WO2020003077A1

WO2020003077A1 - Anti-thrombin antibody molecules for use in patients at risk for gastrointestinal (gi) bleeding

Info

Publication number: WO2020003077A1
Application number: PCT/IB2019/055250
Authority: WO
Inventors: Madhu Chintala
Original assignee: Janssen Pharmaceutica Nv
Priority date: 2018-06-25
Filing date: 2019-06-21
Publication date: 2020-01-02

Abstract

The present invention relates to isolated anti-thrombin antibodies that recognize the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These anti-thrombin antibody molecules may be useful for treating or inhibiting thrombotic and/or embolic disorders and other conditions mediated by thrombin. In particular, the present invention relates to use of the anti-thrombin antibody molecules in patients at risk for gastrointestinal (GI) bleeding.

Description

ANTI-THROMBIN ANTIBODY MOLECULES FOR USE IN PATIENTS AT RISK FOR GASTROINTESTINAL (GI) BLEEDING

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[ 0001 ] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name

“PRD3473WOPCTl_SeqList.txt”, creation date of June 10, 2019 and having a size of 16 KB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

[ 0002 ] The present invention relates to isolated anti-thrombin antibody molecules that recognize the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These anti-thrombin antibody molecules may be useful in the treatment and prevention of thrombotic and/or embolic disorders and other conditions mediated by thrombin. In particular, the present invention relates to use of the anti thrombin antibody molecules in patients at risk for gastrointestinal (GI) bleeding.

BACKGROUND OF THE INVENTION

[ 0003 ] Blood coagulation is a key process in the prevention of bleeding from damaged blood vessels (haemostasis). However, a blood clot that obstructs the flow of blood through a vessel (thrombosis) or breaks away to lodge in a vessel elsewhere in the body (thromboembolism) can be a serious health threat.

[ 0004 ] A number of anticoagulant therapies are available to treat pathological blood coagulation. A common drawback of these therapies is an increased risk of bleeding (Mackman (2008) Nature 451 (7181): 914-918). Many anticoagulant agents have a narrow therapeutic window between the dose that prevents thrombosis and the dose that induces bleeding. This window is often further restricted by variations in the response in individual patients.

[ 0005 ] The present invention relates to the unexpected finding that anti-thrombin antibody molecules which recognise the exosite 1 epitope of thrombin selectively inhibit thrombin without promoting bleeding. These antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other thrombin-mediated conditions.

SUMMARY OF THE INVENTION

[ 0006 ] The general and preferred embodiments are defined, respectively, by the independent and dependent claims appended hereto, which for the sake of brevity are incorporated by reference herein. Other preferred embodiments, features, and advantages of the various aspects of the invention will become apparent from the detailed description below taken in conjunction with the appended drawing figures.

[ 0007 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15.

[ 0008 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: older aged patient, patient previously or currently treated with an anticoagulant, patient currently treated with an antiplatelet agent, patient with GI disorders, patient with a family history of GI disorders, patient with prior GI bleeding, a cancer patient, patient who had a prior stroke, patient with reduced estimated glomerular filtration rate (eGFR), patient with renal impairment, patient with severe liver disease, patient with anemia, and patient with lower bodyweight.

[ 0009 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising: administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

[ 0010 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor.

[ 0011 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

[ 0012 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder in, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC and the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.

[ 0013 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

[ 0014 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to the patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is the FXa inhibitor apixaban.

[ 0015 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent. [ 0016 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

[ 0017 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC), and wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: an older aged patient, a patient previously treated with an anticoagulant, a patient with a GI disorder, a patient with a family history of GI disorders, and a patient with prior GI bleeding.

[ 0018 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC), and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC. [ 0019 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder i, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC and the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.

[ 0020 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

[ 0021 ] In certain embodiments, the present invention provides a composition for use in reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti- thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is the FXa inhibitor apixaban.

[ 0022 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent.

[ 0023 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic, comprising: administering a composition comprising an anti -thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

[ 0024 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC), and wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: an older aged patient, a patient previously treated with an anticoagulant, a patient with a GI disorder, a patient with a family history of GI disorders, and a patient with prior GI bleeding.

[ 0025 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC), and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

[ 0026 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

[ 0027 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC and the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.

[ 0028 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

[ 0029 ] In certain embodiments, the present invention provides a method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering a composition comprising an anti thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC) selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor, and wherein the DOAC is the FXa inhibitor apixaban.

The invention also encompasses the following items:

1. An isolated antibody molecule that specifically binds to the exosite 1 region of thrombin. 2. The antibody molecule according to item 1 that inhibits thrombin activity.

3. The antibody molecule according to item 2 which causes minimal inhibition of haemostasis and/or bleeding.

4. The antibody molecule according to item 2 or item 3 which does not inhibit haemostasis and/or cause bleeding.

5. The antibody molecule according to any one of the preceding items wherein the antibody molecule comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more amino acid

substitutions, deletions or insertions.

6. The antibody molecule according to item 5 wherein the antibody molecule comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or more amino acid substitutions, deletions or insertions.

7. The antibody molecule according to item 5 or item 6 wherein the antibody molecule comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or more amino acid substitutions, deletions or insertions.

8. The antibody molecule according to any one of items 1 to 7 wherein the antibody molecule comprises a VH domain having the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions, deletions or insertions.

9. The antibody molecule according to any one of items 1 to 8 wherein antibody molecule comprises LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7, 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions.

10. The antibody molecule according to any one of items 1 to 9 wherein the antibody molecule comprises a VL domain having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with one or more amino acid substitutions, deletions or insertions. 11. The antibody molecule according to any one of items 1 to 10 comprising a VH domain comprising a HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising a LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9, respectively.

12. The antibody molecule according to item 11 comprising a VH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domain having the amino acid sequence of SEQ ID NO: 6.

13. The antibody molecule according to any one of items 1 to 12 comprising one or more substitutions, deletions or insertions which remove a glycosylation site.

14. The antibody molecule according to item 13 comprising a VL domain having the amino acid sequence of SEQ ID NO: 6 wherein the glycosylation site is mutated out by introducing a substitution at N28 or S30.

15. An antibody molecule which competes with an antibody molecule according to any one of items 5 to 12 for binding to exosite 1.

16. The antibody molecule according to any one of items 1 to 15 which is a whole antibody.

17. The antibody molecule according to item 16 which is an IgA or IgG.

18. The antibody molecule according to any one of items 1 to 15 which is an antibody fragment.

19. A pharmaceutical composition comprising an antibody molecule according to any one of items 1 to 18 and a pharmaceutically acceptable excipient.

20. An antibody molecule according to any one of items 1 to 18 for use in a method of treatment of the human or animal body.

21. An antibody molecule according to any one of items 1 to 18 for use in a method of treatment of a thrombin-mediated condition.

22. Use of an antibody molecule according to any one of items 1 to 18 in the manufacture of a medicament for use in treating a thrombin-mediated condition. 23. A method of treatment of a thrombin-mediated condition comprising

administering an antibody molecule according to any one of items 1 to 18 to an individual in need thereof.

24. An antibody molecule for use according to item 21, use according to item 22 or method according to item 23, wherein the thrombin-mediated condition is a thrombotic condition.

25. An antibody molecule for use, use or method according to item 24 wherein the thrombotic condition is thrombosis or embolism.

26. An antibody molecule for use according to item 21, use according to item 22 or method according to item 23 wherein the thrombin-mediated condition is inflammation, infection, tumour growth, tumour metastasis or dementia.

27. A method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin, the method comprising;

(i) providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3,

wherein the parent VH domain HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid sequence variant of the parent VH domain,

(ii) optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and

(iii) testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for the exosite 1 epitope of thrombin.

28. A method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin, which method comprises:

providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region; combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of said product repertoire to produce product VH domains;

optionally combining said product VH domains with one or more VL domains; selecting an antibody molecule that binds exosite 1 of thrombin, which antibody molecule comprises a product VH domain and optionally a VL domain; and

recovering said antibody molecule or nucleic acid encoding it.

29. An isolated antibody molecule that specifically binds to the exosite 1 region of thrombin comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 7 with one or more amino acid substitutions, deletions or insertions and wherein said LCDR1 has an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6.

30. The antibody molecule according to item 29 that inhibits thrombin activity.

31. The antibody molecule according to item 30 which causes minimal inhibition of haemostasis and/or bleeding.

32. The antibody molecule according to item 30 which does not inhibit haemostasis and/or cause bleeding.

33. The antibody molecule according to item 29 wherein the antibody molecule further comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more amino acid substitutions, deletions or insertions.

34. The antibody molecule according to item 29 wherein the antibody molecule further comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or more amino acid substitutions, deletions or insertions. 35. The antibody molecule according to item 29 wherein the antibody molecule further comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or more amino acid substitutions, deletions or insertions.

36. The antibody molecule according to item 29 wherein the antibody molecule further comprises a VH domain having the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions, deletions or insertions.

37. The antibody molecule according to item 29 wherein the antibody molecule further comprises an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9 respectively, or the sequences of SEQ ID NOs 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions.

38. The antibody molecule according to item 29 wherein the antibody molecule comprises the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A, and optionally one or more additional amino acid substitutions, deletions or insertions.

39. The antibody molecule according to item 29 comprising a VH domain comprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9, respectively.

40. The antibody molecule according to item 39 comprising a VH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domain having the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A.

41. The antibody molecule according to item 29 which is a whole antibody.

42. The antibody molecule according to item 41 which is an IgA or IgG.

43. The antibody molecule according to item 29 which is an antibody fragment.

44. A pharmaceutical composition comprising an antibody molecule according to item 29 and a pharmaceutically acceptable excipient.

45. A method of treatment of a thrombin-mediated condition comprising

administering an antibody molecule according to item 29 to an individual in need thereof. 46. The method of treatment of item 45 wherein the thrombin- mediated condition is a thrombotic condition.

47. The method of treatment of item 45 wherein the thrombotic-mediated condition is thrombosis or embolism.

48. The method of treatment of item 45 wherein the thrombotic-mediated condition is inflammation, infection, tumour growth, tumour metastasis or dementia.

49. A method of treatment of a thrombin-mediated condition comprising

administering a pharmaceutical composition according to item 44 to an individual in need thereof.

50. The method of treatment of item 49 wherein the thrombin- mediated condition is a thrombotic condition.

51. The method of treatment of item 49 wherein the thrombotic-mediated condition is thrombosis or embolism.

52. The method of treatment of item 49 wherein the thrombotic-mediated condition is inflammation, infection, tumour growth, tumour metastasis or dementia.

