non-conservative.

[0199] Another method that can be used alone or in combination with one or more additional methods described herein is to mutate one or more residues present in the VH and / or VL domains of the scFv to introduce one or more substitutions to match the most common residues at the specified positions of the consensus sequences of the VH and / or VL domains of known, screened, and / or identified antibodies. In some aspects, one, two, three, four, five, six, or more than six positions in one or both of the VH domain and / or VL domain of the scFv are introduced with substitutions. The same number of changes can be made in each domain, or different numbers of changes can be made in each domain. In certain aspects, one or more of the changes in the sequence that matches a given consensus sequence are conservative amino acid substitutions from residues present in the unmodified VH and / or VL sequences. In other aspects, one or more of the changes represent non-conservative amino acid substitutions from residues present in the unmodified VH and / or VL sequences. When multiple substitutions are made, in one or both of the VH or VL domains of the scFv, each substitution is independently a conservative or non-conservative substitution. In certain aspects, all substitutions are conservative substitutions. In certain aspects, all substitutions are non-conservative substitutions. In certain aspects, at least one substitution is conservative. In certain aspects, at least one substitution is non-conservative.

[0200] It should be noted that any modification described as being useful for modifying or stabilizing the scFv portion can be used to modify the Fab portion. For example, the variable domains of the Fab can be modified to improve stability, antigen binding, etc. In addition, the Fab or scFv portion can be modified to reduce immunogenicity.

[0201] In some aspects, the antibody can be an scFv comprising a variable light chain portion (VL) that comprises an amino acid sequence listed in any one of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In other embodiments, the scFv comprises a light chain portion that comprises an amino acid sequence listed in any one of SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In some aspects, the scFv comprises a heavy chain portion (VH) that comprises an amino acid sequence listed in any one of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72. In other embodiments, the scFv comprises a heavy chain portion that comprises an amino acid sequence listed in any one of SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72.

[0202] The antibodies disclosed herein can also comprise one or more linker polypeptides. The linker can interconnect the heavy and light chain domains (scFv) or link the antibody or its antigen-binding fragment to another reagent (e.g., a label, an Fc domain, etc.). The linker can vary in length and sequence and is commonly known in the art.

[0203] The serum half-life of an antibody comprising an Fc region can be increased by increasing the binding affinity of the Fc region for FcRn. As used herein, the term "antibody half-life" refers to the pharmacokinetic property of an antibody, which is a measure of the average survival time of antibody molecules after their administration. Antibody half-life can be expressed as the time required to eliminate 50% of a known amount of immunoglobulin from the body of a patient (or other mammal) or from a particular compartment thereof (e.g., as measured in serum, i.e., the circulating half-life), or from other tissues. The half-life can vary from one immunoglobulin or immunoglobulin class to another. Generally, an increase in antibody half-life results in an increase in the mean residence time (MRT) of the administered antibody in the circulation.

[0204] An increase in the half-life can permit a reduction in the dose administered to a patient and a reduction in the frequency of administration. To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated, for example, into an antibody (especially an antibody fragment) as described in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that causes an increase in the in vivo serum half-life of an IgG molecule. Alternatively, the antibodies of the present disclosure having an extended half-life can be generated by modifying amino acid residues identified as being involved in the interaction between Fc and the FcRn receptor (see, e.g., U.S. Patent Nos. 6,821,505 and 7,083,784; and WO09 / 058492). Additionally, the half-life of the antibodies of the present disclosure can be increased by conjugation to PEG or albumin using techniques widely used in the art.

[0205] Antibodies falling within the scope of the present disclosure can be identified by any of the structural and / or functional features identified herein. For example, specific binding characteristics of an antibody (e.g., K 分离 (K off ), KD, IC 50 , specificity / selectivity for ticagrelor and ticagrelor metabolites) can be screened using any of the techniques shown herein or any techniques known in the art.

[0206] Labels, conjugates, and moieties

[0207] For purposes of diagnosis and other assays, the antibodies of the present disclosure can be conjugated to a label, and the antibody and / or one or more of its targets can be detected in these assays. Labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes.

[0208] In certain embodiments, the antibody is conjugated to a fluorophore. The choice of fluorophore attached to the antibody will determine the absorption and fluorescence emission characteristics of the conjugated antibody. Physical properties of fluorophore labels that can be used for antibodies and antibody-binding ligands include, but are not limited to: spectral characteristics (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, or combinations thereof. All of these physical properties can be used to distinguish one fluorophore from another and thus permit multiplex analysis. Other desired properties of fluorescent labels can include cell permeability and low toxicity, for example, if the labeling of the antibody is to be performed in cells or model organisms (e.g., live animals).

[0209] In some aspects, enzymes are markers and conjugated to antibodies. Enzymes are desirable markers because amplification of the detectable signal can be achieved, resulting in increased assay sensitivity. The enzyme itself does not produce a detectable response, but when it is contacted with an appropriate substrate, it acts to break down the substrate such that the converted substrate produces a fluorescent, colorimetric, or luminescent signal. Since one enzyme on the labeling reagent can cause multiple substrates to be converted into detectable signals, the enzyme amplifies the detectable signal. Enzyme substrates are selected to produce a preferred measurable product, e.g., colorimetric, fluorescent, or chemiluminescent. Such substrates are widely used in the art and are well known to those skilled in the art, including, for example, oxidoreductases such as horseradish peroxidase and substrates such as 3,3'-diaminobenzidine (DAB); phosphatases, e.g., acid phosphatase, alkaline phosphatase, and substrates such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, e.g., β-galactosidase, β-glucuronidase, or β-glucosidase, and substrates such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal); additional enzymes include hydrolases such as cholinesterase and peptidase, oxidases such as glucose oxidase and cytochrome oxidase, and reductases, and suitable substrates for these enzymes are known.

[0210] Enzymes and their appropriate substrates that produce chemiluminescence are suitable for some assays. These include, but are not limited to, luciferase and aequorin in their native and recombinant forms. Chemiluminescent substrates for phosphatases, glycosidases, and oxidases, such as those containing stable dioxetanes, luminol, isoluminol, and acridinium esters, are also useful.

[0211] In another aspect, haptens such as biotin are also utilized as markers. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal and it can act as a tag to be used in affinity chromatography for separation purposes. For detection purposes, an enzyme conjugate that has an affinity for biotin, such as avidin-HRP, is used. Subsequently, a peroxidase substrate is added to produce a detectable signal.

[0212] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and the like.

[0213] In some aspects, a fluorescent protein can be conjugated to an antibody as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliproteins and their derivatives. Fluorescent proteins, particularly phycobiliproteins, are especially useful for generating tandem dye-labeled labeling reagents. For the purpose of obtaining a larger Stokes shift, these tandem dyes contain a fluorescent protein and a fluorophore, where the emission spectrum is more displaced from the wavelength of the absorption spectrum of the fluorescent protein.

[0214] In some aspects, the label is a radioisotope. Examples of suitable radioactive materials include, but are not limited to: iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113 mIn, 115 mIn), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 135 Xe), fluorine ( 18 F), 153 SM, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 pr, 105 Rh and 97 Ru.

[0215] In some aspects, a drug can be conjugated to an antibody. For example, an antibody containing an scFv can be conjugated to a drug for the treatment of cardiovascular diseases and / or acute coronary syndrome.

[0216] In certain embodiments, drugs and other molecules can be targeted to antibodies by site-specific conjugation. For example, an antibody can comprise engineered cysteine domains (including one or more cysteines to the binding unit and / or the Fc domain), which results in free sulfhydryl groups for conjugation reactions. In some aspects, the antibody is engineered to incorporate specific conjugation sites.

[0217] Nucleic acid molecules encoding antibodies

[0218] The present disclosure provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. One aspect of the present disclosure provides nucleic acid molecules encoding any of the antibodies specifically described herein. The nucleic acid molecules can encode the heavy and / or light chain variable regions of an antibody.

[0219] In some aspects, the antibody is a Fab or scFv, wherein the nucleic acid portion encoding the Fab or scFv comprises a nucleotide sequence encoding a VL domain and a nucleotide sequence encoding a VH, and wherein the nucleotide sequence encoding the VL domain is optionally linked to the nucleotide sequence encoding the VH domain by a nucleotide sequence encoding a flexible polypeptide linker.

[0220] Another aspect provides host cells transformed with any of the nucleic acid molecules described herein. In another aspect of the present disclosure, host cells are provided that comprise a vector, the vector comprising a nucleic acid molecule as described herein. In one aspect, the host cell can comprise more than one vector.

[0221] This disclosure encompasses nucleic acid molecules encoding any antibody of this disclosure, as well as the light or heavy chain of an antibody. For example, this disclosure encompasses nucleic acid molecules that include nucleotide sequences encoding one or more of the following: SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, and SEQ ID NO: 77. This disclosure further encompasses nucleic acid molecules encoding any antibody of this disclosure that further comprise additional regions (e.g., Fc or modified Fc). In some embodiments, the nucleic acid molecule can be selected from one or more of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71, or SEQ ID NO: 76. In other embodiments, this disclosure provides a vector that includes a nucleic acid molecule selected from one or more of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71, or SEQ ID NO: 76.

[0222] Methods for generating antibodies, Fabs, and scFvs

[0223] This disclosure provides methods for generating the antibodies and fragments thereof described herein. In some aspects, antigen-binding fragments of antibodies that recognize ticagrelor and specific epitopes of ticagrelor and / or TAM disclosed herein can be generated by any technique known to those skilled in the art. For example, Fab and F(ab’)2 fragments can be generated from antibodies by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (for generating Fab fragments) or pepsin (for generating F(ab’)2 fragments). Additionally, antibodies including scFv and Fab as described herein can be generated using a variety of phage display methods known in the art.

[0224] Generally, in phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Specifically, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or murine cDNA library of lymphoid tissue). The DNA encoding these VH and VL domains is recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into Escherichia coli, and the E. coli is infected with helper phage. The phage used in these methods can be filamentous phage including fd and M13, and the VH and VL domains can be recombinantly fused to phage gene III or gene VIII. Phage expressing antigen-binding domains that bind ticagrelor and / or TAM can be selected or identified with an antigen, e.g., using a labeled antigen or an antigen bound or captured on a solid surface or bead. Similarly, binding domains that bind antigens / haptens other than ticagrelor and / or TAM can be identified for de-selection. Examples of phage display methods useful for making the antibodies of the present invention include those disclosed in the following: Brinkman et al., 1995, Journal of Immunological Methods 182:41-50; Ames et al., 1995, Journal of Immunological Methods 184:177-186; Kettleborough et al., 1994, European Journal of Immunology 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT / GB 9I / O1 134; PCT Publication Nos. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and WO 97 / 13844; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0225] As described in the above references, after phage selection, the antibody-encoding regions from the phages can be isolated and used to generate full antibodies, including human antibodies, or any other desired antigen-binding fragments (scFvs and Fabs), and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described in detail below. Techniques for the recombinant production of Fab, Fab’, and F(ab′)2 fragments can also be utilized using methods known in the art, such as those disclosed in the following: PCT Publication No. WO 92 / 22324; Mullinax et al., 1992, Biotechnology 12(6):864-869; Sawai et al., 1995, American Journal of Reproductive Immunology 34:26-34; and Better et al., 1988, Science 240:1041-1043 (the above references are incorporated herein by reference in their entirety).

[0226] In some aspects, the nucleic acids disclosed herein can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The nucleic acid sequences encoding the antibody light and heavy chains can be cloned in the same expression vector in any orientation (e.g., light chain before the heavy chain, or vice versa), or they can be cloned in two different vectors. If expressed using one vector, the two coding genes can have their own genetic elements (e.g., promoter, RBS, leader sequence, termination, polyA, etc.), or they can be cloned with a set of genetic elements but linked to a cis-acting element. The regulatory nucleotide sequences are generally suitable for the host cell in which expression is desired. Many types of suitable expression vectors and suitable regulatory sequences for a variety of host cells are known in the art. Typically, the one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by this disclosure. The promoter can be a naturally occurring promoter or a hybrid promoter that combines more than one promoter component. The expression construct can be present on an episome (e.g., plasmid) in the cell, or the expression construct can be inserted into the chromosome.

[0227] In some aspects, the expression vector contains a selectable marker gene to allow selection of the transformed host cells. Selectable marker genes are well known in the art and will vary with the host cells used. In some aspects, the present disclosure relates to an expression vector comprising a nucleotide sequence that encodes a polypeptide and is operably linked to at least one regulatory sequence. Regulatory sequences are well recognized in the art and are selected to direct the expression of the encoded polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary non-limiting regulatory sequences are described in Goeddel, Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). It should be understood that the design of the expression vector can depend on such factors as the choice of host cell to be transformed and / or the type of protein desired to be expressed. Also, consideration should be given to the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers).

[0228] Methods for generating the antibodies of the present disclosure can include, for example, host cells transfected with one or more expression vectors that encode the antibody (e.g., a single vector encoding the heavy and light chains or their variable regions; or two vectors, one encoding the heavy chain and one encoding the light chain or their variable regions), and the host cells can be cultured under appropriate conditions to allow expression of the antibody to occur. The antibody can be secreted and isolated from a mixture of the cells and the culture medium containing the antibody. Alternatively, the antibody can be retained in the cytoplasmic or membrane fractions and the cells are harvested, lysed, and the protein is isolated. Cell culture includes the host cells, the culture medium, and other by-products. Suitable culture media for cell culture are well known in the art. The antibody can be isolated from the cell culture medium, the host cells, or both using techniques known in the art for purifying proteins, antibodies, and antigen-binding antibody fragments thereof, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification. In some aspects, the antibody is prepared as an antigen-binding fragment of the antibody comprising the variable regions of the heavy and light chains, which can increase solubility and facilitate purification.

[0229] Recombinant nucleic acids can be produced by ligating a cloned gene or a portion thereof to a vector that is suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vectors for the production of recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli. In some aspects, mammalian expression vectors contain prokaryotic sequences that facilitate the propagation of the vector in bacteria, as well as one or more eukaryotic transcription units for expression in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids (such as pBR322) to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses can be used for transient expression of proteins in eukaryotic cells, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205). The various methods used in the preparation of these plasmids and the transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, Second Edition, Sambrook, Fritsch, and Maniatis, eds., (Cold Spring Harbor Laboratory Press, 1989), Chapters 16 and 17. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include PVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as pBlueBac III containing β-gal).

[0230] Techniques for preparing fusion genes are well known. In essence, the joining of various nucleic acid fragments encoding different polypeptide / antibody sequences is carried out according to conventional techniques, using blunt or staggered ends for ligation, restriction endonuclease digestion to provide appropriate ends, adding sticky ends as appropriate, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In other respects, fusion genes can be synthesized by conventional techniques, including DNA automated synthesizers. Alternatively, gene fragments can be PCR amplified using anchored primers, which generates complementary overhangs between two consecutive nucleic acid fragments, and the consecutive gene fragments can then be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons: 1992).