53. A method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin, the method comprising;

(ii) combining the VH domain thus provided with a VL domain having an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6 to provide one or more VH/VL combinations; and

(iii) testing the VH/VL combination or combinations to identify an antibody antigen binding domain for the exosite 1 epitope of thrombin. 54. A method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin, which method comprises:

providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH

domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region;

combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of said product repertoire to produce product VH domains;

combining said product VH domains with a VL domain having an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6; selecting an antibody molecule that binds exosite 1 of thrombin, which antibody molecule comprises a product VH domain and a VL domain having an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6; and

recovering said antibody molecule or nucleic acid encoding it.

[ 0030 ] The present invention further provides recombinant expression vectors engineered to express the antibodies of the present invention as described above, including for example those antibodies having the S30A substitution. Such expression vectors and their uses are well known to those of skill in the art. In an embodiment of the invention the expression vector may be one designed for expression of a protein of interest, such as an antibody molecule, or fragment thereof, in prokaryotic cells such as bacteria or eukaryotic cells such as mammalian cells. In a specific embodiment of the invention the expression vector may provide for protein expression in CHO cells. [ 0031 ] The invention encompasses the additional following items:

55. A recombinant expression vector encoding for an isolated antibody molecule that specifically binds to the exosite 1 region of thrombin.

56. The recombinant expression vector according to item 55 comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 7 with one or more amino acid substitutions, deletions or insertions and wherein said LCDR1 has an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6.

57. The recombinant expression vector according to item 56 wherein the antibody molecule further comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more amino acid substitutions, deletions or insertions.

58. The recombinant expression vector according to item 56 wherein the antibody molecule further comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or more amino acid substitutions, deletions or insertions.

59. The recombinant expression vector according to item 56 wherein the antibody molecule further comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or more amino acid substitutions, deletions or insertions.

60. The recombinant expression vector according to item 56 wherein the antibody molecule further comprises a VH domain having the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or more amino acid

substitutions, deletions or insertions.

61. The recombinant expression vector according to item 56 wherein the antibody molecule further comprises an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9 respectively, or the sequences of SEQ ID NOs 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions.

62. The recombinant expression vector according to item 56 wherein the antibody molecule comprises the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A, and optionally one or more additional amino acid substitutions, deletions or insertions. 63. The recombinant expression vector according to item 56 comprising a VH domain comprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7 and 8, respectively.

64. The recombinant expression vector according to item 63 comprising a VH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domain having the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A.

[ 0032 ] The present invention is also directed to recombinant cells engineered to express the antibodies of the present invention as described above, including for example those antibodies having the S30A substitution. In an embodiment of the invention, such recombinant cells may comprise recombinant expression vectors that provide for the expression of the antibody molecules of the present invention in such cells.

[ 0033 ] Recombinant cells may be prokaryotic cells such as bacteria, as well as eukaryotic cells such as mammalian cells. In a specific embodiment of the invention, the recombinant cells may be CHO cells such as those described in the working examples of the specification.

[ 0034 ] The invention encompasses the additional following items:

65. A recombinant cell expressing an antibody molecule that specifically binds to the exosite 1 region of thrombin.

66. The recombinant cell according to item 65 expressing an antibody comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 7 with one or more amino acid substitutions, deletions or insertions and wherein said LCDR1 has an amino acid substitution of alanine for serine at the residue corresponding to S30 of SEQ ID NO: 6.

67. The recombinant cell according to item 66 wherein the antibody molecule further comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more amino acid substitutions, deletions or insertions.

68. The recombinant cell according to item 66 wherein the antibody molecule further comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or more amino acid substitutions, deletions or insertions. 69. The recombinant cell according to item 66 wherein the antibody molecule further comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or more amino acid substitutions, deletions or insertions.

70. The recombinant cell according to item 66 wherein the antibody molecule further comprises a VH domain having the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions, deletions or insertions.

71. The recombinant cell according to item 66 wherein the antibody molecule further comprises an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9 respectively, or the sequences of SEQ ID NOs 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions.

72. The recombinant cell according to item 66 wherein the antibody molecule comprises the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A, and optionally one or more additional amino acid substitutions, deletions or insertions.

73. The recombinant cell according to item 66 comprising a VH domain comprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9, respectively.

74. The recombinant cell according to item 73 comprising a VH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domain having the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution of S30A.

75. A recombinant cell comprising the expression vector according to items 55-64.

[ 0035 ] An aspect of the invention provides an isolated antibody molecule that specifically binds to exosite 1 of thrombin.

[ 0036 ] Isolated anti-exosite 1 antibody molecules may inhibit thrombin in vivo without promoting or substantially promoting bleeding or haemorrhage, i.e. the antibody molecules do not inhibit or substantially inhibit normal physiological responses to vascular injury (i.e. haemostasis). For example, haemostasis may not be inhibited or may be minimally inhibited by the antibody molecules (i.e. inhibited to an insignificant extent which does not affect the well-being of patient or require further intervention). Bleeding may not be increased or may be minimally increased by the antibody molecules.

[ 0037 ] Exosite 1 (also known as 'anion binding exosite G and the 'fibrinogen recognition exosite') is a well-characterised secondary binding site on the thrombin molecule (see for example James A. Huntington, 2008, Structural Insights into the Life History of Thrombin, in Recent Advances in Thrombosis and Hemostasis 2008, editors; K. Tanaka and E.W. Davie, Springer Japan KK, Tokyo, pp. 80-106). Exosite 1 is formed in mature thrombin but is not formed in prothrombin (see for example Anderson et al (2000) JBC 2775 16428-16434).

[ 0038 ] Exosite 1 is involved in recognising thrombin substrates, such as fibrinogen, but is remote from the catalytic active site. Various thrombin binding factors bind to exosite 1, including the anticoagulant dodecapeptide hirugen (Naski et al 1990 JBC 265 13484-13489), factor V, factor VIII, thrombomodulin (cofactor for protein C and TAFI activation), fibrinogen, PAR1 and fibrin (the co-factor for factor XIII activation).

[ 0039 ] An anti -exosite 1 antibody may bind to exosite 1 of mature human thrombin. The sequence of human preprothrombin is set out in SEQ ID NO: 1. Human prothrombin has the sequence of residues 44 to 622 of SEQ ID NO: 1. Mature human thrombin has the sequence of residues 314-363 (light chain) and residues 364 to 622 (heavy chain).

[ 0040 ] In some embodiments, an anti-exosite 1 antibody may also bind to exosite 1 of mature thrombin from other species. Thrombin sequences from other species are known in the art and available on public databases such as Genbank. The corresponding residues in thrombin sequences from other species may be easily identified using sequence alignment tools.

[ 0041 ] The numbering scheme for thrombin residues set out herein is conventional in the art and is based on the chymotrypsin template (Bode W et al EMBO J. 1989 Nov;

8(11) :3467-75). Thrombin has insertion loops relative to chymotrypsin that are lettered sequentially using lower case letters.

[ 0042 ] Exosite 1 of mature human thrombin is underlined in SEQ ID NO: 1 and may include the following residues: M32, F34, R35, K36, S36a, P37, Q38, E39, L40, L65, R67, S72, R73, T74, R75, Y76, R77a, N78, EB O, K81, 182, S83, M84, K109, KllO, Kl49e, G150, Q 151, S153 and V154. In some embodiments, other thrombin residues which are located close to (i.e. within 0.5nm or within lnm) of any one of these residues may also be considered to be part of exosite 1.

[ 0043 ] An anti-exosite 1 antibody may bind to an epitope which comprises 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues of exosite 1. Preferably, an anti-exosite 1 antibody binds to an epitope which consists entirely of exosite 1 residues.

[ 0044 ] For example, an anti-exosite 1 antibody may bind to an epitope which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 residues selected from the group consisting of M32, F34, S36a, P37, Q38, E39, L40, L65, R67, R73, T74, R75,

Y76, R77a, 182 and Q151 of human thrombin or the equivalent residues in thrombin from another species. In some preferred embodiments, the epitope may comprise the thrombin residues Q38, R73, T74, Y76 and R77a and optionally one or more additional residues.

[ 0045 ] Anti-exosite 1 antibody molecules as described herein are specific for thrombin exosite 1 and bind to this epitope with high affinity relative to other epitopes, for example epitopes from mammalian proteins other than mature thrombin. For example, an anti exosite 1 antibody molecule may display a binding affinity for thrombin exosite 1 which is at least 500 fold, at least 1000 fold or at least 2000 fold greater than other epitopes.

[ 0046 ] Preferably, an antibody molecule as described herein which is specific for exosite 1 may bind to mature thrombin but display no binding or substantially no binding to prothrombin.

[ 0047 ] Without being bound by any theory, anti-exosite 1 antibodies may be unable to access thrombin within the core of a haemostatic clot, and are therefore unable to affect haemostasis by interrupting normal thrombin function at sites of vascular injury.

However, because the anti-exosite 1 antibodies still bind to thrombin on the surface of the clot and in the outer shell of the clot, thrombosis is prevented, i.e. non-haemostatic clot extension is prevented.

[ 0048 ] An anti-exosite 1 antibody molecule may have a dissociation constant for exosite 1 of less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than lOnM, or less than lnM. For example, an antibody molecule may have an affinity for exosite 1 of 0.1 to 50 nM, e.g. 0.5 to 10 nM. A suitable anti-exosite 1 antibody molecule may, for example, have an affinity for thrombin exosite 1 of about 1 nM. [ 0049 ] Binding kinetics and affinity (expressed as the equilibrium dissociation constant, Kd) of the anti-exosite 1 antibody molecules may be determined using standard techniques, such as surface plasmon resonance e.g. using BIAcore analysis.

[ 0050 ] An anti -exosite 1 antibody molecule as described herein may be an immunoglobulin or fragment thereof, and may be natural or partly or wholly synthetically produced, for example a recombinant molecule.

[ 0051 ] Anti-exosite 1 antibody molecules may include any polypeptide or protein comprising an antibody antigen-binding site, including Fab, Fab2, Fab3, diabodies, triabodies, tetrabodies, minibodies and single-domain antibodies, including nanobodies, as well as whole antibodies of any isotype or sub-class. Antibody molecules and methods for their construction and use are described, in for example Holbger & Hudson, Nature Biotechnology 23(9) : 1126-1136 (2005).

[ 0052 ] In some preferred embodiments, the anti-exosite 1 antibody molecule may be a whole antibody. For example, the anti-exosite 1 antibody molecule may be an IgG, IgA, IgE or IgM or any of the isotype sub-classes, particularly IgGl and IgG4.