[0231] In some aspects, expression vectors expressing any of the nucleic acids described herein can be used to express antibodies in host cells. For example, antibodies can be expressed in bacterial cells (e.g., Escherichia coli), insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0232] Once the expression vector has been transferred into the host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce the antibody. Accordingly, the present disclosure includes host cells containing a polynucleotide encoding an antibody or a fragment thereof, the polynucleotide being operably linked to a heterologous promoter. In certain aspects, both the heavy and light chains and / or the variable regions of the heavy and light chains can be co-expressed (from the same or different vectors) in the host cell to express the whole antibody. In certain aspects, both the heavy and light chains of the antibody are expressed by a single promoter. In certain aspects, the heavy and light chains of the antibody are expressed by multiple promoters. In certain aspects, the heavy and light chains of the antibody are encoded on a single vector. In certain aspects, the heavy and light chains of the antibody are encoded on multiple vectors.

[0233] Mammalian cell lines that are available as hosts for expressing recombinant antibodies are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a variety of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed antibody or portions thereof. To this end, eukaryotic host cells with the cellular machinery for the appropriate processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells. In one aspect, human cell lines generated by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. In one aspect, the human cell line PER.C6. (Crucell, The Netherlands) can be used to recombinantly produce monoclonal antibodies.

[0234] Additional cell lines that can be used as hosts for expressing recombinant antibodies include but are not limited to insect cells (e.g., Sf21 / Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g., Saccharomyces cerevisiae, Pichia, US 7326681; etc.), plant cells (US 20080066200); and chicken cells (WO 2008142124).

[0235] In some aspects, the antibodies of the present disclosure are stably expressed in cell lines. Stable expression can be used for the long-term, high-yield production of recombinant proteins, antibodies, and their antigen-binding fragments. For example, cell lines that stably express antibody molecules can be generated. Host cells can be transformed with a suitably engineered vector that contains expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. After introduction of the foreign DNA, the cells can be allowed to grow in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the growth of cells that have stably integrated the plasmid into their chromosomes and form foci, which can then be cloned and expanded into cell lines. Methods for generating stable cell lines with high yields are well known in the art, and the reagents are generally commercially available.

[0236] In some aspects, the antibodies of the present disclosure are transiently expressed in cell lines. Transient transfection is a method in which the nucleic acid introduced into the cell does not integrate into the genome or chromosomal DNA of the cell. In fact, the nucleic acid is maintained as an extrachromosomal element in the cell, e.g., as an episome. The transcription process of the episomal nucleic acid is not affected, and the protein encoded by the episomal nucleic acid is produced.

[0237] Stable or transiently transfected cell lines are maintained in cell culture media and conditions well known in the art for monoclonal antibody expression and production. In some aspects, mammalian cell culture media are based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other aspects, the cell culture media are modified to support the growth of both cell growth and bioprotein expression. As used herein, the terms "cell culture media", "media", and "media formulations" refer to nutrient solutions used for the maintenance, growth, reproduction, or expansion of cells in an artificial in vitro environment outside a multicellular organism or tissue. The cell culture media can be optimized for specific cell culture uses, including, for example, cell culture growth media formulated to promote cell growth or cell culture production media formulated to promote recombinant protein production. The terms nutrients, components, and constituents are used interchangeably herein and refer to the constituents that make up the cell culture media.

[0238] Once the molecule has been produced, it can be purified by any method known in the art for the purification of immunoglobulin molecules and their fragments, e.g., by chromatography (e.g., ion exchange, affinity (especially by affinity for a specific antigen, protein A or protein G), and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins, antibodies, and / or antibody fragments. In addition, the molecules or fragments thereof of the present disclosure can be fused to heterologous polypeptide sequences described herein or otherwise known in the art (referred to herein as "tags", such as histidine tags) to facilitate purification.

[0239] When recombinant techniques are used, the molecule can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the molecule is produced intracellularly, then as a first step, particulate debris of the host cells or lysed fragments are removed, e.g., by centrifugation or ultrafiltration. Carter et al., Bio / Technology, 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. In the case where the molecule is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0240] Compositions prepared from cells can be purified alone or in combination with other purification steps using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the molecule and will be understood by those skilled in the art. The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinylbenzene) permit faster flow rates and shorter processing times than can be achieved with agarose. Other protein purification techniques can also be used depending on the molecule to be recovered, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0241] After any one or more initial purification steps, a mixture containing the molecule of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and 4.5 and performed at low salt concentration (e.g., from about 0 to 0.25 M salt).

[0242] Antibodies can be prepared and purified using any one or combination of techniques described above and / or in the examples. Regardless of how the antibodies are purified, in order to confirm the functional binding of the antibodies of the present disclosure, binding assays can be performed (before and / or after purification). For example, a dual ELISA assay can be used. In some aspects, a first antigen (e.g., ticagrelor or a competitor thereof) is coated on wells and the bound immobilized antibodies are prepared for detection.

[0243] Pharmaceutical formulations

[0244] In certain aspects, the present disclosure provides pharmaceutical compositions. Such pharmaceutical compositions can be compositions comprising a nucleic acid molecule encoding an antibody. Such pharmaceutical compositions can also be compositions comprising an antibody, or a combination of antibodies, and a pharmaceutically acceptable excipient. In certain aspects, the pharmaceutical compositions of the present disclosure are used as medicaments.

[0245] In some aspects, an antibody or combination of antibodies (or a nucleic acid molecule encoding an antibody or antibody combination) can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier, excipient, or stabilizer. In some aspects, such pharmaceutical compositions are suitable for administration to a human or non-human animal via any one or more routes of administration known in the art. As will be understood by one of skill in the art, the route and / or mode of administration will vary with the desired outcome. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations routinely may contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that can be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweetening agents, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming balancing ions (such as sodium), and the like. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy", 21st Edition, Lippincott Williams & Wilkins (2005) and "Physician's Desk Reference", 60th Edition, Medical Economics, Montvale, New Jersey (2005). A pharmaceutically acceptable carrier can be selected that is suitable for the desired or required mode of administration, solubility, and / or stability.

[0246] The formulations described herein contain an active agent (such as an antibody or antibody fragment, such as Fab or scFv) at a concentration in w / v that results in a suitable dose for the desired application. In some aspects, the active agent is present in the formulation at a concentration of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, or from 1 mg / ml to about 25 mg / ml. In some aspects, the active agent is present at a concentration of about 25 mg / ml. In some aspects, the concentration of the active agent in the formulation can vary from about 0.1 wt% to about 100 wt%. In some aspects, the concentration of the active agent ranges from 0.003 to 1.0 molar concentration.

[0247] In one aspect, the formulations of the present disclosure are pyrogen-free formulations that are substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are restricted within microorganisms and are released only when the microorganisms are disrupted or die. Pyrogenic substances also include heat-stable substances (glycoproteins) from the outer membranes of bacteria and other microorganisms that induce fever. If either of these two substances is administered to humans, it can cause fever, hypotension, and shock. Due to the potential harmful effects, even low amounts of endotoxins must be removed from drug solutions administered intravenously. The Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per kilogram of body weight per dose within a single hour for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223 (2000)). In certain specific aspects, the endotoxin and pyrogen levels in the composition are less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg.

[0248] When used for in vivo administration, the formulations of the present disclosure should be sterile. The formulations of the present disclosure can be sterilized by various sterilization methods, including sterile filtration, radiation, and the like. In one aspect, the formulation is filter-sterilized using a pre-sterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices described in “Remington: The Science and Practice of Pharmacy,” 21st Edition, Lippincott Williams & Wilkins Publishers (2005).

[0249] The therapeutic compositions of the present disclosure can be formulated for specific routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Formulations suitable for topical or transdermal administration of the present disclosure include powders, sprays, ointments, pastes, emulsions, lotions, gels, solutions, patches, and inhalants. The antibodies can be mixed with pharmaceutically acceptable carriers under sterile conditions and with any preservatives, buffers, or propellants that may be required (U.S. Patent Nos. 7,378,110; 7,258,873; 7,135,180; U.S. Patent Application Publication Nos. 2004-0042972; and 2004-0042971).

[0250] These formulations may suitably be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure may vary so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient (e.g., a "therapeutically effective amount"). The selected dosage level will depend upon various pharmacokinetic factors, including the activity of the specific composition employed, the route of administration, the time of administration, the excretion rate of the specific compound employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the specific composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and like factors well known in the medical arts. A suitable dosage range may be from about 0.0001 to about 100 mg / kg body weight or greater, such as about 0.1, 1, 10, or 50 mg / kg body weight, with about 1 to about 10 mg / kg body weight being suitable.

[0251] It should be noted that the present disclosure similarly encompasses: Formulations suitable for diagnostic and research use can also be prepared. The concentration of the active agent in such formulations and the presence or absence of excipients and / or pyrogens can be selected based on the specific application and intended use.

[0252] Use

[0253] The antibodies disclosed herein can be used in therapeutic methods, including combination therapies, for neutralizing the activities of inhibitors of platelet activation, aggregation, and degranulation, platelet disaggregation promoters, and antithrombotic agents. Accordingly, the antibodies described herein can be used in a number of applications related to the administration of ticagrelor (including concomitant methods), and are suitable for neutralizing the effects of ticagrelor and / or one or more metabolites of ticagrelor. In such methods, the antibody can optionally reversibly reduce, neutralize, eliminate, or otherwise inhibit the activity of ticagrelor, and treat or prevent a number of effects, conditions, and / or symptoms related to the administration of ticagrelor and / or resulting from treatment with ticagrelor-containing therapies.

[0254] The antibody can be administered to a patient being treated, or in need of treatment or prophylaxis of a treatable indication and / or having an indication suitable for ticagrelor (Brilinta), the indications including, for example, unstable angina, primary arterial thrombotic complications of atherosclerosis such as thrombotic or embolic stroke, transient ischemic attack, peripheral vascular disease, myocardial infarction with or without thrombolysis, arterial complications due to atherosclerotic disease (e.g., angioplasty, including coronary angioplasty (PTCA), endarterectomy, stent placement, coronary and other vascular graft surgery), thrombotic complications of surgery or mechanical injury (e.g., tissue salvage after accidental or surgical trauma, reconstructive surgery including skin and muscle flaps), conditions having a component of disseminated thrombosis / platelet consumption (e.g., disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, thrombotic complications of sepsis, adult respiratory distress syndrome, antiphospholipid syndrome, heparin-induced thrombocytopenia, and preeclampsia / eclampsia), or venous thrombosis (such as deep vein thrombosis), venous occlusive disease, blood disorders (such as myeloproliferative disorders, including thrombocytosis, sickle cell disease); or to prevent mechanically induced platelet activation in vivo, such as cardiopulmonary bypass and extracorporeal membrane oxygenation (to prevent microthromboembolism), extracorporeal mechanically induced platelet activation, such as for the preservation of blood products such as platelet concentrates, or shunt occlusion such as renal dialysis and plasma exchange, thrombosis secondary to vascular injury / inflammation such as vasculitis, arteritis, glomerulonephritis, inflammatory bowel disease, and organ transplant rejection, conditions (such as migraine).

[0255] In some embodiments, the antibodies disclosed herein can be administered to patients who are receiving or have received treatment with ticagrelor, as well as patients in need of treatment or about to need treatment, for bleeding or potential bleeding associated with coronary artery bypass grafting (CABG), cardiothoracic surgery, mediastinal reexploration, postoperative stroke, mechanical ventilation, prolonged stay in the intensive care unit, emergency non-cardiac surgery (such as neurosurgery or ophthalmic surgery, spinal surgery, intracranial surgery, orbital surgery, plastic surgery, nephrectomy, hemicolectomy, etc.). Accordingly, the methods provided herein can include administering the antibody as a co-therapeutic agent (simultaneously) with ticagrelor, or within a period of time after ticagrelor administration (e.g., minutes, hours or days). For example, in some embodiments, the method can include administering the antibody to a patient who has received ticagrelor within 10 - 120 minutes of ticagrelor administration. In some embodiments, the method can include administering the antibody to a patient who has received ticagrelor within 1 - 48 hours of ticagrelor administration. In some embodiments, the antibody is administered to a subject who has received ticagrelor within a time amount that does not allow for metabolism and elimination of ticagrelor and / or its metabolites from the subject.

[0256] In some embodiments, the present disclosure provides methods of inhibiting the action of ticagrelor or its active metabolite on the (P2Y 12 ) receptor in a patient.

[0257] In some embodiments, the present disclosure provides methods of inhibiting the binding of ticagrelor or its active metabolite to the P2Y 12 receptor in a patient's body.

[0258] In some embodiments, the present disclosure provides methods of activating ADP-induced platelet aggregation in a patient who has received ticagrelor.

[0259] The antibodies of the present disclosure, such as those exemplified in the examples, can also be used for diagnostic purposes. For example, one or more target factors (ticagrelor or its metabolites) can be detected in a subject's tissue or cells to determine or screen for the circulating amount of ticagrelor in the subject. A diagnostic kit can include one or more antibodies, as well as a detection system for indicating the reaction of the antibody with ticagrelor or its metabolite (if present).

[0260] Accordingly, the present disclosure encompasses many uses of the antibodies, including therapeutic, diagnostic, and research uses. Diagnostic and research uses can be in vivo or ex vivo.

[0261] Kit

[0262] Another aspect of the present disclosure is a kit. In one aspect, the kit comprises any composition or pharmaceutical composition of the nucleic acids, antibodies, expression vectors, or host cells described above, and instructions or a label for guiding proper use or administration. Optionally, the kit may also include one or more containers and / or syringes or other devices to facilitate delivery or use. The present disclosure contemplates that all or any subset of the components for performing research assays, diagnostic assays, and / or for administering a therapeutically effective amount may be encapsulated in the kit. Similarly, the kit may include instructions for preparing an antibody by, for example, culturing a host cell expressing a nucleic acid encoding an antibody of the present disclosure under suitable conditions. As a further example, a kit for the therapeutic administration of an antibody of the present disclosure may comprise a solution of a pharmaceutical formulation containing the antibody or a lyophilized formulation of the antibody, and instructions for administering the composition to a patient in need thereof, and / or instructions for reconstituting the lyophilized product. In certain embodiments, the kit further comprises ticagrelor in a formulation suitable for administration to a subject (e.g., BRILINTA TM (BRILINTA TM , BRILIQUE TM ))). In such embodiments, the kit may further include instructions for administering the antibody and ticagrelor formulations to a patient in need of treatment with the antibody, ticagrelor, or both the antibody and ticagrelor.

[0263] The present disclosure also encompasses a finished packaged and labeled pharmaceutical product. Such a manufactured article includes a suitable unit dosage form in a suitable vessel or container (e.g., a glass vial or other sealed container). In the case of a dosage form suitable for parenteral administration, the active ingredients, such as the antibody and / or ticagrelor formulation described above, are sterile and suitable for administration as a particle-free solution. In certain aspects, the formulation is suitable for an injectable route of administration. In some embodiments, the administration is subcutaneous administration. In some embodiments, the administration is intravenous administration. Thus, routes of administration including injection or infusion to a human or animal are contemplated.

[0264] In a particular aspect, the formulation of the present disclosure is formulated as a sterile liquid in a single-dose vial. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, blister packs (containing one or more pills). Optionally, associated with such one or more containers may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of drugs or biologics, the notice reflecting the approval of the government agency for the manufacture, use, or sale for human diagnosis and / or administration.

[0265] As with any pharmaceutical product, the packaging materials and containers are designed to protect the stability of the product during storage and shipment. In addition, the products of the present disclosure include instructions for use or other information materials advising physicians, technicians, or patients on how to appropriately prevent or treat the disease or condition being discussed. In other words, the manufactured article includes instructions tools indicating or suggesting a dosing regimen (including but not limited to actual dosage, monitoring procedures, etc.), as well as other monitoring information.