[ 0053 ] The anti -exosite 1 antibody molecules may be monoclonal antibodies. In other preferred embodiments, the anti-exosite 1 antibody molecule may be an antibody fragment.

[ 0054 ] Anti-exosite 1 antibody molecules may be chimeric, humanised or human antibodies.

[ 0055 ] Anti-exosite 1 antibody molecules as described herein may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides and/or serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies may also be employed.

[ 0056 ] Anti -exosite 1 antibody molecules may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof.

[ 0057 ] Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et ak, 1992, Nature 357: 80-82). Isolation of antibodies and/or antibody- producing cells from an animal may be accompanied by a step of sacrificing the animal.

[ 0058 ] As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance, see W092/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

[ 0059 ] Other anti -exosite 1 antibody molecules may be identified by screening patient serum for antibodies which bind to exosite 1.

[ 0060 ] In some embodiments, anti-thrombin antibody molecules may be produced by any convenient means, for example a method described above, and then screened for differential binding to mature thrombin relative to thrombin with an exosite 1 mutation, gamma thrombin (exosite 1 defective due to autolysis at R75 and R77a) or prothrombin. Suitable screening methods are well-known in the art.

[ 0061 ] An antibody which displays increased binding to mature thrombin, relative to non-thrombin proteins, thrombin with an exosite 1 mutation, gamma-thrombin or prothrombin, for example an antibody which binds to mature thrombin but does not bind to thrombin with an exosite I mutation, gamma thrombin or prothrombin, may be identified as an anti -exosite 1 antibody molecule.

[ 0062 ] After production and/or isolation, the biological activity of an anti-exosite 1 antibody molecule may be tested. For example, the ability of the antibody molecule to inhibit thrombin substrate, cofactor or inhibitor binding and/or cleavage by thrombin may be determined and/or the ability of the antibody molecule to inhibit thrombosis without promoting bleeding may be determined.

[ 0063 ] Suitable antibody molecules may be tested for activity using a fibrinogen clotting or thrombin time assay. Suitable assays are well-known in the art.

[ 0064 ] The effect of an antibody molecule on coagulation and bleeding may be determined using standard techniques. For example, the effect of an antibody molecule on thrombosis may be determined in an animal model, such as a mouse model with ferric chloride induced clots in blood vessels. Effects on haemostasis may also be determined in an animal model, for example, by measuring tail bleed of a mouse.

[ 0065 ] Antibody molecules normally comprise an antigen binding domain comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), although antigen binding domains comprising only a heavy chain variable domain (VH) are also possible (e.g. camelid or shark antibodies).

[ 0066 ] Each of the VH and VL domains typically comprise three complementarity determining regions (CDRs) responsible for antigen binding, interspersed by framework regions.

[ 0067 ] In some embodiments, binding to exosite 1 may occur wholly or substantially through the VHCDR3 of the anti-exosite 1 antibody molecule.

[ 0068 ] For example, an anti-exosite 1 antibody molecule may comprise a VH domain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 5 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions. In some embodiments, the HCDR3 may comprise the amino acid residues at positions 4 to 9 of SEQ ID NO: 5 (SEFEPF), or more preferably the amino acid residues at positions 2, and 4 to 10 of SEQ ID NO: 5 (D and SEFEPFS) with substitutions, deletions or insertions at one or more other positions in SEQ ID NO :5. The HCDR3 may be the only region of the antibody molecule that interacts with a thrombin exosite 1 epitope or substantially the only region. The HCDR3 may therefore determine the specificity and/or affinity of the antibody molecule for the exosite 1 region of thrombin.

[ 0069 ] The VH domain of an anti-exosite 1 antibody molecule may additionally comprise an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the sequence of SEQ ID NO: 4 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. In some embodiments, the HCDR2 may comprise the amino acid residues at positions 3 to 7 of SEQ ID NO: 4 (DPQDG) or the amino acid residues at positions 2 and 4 to 7 of SEQ ID NO: 4 (L and PQDG) of SEQ ID NO: 4, with substitutions, deletions or insertions at one or more other positions in SEQ ID NO: 4. [ 0070 ] The VH domain of an anti -exosite 1 antibody molecule may further comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 3 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. In some embodiments, the HCDR1 may comprise amino acid residue T at position 5 of SEQ ID NO: 3 with substitutions, deletions or insertions at one or more other positions in SEQ ID NO: 3.

[ 0071 ] In some embodiments, an antibody molecule may comprise a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5 respectively. For example, an antibody molecule may comprise a VH domain having the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 2.

[ 0072 ] The anti-exosite 1 antibody molecule may further comprise a VL domain, for example a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7, 8 and 9 respectively with, independently, 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions. For example, an antibody molecule may comprise a VL domain having the sequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 6 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 6.

[ 0073 ] In some embodiments, the VL domain may comprise Tyr49.

[ 0074 ] The anti-exosite 1 antibody molecule may for example comprise one or more amino acid substitutions, deletions or insertions which improve one or more properties of the antibody, for example affinity, functional half-life, on and off rates.

[ 0075 ] The techniques that are required in order to introduce substitutions, deletions or insertions within amino acid sequences of CDRs, antibody VH or VL domains and antibodies are generally available in the art. Variant sequences may be made, with substitutions, deletions or insertions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind exosite 1 of thrombin and/or for any other desired property. [ 0076 ] In some embodiments, anti -exosite 1 antibody molecule may comprise a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4, and 5, respectively, and a VL domain comprising a LCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9, respectively.

[ 0077 ] For example, the VH and VL domains may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 respectively; or may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 comprising, independently 1 or more, for example 2,

3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions.

[ 0078 ] In some embodiments, an antibody may comprise one or more substitutions, deletions or insertions which remove a glycosylation site. For example, a glycosylation site in VL domain of SEQ ID NO 6 may be mutated out by introducing a substitution at either N28 or S30.

[ 0079 ] The anti-exosite 1 antibody molecule may be in any format, as described above. In some preferred embodiments, the anti -exosite 1 antibody molecule may be a whole antibody, for example an IgG, such as IgGl or IgG4, IgA, IgE or IgM.

[ 0080 ] An anti-exosite 1 antibody molecule of the invention may be one which competes for binding to exosite 1 with an antibody molecule described above, for example an antibody molecule which

(i) binds thrombin exosite 1 and

(ii) comprises a VH domain of SEQ ID NO: 2 and/or VL domain of SEQ ID NO: 6; an HCDR3 of SEQ ID NO: 5; an HCDR1, HCDR2, LCDR1, LCDR2, or LCDR3 of SEQ ID NOS: 3, 4, 7, 8 or 9 respectively; a VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 respectively; and/or a VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 and a VL domain comprising LCDR1, LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8 and 9 respectively.

[ 0081 ] Competition between antibody molecules may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules, to enable identification of antibody molecules which bind the same epitope or an overlapping epitope. Such methods are readily known to one of ordinary skill in the art. Thus, a further aspect of the present invention provides an antibody molecule comprising an antibody antigen-binding site that competes with an antibody molecule, for example an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the parent antibody described above for binding to exosite 1 of thrombin. A suitable antibody molecule may comprise an antibody antigen- binding site which competes with an antibody antigen-binding site for binding to exosite 1 wherein the antibody antigen- binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDR1, LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8, and 9 respectively, for example the VH and VL domains of SEQ ID NOS: 2 and 6.

[ 0082 ] An anti-exosite 1 antibody molecule as described herein may inhibit the binding of thrombin-binding factors, including factors which bind to exosite 1. For example, an antibody molecule may competitively or non-competitively inhibit the binding of one or more of fV, fVIII, thrombomodulin, fibrinogen or fibrin, PAR1 and/or hirugen and hirudin analogues to thrombin.

[ 0083 ] An anti-exosite 1 antibody molecule as described herein may inhibit one or more activities of thrombin. For example, an anti-exosite 1 antibody molecule may inhibit the hydrolytic cleavage of one or more thrombin substrates, such as fibrinogen, platelet receptor PAR-l and coagulation factor FVIII. For example, binding of the antibody molecule to thrombin may result in an at least 5-fold, at least lO-fold, or at least l5-fold decrease in the hydrolysis of fibrinogen, PAR-l, coagulation factor FVIII and/or another thrombin substrates, such as factor V, factor XIII in the presence of fibrin, and protein C and/or TAFI in the presence of thrombomodulin. In some embodiments, binding of thrombin by the anti-exosite 1 antibody molecule may result in no detectable cleavage of the thrombin substrate by thrombin.

[ 0084 ] Techniques for measuring thrombin activity, for example by measuring the hydrolysis of thrombin substrates in vitro are standard in the art and are described herein.

[ 0085 ] Anti-exosite 1 antibody molecules may be further modified by chemical modification, for example by PEGylation, or by incorporation in a liposome, to improve their pharmaceutical properties, for example by increasing in vivo half-life. [ 0086 ] The effect of an anti-exosite 1 antibody molecule on coagulation and bleeding may be determined using standard techniques. For example, the effect of an antibody on a thrombosis model may be determined. Suitable models include ferric chloride clot induction in blood vessels in a murine model, followed by a tail bleed to test normal haemostasis. Other suitable thrombosis models are well known in the art (see for example Westrick et al ATVB (2007) 27:2079-2093)

[ 0087 ] Anti-exosite 1 antibody molecules may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

[ 0088 ] A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the anti-exosite 1 antibody molecule, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the anti-exosite 1 antibody molecule.

[ 0089 ] In some embodiments, anti-exosite 1 antibody molecules may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antibody molecules may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[ 0090 ] Anti-exosite 1 antibody molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule. Thus, pharmaceutical compositions may comprise, in addition to the anti-exosite 1 antibody molecule, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the anti-exosite 1 antibody molecule. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

[ 0091 ] For parenteral, for example sub-cutaneous or intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the anti -exosite 1 antibody molecule may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer' s Injection.

[ 0092 ] Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as

octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEENTM' PLURONICSTM or polyethylene glycol (PEG).

[ 0093 ] A pharmaceutical composition comprising an anti-exosite 1 antibody molecule may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[ 0094 ] An anti-exosite 1 antibody molecule as described herein may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset). The method of treatment may comprise administering an anti -exosite 1 antibody molecule to an individual in need thereof.

[ 0095 ] Administration is normally in a "therapeutically effective amount" or "effective amount”, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of

administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922) Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount, effective amount, or suitable dose of an antibody molecule may be determined by comparing it’s in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment) and the nature of any detectable label or other molecule attached to the antibody.