[0266] Kits for diagnostic assays can contain solutions containing antibodies or lyophilized preparations of antibodies of the present disclosure, as well as reagents for detecting such antibodies, wherein the antibody specifically binds to ticagrelor and / or its metabolites. The antibodies can be labeled according to methods known in the art and described herein, including but not limited to, for example, labeling with small molecule fluorescent tags, proteins such as biotin, GFP, or other fluorescent proteins, or epitope sequences such as his or myc. Similarly, primary antibodies for detecting the antibodies can be included in the kit. The primary antibody can be directed against a sequence on the antibody or against a label, tag, or epitope of the labeled antibody. The primary antibody can in turn be labeled for detection, or if further signal amplification is required, the primary antibody can be detected by a secondary antibody, which can also be included in the kit.

[0267] Kits for research use are also contemplated. Such kits can, for example, be similar to kits intended for diagnostic or therapeutic use, but also include a label designating that the kit and its use are limited to research purposes.

[0268] Examples

[0269] List of Abbreviations

[0270] Abbreviation Description

[0271] ACN Acetonitrile

[0272] br Broad peak

[0273] BSA Bovine serum albumin

[0274] CV Column volume

[0275] d Doublet

[0276] dd Double doublet

[0277] DCM Dichloromethane

[0278] DMF N,N-Dimethylformamide

[0279] DMSO Dimethyl sulfoxide

[0280] DPBS Dulbecco's phosphate buffered saline

[0281] EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0282] EtOAc Ethyl acetate

[0283] FA Formic acid

[0284] HOAc Acetic acid

[0285] HPLC High performance liquid chromatography

[0286] HRMS High resolution mass spectrometry

[0287] HTS High throughput screening

[0288] Homogeneous time-resolved fluorescence

[0289] Filter aid, calcined flux, treated with sodium carbonate

[0290] Hz Hertz

[0291] J Coupling constant

[0292] LC Liquid chromatography

[0293] m Multiplet

[0294] MS Mass spectrometry

[0295] NMR Nuclear magnetic resonance

[0296] OAc Acetate

[0297] Pd / C Palladium on carbon

[0298] pM Picomole

[0299] PK / PD Pharmacokinetics / Pharmacodynamics

[0300] KF Potassium fluoride

[0301] q Quartet

[0302] r.t. Room temperature

[0303] s Singlet

[0304] sat. Saturated

[0305] scFv Single-chain fragment variable

[0306] t Triplet

[0307] TFA Trifluoroacetic acid

[0308] TEA Triethylamine

[0309] TBME tert-butyl methyl ether

[0310] THF tetrahydrofuran

[0311] TIM Ticagrelor inactive metabolite

[0312] TLC thin layer chromatography

[0313] TR-FRET time-resolved fluorescence resonance energy transfer

[0314] Example 1: Preparation and Characterization of Haptens

[0315] This example describes methods for the synthesis, optimization, isolation, and characterization of several haptens for generating exemplary antibodies as described herein. The haptens include ticagrelor, ticagrelor metabolites (TAM and TIM), biotinylated ticagrelor, and biotinylated adenosine (see, for example, the chemical structures of the non-biotinylated haptens in Figure 2). Ticagrelor was synthesized as described in International Patent Application WO 2000 / 034283 (Guile et al., 2000), and TAM was synthesized as described in International Patent Application WO1999 / 005143 (Guile et al., 1999), each incorporated herein by reference in its entirety.

[0316] Normal-phase chromatography was performed using Biotage silica 40S, 40M, 12i or Merck silica 60 (0.063 - 0.200 mm). Flash chromatography was performed using a standard glass column or plastic column or on a Biotage Horizon system. Chemical shifts were given in ppm using a solvent as an internal standard. Heteroatoms such as N H and O H were detected only in the NMR, and protons on protons, so that proton may be missing.

[0317] Example 1.1: Biotinylated Ticagrelor

[0318] N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy)ethyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide (1.1)

[0319]

[0320] (i) Preparation of 2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethyl methanesulfonate (1.a)

[0321]

[0322] Methanesulfonyl chloride (0.086 mL, 1.10 mmol) was added dropwise at 0 °C to a solution of 2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethanol (see Springthorpe, B. et al., Bioorg. Med. Chem. Lett., 2007, 17, 6013 - 6018) (0.563 g, 1.0 mmol) and TEA (0.209 mL, 1.50 mmol) in DCM (5 mL). The mixture was stirred from 0 °C to 5 °C for 3 h. The reaction mixture was diluted with DCM (30 mL) and washed with water (5 mL). The mixture was dried by passing through a phase separator. The solvent was evaporated and co-evaporated with toluene to give the title compound (1.a) (714 mg, 111%) as a yellow viscous oil, which was used directly as a crude product without further purification.

[0323] 1 H NMR (400 MHz, CDCl3) δ 1.02 - 1.47 (m, 1H), 1.55 - 1.72 (d, 2H), 2.20 - 2.71 (m, 1H), 2.97 (dd, 2H), 3 - 3.19 (m), 3.57 - 3.68 (m, 1H), 3.69 - 3.79 (m, 1H), 4.02 - 4.24 (m, 1H), 4.78 (s, 3H), 5.13 (s, 3H), 5.57 (d, 2H), 6.50 (d, 2H), 7.03 (s, 1H), 7.07 (s, 1H).

[0324] 19 F NMR (376 MHz, CDCl3) δ -141.37 (J = 21.3), -138.10 (J = 21.3).

[0325] (ii) Preparation of 3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (1.b)

[0326]

[0327] A mixture of 2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethyl methanesulfonate (1.a) (0.641 g, 1 mmol) and sodium azide (0.070 mL, 2.00 mmol) in DMF (7 mL) was heated to 60 °C and maintained for 15.5 h under a nitrogen atmosphere. A white precipitate formed. Water (20 mL) was added, and the product was extracted twice with TBME (100 + 40 mL). The organic phase was dried over Na2SO4. The organic phase was filtered and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on a 2 × 8 cm silica gel column using heptane / EtOAc 1 / 1 as the eluent (TLC using heptane / EtOAc 1 / 1 (Rf product = 0.5)). The relevant fractions were collected and the solvent was evaporated to give the title compound (1.b) as a clear viscous oil (514 mg, 87%).

[0328] 1 1H NMR (400 MHz, CDCl3) δ 1.00 - 1.46 (m, 2H), 1.42 (q, 1H), 1.59 - 1.73 (m, 1H), 2.17 (dd, 2H), 2.68 (t, 2H), 2.96 (s, 3H), 3.19 - 3.33 (m, 4H), 3.52 - 3.63 (m, 1H), 3.72 (s, 3H), 4.03 (s, 1H), 4.79 (d, 1H), 5.13 (s, 1H), 5.54 (d, 2H), 6.43 (s, 1H), 6.96 (d, 2H).

[0329] (iii) Preparation of intermediate 3-((3aS,4R,6S,6aR)-6-(2-aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (1.c)

[0330]

[0331] 3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (1.b) (62.0 mg, 0.11 mmol) in EtOH (99.5%) (2 mL) was added to Pd / C (5% Pd, 50 wt% Pd / C, 22.46 mg, 5.28 μmol) and the mixture was hydrogenated at standard atmospheric pressure for 2 h. The reaction mixture was filtered through a filter, and the plug was further rinsed with EtOH (99.5%). The solvent was removed under reduced pressure, the residue was redissolved in DCM (2 × 2 mL), and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on a 2 × 8 cm silica gel column using DCM / NH3 (saturated) in MeOH 95 / 5 as the eluent. The relevant fractions were collected to give the title compound (1.c) (41 mg, 69%).

[0332] 1 H NMR (400 MHz; CDCl3): δ 0.98 (d, 3H), 1.28 (d, 3H), 1.54 (t, 3H), 1.62 1.81 (m, 1H), 2.15 (s, 3H), 2.48 (dt, 1H), 2.81 (d, 1H), 3.07 (d, 1H), 3.34 (dd, 1H), 3.53 (d, 1H), 3.99 (dd, 1H), 4.79 (dd, 1H), 5.12 (dd, 1H), 5.52 (d, 1H), 7.02 (t, 1H), 7.09 - 7.67 (m, 2H), 7.23 (s, 1H).

[0333] 19 F NMR (376 MHz, CDCl3) δ -141.43 (J = 21.3), -138.13 (J = 21.3).

[0334] (iV) Preparation of intermediate (1S,2S,3S,5R)-3-(2-aminoethoxy)-5-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2-diol (1.d)

[0335]

[0336] A mixture of pre-cooled TFA (8 mL, 103.84 mmol) and water (0.88 mL, 48.85 mmol) at ice / water bath temperature was added to a pre-cooled flask containing 3-((3aS,4R,6S,6aR)-6-(2-aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (1.c) (340 mg, 0.61 mmol). The reaction mixture was stirred at 0 °C - 5 °C for 1 h. The solvent was removed under reduced pressure and the residue was dissolved in DCM (100 mL) and washed with NaHCO3 (saturated, 10 mL). Brine (5 mL) was added to the aqueous phase and the mixture was extracted with EtOAc (30 mL). The combined organic phases were dried over Na2SO4. Filtered and then the solvent was evaporated to give the crude product as an off-white solid. The compound was purified by preparative HPLC on a XBridge C18 column (10 μm 250x50ID mm) using a gradient of 35% - 75% ACN in H2O / ACN / MeCN / AcOH 95 / 5 / 0.2 buffer at a flow rate of 100 mL / min over 20 minutes. The compounds were detected by UV at 298 nm. The peak fractions were evaporated to dryness under reduced pressure. The residue was dissolved in DCM and filtered through a phase separator. The solvent was removed under reduced pressure to give the title compound (1.d) (213 mg, 67.5%). LC / MS: m / z 522.3 [M+H] + 。

[0337] (v) Preparation of compound N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy)ethyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide. (1.1)

[0338] 2,5-Dioxopyrrolidin-1-yl 6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanoate (21.77 mg, 0.04 mmol) was added to a solution of (1S,2S,3S,5R)-3-(2-aminoethoxy)-5-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2-diol (20 mg, 0.04 mmol) in dry DMF (1.0 mL), and the mixture was placed under a nitrogen atmosphere and stirred at room temperature for 6 h. The solvent was removed at 40 °C under reduced pressure. The compound was purified by preparative HPLC on a Kromasil C18 column (10 μm 250x20ID mm) using a gradient of 20%-60% ACN in H2O / ACN / FA 95 / 5 / 0.2 buffer at a flow rate of 19 mL / min over 20 minutes. The compounds were detected by UV at 298 nm. The peak fractions were collected, concentrated, and lyophilized to give the title compound (1.1) (21.4 mg, 57.3%).

[0339] 1 1H NMR (600 MHz, DMSO): There are two rotamers (ratio 5:1). Signals from the major rotamer are δ 0.81 (t, 3H), 1.15 - 1.64 (m, 20H), 2.03 (ddd, 7H) ddd, 1H), 2.57 (d, 1H), 2.59 - 2.67 (m, 1H), 2.77 - 2.89 (m, 2H), 2.93 (dd, 1H), 2.96 - 3.01 (m, 4H), 3.05 - 3.12 (m, 1H), 3.15 (td, 1H), 3.18 - 3.25 (m, 2H), 3.39 - 3.46 (m, 1H)1H), 4.08 - 4.14 (m, 1H), 4.30 (dd, 1H), 4.54 (dd, 1H), 4.95 (q, 1H), 5.06 (s, 1H), 5.13, 1H), 6.42 (s, 1H), 7.07 (d, 1H), 7.31 (ddt, 2H), 7.71 (dt, 2H), 7.82 (t, 1H), 9.36 (d, 1H). Selected signals from the minor rotamer are δ 0.98 (CH3), 8.95 (ATNH).

[0340] HRMS calculated for [[C45H65F2N11O7S2]+ 974.4556; found: 974.4585 (M + H)+

[0341] Example 1.2: Biotinylated Adenosine

[0342] N-(((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide (1.2)

[0343]

[0344] (i) Preparation of N-(((3aR,4R,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide (1.e)

[0345]

[0346] At room temperature, DMF (2 mL) was added to 2,5-dioxopyrrolidin-1-yl 6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanoate (55.6 mg, 0.10 mmol) and 9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine (30 mg, 0.10 mmol) (see Austin, D.J. and Liu, F. Tetrahedron Lett. 2001, 3153 - 3154), and the reaction mixture was placed under a nitrogen atmosphere and stirred for 1 hour 45 minutes to give (1.e). The solvent was then removed under reduced pressure. Used as a crude product without further purification. LC / MS: m / z = 759 [M - H] - , 757 [M + H] + .

[0347] (ii) Preparation of the final compound N-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide (1.2)

[0348] A mixture of TFA (1.8 mL, 23.36 mmol) and water (0.2 mL, 11.10 mmol) was added to the crude N-(((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxin-4-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide (1.e) (76 mg, 0.1 mmol) and the reaction mixture was stirred at 0 °C for 1 hour 25 minutes. The solvent was removed under reduced pressure and the residue was dissolved in DMSO. The compound was purified by preparative HPLC on a XBridge C18 column (10 μm 250x19 ID mm) using a gradient of 5%-45% ACN in H2O / ACN / NH3 95 / 5 / 0.2 buffer at a flow rate of 19 mL / min over 20 minutes. The compounds were detected by UV at 259 nm. The peak fractions were concentrated and lyophilized to give the title compound (1.2) (50 mg, 69.6%), as a white fluffy solid.

[0349] 11H NMR (600 MHz, DMSO, 40 °C) δ 1.17 - 1.26 (m, 4H), 1.27 - 1.4 (m, 6H), 1.43 - 1.54 (m, 7H), 1.62 (ddt, 1H), 1.99 - 2.06 (m, 4H), 2.12 (t, 2H), 2.58 (d, 1H), 2.82 (dt, 1H), 2.96 - 3.05 (m, 4H), 3.05 - 3.14 (m, 1H), 3.36 (dt, 1H), 3.44 (dt, 1H), 3.96 (dd, 1H), 4.04 (dd, 1H), 4.11 - 4.15 (m, 1H), 4.29 - 4.33 (m, 1H), 4.67 (dd, 1H), 5.16 (d, 1H), 5.38 (d, 1H), 5.84 (d, 1H), 6.29 (s, 1H), 6.33 (d, 1H), 7.25 (s, 2H), 7.64 (dt, 2H), 8.11 (t, 1H), 8.16 (s, 1H), 8.31 (s, 1H). HRMS calculated for [[C32H50N10O7S]+ 719.3657; found: 719.3667 (M + H) +

[0350] Example 2: Isolation and Identification of Anti-Ticagrelor / TAM Antibodies

[0351] This example illustrates strategies and techniques for producing antibodies to ticagrelor and its metabolites, compounds that have structural similarity to ATP and contain an adenosine-like core (Springthorpe et al., 2007, Bioorg. Med. Chem. Lett. 17: 6013 - 6018). The chemical structures of ticagrelor / ticagrelor active metabolite (TAM) and ticagrelor inactive metabolite (TIM) are shown in Figure 2. As discussed above, the antibodies disclosed and generated herein can bind and neutralize ticagrelor and TAM, and can bind TIM, but do not bind or significantly inhibit other structurally related compounds such as adenosine. Although binding activity to TIM is an optional feature of the antibodies disclosed herein, it is expected that antibodies that display binding activity to TIM do not affect the required antibody / detoxifying agent dose, since TIM typically represents a small or insignificant fraction of the ticagrelor metabolites.

[0352] Target the common epitope, i.e., the unique R groups (difluorophenyl-cyclopropyl and thiopropyl substituents) of ticagrelor and TAM, to confer antibody binding specificity and selectivity to these compounds. The epitope of interest is enclosed by the dashed line in Figure 2. Direct the antibody epitope to the difluorophenyl-cyclopropyl and thiopropyl substituents using the haptens described in Example 1. As described in Example 1, the linker of the biotinylated haptens (biotinylated ticagrelor and biotinylated adenosine) is located on the diol group. This strategy allows the generation of antibodies with binding specificity for unmodified difluorophenyl-cyclopropyl and thiopropyl for biotinylated ticagrelor / TAM and also enables the screening and de-selection of an antibody library that binds adenosine.

[0353] Using known techniques, a human scFv phage display library is used to generate scFv antibodies, and specific scFvs are isolated from the library in a series of repeated selection cycles against biotinylated ticagrelor and a series of repeated de-selection cycles against biotinylated adenosine, substantially as described below: Lloyd et al., 2009, Protein Engineering Design and Selection (PEDS) 22:159-168, which is incorporated herein by reference. Many individual clones from the outputs of the 2nd and 3rd round of selections are chosen, and the scFvs are expressed in the bacterial periplasm and screened for specificity in three parallel biochemical assays. The screening tests are against: i) binding to biotinylated ticagrelor (assay 1), ii) binding to biotinylated adenosine (assay 2), and iii) binding to biotinylated ticagrelor in the presence of 50-fold excess of unmodified ticagrelor (assay 3) to determine specificity for ticagrelor rather than the biotinylated linker.

[0354] Assays 1, 2, and 3 are performed using the same general techniques and strategies. Briefly, HTS of crude periplasmic scFv samples for binding to biotinylated ticagrelor or biotinylated adenosine uses the assay technique. (homogeneous time-resolved fluorescence) based on the principle of TR-FRET (time-resolved fluorescence resonance energy transfer). Briefly, TR-FRET utilizes the transfer of energy from a donor fluorophore (in this case a cryptate of europium) to an acceptor fluorophore (in this case XL 665 ). If the donor and acceptor fluorophores are close enough (about <10m), then the excitation (337nm) of the europium cryptate donor results in energy transfer to XL 665The receptor then emits a fluorescent signal at 665 nm. This technique can be used to sensitively measure biomolecular interactions by attaching donor and acceptor fluorophores (directly or indirectly) to each binding partner in a specific interaction. The HTS format (assay 1) for the binding of scFv to biotinylated ticagrelor is as follows and relies on the presence of chemical tags on both biotinylated ticagrelor and his-tagged periplasmic scFv:

[0355] Europium chelate streptavidin: biotinylated ticagrelor: scFv-His: anti-His-XL 665

[0356] The assay was performed in a buffer containing DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4 M), and Tween 20 (Sigma P9416) (0.05%) with a 10 μl assay volume using a black, low-volume 384-well assay plate (Corning / Costar 3676). The assay was set up by adding 5 μl of biotinylated ticagrelor (60 nM, final concentration 30 nM), 2 μl of periplasmic scFv sample (final concentration 20%), and 3 μl of a solution containing europium cryptate-labeled streptavidin (CisBio 610SAKLB) (4.2 nM, final concentration 1.26 nM) and both XL 665 labeled anti-His antibody (CisBio 61HISXLB) (40 nM, final concentration 12 nM). A negative binding control well was set up that contained all of the above assay components except that 2 μl of assay buffer was added instead of the periplasmic scFv. The assay plate was incubated at room temperature for 4 hours and then read on an Envision plate reader using the standard HTRF read protocol where the samples were excited at 337 nm and the time-resolved fluorescence emission was measured at 620 nm and 665 nM.

[0357] The raw 665 nm and 620 nm counts were first converted to a 665 nm / 620 nm ratio and the results were subsequently expressed as ΔF(%) values. ΔF was calculated according to the following equation:

[0358] ΔF(%) = {((sample 665 / 620 ratio) - (negative 665 / 620 ratio)) / (negative 665 / 620 ratio)} × 100

[0359] (The negative ratio was obtained from the negative binding control well). An scFv providing a ΔF value greater than 100% was defined as a hit in this assay.

[0360] HTS of the crude periplasmic scFv samples was performed using the same protocol as above to bind biotinylated adenosine (final assay concentration 30 nM). The format of Assay 2 can be described as follows:

[0361] Europium chelate streptavidin: biotinylated adenosine: scFv-His: anti-His-XL 665

[0362] HTS for Assay 3 was performed using the same protocol as above, which identified crude periplasmic scFv samples that showed reduced binding to biotinylated ticagrelor in the presence of excess free unmodified ticagrelor. This protocol was modified from Assay 1 because Assay 3 was performed in the presence of a 50-fold molar excess of free unmodified ticagrelor (1500 nM).

[0363] Hits were defined as binding to biotinylated ticagrelor (ΔF > 100% in Assay 1), not binding to biotinylated adenosine (ΔF < 25% in Assay 2), and a > 50% reduction in binding to biotinylated ticagrelor in the presence of excess free unmodified ticagrelor. Exemplary correlations of data from Assays 1 and 3 are shown in Figure 3 . Many scFvs showed limited inhibition in the presence of excess free unmodified ticagrelor, which means they have binding interactions with some components and linkers of ticagrelor. A subset of scFvs was identified in which binding to biotinylated ticagrelor was inhibited (> 50%) in the presence of excess free unmodified ticagrelor. The scFvs were ranked based on the % inhibition of binding observed in Assay 3 versus Assay 1 (50% - 80%, 80% - 90%, > 90%), where scFvs giving > 90% inhibition (including TICA0072) were prioritized for further characterization. Sequence-unique scFv hits were converted to Fabs and expressed in CHO cells using standard techniques.

[0364] Fab Expression and Purification

[0365] Separate HC and LC expression plasmids were used for transient transfection, based on the expression vector described by Persic et al. in 1997. The vector was modified to contain the Epstein–Barr virus origin of replication (OriP). The Fab (HC) vector contained only constant region 1 (CH1) and the hinge region, with CH2 and CH3 removed. HC and LC DNA were added to 150 mM NaCl and 25-kDa linear PEI (Polysciences Europe, Germany, 23966) according to the manufacturer's recommendations. The DNA–PEI complex was then added to Chinese hamster ovary wild-type (CHO wt) cells (Daramola O et al., 2014) derived from the CHOK1 cell line (ECACC No: 85051005) suitable for suspension culture. After 7 days, the cells were harvested by centrifugation and the supernatant was filtered. The cell culture supernatant containing the Fab protein was directly loaded onto a chromatography column packed with 5 ml CaptureSelect IgG-CH1 (Life Technologies, Carlsbad, USA) at a flow rate of 5 ml / min using an Akta purifier (GE Healthcare). The column was equilibrated and washed with phosphate-buffered saline (PBS) pH 7.2, and eluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 3.5 (CaptureSelect IgG-CH1) according to the resin manufacturer's instructions. The eluted Fab was adjusted to pH 5.5 and filtered (0.22 μm Steriflip, Millipore EMD, USA) before analysis. Protein concentration was determined by absorbance at 280 nm using a DU520 UV / Vis spectrophotometer (Beckman Coulter, Brea, USA). Sample purity was determined using a TSKgel G3000SWx1 column (Tosoh Bioscience, Tokyo, Japan) running at 1.0 ml / min and an 1100 HPLC system (Agilent Technologies, Santa Clara, USA).

[0366] Example 3: Ticagrelor / TAM Fab

[0367] Query the structural database containing commercially available drugs ("DrugsDB", Oprea TI et al., 2011, Mol. Inv. 30(2-3), 100-111, incorporated herein by reference) to identify molecules with a certain structural similarity to ticagrelor. Once identified, these structurally similar molecules can be used to test the binding specificity of the Fab over adenosine and its phosphorylated forms (e.g., ADP and ATP). Based on the ticagrelor x-ray and NMR structures, query the database for molecules with 2D fingerprint similarity, 3D shape, and electrostatic similarity to ticagrelor. From this computer analysis, a set of 12 compounds was selected, which included six potential combination drugs. The structures of these compounds are shown in Figure 4 in.

[0368] Specificity was investigated in a competitive binding assay format, in which the ability of each test compound to competitively inhibit the interaction of biotinylated ticagrelor with the relevant Fab was tested. Using the competitive assay format, where the goal is to measure the competition of biotinylated ticagrelor binding to each His-Fab by a set of test compounds:

[0369] Europium chelate anti-His antibody: test His-Fab: biotinylated ticagrelor: XL 665 labeled streptavidin.

[0370] This basic assay format was used to evaluate the selectivity profile of the lead Fabs from both the lead isolation and lead optimization phases, and for the purposes of these studies, the His-Fab expression vector was used to produce the Fabs.

[0371] The assay was performed in a 20 μl assay volume in a black, low-volume 384-well assay plate (Corning / Costar 3676) in a buffer containing DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4 M), and BSA (PAAK05-013) (0.1%). The assay setup included the addition of 5 μl of biotinylated ticagrelor, 5 μl of titrations of each test selectivity compound, 5 μl of the relevant His-Fab, and 5 μl of the europium-containing cryptate-labeled anti-His antibody (CisBio 61HISKLB) (5.33 nM, final concentration 1.33 nM) and XL 665A combined solution of labeled streptavidin (CisBio 611SAXLB) (40 nM, final concentration 10 nM). A total binding control well was set up, which contained all of the above assay components except that 5 μl of assay buffer was added instead of the addition of the test selective compound. A negative binding control well was set up, which contained all of the assay components included in the total binding control well except that 5 μl of assay buffer was added instead of the addition of His-Fab. The test compound was serially titrated as 1 / 2 or 1 / 3, depending on the specific experiment, and the top final assay compound concentration was optimized based on compound specificity. The concentrations of biotinylated ticagrelor and His-Fab were optimized based on Fab specificity in separate experiments. For the four Fabs (TICA0010, TICA0049, TICA0053, and TICA0072) studied at the end of the lead isolation phase, the final assay reagent concentrations used are listed in Table 1 below:

[0372] Table 1

[0373] Antibody ID [Antibody] (nM) [Biotinylated Ticagrelor] (nM) TICA0039 16.0 139.9 TICA0049 16.0 37.9 TICA0053 16.0 70.7 TICA0072 8.0 17.6

[0374] For the two Fabs (TICA0162 and TICA0212) studied at the end of the lead optimization phase, the final assay reagent concentrations used in both cases were 5 nM biotinylated ticagrelor and 1 nM His Fab. Several test selective compounds to be used in these experiments were dissolved in 100% DMSO, and a carrier-related decrease in the assay signal could start to occur at concentrations above approximately 1% DMSO. To normalize the subsequent data to correct for any such carrier-related effects, parallel titrations of DMSO alone were included in most experiments, where the final assay DMSO concentration was opposite the test compound serial dilutions. At the end of the setup procedure, the assay was incubated at room temperature for 3 hours and then read on an Envision plate reader using a standard read protocol.

[0375] For subsequent data analysis, the raw 665 nm and 620 nM counts were first converted to a 665 nm / 620 nm ratio, and then the %ΔF value was calculated using the above ratio according to the equation described in Assay 1. The negative ratio used in the calculation of %ΔF was from the negative binding control well. Then the % specific binding value was calculated using the %ΔF value according to the following equation:

[0376] Specific binding (%) = { (Sample ΔF - Negative binding ΔF) / (Total binding ΔF - Negative binding ΔF)} × 100

[0377] For the standard calculation of % specific binding, the total binding ΔF was obtained from the total binding control well, which contained all of the above components of the assay but did not include any competing test compound.

[0378] In those experiments where DMSO normalization was applied, the % specific binding normalized to DMSO was essentially calculated according to the above equation, except that in this case the total binding ΔF was obtained from the wells containing all components from the total binding control wells, and DMSO at equivalent concentration was obtained from the relevant sample wells.

[0379] The results of this type of initial experiment are summarized in Table 2. In three of the four initial Fabs studied (TICA0010, TICA0049 and TICA0053), the compound cangrelor showed competitive inhibition of the binding of the Fab to biotinylated ticagrelor. In the case of TICA0049, pantoprazole and linezolid showed partial competitive inhibition. As shown in Table 2, TICA0072 Fab did not show inhibition against eleven of the twelve compounds, and showed weak partial inhibition against pantoprazole.

[0380] Inhibition by unmodified ticagrelor and TAM was observed for all four Fabs tested in this first series of experiments, with IC 50 values falling in the range of 0.1 μM to 0.5 μM. Two of the four Fabs (TICA0049 and TICA0072) showed competitive inhibition against TIM, however, the IC 50 value > 50 μM, indicating a greatly reduced affinity for TIM relative to unmodified ticagrelor and TAM. Based on the results in Table 2, TICA0072 was identified as having the most favorable selectivity profile. Based on the criterion that this Fab showed the least binding to cangrelor among the remaining three Fabs, TICA0049 was identified as a potential backup.

[0381] Table 2. Relative IC Figure 4 values of inhibition of the binding of biotinylated ticagrelor to each of the tested Fabs by each of the 12 test compounds ( 50 ), as well as unmodified ticagrelor, TAM and TIM.

[0382]

[0383]

[0384] NI indicates no inhibition.

[0385] In the second series of experiments, further selectivity data were generated for TICA0049 and TICA0072 Fabs, where Figure 4A subset of 4 out of the 12 compounds listed in 50 was retested together with ticagrelor, TAM, and TIM, as well as several adenosine-related compounds. Here, in a refinement of the earlier study in Table 2, experiments were designed to be able to standardize the percent specific binding values to correct for any non-specific carrier-related effects due to DMSO. Exemplary plot data from this second series of experiments, as well as the IC Figure 5 values tabulated in

[0386] Table 3. Figure 4 Examples of IC 50 results for a subset of four of the twelve compounds listed in

[0387] Compound TICA0049 TICA0072 Adenosine NI NI ADP NI NI 2MeS ADP NI NI ATP NI NI 2 MeS ATP NI NI Bucladesine 864.9 NI Linezolid 902.0 NI Cangrelor 188.4 NI Pantoprazole 243.5 1546.0 Ticagrelor 0.368 0.356 TAM 0.366 0.483 TIM 74.8 119.5

[0388] ticagrelor, TAM, TIM, and several adenosine family compounds in a competitive binding selectivity study (DMSO-normalized) 50 As in the earlier experiment, TICA0072 showed the most favorable selectivity profile, where only pantoprazole showed weak partial inhibition (IC 50 > 1500 μM) within the four compounds tested. In the case of TICA0049, significant inhibition of canagrelor and pantoprazole was observed, and weak inhibition of linezolid and bucladesine was observed. It should be noted that for some of the test compounds, the small differences in the absolute IC

[0389] values between the first and second series of experiments did not fundamentally alter the overall conclusions. Such differences may stem from the fact that DMSO normalization was incorporated into the second series of experiments, combined with the fact that in some cases, we were attempting to measure very weak inhibition. 50 For both TICA0049 and TICA0072, the measured IC 50 values for ticagrelor and TAM were in the range of 0.3 μM to 0.5 μM, and the IC

[0390] values for TIM were 2 log values higher at 74.8 μM and 119.5 μM, respectively. For both TICA0049 and TICA0072 Fab, trace inhibition of adenosine, ADP, ATP, and the methyl-thio derivatives of the latter two compounds was observed, however this was only detected at the highest compound concentrations tested and was not considered significant.