[ 0096 ] A typical antibody dose will be in the range 100 pg to 1 g for systemic applications, and 1 pg to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the antibody will be a whole antibody, e.g. the IgGl or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmocokinetic and pharmacodynamic properties of the antibody composition, the route of administration and the nature of the condition being treated.

[ 0097 ] Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g. about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks.

Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure.

Suitable formulations and routes of administration are described above. [ 0098 ] In some embodiments, anti-exosite 1 antibody molecules as described herein may be administered as sub-cutaneous injections. Sub-cutaneous injections may be administered using an auto-injector, for example for long term prophylaxis/treatment.

[ 0099 ] In some preferred embodiments, the therapeutic effect of the anti-exosite 1 antibody molecule may persist for several half- lives, depending on the dose. For example, the therapeutic effect of a single dose of anti-exosite 1 antibody molecule may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

[ 00100 ] Anti -exosite 1 antibody molecules described herein inhibit thrombin and may be useful in the treatment of thrombin- mediated conditions.

[ 00101 ] Haemostasis is the normal coagulation response i.e. the prevention of bleeding or haemorrhage, for example from a damaged blood vessel. Haemostasis arrests bleeding and haemorrhage from blood vessels in the body.

[ 00102 ] Anti -exosite 1 antibody molecules may have no effect or substantially no effect on haemostasis i.e. they do not promote bleeding or haemorrhage.

[ 00103 ] Aspects of the invention provide; an anti -exosite 1 antibody molecule as described herein for use in a method of treatment of the human or animal body; an anti exosite 1 antibody molecule as described herein for use in a method of treatment of a thrombin-mediated disorder; the use of an anti-exosite 1 antibody molecule as described herein in the manufacture of a medicament for the treatment of a thrombin-mediated condition; and a method of treatment of a thrombin-mediated condition comprising administering an anti-exosite 1 antibody molecule as described herein to an individual in need thereof.

[ 00104 ] Inhibition of thrombin by anti -exosite 1 antibodies as described herein may be of clinical benefit in the treatment of any thrombin-mediated condition. A thrombin- mediated condition may include disorders associated with the formation or activity of thrombin.

[ 00105 ] Thrombin plays a key role in haemostasis, coagulation and thrombosis. Thrombin-mediated conditions include thrombotic conditions, such as thrombosis, embolism, and stroke. [ 00106 ] Thrombosis is coagulation which is in excess of what is required for haemostasis (i.e. excessive coagulation), or which is not required for haemostasis (i.e. extra-haemostatic or non-haemostatic coagulation).

[ 00107 ] Thrombosis is blood clotting within the blood vessel lumen. It is characterised by the formation of a clot (thrombus) that is in excess of requirement or not required for haemostasis. The clot may impede blood flow through the blood vessel leading to medical complications. A clot may break away from its site of formation, leading to embolism elsewhere in the circulatory system. In the arterial system, thrombosis is typically the result of atherosclerotic plaque rupture.

[ 00108 ] In some embodiments, thrombosis may occur after an initial physiological haemostatic response, for example damage to endothelial cells in a blood vessel. In other embodiments, thrombosis may occur in the absence of any physiological haemostatic response.

[ 00109 ] Thrombosis may occur in individuals with an intrinsic tendency to thrombosis (i.e. thrombophilia) or in 'normal' individuals with no intrinsic tendency to thrombosis, for example in response to an extrinsic stimulus.

[ 00110 ] Thrombosis and embolism may occur in any vein, artery or other blood vessel within the circulatory system and may include microvascular thrombosis.

[ 00111 ] Thrombosis and embolism may be associated with surgery (either during surgery or afterwards) or the insertion of foreign objects, such as coronary stents, into a patient.

[ 00112 ] For example, anti-exosite 1 antibodies as described herein may be useful in the surgical and other procedures in which blood is exposed to artificial surfaces, such as open heart surgery and dialysis.

[ 00113 ] Thrombotic conditions may include thrombophilia, thrombotic stroke and coronary artery occlusion.

[ 00114 ] Patients suitable for treatment as described herein include patients with conditions in which thrombosis is a symptom or a side-effect of treatment or which confer an increased risk of thrombosis or patients who are predisposed to or at increased risk of thrombosis, relative to the general population. For example, an anti-exosite 1 antibody molecule as described herein may also be useful in the treatment or prevention of venous thrombosis in cancer patients, and in the treatment or prevention of hospital -acquired thrombosis, which is responsible for 50% of cases of venous thromboembolism.

[ 00115 ] Anti-exosite 1 antibody molecules as described herein may exert a therapeutic or other beneficial effect on thrombin- mediated conditions, such as thrombotic conditions, without substantially inhibiting or impeding haemostasis. For example, the risk of haemorrhage in patients treated with anti -exosite 1 antibody molecules may not be increased or substantially increased relative to untreated individuals.

[ 00116 ] Individuals treated with conventional anticoagulants, such as natural and synthetic heparins, warfarin, direct serine protease inhibitors (e.g. argatroban, dabigatran, apixaban, and rivaroxaban), hirudin and its derivatives (e.g. lepirudin and bivalirudin), and anti-platelet drugs (e.g. clopidogrel, ticlopidine and abciximab) cause bleeding. The risk of bleeding in patients treated with anti-exosite 1 antibody molecules as described herein may be reduced relative to individuals treated with conventional anticoagulants.

[ 00117 ] Thrombin-mediated conditions include non-thrombotic conditions associated with thrombin activity, including inflammation, infection, tumour growth and metastasis, organ rejection and dementia (vascular and non-vascular, e.g. Alzheimer 's disease)

(Licari et al J Vet Emerg Crit Care (San Antonio). 2009 Feb;l9(l) : 11-22; Tsopanoglou et al Eur Cytokine Netw. 2009 Dec l;20(4) : 171-9).

[ 00118 ] Anti -exosite 1 antibody molecules as described herein may also be useful in in vitro testing, for example in the analysis and characterisation of coagulation, for example in a sample obtained from a patient.

[ 00119 ] Anti -exosite 1 antibody molecules may be useful in the measurement of thrombin generation. Assays of thrombin generation are technically problematic because the conversion of fibrinogen to fibrin causes turbidity, which precludes the use of a simple chromogenic end-point.

[ 00120 ] The addition of an anti-exosite 1 antibody molecule as described herein to a sample of blood prevents or inhibits fibrin formation and hence turbidity and permits thrombin generation to be measured using a chromogenic substrate, without the need for a defibrination step.

[ 00121 ] For example, a method of measuring thrombin generation may comprise contacting a blood sample with a chromogenic thrombin substrate in the presence of an anti-exosite 1 antibody molecule as described herein and measuring the chromogenic signal from the substrate; wherein the chromogenic signal is indicative of thrombin generation in the sample.

[ 00122 ] The chromogenic signal may be measured directly without defibrination of the sample.

[ 00123 ] Suitable substrates are well known in the art and include S2238 (H-D-Phe- Pip-Arg-pNa), -Ala-Gly-Arg-p-nitroanilide diacetate (Prasa, D. et al. (1997) Thromb. Ha emost. 78, 1215; Sigma Aldrich Inc) and Tos-Gly-Pro-Arg-pNa.AcOH (Biophen CS- 01 (81); Aniara Inc OH USA).

[ 00124 ] Anti -exosite 1 antibody molecules may also be useful in inhibiting or preventing the coagulation of blood as described above in extracorporeal circulations, such as haemodialysis and extracorporeal membrane oxygenation.

[ 00125 ] For example, a method of inhibiting or preventing blood coagulation in vitro or ex vivo may comprise introducing an anti-exosite 1 antibody molecule as described herein to a blood sample. The blood sample may be introduced into an extracorporeal circulation system before, simultaneous with or after the introduction of the anti-exosite 1 antibody and optionally subjected to treatment such as haemodialysis or oxygenation. In some embodiments, the treated blood may be subsequently administered to an individual. Other embodiments provide an anti-exosite 1 antibody molecule as described herein for use in a method of inhibiting or preventing blood coagulation in a blood sample ex vivo and the use of an anti-exosite 1 antibody molecule as described herein in the manufacture of a medicament for use in a method of inhibiting or preventing blood coagulation in a blood sample ex vivo.

[ 00126 ] Other aspects of the invention relate to the production of antibody molecules which bind to the exosite 1 epitope of thrombin and may be useful, for example in the treatment of pathological blood coagulation or thrombosis.

[ 00127 ] A method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin, may comprise;

providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid sequence variant of the parent VH domain, and;

optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and

testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for the exosite 1 epitope of thrombin.

[ 00128 ] A VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDR3 sequence of SEQ ID NO: 5 or a variant with the addition, deletion, substitution or insertion of one, two, three or more amino acids.

[ 00129 ] The VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDR1 and HCDR2 sequences of SEQ ID NOS: 3 and 4 respectively, or variants of these sequences with the addition, deletion, substitution or insertion of one, two, three or more amino acids.

[ 00130 ] A method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin may comprise:

providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region;

recovering said antibody molecule or nucleic acid encoding it.

[ 00131 ] Suitable techniques for the maturation and optimisation of antibody molecules are well-known in the art.

[ 00132 ] Antibody antigen-binding domains and antibody molecules for the exosite 1 epitope of thrombin may be tested as described above. For example, the ability to bind to thrombin and/or inhibit the cleavage of thrombin substrates may be determined.

[ 00133 ] The effect of an antibody molecule on coagulation and bleeding may be determined using standard techniques. For example, a mouse thrombosis model of ferric chloride clot induction in a blood vessel, such as the femoral vein or carotid artery, followed by a tail bleed to test normal haemostasis, may be employed.

[ 00134 ] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[ 00135 ] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[ 00136 ] Unless stated otherwise, antibody residues are numbered herein in accordance with the Kabat numbering scheme.

[ 00137 ] "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and A and B, just as if each is set out individually herein.

[ 00138 ] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[ 00139 ] Thus, the features set out above are disclosed in all combinations and permutations.

de BRIEF DESCRIPTION OF THE DRAWINGS

[ 00140 ] Certain aspects and embodiments of the present invention will now be illustrated by way of example only, with reference to the figures described below.

[ 00141 ] Figure 1 shows the binding and elution of the IgA on human thrombin- Sepharose column. Figure 1A shows an elution profile for IgA (narrow peak) from a thrombin-Sepharose column using a pH gradient (neutral to low, indicated by upward sloping line). Figure 1B shows a native blue gel showing total IgA load, flow-through from the human thrombin column and eluate following elution at low pH.