[0391] Conclusion: TICA0072 is considered to be the only Fab with specificity for ticagrelor and TAM.

[0392] The affinity of the anti - ticagrelor Fab generated above was determined using Biolayer interferometry on an Octet Red384. For the affinity measurement, the anti - ticagrelor Fab antibody was diluted to a concentration 2 - fold the final assay concentration in assay buffer (PBS, 0.05% Tween20, 0.02% BSA), such as 200 nM. A 10 - point 2 - fold serial dilution of ticagrelor was prepared in a Greiner polypropylene 96 - well plate. Then, equal volumes (e.g., 70 μL plus 70 μL) of the diluted antibody and free ticagrelor were transferred to a second Greiner polypropylene plate. The samples were mixed by pipetting, covered with a plate sealer, and allowed to equilibrate at room temperature for 3 - 5 days. After equilibration, 60 μL of the antibody / ticagrelor titration was transferred in duplicate to a 384 - well black, angled - bottom polypropylene plate. Biotinylated ticagrelor was diluted to 250 nM in assay buffer and added to the alternate wells of the first 2 columns of the 384 - well assay plate, and the remaining wells contained only assay buffer. The streptavidin biosensor was pre - soaked in assay buffer for at least 10 minutes. Then, the sample plate and the biosensor were loaded onto the stage of the Octet Red384.

[0393] All assays were performed at room temperature. After a 60 - second baseline equilibration in assay buffer, biotinylated ticagrelor was loaded onto the streptavidin biosensor for 300 seconds, and then assay buffer was added for 600 seconds to establish a new baseline. Then, the antibody / ticagrelor mixture was allowed to associate with the biotinylated ticagrelor sensor surface for 30 - 600 seconds, depending on the concentration of the antibody used. The resulting association phase data were analyzed using the Octet Red data analysis software. The signal was compared to the baseline, and the reference sensor signal (no - antibody control) for each sample was subtracted. Then, the data were output using KinExA n - curve analysis software for analysis. Using Constant Partner analysis, the equilibrium KD of the anti - ticagrelor Fab antibody was determined. The data showed that Fab TICA0072 and TICA0049 had affinities for ticagrelor of 7.4 nM and 11.6 nM, respectively (Table 4).

[0394] Table 4. Equilibrium affinity analysis of anti - ticagrelor Fab

[0395] Antibody ID Hapten Equilibrium KD 95% Confidence Interval TICA0072 Ticagrelor 7.43 nM 1.75 - 21.46 nM TICA0049 Ticagrelor 11.6 nM 1.7 - 66.5 nM

[0396] Example 5: Optimization of anti - ticagrelor / TAM antibody TICA0072

[0397] Optimize antibody TICA0072 using affinity-based phage selection. By using standard molecular weight biotechnology as described (Clackson and Lowman, 2004, Practical Approach Series 266), oligonucleotide-directed mutagenesis of variable heavy chain (VH) complementarity-determining region (CDR) 1, 2, or 3 or variable light chain (VL) CDR 1, 2, or 3 generates a large scFv library derived from a lead scFv sequence. The library is subjected to affinity-based phage display selection to select variants with higher affinity for ticagrelor and TAM. Briefly, essentially as previously described (Thompson et al., 1996, J Mol Biol. 256(1):77-88), scFv-phage particles are incubated in a solution of biotinylated ticagrelor at reducing concentrations (a typical example is 20 nM to 20 pM, over four rounds of selection). Periplasmic extracts containing crude scFv are prepared from a representative number of individual scFvs from the output of CDR-targeted selection and screened in an epitope competition assay format designed to screen for improved affinity relative to TICA072.

[0398] Briefly, to screen for scFv and Fab variants with improved affinity, by testing scFv variants, the epitope competition assay is completed based on competition of the interaction between the parental TICA0072 IgG and biotinylated ticagrelor. This assay is used as a primary single-point HTS to screen crude periplasmic extract scFv samples and as a multi-point secondary profiling assay to measure the improvement in IC 50 values of both purified scFv and Fab variants relative to the parental TICA0072. Although epitope competition assays (such as the assay described herein) are not typically used to determine absolute affinity values, this assay can be used as a basis for HTS-based affinity. In addition, the fold improvement in IC 50 of purified scFv / Fab variants (relative to the parental scFv / Fab) can provide a good indication of the overall fold increase in affinity and can represent an effective way to rank the affinity of scFv / Fab variants in a lead optimization campaign. The format of the parental TICA0072 IgG based on the epitope competition assay described herein is as follows:

[0399] Europium-labeled streptavidin: biotinylated ticagrelor: TICA0072 IgG: XL 665 XL-labeled anti-human Fc antibody

[0400] The assay was performed in a black, low-volume 384-well assay plate (Coming / Corning 3676) in a buffer containing DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4 M), and Tween 20 (Sigma P9416) (0.05%). For single-point testing of crude periplasmic scFv variants, a 10 μl assay volume was used, whereas a 20 μl assay volume was used when testing purified scFv and Fab in multi-point secondary IC 50 profiling assays.

[0401] For single-point HTS, the assay was set up by adding a combined solution of 3 μl of TICA0072IgG (53.3 nM, final concentration 16 nM), 2 μl of crude periplasmic extract scFv sample, 2.5 μl of biotinylated ticagrelor (8 nM, final concentration 2 nM), and 2.5 μl of a solution containing europium-labeled streptavidin (CisBio 610SAKLB) (3 nM, for a final assay concentration of 0.75 nM) and XL 665 -labeled anti-human Fc antibody (CisBio 61HFCXLB) (30 nM, final assay concentration 7.5 nM). The parental TICA0072 crude periplasmic scFv was used as a reference, and HTS was configured to identify variants that provided improved inhibition relative to the parental. The total binding control wells contained all assay components except that 2 μl of assay buffer was added instead of the scFv sample. The negative binding control wells contained all components of the total binding control wells except that 3 μl of assay buffer was added instead of TICA0072IgG.

[0402] For multi-point IC 50 testing of purified scFv / Fab variants, the assay was set up by adding a combined solution of 5 μl of TICA0072IgG (53.3 nM, final concentration 16 nM), 5 μl of a 1 / 3 titration of the purified test scFv or Fab variant, 5 μl of biotinylated ticagrelor (8 nM, final concentration 2 nM (scFv profiling); 4 nM, final assay concentration 1 nM (Fab profiling)), and 5 μl of a solution containing europium-labeled streptavidin (CisBio 610SAKLB) (3 nM, for a final assay concentration of 0.75 nM) and XL 665 -labeled anti-human Fc antibody (CisBio 61HFCXLB) (30 nM, final assay concentration 7.5 nM). Purified parental TICA0072 (scFv or Fab) was used as a reference in all experiments such that the IC 50The improvement can be expressed as a multiple improvement relative to the parental TICA0072. The summary binding control wells contain all assay components except that 5 μl of assay buffer is added instead of the purified scFv or Fab sample. The negative binding control wells contain all components of the total binding control wells except that 5 μl of assay buffer is added instead of TICA0072 IgG.

[0403] In the single-point HTS and multi-point IC assays 50 in the profiling version, the plate was incubated at room temperature for 3 hours and then read on an EnVision plate reader using the standard HTRF read protocol where the samples were excited at 337 nm and the time-resolved fluorescence emission was measured at 620 nm and 665 nM.

[0404] ΔF(%) and % specific binding were calculated using the raw 665 nm and 620 nm counts according to the equations described earlier in Assays 1 and 4 respectively. For the multi-point secondary profiling experiments, the IC 50 values were determined using Graphpad Prism software with a sigmoidal dose-response (variable slope) curve fit (4-parameter log equation).

[0405] DNA sequencing was performed on the hits identified in the screen, i.e., scFv variants that showed a significant improvement in inhibitory effect compared to the parental TICA0072 scFv, and the unique variants from the variable heavy chain CDR1, CDR2 or CDR3 and variable light chain library CDR1, CDR2 or CDR3 outputs were produced as purified scFv and retested in the same assay to determine the concentration-response IC 50 curve. The scFv variant showing the maximum improvement in the IC 50 value was then produced as a Fab and tested in the second-generation epitope competition assay described below.

[0406] Second-generation epitope competition assay for screening / grading the highest affinity Fabs

[0407] To effectively distinguish very high affinity purified Fabs at the end of the lead optimization campaign, a further epitope competition assay was performed. However, in this case, the assay was based on the competitive inhibition of the binding of biotinylated ticagrelor to a TICA0072 lineage IgG (TICA0159) optimized for intermediate affinity as opposed to the parental TICA0072 IgG. This assay was only used for the multi-point IC 50 profiling format (as opposed to the HTS format) and was performed essentially the same as the method given in Assay 5 (above) for the multi-point IC of purified Fab variants in the parental TICA0072 IgG based on the epitope competition assay. 50Spectrum analysis. The only difference was that TICA0159IgG was used instead of TICA0072IgG at the appropriate point in the assay setup, but the final assay concentration was the same, 16 nM. In all other respects, the assay was performed exactly as described above for the multi-point secondary spectrum analysis version of the purified Fab.

[0408] The second-generation assay used the partially optimized antibody TICA0159 in place of TICA0072 to enable more efficient discrimination and ranking of the highest-affinity Fabs than was possible in the epitope competition-based assay with TICA0072.

[0409] The most improved VH was identified as the CDR3 variant TICA0162. The most improved VL was identified as the CDR3 variant TICA0152. To generate further affinity improvements, the different CDRs from the improved antibodies were recombined into new Fabs using standard molecular biology techniques. From this recombination work, it was found that the combination of TICA0162 and TICA0152 produced a new Fab, TICA0212, with a further improved epitope competition profile. The plotting competition curves of TICA0072, TICA0152, TICA0162, and TICA0212 Fabs in the second-generation epitope competition assay are shown in Figure 6 where the measured IC 50 values are shown in Table 5. TICA0212 showed an approximately 2-log increase in IC 50 relative to the parental TICA0072 Fab.

[0410] Table 5: IC 50 data for the optimized ticagrelor Fabs listed in the second-generation epitope competition assay.

[0411] Fab <![CDATA[IC 50 (nM)]]> Fold Improvement TICA0072 1714.0 0 TICA0152 73.5 23.3 TICA0 162 16.3 105.2 TICA0212 12.0 142.8

[0412] Example 6: Affinity measurement of optimized ticagrelor / TAM Fab

[0413] The affinity of the anti - ticagrelor / TAM Fab generated in Example 5 was determined using a KinExA 3200. For KinExA affinity measurements, beads (600 mg dihydrozolone beads) were first prepared by reacting them with 1 mg streptavidin overnight in 50 mM NaHCO3. After blocking with 2 variations of Tris buffer (1 M Tris pH 8.7, 10 mg / mL BSA), the streptavidin - coated beads were resuspended in a total volume of 8 mL. The beads (1.33 mL, equivalent to 100 mg of the initial dry dihydrozolone beads) were washed thoroughly with PBS and then allowed to bind approximately 2.5 μg of biotin - ticagrelor in 1 mL of PBS for 10 minutes with occasional stirring. The resulting biotin - ticagrelor - coated beads were washed with PBS and then resuspended in 50 mL of PBS containing 0.1% BSA and 0.02% NaN3 and stored at room temperature until transferred to the KinExA bead vial.

[0414] Antibody / ticagrelor sample preparation was carried out essentially as described in the previous method. The anti - ticagrelor Fab antibody was diluted to a concentration 2 - fold the final assay concentration in the assay buffer (PBS, 0.05% Tween 20, 0.02% BSA, 0.02% NaN3), such as 200 nM. A 10 - point 2 - fold serial dilution of ticagrelor was prepared in a Falcon 50 mL polypropylene tube. Then, equal volumes, e.g., 5 mL plus 5 mL, of the diluted antibody and free ticagrelor were transferred to a second Falcon polypropylene tube. The samples were mixed by pipetting, covered with a plate sealer, and allowed to equilibrate at room temperature for 3 - 5 days. After equilibration, the sample tubes were transferred to the KinExA 3200 for analysis.

[0415] All assays were performed at room temperature. The antibody / ticagrelor mixture was sampled and mixed with the biotin - ticagrelor beads while washing away the unbound free ticagrelor. Then, the bound antibody was detected using a DyLight649 - labeled mouse anti - human heavy and light chain antibody. The sample volume (300 - 1300 μL) and injection time (90 - 120 s) varied with concentration, and there were 2 - 3 readings per sample. The data were analyzed using the KinExA n - curve analysis software. The equilibrium KD of the anti - ticagrelor Fab antibody was determined using a constant match analysis.

[0416] As in the previous example, Fab was equilibrated at room temperature in the presence of 20-fold excess concentrations of unmodified ticagrelor or TAM. Biotinylated ticagrelor was loaded onto the surface of streptavidin-coated beads. After equilibration, the remaining free antibody was allowed to bind to biotinylated ticagrelor. Bound antibody was then detected using DyLight649-labeled mouse anti-human heavy and light chain detection antibodies. Titrations of free ticagrelor or TAM were performed with at least three different fixed concentrations of antibody to generate independent titration profiles with at least a 10-fold apparent KD shift. Data were analyzed using KinExA Pro n-curve analysis software to determine the equilibrium KD of free ticagrelor or TAM. The affinities of Fab TICA0212 for unmodified ticagrelor and TAM were approximately 20 pM (Table 6). From the equilibrium data, TICA0212 demonstrated equivalent high affinities (approximately 20 pM) for binding to ticagrelor and TAM.

[0417] Table 6: Equilibrium affinity analysis of anti-ticagrelor / TAM Fab

[0418]

[0419] Changes in sequence residues from the parent TICA0072 are bolded, and Kabat numbering identifying certain residues in TICA0072 is provided.

[0420] Example 7: Specificity of anti-ticagrelor / TAM Fab TICA0162 and TICA0212

[0421] The specificities of Fab, TICA0162, and TICA0212 were tested as in Example 3 and normalized against DMSO. A summary table of all available selectivity data for TICA0162 and TICA0212 is included in Table 7. Additionally, the data plotted in the example are from experiments in which Figure 4 five of the twelve compounds listed in Figure 7 were tested together with ticagrelor, TAM, TIM, and

[0422] several adenosine family compounds shown in 50 .

[0423]

[0424]

[0425] NI indicates no inhibition.

[0426] As the data show, TICA0212 (MEDI2452) has substantially equal binding specificity for ticagrelor and TAM, and weak binding to TIM. In addition, MEDI2452 / TICA0212 did not show significant binding to any other structurally related drugs or adenosine-related compounds.

[0427] Example 8: Protein Crystallography

[0428] TICA0072 with a C-terminal his-tag was concentrated to 9 mg / ml in PBS. Complex formation was achieved by adding 1 mM ticagrelor dissolved in DMSO. The complex was incubated at room temperature for 2 hours, and then a crystallization trial was set up using the sitting-drop vapor diffusion method. Extensive screening was carried out using 3 commercial screens, and several hits were obtained. Optimal diffraction crystals were obtained from grid optimization of the hits from (Molecular Dimensions, UK) Crystals for structure determination were grown by mixing equal volumes of the TICA0072-ticagrelor complex and a stock solution of 12.8% PEG 3350, 12.8% PEG 1000, 12.8% MPD, 1.7% 1,6-hexanediol, 1.7% 1-butanol, 1.7% 1,2-propanediol, 1.7% 2-propanol, 1.7% 1,4-butanediol, 1.7% 1,3-propanediol, 25 mM imidazole, 25 mM sodium cacodylate, 25 mM MES, and 25 mM Bis-Tris at pH 6.5 at 20 °C. The crystals were flash-frozen in liquid nitrogen without adding any cryoprotectant.

[0429] TICA0212 / MEDI2452 in PBS at a concentration of approximately 15 mg / ml was mixed with ticagrelor to a concentration of 1 mM and incubated at room temperature for 2 hours before extensive screening. No spontaneous hits were obtained. Seeds from the TICA0072-ticagrelor crystals were prepared by crushing several crystals in 30 ul of the well solution and used for MMS (microbatch matrix screening) to extensive commercial screens. Several hits were obtained, but crystals for structure determination were grown at 20 °C under conditions of 20% glycerol, 20% PEG4000, 10% 2-propanol, 0.1 M NaCl, and 0.5 M sodium acetate at pH 4.6. Drops were set up with 0.2 μl of protein, 0.18 μl of well solution, and 0.02 μl of the seed stock solution. The crystals were flash-frozen in liquid nitrogen without adding any cryoprotectant.

[0430] Data were collected at beamline ID23-1 of the European Synchrotron Radiation Facility (Grenoble, France). The data were processed, scaled and further reduced using the AutoProc workflow [Vonrhein, C. et al., Acta Cryst., 2011; D67: 293-302], and the statistics are shown in Table 8. For TICA0072, initial phasing was carried out by molecular replacement using a high-resolution Fab structure (PDB id code 1aqk, [Faber, C. et al., Immunotechnology, 1998; 3: 253-70]) as the starting model. For TICA0212 / MEDI2452, the structure of TICA0072 was used as the starting model. Model building was carried out using Coot [Bricogne, G. et al., (2011), BUSTER version 2.11.4, Global Phasing Ltd, Cambridge, UK], and refinement was carried out using autobuster [Emsley, P. et al., Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, D60, 2126-2132]. The statistics for the final model are shown in Table 8.

[0431]

[0432]

[0433] The structure of the complex of TICA0072 with ticagrelor was determined to be Resolution ( Figure 10 ). The CDRs form a highly concave surface, and ticagrelor inserts deeply into the interface between the VH and VL domains. This type of binding is typically observed in small haptens. All CDRs except VL CDR2 contribute directly to ticagrelor binding, and most of VH CDR3 is disordered. The difluorophenyl group of ticagrelor lies in a cavity lined with hydrophobic residues that includes the vernier residues VH Trp47, VL Phe98 and VH CDR3 residue Leu100L. The key residue interacting with ticagrelor is VL Trp91, which is involved in π-stacking against the adenosine-like core and hydrogen bonding to one of the ribose hydroxyls of the cyclopentyl moiety. Additional interactions with the adenosine-like core are provided by VH CDR1 His35 and VH CDR3 Tyr99. The thiopropyl substituent superimposes on the backbone of the VH CDR2 loop. The hydroxyethyl substituent on the cyclopentyl moiety protrudes into the solvent and does not make any interactions with the Fab.

[0434] In the structure of the affinity-improved Fab, TICA0212 / MEDI2452, ticagrelor binding is similar to that of TICA0072, retaining all of the above interactions but with some important differences ( Figure 10 B). The combination of the VL CDR3 mutations Asp92Leu and Ser94Asp breaks the hydrogen bonds within the VL CDR3 loop to create a more "relaxed" structure. The new conformation is associated with a 15° tilt of the pyrimidine ring (with respect to the attachment of the cyclopropyl-difluorophenyl substituent). The new position of the pyrimidine ring brings it closer to VH CDR3 Tyr99 in TICA0212 / MEDI2452 compared to Fab 72 by about In addition, VL Ile93Tyr introduces a hydrogen bond donor that enables interaction with the VH CDR3 loop, further defining the binding site.

[0435] The crystal structure of TICA0212 / MEDI2452 shows ticagrelor bound in a deep crevice between the V H and V L interfaces. The design strategy of labeling ticagrelor with biotin via a triamide linker to the hydroxyethyl group was confirmed as the crystal structure demonstrated that the hydroxyethyl group does not participate in any interactions with TICA0212 / MEDI2452. This is further supported by the fact that the Fab also binds TAM lacking the hydroxyethyl group with the same affinity. TICA0212 / MEDI2452 shows weak binding to TIM, which lacks the cyclopropyl-difluorophenyl group. In the TICA0212 / MEDI2452-ticagrelor complex, the cyclopropyl-difluorophenyl group is buried at the bottom of a hydrophobic pocket and must play a key structural role in aligning the adenosine-like core of ticagrelor with the V L CDR3 residue Trp L91. Since the chemical starting point of ticagrelor is ATP and it retains the adenosine-like core, a key property of antidote specificity is demonstrating no binding of adenosine. The lead isolation strategy involved high-throughput and detailed specificity analysis, and no binding of adenosine was detected by competitive or direct binding assays. From structural analysis, it was expected that the purine ring and ribose group of adenosine could mimic the interactions of the adenosine-like core of ticagrelor. However, the lack of binding can be explained by the absence of two hydrophobic R groups (cyclopropyl-difluorophenyl and thiopropyl), which significantly reduces the relative shape complementarity and hydrophobicity of the binding interaction.

[0436] Analysis of the structures of the parent TICA0072 and TICA0212 / MEDI2452 shows the significance of some of the changes introduced during affinity maturation. V LMutations in CDR3 appear to have a particularly high impact, resulting in different loop conformations and additional hydrogen bonds in TICA0212 / MEDI2452 to define the binding cavity. In contrast, V H The contribution of mutations in CDR3 is less obvious structurally. These observations from the structure are partly due to antibodies containing only V L CDR3 (TICA0152) or V H Data from modified antibodies containing only modifications in CDR3 (TICA0162) confirm that these modifications result in 200-fold and 50-fold improvements over TICA0072, respectively. However, although V L CDR3 changes appear to have a greater effect, both sets of mutations result in significant improvements. It should be noted that crystal structures are static pictures of the complex and cannot capture any protein and ligand dynamics involved in binding.

[0437] Example 9: In the presence of ticagrelor or TAM, TICA0212 / MEDI2452 restores platelet aggregation in vitro in a concentration-dependent manner

[0438] The degree and potency of TICA0212 / MEDI2452 to reverse ticagrelor- or TAM-mediated inhibition of ADP-induced platelet aggregation were determined in human platelet-rich plasma (PRP) using a light transmission aggregometry assay.

[0439] For in vitro human PRP assays, blood was collected by venipuncture of the cephalic vein from fasting healthy volunteers. The first 2 mL of blood was discarded, and then aliquots were collected into tubes containing 0.109 M sodium citrate, 1+9 (citrate + blood) to a final concentration of 10.9 mM. The anticoagulated human blood was centrifuged at 240 × g for 15 minutes. The PRP was carefully removed and transferred to a clean vial. Platelet-poor plasma (PPP) was prepared by centrifuging the PRP at 2000 × g for 15 minutes. Light transmission aggregometry (LTA) was evaluated in the PRP using a platelet aggregometer (PAP-8E, Bio / Data Corporation, Horsham, PA, USA). Zero percent aggregation was defined as the light transmittance of the PRP, and 100% aggregation was defined as the light transmittance of the PPP.

[0440] PRP was pre-incubated with 1 μM ticagrelor or TAM for 1 hour and then co-incubated with different concentrations of TICA0212 or isotype control Fab for 30 minutes. Platelet aggregation was initiated by the addition of 20 μM ADP and continuously recorded for 6 minutes. Data on the degree of final aggregation (FA) at 6 minutes were analyzed.

[0441] Calculate the concentration of TICA0212 / MEDI2452 that gives half-maximal reversal (IC 50)。TICA0212 / MEDI2452 produced concentration-dependent reversal of 1 μM ticagrelor and inhibition of 1 μM TAM-mediated platelet aggregation induced by 20 μM ADP. The mean (n = 5) IC 50 values were calculated to be 0.64 and 0.78 μM, respectively (Figure 8). When evaluated under 1:1 conditions (1 μM TICA0212 / MEDI2452: 1 μM ticagrelor or TAM), the mean reversal degrees were 78% and 62%, respectively. Isotype control Fab did not cause significant reversal of ticagrelor- and TAM-induced ADP-induced platelet aggregation. For example, after 30 minutes of incubation, isotype control Fab resulted in -3% and 2% reversal of ticagrelor and TAM, respectively.

[0442] Thus, the data showed that TICA0212 / MEDI2452 could reverse the inhibition of ADP-induced platelet aggregation mediated by ticagrelor and TAM in a concentration-dependent manner in vitro. The maximum and near-complete reversal effects were obtained when evaluated in a 1:1 experimental setting, and this effect would be predictable when TICA0212 / MEDI2452 bound to ticagrelor or TAM in a 1:1 stoichiometry.

[0443] Example 10: TICA0212 / MEDI2452 effectively and rapidly restored platelet aggregation in vitro in the presence of ticagrelor or TAM

[0444] The onset time of TICA0212 / MEDI2452 to reverse ticagrelor or TAM was determined in human platelet-rich plasma (PRP) using a light transmission aggregometry as in Example 8. PRP was pre-incubated with 1 μM ticagrelor or TAM for 1 hour, then 1 μM TICA0212 / MEDI2452 was added and co-incubated for 5, 10, 15, 30, and 60 minutes, or isotype control Fab was added for 30 minutes. Platelet aggregation was initiated by adding 20 μM ADP, and the aggregation was continuously recorded for 6 minutes. Data on the degree of final aggregation (FA) at 6 minutes were analyzed.

[0445] TICA0212 / MEDI2452 produced a similar degree of reversal of ticagrelor-mediated inhibition, regardless of the co-incubation time, as the mean (n = 3) degree of reversal was 85%, 69%, 74%, 80% and 81% after 5, 10, 15, 30 and 60 minutes, respectively. Similarly, the mean (n = 3) degree of TAM reversal induced by TICA0212 / MEDI2452 was 53%, 56%, 58%, 69% and 74% after 5, 10, 15, 30 and 60 minutes, respectively. TICA0212 / MEDI2452 rapidly and effectively reversed ticagrelor and mediated inhibition of ADP-induced platelet aggregation. After co-incubation with isotype control Fab for 30 minutes, there was no reversal, mean (n = 3) -2%. From this data, it was found that when evaluated in a 1:1 experimental setting, TICA0212 / MEDI2452 was shown to rapidly and effectively reverse ticagrelor- and TAM-mediated inhibition of ADP-induced aggregation.

[0446] Example 11: TICA0212 / MEDI2452 effectively and rapidly restores platelet aggregation after in vivo administration to ticagrelor-treated mice

[0447] After intravenous (i.v.) administration of ticagrelor to mice, the onset rate and degree of reversal (impedance aggregation assay) of TICA0212 / MEDI2452-mediated inhibition of ticagrelor-mediated ADP-induced whole blood platelet aggregation were determined ex vivo. Mice were injected intravenously with ticagrelor, a bolus of 1200 μg / kg was given over 5 minutes, followed by a continuous infusion of 30 μg / kg / min for 15 minutes. After termination of the ticagrelor infusion, when the ticagrelor plasma exposure was measured to be an average of 1.4 μM, mice were given a bolus injection of 250 mg / kg of TICA0212 / MEDI2452 intravenously. Mice were sacrificed at 5, 30 and 60 minutes after administration of TICA0212 / MEDI2452, and blood samples were collected. In this study, the ADP-induced aggregation response was measured for 6 minutes, and the data were expressed as the mean area under the curve (AU*min) of aggregation units (AU) recorded over time.

[0448] In performing the impedance aggregation assay, mice were sacrificed and blood samples were collected into 7 μM hirudin. Blood (175 μL) was added to pre-warmed NaCl2 (37 °C, 175 μL) in a multi-plate microtest unit, mixed for 3 minutes, and then 12 μL of ADP was added to a final concentration of 6.5 μM. The disposable multi-plate microtest unit contained a stir bar and had two separate pairs of electrodes immersed in the blood sample.

[0449] When an agonist (ADP) is added and shear is induced by stirring, platelets will begin to adhere and aggregate on the electrode. This results in an increased impedance on the electrode, which is continuously recorded over time by a multi-layer impedance meter (DynaByte, Munich, Germany).

[0450] Treatment with ticagrelor induced almost complete inhibition of ADP-induced aggregation, as the mean (n = 4) aggregation responses decreased from 432 to 2, from 474 to 6, and from 494 to 14 AU*min at 5, 30, and 60 minutes after ticagrelor infusion and PBS bolus( Figure 9 ). TICA0212 / MEDI2452 mediated reversal of ticagrelor-mediated inhibition in vivo, as the mean (n = 4) aggregation responses increased from 2 to 147, from 6 to 448, and from 14 to 413 AU*min at 5, 30, and 60 minutes after ticagrelor infusion and TICA0212 bolus( Figure 9 ). No reversal was observed 30 minutes after administration of the isotype control, as the mean (n = 4) aggregation response remained unchanged at 6 to 4 AU*min.

[0451] These data indicate that when ticagrelor plasma at a concentration of 1.4 μM is administered intravenously (which provides complete inhibition of ADP-induced aggregation), TICA0212 / MEDI2452 can rapidly and effectively restore ADP-induced platelet aggregation.

[0452] Example 12: Mouse bleeding in ticagrelor-treated mice

[0453] Since one of the intended indications for TICA0212 / MEDI2452 is as an antidote for ticagrelor patients requiring urgent surgery, TICA0212 / MEDI2452 was evaluated in a mouse bleeding experiment, which was designed to mimic the clinical setting where complete reversal should be achieved prior to the start of surgery.

[0454] Mice were pretreated with continuous infusion of ticagrelor (300 μg / kg / min) or vehicle for 20 minutes. After stopping the infusion, at t = 0, a bolus dose of TICA0212 / MEDI2452 (600 mg / kg) or vehicle (histidine sucrose buffer) was administered over 45 seconds, and at t = 30 minutes, bleeding was induced by cutting 5 mm from the tip of the tail. The tip of the tail was rinsed with water (2 mL / min), and the blood and water mixture was collected in a chamber where a stirrer mixed the liquid to enhance hemolysis and produce a homogeneous solution. When collecting the terminal blood sample from the abdominal aorta for platelet aggregation, light transmission was recorded at 525 nm for 30 minutes. The light transmittance was converted to absorbance and used to calculate the blood loss and total bleeding time (BT, s) as the area under the absorbance curve (AUC, absorbance * s) by plotting the absorbance over time. All transmittances below 95% were defined as bleeding. Samples were collected for platelet aggregation and total and free plasma exposure at the end of ticagrelor infusion (t = 0) and at tail cutting (t = 30 minutes).