[ 00142 ] Figure 2 shows a non-reducing SOS-PAGE gel which indicates that the IgA binds thrombin but not prothrombin. In this pull- down assay, lectin agarose is used to bind to IgA in the presence of thrombin or prothrombin. The supernatant is then run on an SOS gel. Lane 1 is size standards; lane 2 shows a depletion of thrombin from the supernatant; Lane 3 shows that depletion is dependent on the presence of the IgA; Lanes 3 and 4 show that prothrombin is not depleted, and therefore does not bind to the IgA.

[ 00143 ] Figure 3 shows the relative rate of S2238 cleavage by thrombin in the presence or absence of IgA (i.e. a single slope of Abs405 with time for S2238 hydrolysis). This indicates that the IgA does not bind at the thrombin active site.

[ 00144 ] Figure 4 shows the results of binding studies which indicate that the IgA competes with the fluorescently labelled dodecapeptide hirugen for binding to thrombin.

[ 00145 ] Figure 5 shows the effect of the IgA on the cleavage of S2238 by thrombin. This analysis allows the estimate of Kd for the IgA-thrombin interaction of 12hM.

[ 00146 ] Figure 6 shows an SOS-PAGE gel of whole IgA and Fab fragments under reducing and non-reducing (ox) conditions. The non-reduced IgA is shown to have a molecular weight of between 100-200 kDa and the non-reduced Fab has a molecular weight of about 50kDa.

[ 00147 ] Figure 7 shows the crystal structure of Thrombin-Fab complex showing interaction between the exosite 1 of thrombin and HCDR3 of the Fab fragment.

[ 00148 ] Figure 8 shows detail of crystal structure showing interaction between specific residues of thrombin exosite 1 and HCDR3 of the Fab fragment. [ 00149 ] Figure 9 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen taken at between 2 and 30 minutes. 1 O Oul of PBS was administered (vehicle control)

[ 00150 ] Figure 10 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 40nM (final concentration in mouse blood, equivalent to a dose of approximately 0.6 mg/Kg) anti-exosite 1 IgA (IOOmI in PBS).

[ 00151 ] Figure 11 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 80nM (final concentration in mouse blood, equivalent to a dose of approximately 1.2 mg/Kg) anti-exosite 1 IgA (IOOmI in PBS), and a region outside of injury site for comparison.

[ 00152 ] Figure 12 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 200nM (final concentration in mouse blood, equivalent to a dose of approximately 3 mg/Kg) anti-exosite 1 IgA (IOOmI in PBS), and a region outside of injury site for comparison.

[ 00153 ] Figure 13 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 400nM (final concentration in mouse blood, equivalent to a dose of approximately 6 mg/Kg) anti-exosite 1 IgA (IOOmI in PBS).

[ 00154 ] Figure 14 shows fluorescence microscopy images of FeCb induced blood clots in femoral vein injuries in C57BL/6 mice treated with FITC labelled fibrinogen and 4mM (final concentration in mouse blood, equivalent to a dose of approximately 60 mg/Kg) anti-exosite 1 IgA (IOOmI in PBS).

[ 00155 ] Figure 15 shows a quantitation of the dose response to anti-exosite 1 IgA from the fluorescent images shown in figures 9 to 13.

[ 00156 ] Figure 16 shows tail bleed times in control C57BL/6 mice and in mice treated with increasing amounts of anti-exosite 1 IgA. The second average excludes the outlier. [ 00157 ] Figure 17 shows the results of tail clip assays on wild-type male C57BL/6 mice (n=5) after injection into tail vein with either IgA or PBS. 15 min after injection, tails were cut at diameter of 3mm and blood loss monitored over lOmin.

[ 00158 ] Figure 18 (18A to 18D) show the results of an FeCb carotid artery occlusion model on 9 week old WT C57BL/6 male mice injected as previously with 400nM anti thrombin IgA (final concentration in blood, equivalent to a dose of approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCb for 2 min. Figure 18A shows results for a typical PBS-injected mice (occlusion in 20min) and figures 18B, 18C and 18D show examples of results for mice treated with 400nM anti -thrombin IgA (no occlusion).

[ 00159 ] Figure 19 shows thrombin times (i.e. clotting of pooled plasma) with increasing concentrations of IgG and IgA of the invention, upon addition of 20nM human thrombin.

[ 00160 ] Figure 20 shows the binding of synthetic IgG to immobilized thrombin (on ForteBio Octet Red instrument).

[ 00161 ] Figure 21 shows a typical Octet trace for the binding of 24nM S 195A thrombin to immobilized IgG showing the on phase, followed by an off phase. The black line is the fit.

[ 00162 ] Figure 22 shows an Octet trace of 500nM prothrombin with a tip loaded with immobilized IgG. The same conditions were used as the experiment with thrombin in Fig. 21. There is no evidence of binding, even at this high concentration.

[ 00163 ] Figure 23 shows the ELISA binding curves for anti -exosite 1 IgG and an IgG S30A variant binding to thrombin.

[ 00164 ] Figure 24 shows the potency of IgG and IgG S30A in an ex vivo activated partial thromboplastin time (APTT) coagulation assay.

[ 00165 ] Figure 25 shows time to stop bleeding for 30 seconds data for IgG S30A and IgG in the rat tail clip bleeding model.

[ 00166 ] Figure 26 shows total bleeding time data for IgG S30A and IgG in the rat tail clip bleeding model.

[ 00167 ] Figure 27 shows total hemoglobin lost data for IgG S30A and IgG in the rat tail clip bleeding model. [ 00168 ] Figure 28 shows data on the prevention of thrombus formation by IgG S30A and IgG in the rat venous thrombosis model using ferric chloride (FeCb) at 2.5% concentration.

[ 00169 ] Figure 29 shows data on the prevention of thrombus formation by IgG S30A and IgG in the rat venous thrombosis model using ferric chloride (FeCb) at 5% concentration.

[ 00170 ] Figure 30 shows bar graphs of the thrombosis weight (mg) results from the rat AV shunt model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin. Doses are mg/kg (mpk) except for heparin which is U/kg. (n=l2 for the control and n=6 for all other groups)

[ 00171 ] Figure 31 shows bar graphs of the blood coagulation test results from the rat AV shunt model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin. For the AV shunt model, the tests included Thrombin Time (TT), activated Partial Thrombin Time (aPTT), Prothrombin Time (PT), and Ecarin Clotting Time (ECT). Doses are mg/kg except for heparin which is U/kg.

[ 00172 ] Figure 32 shows graphs of the plasma concentrations in the rat AV shunt model with different doses of Apixaban, Dabigatran, and Bivalirudin. Plasma concentrations are on the y-axis in mg/ml and dose levels are on the x-axis in mg/kg by intravenous administration (IV).

[ 00173 ] Figure 33 shows representations of thrombin and different thrombin binding sites, including the catalytic site, exosite 1 and exosite 2 and also shows the different binding modes for Hirudin, Bivalrudin, Dabigatran, and JNJ-64179375.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[ 00174 ] It is to be understood at the outset, that the figures and examples provided herein are to exemplify, and not to limit the invention and its various embodiments.

Definitions

[ 00175 ] According to the invention as defined herein, the term“safe”, as it relates to a dose, dosage regimen or treatment with an anti-thrombin antibody JNJ-64179375, refers to a favorable risk: benefit ratio with a relatively low or reduced frequency and/or low or reduced severity of adverse events, including reduced adverse gastrointestinal (GI) bleeding events, reduced infusion or hypersensitivity reactions, or reduced wound or joint complications compared to the standard of care or to another comparator. In particular, the present invention relates to a dose, dosage regimen or treatment with reduced treatment-related adverse GI bleeding events in a patient at risk for GI bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder. In some embodiments of the invention, the reduced treatment-related adverse GI bleeding events of the present invention are compared to treatment-related adverse GI bleeding events caused by a direct acting oral anticoagulant (DOAC). This is especially important because GI bleeding is the most common bleeding complication for patients receiving DOAC therapy. [Edupuganti et al. AJCEM. Vol. 5, No. 3, 2017, pp. 64-68.] The population of patients with risk factors for GI bleeding include, e.g., older aged patients, patients previously or currently treated with anticoagulants, patients currently treated with antiplatelet agents, patients with GI disorders, patients with a family history of GI disorders, patients with prior GI bleeding, cancer patients, patients who had a prior stroke, patients with reduced estimated glomerular filtration rate (eGFR), patients with renal impairment, patients with severe liver disease, patients with anemia, and patients with lower bodyweight.

[ 00176 ] As used herein, GI disorders include, e.g., ulcers, polyps, inflammation in the GI system, and malignant or benign gastrointestinal tumors. In certain embodiments, older aged patients include patients with ages >55, >60, >65, >70, >75, >80, or >85. In certain embodiments, older aged patients include patients with ages >75, >80, or >85. In certain embodiments, older aged patients include patients with ages >75. For example, compared to warfarin, the likelihood of GI bleeding in patients treated with dabigatran l50mg twice daily was ~50% higher in patients ages >75 compared to patients with ages <75. [Abraham. Am. J. of Gastroenterology Suppl. 2016 Vol. 3:2-12.; Romanelli et al. Circ Cardiovasc Qual Outcomes 20l6;9: 126-134.] In certain embodiments, patients with lower bodyweight include patients with a body weight <60kgs or <50kgs. For example, after the introduction of dabigatran in New Zealand, more than half of the reported bleeding events occurred in patients with a body weight <60kgs and in another study patients with body weights <50kgs were shown to have a higher risk of bleeding complications with apixaban compared with patients who weighed 65-85kgs. [Abraham. Am. J. of Gastroenterology Suppl. 2016 Vol. 3:2-12.; Harper et al. N Engl JMed 2012;366:864-866.; Gong and Kim. Can J Cardiol 20l3;29 (7 Suppl): S24-S33.] In certain embodiments, the patients at risk for GI bleeding are patients with renal impairment and reduced estimated glomerular filtration rate (eGFR). For example, reduced eGFR is associated with an increased risk of ischemic stroke and systemic embolism in atrial fibrillation and is also known to increase anticoagulant-related bleeding. In addition, reduced renal excretion of the DOAC affects half-life, such that the plasma concentration of the DOAC rises along with the risk of bleeding. [Abraham. Am.

J. of Gastroenterology Suppl. 2016 Vol. 3:2-12.; Go et al. Circulation 2009;l 19: 1363— 1369.] In certain embodiments, the patients at risk for GI bleeding are patients currently treated with antiplatelet agents. For example, concurrent use of aspirin or other NSAIDs with DOACs or warfarin has been shown to increase risk of bleeding, including major bleeding. [Abraham. Am. J. of Gastroenterology Suppl. 2016 Vol. 3:2-12.; Steinberg et al. Circulation 2013;128:721-728.; Dans et al. Circulation 2013;127:634-640.] In certain embodiments, the patients at risk for GI bleeding are cancer patients and the risk of bleeding is influenced by a number of different factors, e.g., cancer type and stage, chemotherapy, surgical interventions, and thrombocytopenia. [Kamphuisen and Beyer- Westendorf J. Thromb Res. 2014 May;l33 Suppl 2:S49-55.; Naresh et al. Blood 2012; 120:3408.] In certain embodiments, the patients at risk for GI bleeding are patients with anemia, e.g., because anemia is indicative of having a history of bleeding or a predisposition to bleeding. [Habert. Int J Gen Med. 2016 Oct 11;9:337-347.] In certain other embodiments, the patient population with risk factors that put them at risk of GI bleeding includes patients that may be advised not to take other anticoagulants such as direct acting oral anticoagulants (DOACs), e.g., factor Xa (FXa) inhibitors (e.g., apixaban), thrombin inhibitors (e.g., dabigatran), or factor XIa (FXIa) inhibitors. Other anticoagulants include, for example, natural and synthetic heparins, warfarin, hirudin, and derivatives of hirudin (e.g. lepirudin and bivalirudin). For example, there are restrictions for use of DOACs in patients with liver disease that are based on Child-Pugh classification system, e.g., DOACs should not be used with Child-Pugh C disease or with cirrhosis given the likelihood of luminal evidence of portal hypertension (i.e., portal hypertensive gastropathy, varices, etc.) and impaired ability to metabolize the drug, further increases the risk of GI bleeding. [Abraham. Am. J. of Gastroenterology Suppl. 2016 Vol. 3:2-12.; Graff and Harder. Clin Pharmacokinet 2013;52:243-254.] [ 00177 ] As used herein, adverse gastrointestinal (GI) bleeding events are considered “treatment-related”, if the attribution of the adverse event is possible, probable, or very likely associated with the treatment based on the following attribution definitions.

Attribution Definitions:

• Not Related: An adverse event that is not related to the use of the drug.

• Doubtful: An adverse event for which an alternative explanation is more likely, e.g., concomitant drug(s), concomitant disease(s), or the relationship in time suggests that a causal relationship is unlikely.

• Possible: An adverse event that might be due to the use of the drug. An alternative explanation, e.g., concomitant drug(s), concomitant disease(s), is inconclusive. The relationship in time is reasonable; therefore, the causal relationship cannot be excluded.

• Probable: An adverse event that might be due to the use of the drug. The relationship in time is suggestive (e.g., confirmed by dechallenge). An alternative explanation is less likely, e.g., concomitant drug(s), concomitant disease(s).

• Very Likely: An adverse event that is listed as a possible adverse reaction and cannot be reasonably explained by an alternative explanation, e.g., concomitant drug(s), concomitant disease(s). The relationship in time is very suggestive (e.g., it is confirmed by dechallenge and rechallenge).

[ 00178 ] Non-limiting examples of a patient or patients“in need of a treatment for inhibiting a thrombotic and/or embolic disorder” include, e.g., patient populations listed below.

Patients with“Venous” indications - Traditional oral anticoagulant pathway

• Orthopedic surgery prophylaxis, e.g., TKR and THR

• Hospitalized medically ill prophylaxis

• DVT/PE Rx and extended prevention

• Stroke prevention in AF

Patients with“Arterial” indications - Oral anticoagulation not (yet) standard of care

• Acute ACS

• Secondary Prevention post ACS • Heart failure

• Non-AF Ischemic stroke

• Secondary Prevention Stable CAD or PAD

Other Specialty Patient Populations

• Severe renal impairment (ESRD-dialysis)

• AF with recent ICH

• Cancer patients (thrombosis prevention or Rx)

• Mechanical heart valves

• Post general surgery

Abbreviations

ACS acute coronary syndrome

AF atrial fibrillation

CAD coronary artery disease

DOAC direct-acting oral anticoagulant

DVT deep vein thrombosis

ESRD end stage renal disease

ICH intracranial hemorrhage

PAD peripheral artery disease

PE pulmonary embolism

Rx prescription

TKR total knee replacement

THR total hip replacement

[ 00179 ] As used herein, the term“antiplatelet agents” or“platelet inhibitory agents”, refers to agents that inhibit platelet function, for example by inhibiting the aggregation, adhesion, or granular secretion of platelets. Agents include, for example, but are not limited to, the various known non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam, and pharmaceutically acceptable salts or prodrugs thereof. Of the NSAIDS, aspirin (acetylsalicyclic acid or ASA) is preferred. Other suitable platelet inhibitory agents include Ilb/IIIa antagonists (e.g., tirofiban, eptifibatide, and abciximab), thromboxane-A2 -receptor antagonists (e.g., ifetroban), thromboxane-A2-synthetase inhibitors, PDE-III inhibitors (e.g., dipyridamole), thrombin receptor antagonists that are also referred to as PAR-l antagonists, e.g., Vorapaxar (trade name Zontivity), P2Y12 inhibitors, e.g., Ticagrelor (trade name Brilinta and others), clopidogrel (brand name Plavix among others), and Cangrelor (trade name Kengreal in the US and Kengrexal in Europe), and pharmaceutically acceptable salts or prodrugs thereof. Clopidogrel acts by irreversibly inhibiting the P2Y12 subtype of ADP receptor, which is important in activation of platelets and eventual cross-linking by the protein fibrin. Cangrelor is a P2Y12 inhibitor for intravenous application. Non-limiting examples of antiplatelet agents also include, prasugrel (Effient), Dipyridamole, dipyridamole/aspirin (Aggrenox), and ticlodipine (Ticlid).

[ 00180 ] As used herein,“reduced adverse GI bleeding events” relates to reduced frequency of adverse GI bleeding events from treatment with JNJ-64179375 compared to treatment with other anticoagulants, including for example direct acting oral

anticoagulants (DOACs), e.g., factor Xa (FXa) inhibitors (e.g., apixaban), thrombin inhibitors (e.g., dabigatran), or factor XIa (FXIa) inhibitors. As used herein, frequency as it relates to adverse GI bleeding events, can be expressed as a rate, e.g., percentage of subjects with at least one bleeding event per year. As defined herein, the term“adverse GI bleeding events” includes, for example, major GI bleeding, minor GI bleeding, and the individual components of the composite endpoint of any GI bleeding event. [Lassen et al. J Thromb Haemost. 2007;5:2368-2375.] In certain embodiments, reduced adverse GI bleeding events are reduced major GI bleeding events.

[ 00181 ] Bleeding events are standard primary safety endpoints in studies of anticoagulants, but there is substantial heterogeneity in bleeding definitions. [Schulman et al. J Thromb Haemost. 20l0;8(l):202-204.; Mehran et al. Circulation. 2011 Jun

14;123(23):2736-47.] See, for example, published guidelines describe how to define major bleeding events, e.g., the Control of Anticoagulation Subcommittee of the

International Society on Thrombosis and Haemostasis (ISTH) recommends the following criteria for major bleeding: fatal bleeding, and/or symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intraarticular or pericardial, or intramuscular with compartment syndrome, and/or bleeding causing a fall in hemoglobin level of 2g/dl ) (1.24 mmol/L) or more, or leading to transfusion of two or more units of whole blood or red cells. [Schulman et al. J Thromb Haemost.

20l0;8(l):202-204.] See, also, for example, the Thrombolysis in Myocardial Infarction (TIMI) bleeding criteria that have been in use for decades and have been reported in most cardiovascular trials (major bleeding: blood Hgb drops >5 g/dl; minor: blood Hgb drops >3 to <5 g/dl). [Mehran et al. Circulation. 2011 Jun 14;123(23):2736-47.; Rao et al. Am J Cardiol. 2005 Nov l;96(9): 1200-6.; Bovill Eet al. Ann Intern Med. 1991 Aug

15; 115(4):256-65.] Standardized definitions of major and nonmajor clinically relevant events can also be found, for example, in the Phase 3 studies of apixaban. [Lassen et al. N Engl J Med. 2009;36l(6):594-604.; Lassen et al. Lancet. 2010;375:807-815.] In addition, the labels of commercially available anticoagulants often include definitions for major bleeding. Lor example, the label for Eliquis (Apixaban) defines major bleeding as clinically overt bleeding that was accompanied by one or more of the following: a decrease in hemoglobin of 2 g/dL or more; a transfusion of 2 or more units of packed red blood cells; bleeding that occurred in at least one of the following critical sites:

intracranial, intraspinal, intraocular, pericardial, intra-articular, intramuscular with compartment syndrome, retroperitoneal; or bleeding that was fatal. Intracranial hemorrhage included intracerebral (hemorrhagic stroke), subarachnoid, and subdural bleeds. In addition, the label for Pradaxa (dabigatran) defines major bleeding as bleeding accompanied by one or more of the following: a decrease in hemoglobin of >2 g/dL, a transfusion of >2 units of packed red blood cells, bleeding at a critical site or with a fatal outcome. Intracranial hemorrhage included intracerebral (hemorrhagic stroke), subarachnoid, and subdural bleeds. As used herein, bleeding events are based on the ISTH bleeding scale, e.g.,“major bleeding” in non-surgical patients is defined as, 1. fatal bleeding and/or; 2. symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intraarticular or pericardial, or intramuscular with compartment syndrome; and/or 3. bleeding causing a fall in hemoglobin level of 2 g/dL (1.24 mmol/L) or more, or leading to transfusion of two or more units of whole blood or red cells. These same three criteria for non-surgical site bleeding are also consistent with the ISTH criteria used for surgical studies. [Schulman et al . JThromb Haemost.

20l0;8(l):202-204.]