[0455] The study was approved by the Animal Research Ethics Committee of the University of Gothenburg, Sweden. Mice were anesthetized with isoflurane gas ( from Abbot Scandinavia AB, Sweden). A catheter was inserted into the left jugular vein for administration of vehicle or drug. Body temperature was maintained at 38 °C by external heating.

[0456] To translate the effect of TICA0212 / MEDI2452 on platelet aggregation into its potential effect on bleeding, a prophylactic mouse tail bleeding study was conducted. Ticagrelor was infused to achieve mean total ticagrelor and TAM plasma concentrations of 7.6 and 0.3 μM, respectively, to provide a significant drug-dependent bleeding window. TICA0212 / MEDI2452 was administered as a single bolus of 600 mg / kg over 30 seconds immediately after stopping ticagrelor infusion. After 30 minutes, ADP-induced aggregation was fully normalized and the mean free plasma concentration of ticagrelor decreased from 4.7 nM to below 0.03 nM (lower limit of quantification). Bleeding was initiated by tail cutting and monitored for 30 minutes. In vehicle-treated mice, the mean total ticagrelor and TAM plasma concentrations at tail cutting were 2.4 and 0.6 μM. The total blood loss and bleeding time were significantly (p < 0.05) enhanced by ticagrelor, by approximately 3.8-fold and 1.6-fold, respectively. Relative to ticagrelor alone, TICA0212 / MEDI2452 significantly (p < 0.05) reversed the blood loss and bleeding time back to levels not significantly different from those of mice not treated with ticagrelor ( Figure 12)。In this prophylactic setting, TICA0212 / MEDI2452 normalizes ticagrelor-dependent bleeding. The 30-minute onset time translated from the ex vivo model to this in vivo model demonstrates that TICA0212 / MEDI2452 can normalize blood loss and bleeding time to those of mice not treated with ticagrelor.

[0457] Example 13: Total plasma concentration of ticagrelor and TAM

[0458] Blood samples were collected in tubes with EDTA anticoagulant and centrifuged at 10,000×g for 5 minutes at room temperature to prepare plasma. The plasma concentrations of ticagrelor and TAM were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) with protein precipitation, as described in the published method [Sillén H., et al., Analytical Techniques in the Life Sciences, J Chromatogr B Analyt Technol Biomed Life Sci, 2010; 878: 2299-306], with the following deviations. Plasma, 50 μL, was subjected to protein precipitation with 180 μL of internal standard (D7-ZD6140) in acetonitrile. The liquid chromatography system and mass spectrometer were an Acquity ultra performance LC, coupled with a Waters Xevo TQ-S mass spectrometer. Chromatographic separation was performed on an Acquity BEH C18 column (2.1×50 mm, particle size 1.7 μm). Negative electrospray ionization was used. Eluent A was water with 10 mmol / L ammonium acetate, pH 5, and eluent B was acetonitrile with 10 mmol / L ammonium acetate. The injection volume was 1 to 5 μL, and the analytical gradient started at 4% B, increased to 95% in 1.5 minutes, was held for 2.3 minutes, then returned to the initial conditions in 2.4 minutes, followed by 0.3 minutes of re-equilibration. Quality control samples were not used in the analysis. The lower limit of quantification (LLOQ) was 0.005 μmol / L, and the calibration range was 0.005 to 15.0 μM.

[0459] The total plasma concentrations of ticagrelor and TAM in the presence of TICA0212 / MEDI2452 were determined by adding 1% formic acid (FA:sample, 1:5), followed by protein precipitation and LC-MS / MS as described above. Formic acid was added to the sample to facilitate the dissociation of ticagrelor and TICA0212 / MEDI2452.

[0460] Example 14: Free plasma concentration of ticagrelor

[0461] Optimize the method based on the previously published method [Sillén H. et al., Analytical Techniques in the Determination of Biological Life Sciences (J Chromatogr B Analyt Technol Biomed Life Sci), August 1, 2011; 879(23): 2315 - 22]. Soak the dialysis membrane (Spectrum Laboratories, Inc) that allows molecules with a mass lower than 6 to 8 kDa to pass through in ELGA water for 10 to 15 minutes, and place it between dialysis plates (prepared in the laboratory). Add plasma (130 μL) to one side of the dialysis plate, and add 130 μL of phosphate buffer (pH 7.0) to the other side. Seal the holes on both sides with lids, and place aluminum plates on top of each side of the plate to avoid leakage. Then place the plate vertically on an orbital shaker at 37 °C and 100 rpm / min for 24 hours. Terminate dialysis by transferring 50 μL of the retainate from the plasma side and 75 μL of the dialysate from the buffer side to a protein LoBind PCR clean 96 - deep well plate in acetonitrile, and each of the 96 - deep well plates contains 150 μL and 75 μL of internal standard (D7 - ZD6140). Mix the plate for 1 minute, and then centrifuge at 1500×g at 4 °C for 20 minutes. After centrifugation, transfer 50 μL of the supernatant from the precipitated retainate and dilute it with 50 μL of ELGA H2O before LC - MS / MS (Waters Acquity Ultra Performance LC combined with Xevo TQ - S mass spectrometer) analysis. No quality control samples were used in the analysis. The calibration range of the retainate is 0.4 to 1000 nmol / L, and the calibration range of the dialysate is 0.003 to 50 nmol / L. The LLOQ of ticagrelor in the dialysate is 0.03 nmol / L.

[0462] Table 9 below provides an overview of the exemplary antibodies described in the above examples and is used to illustrate certain embodiments of the technology disclosed herein.

[0463] Overview of antibody / scFv / Fab sequences

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473] References and Incorporation by Reference

[0474] 1. Van Giezen JJ, Nilsson L, Bemtsson P, et al. Ticagrelor binds independently of ADP to human P2Y(12) but antagonizes ADP-induced receptor signaling and platelet aggregation. J Thromb Haemost. 2009;7(9):1556-1565

[0475] 2. Wallentin L, Becker Rc, Budaj A, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009;361:1045-1057.

[0476] 3. Amsterdam EA, Wenger NK, Brindis RG, et al. 2014 ACC / AHA guideline for the management of patients with non-ST-elevation acute coronary syndromes: a report of the American College of Cardiology / American Heart Association task force on practice guidelines. Circulation. 2014;000:000-000

[0477] 4. Storey RF, Angiolillo DJ, Patil SB, et al. Inhibitory effects of ticagrelor on platelet function compared with clopidogrel in patients with acute coronary syndromes: the PLATO (platelet inhibition and patient outcomes) platelet study. J Am Coll Cardiol. 2010;56(18):1456-1462.

[0478] 5. Taylor G, Osinski D, Thevenin A, et al. Is platelet transfusion effective in restoring platelet reactivity in patients responsive to aspirin and / or clopidogrel before urgent surgery? J Trauma Acute Care Surg. 2013;74:1367-1369.

[0479] 6. Prüller F, Drexler C, Archan S, Macher S, Raggam RB, Mahla E. Restoration of low platelet reactivity by transfusion of stored platelets: an in vivo study in healthy volunteers. J Thromb Haemost 2011;9(8):1670-1673.

[0480] 7. Hansson EC, Shams Hakimi C, Astron Olsen K -Olsson K) et al., Effect of ex vivo platelet supplementation on platelet aggregation in blood samples from patients treated with acetylsalicylic acid, clopidogrel or ticagrelor, Br J Anaesth 2014; 112(3): 570-575.

[0481] 8. Thiele T, Sümnig A, Hron G. Platelet transfusion for reversal of dual antiplatelet therapy in patients requiring urgent surgery: a pilot study. J Thromb Haemost 2012;10:968-971.

[0482] 9. Pehrsson S, Hansson K, Nylander S. Ticagrelor-induced bleeding in mice can be regulated by FVIIa and FII reversal. J Am Coll Cardiol, 2013;61(10S):E212.

[0483] 10. Dolgin E antidote edges closer to reversing effects of new blood thinner, Nat Med 2013: 193, 251.

[0484] 11. Crowther MA, Ageno W, Garcia D, et al. Oral vitamin K versus placebo for correction of overanticoagulation in patients receiving warfarin: a randomized trial. Ann Intern Med 2009;150:293–300.

[0485] 12. Schiele F, van Ryn J, Canada K, et al. Specific antidote for dabigatran: functional and structural characteristics. Blood 2013;121:3554 - 3562.

[0486] 13. Lu G, DeGuzman FR, Hollenbach SJ, et al. Specific antidotes for reversing anticoagulation by direct and indirect inhibitors of factor Xa. Nat Med 2013;19:446 - 451.

[0487] 14. Storey RF, Husted S, Harrington RA, et al. AZD6140, a reversible oral P2Y12 receptor antagonist, inhibits platelet aggregation compared with clopidogrel in patients with acute coronary syndromes. J Am Coll Cardiol 2007;50:1852 - 1856.

[0488] 15. Husted SE, Storey RF, Bliden K, et al. Pharmacokinetics and pharmacodynamics of ticagrelor in patients with stable coronary artery disease: results from the ONSET - OFFSET and RESPOND studies. Clin Pharmacokinet. 2012;51(6):397 - 409.

[0489] 16. Teng R, Oliver S, Hayes MA, Butler K. Absorption, distribution, metabolism, and excretion of ticagrelor in healthy subjects. Drug Metab Dispos. 2010;38(9):1514 - 1521.

[0490] 17. Daramola O, Stevenson J, Dean G, et al. High - yield CHO transient system: co - expression of genes encoding EBNA - 1 and GS enhances transient protein expression. Biotechnol Prog 2014;30(1):132 - 141.

[0491] 18. Oprea TI, Nielsen SK, Ursu O, et al. A new platform for computer-aided drug repurposing by linking drugs, targets, and clinical outcomes into an integrated network. Mol Inform 2011;30:100-111.

[0492] 19. Springmorpe B, Bailey A, Barton P, et al. From ATP to AZD6140: discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis. Bioorg Med Chem Lett 2007;17:6013-6018.

[0493] 20. Fanning SW1, Horn JR. Anti-hapten camel antibodies reveal cryptic binding sites with significant energetic contributions from non-hypervariable loops. Protein Sci. 2011;20:1196-1207.

[0494] 21. Zhang K, Zhang J, Gao ZG, et al. Structure of the human P2Y12 receptor complexed with an anti-thrombotic drug. Nature 2014;509:115-118.

[0495] 22. Cattaneo M, Schulz R, Nylander S. Adenosine-mediated effects of ticagrelor: evidence and potential clinical relevance. J Am Coll Cardiol 2014;63:2503-2509.

[0496] 23. Sillén H, Cook M, Davis P. Determination of free ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS / MS. J Chromatogr B Analyt Technol Biomed Life Sci 2011;879(23):2315-2322.