[ 00182 ] In certain embodiments, treatment with JNJ-64179375 compared to treatment with other DOACs is associated with significantly reduced adverse GI bleeding events of >5%, >10%, >15%, >20%, >25%, >30%, >35%, >40%, >45%, or > 50%. In certain embodiments, treatment with JNJ-64179375 is associated with significantly reduced adverse GI bleeding events of 35-50% reduction compared to treatment with a DOAC. In some embodiments, the significantly reduced adverse GI bleeding events is a >35% reduction compared to the DOAC. In other embodiments, the significantly reduced the adverse GI bleeding events is a >40% reduction compared to the DOAC. In other embodiments, the significantly reduced adverse GI bleeding events is a >45% reduction compared to the DOAC. In other embodiments, the significantly reduced adverse GI bleeding events is a >50 % reduction compared to the DOAC. In certain embodiments, treatment with JNJ-64179375 compared to treatment with other DOACs is associated with significantly reduced adverse GI bleeding events of 35-50% reduction compared to the FXa inhibitor apixaban. In certain embodiments, treatment with JNJ-64179375 is associated with significantly reduced adverse GI bleeding events of 35-50% reduction compared to the thrombin inhibitor dabigatran.

[ 00183 ] An“adverse event” is any untoward medical occurrence in a clinical study subject administered a medicinal product. Treatment-emergent adverse events are adverse events with onset during the treatment phase or that are a consequence of a preexisting condition that has worsened since baseline, but an adverse event does not necessarily have a causal relationship with the treatment. An adverse event can therefore be any unfavorable and unintended sign (including an abnormal finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not related to that medicinal product. (Definition per International Conference on Harmonisation [ICH]) This includes any occurrence that is new in onset or aggravated in severity or frequency from the baseline condition, or abnormal results of diagnostic procedures, including laboratory test abnormalities. A laboratory test abnormality that is considered by the investigator to be clinically relevant (e.g., causing the subject to discontinue the study drug, requiring treatment, or causing apparent clinical manifestations) should be reported as an adverse event.

[ 00184 ] As defined herein, the term“therapeutic index” (TI) (also referred to as “therapeutic ratio” or“therapeutic window”) is a comparison of the amount of a therapeutic agent that causes the therapeutic effect (e.g., inhibition of a thrombin- mediated condition) to the amount that causes adverse bleeding events (e.g., major bleeding, minor clinically relevant bleeding, and/or individual components of the composite endpoint of any bleeding event).

[ 00185 ] According to the invention as defined herein, the terms“effective”,

“efficacy”, or“therapeutically effective” as they relate to terms such as amounts, dose, dosage regimen, or treatment with an anti-thrombin antibody JNJ-64179375, refer to the treatment or inhibition of a thrombotic and/or embolic disorder. Such inhibition can be observed, for example, as a reduction in the frequency of occurrence and/or severity of the thrombin-mediated condition in patients treated with the anti-thrombin antibody.

[ 00186 ] It should also be understood that the terms“about,”“approximately,” “generally,”“substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/ characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[ 00187 ] More in particular, according to the invention as described herein, effective is intended to refer to agents that are effective to treat a desired disease or condition, e.g., JNJ-64179375 and compositions comprising JNJ-64179375 that are used to treat a thrombotic and/or embolic disorder. In general, a thrombotic and/or embolic disorder is a circulatory disease or condition caused by thrombosis or embolism which can involve the effects of platelet activation and/or platelet aggregation. The term "thrombotic and/or embolic disorder" as used herein includes arterial cardiovascular thrombotic and/or embolic disorders, venous cardiovascular thrombotic and/or embolic disorders, arterial cerebrovascular thrombotic and/or embolic disorders, and venous cerebrovascular thrombotic and/or embolic disorders. Non-limiting examples of "thrombotic and/or embolic disorders" include, for example, unstable angina, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis. It is noted that thrombosis includes occlusion (e.g., after a bypass) and reocclusion (e.g., during or after percutaneous transluminal coronary angioplasty). The term thrombotic and/or embolic disorders also includes conditions such as acute coronary syndrome, coronary artery disease, peripheral artery disease, unstable angina, refractory angina, occlusive coronary thrombus occurring post-thrombolytic therapy or post-coronary angioplasty, a thrombotically mediated cerebrovascular syndrome, embolic stroke, thrombotic stroke, transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus, coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, thromboangiitis obliterans, thrombotic disease associated with heparin-induced thrombocytopenia, thrombotic complications associated with extracorporeal circulation, thrombotic complications associated with instrumentation, and thrombotic complications associated with the fitting of prosthetic devices.

Examples

[ 00188 ] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

1. Antibody Isolation and Characterisation

[ 00189 ] Coagulation screening was carried out on a blood plasma sample from a patient. The coagulation tests were performed on a patient who suffered subdural haematoma following head injury. The haematoma spontaneously resolved without intervention. There was no previous history of bleeding and in the years since the patient presented, there have been no further bleeding episodes. The results are shown in Table 1.

[ 00190 ] The prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were all prolonged in the patient compared to controls, but reptilase time was normal.

[ 00191 ] Thrombin time was not corrected by heparinase, indicating that heparin treatment or contamination was not responsible.

[ 00192 ] Fibrinogen levels were normal in the patient, according to ELISA and Reptilase assays. The Clauss assay gave an artifactually low fibrinogen level due to the presence of the thrombin inhibitor. The PT and APTT clotting times were found to remain prolonged following a mixing test using a 50:50 mix with pooled plasma from normal individuals. This showed the presence of an inhibitor in the sample from the patient.

[ 00193 ] The patient' s blood plasma was found to have a high titre of an IgA. This IgA molecule was found to bind to a human thrombin column (Figure 1). IgA binding lectin- agarose pulled down thrombin in the presence but not the absence of the IgA.

Prothrombin was not pulled down by the lectin-agarose in the presence of the IgA, indicating that the IgA specifically binds to thrombin but not prothrombin (Figure 2).

[ 00194 ] The binding site of the IgA on the thrombin molecule was then investigated.

[ 00195 ] A slightly higher rate of cleavage of S2238 by thrombin was measured in the presence of the IgA, indicating that the IgA does not block the active site of thrombin (Figure 3).

[ 00196 ] The binding of fluorescently labelled hirugen to thrombin is inhibited by the presence of 700 nM of the IgA, indicating that the epitope for the antibody overlaps with the binding site of hirugen on thrombin, namely the exosite 1 of thrombin (Figure 4).

[ 00197 ] The effect of the IgA on the hydrolysis of some of thrombin' s procoagulant substrates was tested. The results are shown in Table 2. These results demonstrate that the IgA molecule isolated from the patient sample inhibits multiple procoagulant activities of thrombin.

[ 00198 ] Inhibition of thrombin by antithrombin (AT) in the presence of the IgA was only marginally affected in both the absence and presence of heparin (Table 3).

[ 00199 ] The dissociation constant (K_d) of the IgA for thrombin was initially estimated based on rate of S2238 hydrolysis to be approximately 12hM (Figure 5). The Kd for the binding of the IgA to S195A thrombin (inactivated by mutation of the catalytic serine) was determined to be 2nM using the ForteBio Octet Red instrument (Table 4).

[ 00200 ] The purified IgA was cleaved with papain (Figure 6), and the Fab fragment was isolated and combined with human PPACK-Thrombin (PPACK is a covalent active site inhibitor). The human PPACK-Thrombin-FAB complex was crystallized and used for structural analysis. The statistics of the structure obtained were as follows: resolution is 1.9.4; Rfactor = 19.43%; Rfree = 23.42%; one complex in the asymmetric unit; Ramachandran: favoured 97.0%, outliers 0%. The crystal structure revealed a close association between the HCDR3 of the IgA Fab and the exosite 1 of thrombin (Figure 7).

[ 00201 ] In particular, residues M32, F34, Q38, E39, L40, L65, R67, R73, T74, R75, Y76, R77a and 182 of the exosite 1 all directly interact with the HCDR3 loop of the IgA Fab (Figure 8).

[ 00202 ] PISA analysis of the antibody-thrombin interface showed that the total buried surface area in the complex is 1075 A. The contact residues in the IgA heavy chain were (Rabat numbering): 30, 51, 52a, 53-55, 96, 98, 99, 100, lOOa, lOOb, lOOc, lOOd). These are all in CDRs: CDRH1-GYTLTEAAIH; CDRH2-GLDPODGETVYAOOFKG: CDRH3- GDFSEFEPFSMDYFHF (underlined residues contacting). CDRH3 was found to be the most important, providing 85% of the buried surface area on the antibody. The light chain made one marginal contact with Tyr49, right before CDRL2 (with Ser36a of thrombin). Some individual contributions to buried surface were: Glu99 54 A², PhelOO 134.8 A², GlulOOa 80.6 A², Phe lOOc 141.7 A².

[ 00203 ] The contact residues in thrombin were found to be (chymotrypsin numbering): 32, 34, 36a-40, 65, 67, 73-76, 77a, 82, and 151. The most important individual contributors to the buried surface were: Gln38 86.4 A², Arg73 44.5 A2, Thr74 60.1 A², Tyr76 78.4 A², Arg77a 86.9 A².

[ 00204 ] The patient did not display increased or abnormal bleeding or haemorrhage, in spite of 3g/l circulating levels of this IgA, demonstrating that the antibody inhibits thrombin without affecting normal haemostasis.

2. The effect of IgA on Animal Thrombosis Models

[ 00205 ] C57BL/6 mice were anaesthetized. A catheter was inserted in the carotid artery (for compound injection). FITC labelled fibrinogen (2mg/ml) was injected via the carotid artery. PBS (control) or IgA was also injected via the carotid artery. The femoral vein was exposed and 10% FeCb applied (saturated blotting paper 3mm in length) for 3 min to induce clotting.

[ 00206 ] Fluorescence microscopy images were taken along the length of injury site at 0, 5, 10, and 20 min post FeCb injury using fluorescence microscopy techniques. [ 00207 ] Clots (fibrin deposits) in the femoral vein were clearly visible as bright areas (figure 9). The lowest dose of the antibody was observed to cause significant inhibition of clotting but as the dose increased, clotting was abolished (figures 10 to 15).

[ 00208 ] The bleeding times of the mice were also measured. Bleeding times were assessed as time to cessation of blood flow after a tari cut. Despite the presence of a single outlier sample, the bleeding time was found to be unaffected by treatment with anti exosite 1 IgA (figure 16).

[ 00209 ] These results show that the anti-exosite 1 IgA antibody is a potent inhibitor of thrombosis but has no effect on bleeding time.

3. Tail clip assays

[ 00210 ] A tail clip assay was performed on wild-type male C57BL/6 mice injected with either 400nM IgA (final concentration in blood, equivalent to a dose of

approximately 6 mg/Kg) or PBS. Blood loss was monitored over lOmins after the tail was cut at 3mm diameter 15 minutes after the injection. Total blood loss was found to be unaffected by treatment with anti-exosite 1 IgA (figure 17).