[0497] All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0498] Although specific aspects of the present disclosure have been discussed, the above description is illustrative rather than restrictive. By reviewing this specification and the following schemes, multiple variations of the present disclosure will become apparent to those skilled in the art. The full scope of the present disclosure should be determined by reference to the schemes, together with the full range of their equivalents, and the specification, together with such variations. Sequence Listing <110> Immune Medical Ltd. <120> Antibodies to ticagrelor and methods of use thereof <130> PHAS-035 / 02US 309646-2264 <140> US 14 / 871,111 <141> 2015-09-30 <150> US 62 / 114,931 <151> 2015-02-11 <150> US 62 / 058,458 <151> 2014-10-01 <160> 80 <170> PatentIn version 3.5 <210> 1 <211> 363 <212> DNA <213> Homo sapiens <400> 1 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aacagagtac 300 gacctgcaac ggcctttcgg gtttgacttc tggggcaagg ggacaatggt caccgtctcg 360 agt 363 <210> 2 <211> 121 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Tyr Asp Leu Gln Arg Pro Phe Gly Phe Asp Phe Trp Gly 100 105 110 Lys Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Ser Tyr Ala Met Ser 1 5 <210> 4 <211> 17 <212> PRT <213> Homo sapiens <400> 4 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 5 <211> 12 <212> PRT <213> Homo sapiens <400> 5 Glu Tyr Asp Leu Gln Arg Pro Phe Gly Phe Asp Phe 1 5 10 <210> 6 <211> 333 <212> DNA <213> Homo sapiens <400> 6 tcctatgtgc tgactcagcc accctcagcg tctggggccc ccgggcagag ggctaccatc 60 tcctgctctg gaagcagctc caacatcgga agtaatcttg tgaactggta ccaacaattc 120 ccaggagagg cccccaagct cctcatcttt agtgacaatc aacgaccctc aggggtccct 180 gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tccgaggatg aggctgatta ttactgtgca acgtgggatg acagactgga tggttatgtg 300 gtattcggcg gagggaccaa gctgaccgtc cta 333 <210> 7 <211> 111 <212> PRT <213> Homo sapiens <400> 7 Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Ala Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Leu Val Asn Trp Tyr Gln Gln Phe Pro Gly Glu Ala Pro Lys Leu Leu 35 40 45 Ile Phe Ser Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg Leu 85 90 95 Asp Gly Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 8 <211> 13 <212> PRT <213> Homo sapiens <400> 8 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Leu Val Asn 1 5 10 <210> 9 <211> 7 <212> PRT <213> Homo sapiens <400> 9 Ser Asp Asn Gln Arg Pro Ser 1 5 <210> 10 <211> 12 <212> PRT <213> Homo sapiens <400> 10 Ala Thr Trp Asp Asp Arg Leu Asp Gly Tyr Val Val 1 5 10 <210> 11 <211> 345 <212> DNA <213> Homo sapiens <400> 11 caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg cttctggata caccttcatt acctatggta ttcactgggt gcgccaggcc 120 cccggacaag ggcttgagtg gatgggatgg atcgaccccg ggcatggtta cacaaaatat 180 tcacagaagt tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac 240 atggagatga gcagcctcag atctgaagac acggctgtgt attactgtgc gagagcggac 300 ctgggtgact actggggccg gggaaccctg gtcaccgtct cgagt 345 <210> 12 <211> 115 <212> PRT <213> Homo sapiens <400> 12 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Thr Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Gly His Gly Tyr Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Asp Leu Gly Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 13 <211> 5 <212> PRT <213> Homo sapiens <400> 13 Thr Tyr Gly Ile His 1 5 <210> 14 <211> 17 <212> PRT <213> Homo sapiens <400> 14 Trp Ile Asp Pro Gly His Gly Tyr Thr Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 15 <211> 6 <212> PRT <213> Homo sapiens <400> 15 Ala Asp Leu Gly Asp Tyr 1 5 <210> 16 <211> 330 <212> DNA <213> Homo sapiens <400> 16 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcagctc caacattggg aagaattatg tttcctggtt ccagcagctc 120 ccaggtacag cccccaaact cctcatttat gacaatcata agcgaccctc agggattcct 180 gaccgattct ctgcctccaa gtctggcacg tcagccaccc tggtcatctc cggtctccag 240 actggggacg aggcccatta ttactgcgga acatgggata ccagactgag tgctggggtg 300 ttcggcggag ggaccaaggt caccgtccta 330 <210> 17 <211> 110 <212> PRT <213> Homo sapiens <400> 17 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Lys Asn 20 25 30 Tyr Val Ser Trp Phe Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn His Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Thr Leu Val Ile Ser Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala His Tyr Tyr Cys Gly Thr Trp Asp Thr Arg Leu 85 90 95 Ser Ala Gly Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 18 <211> 13 <212> PRT <213> Homo sapiens <400> 18 Ser Gly Ser Ser Ser Asn Ile Gly Lys Asn Tyr Val Ser 1 5 10 <210> 19 <211> 7 <212> PRT <213> Homo sapiens <400> 19 Asp Asn His Lys Arg Pro Ser 1 5 <210> 20 <211> 11 <212> PRT <213> Homo sapiens <400> 20 Gly Thr Trp Asp Thr Arg Leu Ser Ala Gly Val 1 5 10 <210> 21 <211> 360 <212> DNA <213> Homo sapiens <400> 21 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgg ccatgatagt 300 agtggttact cctactcctt tgacttctgg gggcggggga ccacggtcac cgtctcgagt 360 <210> 22 <211> 120 <212> PRT <213> Homo sapiens <400> 22 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly His Asp Ser Ser Gly Tyr Ser Tyr Ser Phe Asp Phe Trp Gly Arg 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 23 <211> 5 <212> PRT <213> Homo sapiens <400> 23 Ser Tyr Ala Met Ser 1 5 <210> 24 <211> 17 <212> PRT <213> Homo sapiens <400> 24 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 25 <211> 11 <212> PRT <213> Homo sapiens <400> 25 Asp Ser Ser Gly Tyr Ser Tyr Ser Phe Asp Phe 1 5 10 <210> 26 <211> 330 <212> DNA <213> Homo sapiens <400> 26 cagtctgtgt tgacgcagcc gccctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gcaacatctc caacatcgga agtaacactg tcaactggta tcaacacgtc 120 ccaggagcgg cccccagact cctcatctat gttaatgatc agcggccgtc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tctgaagatg aggctgatta ttactgtgca acgtgggatg acaccctgaa tggaggggtc 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 27 <211> 110 <212> PRT <213> Homo sapiens <400> 27 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Asn Ile Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln His Val Pro Gly Ala Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Val Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Thr Leu 85 90 95 Asn Gly Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 28 <211> 13 <212> PRT <213> Homo sapiens <400> 28 Ser Gly Asn Ile Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 29 <211> 7 <212> PRT <213> Homo sapiens <400> 29 Val Asn Asp Gln Arg Pro Ser 1 5 <210> 30 <211> 11 <212> PRT <213> Homo sapiens <400> 30 Ala Thr Trp Asp Asp Thr Leu Asn Gly Gly Val 1 5 10 <210> 31 <211> 387 <212> DNA <213> Homo sapiens <400> 31 caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60 tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc 180 gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat 240 atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc 300 catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc 360 caaggaaccc tggtcaccgt ctcgagt 387 <210> 32 <211> 129 <212> PRT <213> Homo sapiens <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe 50 55 60 Gln Ala Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Leu Tyr Asp Phe Trp Ser Ala Ser His Pro Pro 100 105 110 Asn Asp Ala Leu Ala Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 33 <211> 5 <212> PRT <213> Homo sapiens <400> 33 Ser Tyr Ser Ile His 1 5 <210> 34 <211> 17 <212> PRT <213> Homo sapiens <400> 34 Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe Gln 1 5 10 15 Ala <210> 35 <211> 20 <212> PRT <213> Homo sapiens <400> 35 Gly Ser His Leu Tyr Asp Phe Trp Ser Ala Ser His Pro Pro Asn Asp 1 5 10 15 Ala Leu Ala Ile 20 <210> 36 <211> 330 <212> DNA <213> Homo sapiens <400> 36 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag 240 gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg 300 ttcggcggag ggaccaaggt caccgtccta 330 <210> 37 <211> 110 <212> PRT <213> Homo sapiens <400> 37 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ile Ser Leu 85 90 95 Ser Ala Gly Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 38 <211> 13 <212> PRT <213> Homo sapiens <400> 38 Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 39 <211> 7 <212> PRT <213> Homo sapiens <400> 39 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 40 <211> 11 <212> PRT <213> Homo sapiens <400> 40 Gly Thr Trp Asp Ile Ser Leu Ser Ala Gly Leu 1 5 10 <210> 41 <211> 387 <212> DNA <213> Homo sapiens <400> 41 caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60 tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc 180 gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat 240 atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggagc 300 ttcgactaca ggttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc 360 caaggaaccc tggtcaccgt ctcgagt 387 <210> 42 <211> 129 <212> PRT <213> Homo sapiens <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe 50 55 60 Gln Ala Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Phe Asp Tyr Arg Phe Trp Ser Ala Ser His Pro Pro 100 105 110 Asn Asp Ala Leu Ala Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 43 <211> 5 <212> PRT <213> Homo sapiens <400> 43 Ser Tyr Ser Ile His 1 5 <210> 44 <211> 17 <212> PRT <213> Homo sapiens <400> 44 Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe Gln 1 5 10 15 Ala <210> 45 <211> 20 <212> PRT <213> Homo sapiens <400> 45 Gly Ser Phe Asp Tyr Arg Phe Trp Ser Ala Ser His Pro Pro Asn Asp 1 5 10 15 Ala Leu Ala Ile 20 <210> 46 <211> 330 <212> DNA <213> Homo sapiens <400> 46 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag 240 gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg 300 ttcggcggag ggaccaaggt caccgtccta 330 <210> 47 <211> 110 <212> PRT <213> Homo sapiens <400> 47 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ile Ser Leu 85 90 95 Ser Ala Gly Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 48 <211> 13 <212> PRT <213> Homo sapiens <400> 48 Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 49 <211> 7 <212> PRT <213> Homo sapiens <400> 49 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 50 <211> 11 <212> PRT <213> Homo sapiens <400> 50 Gly Thr Trp Asp Ile Ser Leu Ser Ala Gly Leu 1 5 10 <210> 51 <211> 387 <212> DNA <213> Homo sapiens <400> 51 caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60 tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc 180 gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat 240 atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc 300 ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc 360 caaggaaccc tggtcaccgt ctcgagt 387 <210> 52 <211> 129 <212> PRT <213> Homo sapiens <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe 50 55 60 Gln Ala Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Phe Asp Tyr Tyr Phe Trp Ser Ala Ser His Pro Pro 100 105 110 Asn Asp Ala Leu Ala Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 53 <211> 5 <212> PRT <213> Homo sapiens <400> 53 Ser Tyr Ser Ile His 1 5 <210> 54 <211> 17 <212> PRT <213> Homo sapiens <400> 54 Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe Gln 1 5 10 15 Ala <210> 55 <211> 20 <212> PRT <213> Homo sapiens <400> 55 Gly Ser Phe Asp Tyr Tyr Phe Trp Ser Ala Ser His Pro Pro Asn Asp 1 5 10 15 Ala Leu Ala Ile 20 <210> 56 <211> 330 <212> DNA <213> Homo sapiens <400> 56 <h2 style=";text-align:left;direction:ltr">cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttcggcggag ggaccaaggt caccgtccta 330<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 57<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 110<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213>智人<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 57<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ile Ser Leu 85 90 95 Ser Ala Gly Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 58 <211> 13 <212> PRT <213> Homo sapiens <400> 58 Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 59 <211> 7 <212> PRT <213> Homo sapiens <400> 59 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 60 <211> 11 <212> PRT <213> Homo sapiens <400> 60 Gly Thr Trp Asp Ile Ser Leu Ser Ala Gly Leu 1 5 10 <210> 61 <211> 387 <212> DNA <213> Homo sapiens <400> 61 caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60 tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc 180 gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat 240 atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc 300 catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc 360 caaggaaccc tggtcaccgt ctcgagt 387 <210> 62 <211> 129 <212> PRT <213> Homo sapiens <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe 50 55 60 Gln Ala Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Leu Tyr Asp Phe Trp Ser Ala Ser His Pro Pro 100 105 110 Asn Asp Ala Leu Ala Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 63 <211> 5 <212> PRT <213> Homo sapiens <400> 63 Ser Tyr Ser Ile His 1 5 <210> 64 <211> 17 <212> PRT <213> Homo sapiens <400> 64 Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe Gln 1 5 10 15 Ala <210> 65 <211> 20 <212> PRT <213> Homo sapiens <400> 65 Gly Ser His Leu Tyr Asp Phe Trp Ser Ala Ser His Pro Pro Asn Asp 1 5 10 15 Ala Leu Ala Ile 20 <210> 66 <211> 330 <212> DNA <213> Homo sapiens <400> 66 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag 240 gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg 300 ttcggcggag ggaccaaggt caccgtccta 330 <210> 67 <211> 110 <212> PRT <213> Homo sapiens <400> 67 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Leu Tyr Asp Arg 85 90 95 Ala Val Gly Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 68 <211> 13 <212> PRT <213> Homo sapiens <400> 68 Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 69 <211> 7 <212> PRT <213> Homo sapiens <400> 69 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 70 <211> 11 <212> PRT <213> Homo sapiens <400> 70 Gly Thr Trp Leu Tyr Asp Arg Ala Val Gly Leu 1 5 10 <210> 71 <211> 387 <212> DNA <213> Homo sapiens <400> 71 caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc 120 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc 180 Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Pro Cys Phe Gly Thr Ile Ser gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat 240 Ala Arg Asp Phe Gln Ala Glu Ser Thr Ile Ser Ala Asp Lys Ser Thr Glu His Ser Tyr atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc 300 Met Glu Leu Ser Ala Glu Ile Glu Asp Thr Ala Val Tyr Ile Thr Cys Glu Arg Ala Ser ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc 360 Phe Asp Tyr Tyr Phe Gly Val Leu Ser Ile Pro Pro Asn Asp Ala Leu Ala Tyr Phe Gly caaggaaccc tggtcaccgt ctcgagt 387 Gln Gly Thr Leu Val Thr Val Ser Glu <210> 72 <211> 129 <212> PRT <213> Homo sapiens <400> 72 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asp Ser Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe 50 55 60 Gln Ala Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Phe Asp Tyr Tyr Phe Trp Ser Ala Ser His Pro Pro 100 105 110 Asn Asp Ala Leu Ala Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 73 <211> 5 <212> PRT <213> Homo sapiens <400> 73 Ser Tyr Ser Ile His 1 5 <210> 74 <211> 17 <212> PRT <213> Homo sapiens <400> 74 Gly Ile Ile Pro Ala Phe Gly Thr Leu Ser Ser Ala Gln Asp Phe Gln 1 5 10 15 Ala <210> 75 <211> 20 <212> PRT <213> Homo sapiens <400> 75 Gly Ser Phe Asp Tyr Tyr Phe Trp Ser Ala Ser His Pro Pro Asn Asp 1 5 10 15 Ala Leu Ala Ile 20 <210> 76 <211> 330 <212> DNA <213> Homo sapiens <400> 76 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag 240 gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg 300 ttcggcggag ggaccaaggt caccgtccta 330 <210> 77 <211> 110 <212> PRT <213> Homo sapiens <400> 77 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Leu Tyr Asp Arg 85 90 95 Ala Val Gly Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 78 <211> 13 <212> PRT <213> Homo sapiens <400> 78 Ser Gly Ser Asn Ser Asp Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 79 <211> 7 <212> PRT <213> Homo sapiens <400> 79 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 80 <211> 11 <212> PRT <213> Homo sapiens <400> 80 Gly Thr Trp Leu Tyr Asp Arg Ala Val Gly Leu 1 5 10

Claims

1. Use of an antibody or an antigen-binding fragment thereof that binds ticagrelor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol) in the preparation of a medicament for treating acute bleeding in a patient who has been administered ticagrelor, wherein the antibody or its antigen-binding fragment comprises the following complementarity-determining region (CDR) combination: VH CDR1 shown in SEQ ID NO:73, VH CDR2 shown in SEQ ID NO:74, VH CDR3 shown in SEQ ID NO:75, VL CDR1 shown in SEQ ID NO:78, VL CDR2 shown in SEQ ID NO:79, and VL CDR3 shown in SEQ ID NO:

80.

2. Use of an antibody or an antigen-binding fragment thereof that binds ticagrelor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol) in the preparation of a medicament for treating severe bleeding in a patient who has undergone or is undergoing surgery and has been administered ticagrelor, wherein the antibody or its antigen-binding fragment comprises the following complementarity-determining region (CDR) combination: VH CDR1 shown in SEQ ID NO:73, VH CDR2 shown in SEQ ID NO:74, VH CDR3 shown in SEQ ID NO:75, VL CDR1 shown in SEQ ID NO:78, VL CDR2 shown in SEQ ID NO:79, and VL CDR3 shown in SEQ ID NO:

80.

3. The use according to claim 1 or 2, wherein the VH has the amino acid sequence of SEQ ID NO:72, and the VL has the amino acid sequence of SEQ ID NO:

77.

4. The use according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is a monoclonal antibody.

5. The use according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is a humanized antibody or a human antibody.

6. The use according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is Fab or F(ab')2.

7. The use according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is an antigen-binding fragment.

8. Use according to claim 7, wherein the antibody or antigen-binding fragment thereof is a Fab.

9. Use according to claim 1 or 2, wherein the patient is in need of treatment or is about to need treatment for bleeding or potential bleeding associated with coronary artery bypass grafting, cardiothoracic surgery, mediastinal re-exploration, postoperative stroke, mechanical ventilation, prolonged stay in the intensive care unit or emergency non-cardiac surgery.

10. Use according to claim 1 or 2, wherein the patient is undergoing treatment, or in need of treatment or prophylaxis for an indication treatable with and / or amenable to ticagrelor.

11. Use according to claim 10, wherein ticagrelor is administered for the treatment of thrombotic or embolic stroke.

12. Use according to claim 10, wherein ticagrelor is administered for the treatment of myocardial infarction with or without thrombolysis.

13. Use according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is administered intravenously.

14. A vector comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the following complementarity determining region (CDR) combination: VH CDR1 shown in SEQ ID NO:73, VH CDR2 shown in SEQ ID NO:74, VH CDR3 shown in SEQ ID NO:75, VL CDR1 shown in SEQ ID NO:78, VL CDR2 shown in SEQ ID NO:79 and VL CDR3 shown in SEQ ID NO:80; wherein said antibody or antigen-binding fragment thereof binds to ticagrelor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol).

15. The vector according to claim 14, wherein the antibody or antigen-binding fragment thereof comprises a combination of heavy chain variable region (VH) and light chain variable region (VL) sequences SEQ ID NO:72 and SEQ ID NO:

77.

16. A host cell comprising the vector according to claim 14, wherein the host cell produces an antibody or antigen-binding fragment thereof that binds to ticagrelor.

17. A method for producing an antibody that binds to ticagrelor, comprising culturing the host cell according to claim 16 under conditions for expressing said antibody.

18. The method according to claim 17, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

19. The vector of claim 14 or 15, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

20. The host cell of claim 16, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

21. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a human antibody.

22. The vector of claim 14 or 15, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a human antibody.

23. The host cell of claim 16, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a human antibody.

24. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is a Fab or F(ab')2.

25. The vector of claim 14 or 15, wherein the antibody or antigen-binding fragment thereof is a Fab or F(ab')2.

26. The host cell of claim 16, wherein the antibody or antigen-binding fragment thereof is a Fab or F(ab')2.

27. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

28. The vector of claim 14 or 15, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

29. The host cell of claim 16, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

30. The method of claim 27, wherein the antigen-binding fragment is a Fab.

31. The vector of claim 28, wherein the antigen-binding fragment is a Fab.

32. The host cell of claim 29, wherein the antigen-binding fragment is a Fab.

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