4. FeCb injury carotid artery occlusion

[ 00211 ] FeCb injury carotid artery occlusion studies were performed on 9 week old WT C57BL/6 male mice. Mice were injected with 400nM anti-IIa IgA (final

concentration in blood, equivalent to a dose of approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCb for 2 min. Blood flow was then monitored by Doppler and the time to occlusion measured. A "clot" was defined as stable occlusive thrombus where blood flow was reduced to values typically less than O.lml/min and stayed reduced. In the control mice, a stable clot was observed to form about 20mins after injury (Figure 18A). However, the majority of mice treated with 400nM anti-IIa IgA were unable to form stable clots and gave traces in which the clots were quickly resolved, repeatedly resolved or never formed. Three representative traces are shown in Figures 18B to 18D.

5. Anti-exosite 1 IgG

[ 00212 ] The IgA molecule identified in the patient described above was re-formatted as an IgG using standard techniques. [ 00213 ] The clotting time of pooled human plasma spiked with increasing amounts of the original IgA and the new IgG was tested upon addition of human thrombin to 20nM (Figure 19). Both parent IgA and the synthetic IgG increased time to clot formation in an identical concentration-dependent manner, implying identical affinities for thrombin.

[ 00214 ] This was confirmed by measuring the binding of synthetic IgG to

immobilized S195A thrombin using a ForteBio™ Octet Red instrument. Thrombin was attached to the probe and the binding of the antibodies (at various concentrations) was monitored.

[ 00215 ] On-rates and off-rates were determined. Both antibodies gave similar on-rates of approximately 3xl0⁵ M ¹ s ¹ and off-rates of approximately 5xl0 ⁴ s ¹, and dissociation constants (Kd) of approximately 2nM. Kds of approximately 2nM were also obtained for the IgA and the IgG by steady-state analysis (Table 4). A representative steady state curve is shown in Figure 20. The properties of the IgA were therefore reproduced on an IgG framework.

[ 00216 ] Binding of prothrombin to the IgG antibody was tested using the Octet system by immobilizing IgG. Thrombin bound to the immobilized IgG with comparable rates and affinities as those obtained using immobilized thrombin (Table 4); prothrombin did not bind to the IgG. Figure 21 is a trace of 24nM thrombin binding to and dissociating from the immobilized IgG. Figure 22 is the same experiment using 500nM prothrombin, and shows no evidence of binding.

6. Anti-exosite 1 IgG S30A variant antibody

6.1 Introduction

[ 00217 ] Glycosylation sites in an antibody can raise issues during manufacture and/or therapeutic use of the antibody. The oligosaccharides added to glycosylation sites are typically heterogenous, for example with complex di-antenary and hybrid

oligosaccharides with sialic acids and galactoses (for Fab oligosaccharides) or with fiicosylated non-galactosylated di-antenary oligosaccharides (for Fe oligosaccharides).

The presence of more than one glycosylation site in an antibody (or active fragment thereof) thus adds further to potential heterogeneity. Removal of incorrectly glycosylated forms of an antibody during the purification process is very difficult and can lead to extended process development activities and reduced yields. [ 00218 ] Therefore, if a glycosylation site in an antibody (or active fragment thereof) is determined not to be required directly or indirectly for antigen binding activity, it may be desirable from a manufacturing and quality control perspective to remove that glycosylation site by engineering.

[ 00219 ] As noted above, it was envisaged that a glycosylation site in VL domain of SEQ ID NO 6 of the antibody of the present invention could be mutated out by introducing a substitution at either N28 or S30.

[ 00220 ] Of the two residues N28 and S30, S30 was targeted for substitution as it was considered, based on crystal structure analysis, less likely to be involved in antibody folding or stability.

6.2 Methods and Materials

[ 00221 ] An "IgG S30A" variant monoclonal antibody was produced using standard site-directed mutagenesis techniques from the anti-exosite IgG antibody ("IgG") described in section 5 above by substituting serine residue 30 (S30) with an alanine (hence, S30A).

[ 00222 ] The IgG S30A variant was expressed for analysis using standard transient expression techniques as described below. In outline, single gene vectors (SGVs) were constructed using GS Xceed vectors (Lonza Biologies, Slough, UK) (pXC IgG4pro DK for the heavy chain constant domain encoding region and pXC Kappa for light chain constant domain encoding region) and the variable domain encoding regions as synthesised by GeneArt AG. The SGVs were amplified and transiently co-transfected into Chinese Hamster Ovary CHOK1SV GS KO cells for initial expression at a volume of 200 ml and then subsequently at a scaled-up volume of 2.5 litres.

[ 00223 ] The methods used will be described below. Where manufacturer' s instructions were followed, this will be indicated.

6.2.1 Vector Construction

[ 00224 ] The sequences of the light and heavy chain variable domain encoding regions were synthesised by GeneArt AG. Light chain variable domain encoding region was sub cloned into pXC Kappa and heavy chain variable domain encoding region into pXC IgG4pro DK vectors respectively using the N-terminal restriction site Hind III and the C- terminal restriction sites BsiWI (light chain) and Apal (heavy chain). In short, the 5 pi of lyophilised shuttle vectors, as produced by GeneArt AG, were resuspended in 50 pl endotoxin free, sterile water. 1 pg of DNA was digested with the relevant restriction enzymes in a total volume of 50 pl and samples were incubated for 2 hours at 37°C. 8.3 mΐ of 6x DNA loading buffer was added and samples electrophoresed at 120 V for 40 min on a 1% w/v agarose gel stained with ethidium bromide. 10 mΐ Lonza Simply Load Tandem DNA ladder was used as reference ladder.

[ 00225 ] The relevant fragments were gel-extracted using a QIAquick gel extraction kit (QIAGEN, 28704) according to a manufacturer' s instructions. Ligations were set-up using Roche' s quick ligation kit with a 1 : 12 ratio of vector backbone to insert DNA, 1 mΐ T4 quick ligase, 10 mΐ of 2x T4 quick ligation buffer, reaction volume adjusted to 21 mΐ with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 10 minutes. 10 mΐ aliquots of the ligation reactions were used to transform One Shot Top 10 Chemically Competent Escherichia coli cells (Invitrogen, C404003) using the heat-shock method according to manufacturer 's instructions. Cells were spread onto ampicillin-containing (50 pg/ml) Luria Bertani agar plates (LB Agar, Sigma- Aldrich L7025) and incubated overnight at 37°C until bacterial colonies were evident. Positive clones were screened by PCR amplification and verified by restriction digest (using a double digest of EcoRI-HL and HindIII-HP) and nucleotide sequencing of the gene of interest through a 3^rd party provider.

6.2.2 DNA Amplification

[ 00226 ] A single bacterial colony was picked into 15 ml Luria Bertani (LB) medium (LB Broth, Sigma-Aldrich, L7275) containing 50 pl/ml ampicillin and incubated at 37°C overnight with shaking at 220 rpm. The resulting starter culture was used to inoculate 1 L Luria Bertani (LB) medium containing 50 pl/mg ampicillin and incubated at 37°C overnight with shaking at 220 rpm. Vector DNA was isolated using the QIAGEN Plasmid Plus Gigaprep system (QIAGEN, 12991). In all instances, DNA concentration was measured using a Nanodrop 1000 spectrophotometer (Thermo-Scientific) and adjusted to 1 mg/ml with EB buffer (10 mM Tris-Cl, pH 8.5).

6.2.3 Routine Culture of CHOK1SV GS KO Cells

[ 00227 ] CHOK1SV GS KO cells were cultured in CD-CHO media (Invitrogen 10743- 029) supplemented with 6 mM glutamine (Invitrogen, 25030-123) Cells were incubated in a shaking incubator at 36.5°C, 5% C02 , 85% humidity, sub-cultured every 3-4 days, 140 rpm. Cells were routinely sending at 2 x 10⁵ cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20.

6.2.4 Transient Transfections of CHOK1SV GS KO Cells

[ 00228 ] Transient transfections were performed using CHOK1SV GS KO cells which had been in culture a minimum two weeks. Cells were sub-cultured 24 h prior to transfection.

[ 00229 ] All transfections were carried out via electroporation using either the Gene Pulse XCell (Bio-Rad), a cuvette based electroporation system for small scale (200 ml) transfections or a Gene Pulse MXCell (Bio-Rad), a plate based system for electroporation for the larger scale (2.5 L) transfection. For each transfection, viable cells were resuspended in pre- warmed media to 2.86 x 10⁷ cells/ml. 80 pg DNA (1 : 1 ratio of heavy and light chain SGVs) and 700 pl cell suspension were aliquoted into each cuvette/well. Cells were electroporated at 300 V, 900 pF for the Gene Pulse XCell system and 300 V, 1300 pF for the Gene Pulse MXCell system. Transfected cells were transferred to pre warmed media in Erlenmeyer flasks and the cuvette/wells rinsed twice with pre-warmed media which was also transferred to the flasks. Transfected cell cultures were incubated in a shaking incubator at 36.5°C, 5% CO², 85% humidity, 140 rpm for 6 days. Cell viability and viable cell concentrations were measured at the time of harvest using a Cedex HiRes automated cell counter (Roche).

6.2.5 Protein A Affinity Chromatography

[ 00230 ] Small (200 ml) and large (2.5 L) scale culture supernatants were harvested and clarified by centrifugation at 2000 rpm for 10 min, then filtered through a 0.22 pm filter. Clarified supernatant was purified using a pre-packed 5 ml HiTrap MabSelect SuRE column (GE Healthcare, 11-0034-94) on an AKTA purifier (10 ml/min). The column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0 (equilibration buffer) for 5 column volumes (CVs). After sample loading, the column was washed with 2 CVs of equilibration buffer followed by 3 CVs of 50 mM sodium phosphate, 1 M sodium chloride pH 7.0 and a repeat wash of 2 CVs of equilibration buffer. The Product was then eluted with 10 mM sodium formate, pH 3.5 over 5 CVs. Protein containing, eluted fractions were immediately pH adjusted to pH 7.2 and filtered through a 0.2 pm filter. 6.2.6 SE-HPLC Analysis

[ 00231 ] Duplicate samples were analysed to SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 pm 9.4 mm ID x 250 mm column (Agilent). Aliquots of sample at a concentration of 1 mg/ml were filtered through a 0.2 pm filter prior to injection. 80 pl aliquots were injected respectively and run at 1 ml/min for 15 minutes. Soluble aggregate levels were analysed using Chemstation (Agilent) software.

6.2.7 SOS-PAGE Analysis

[ 00232 ] Reduced samples were prepared for analysis by mixing with NuPage 4x LOS sample buffer (Invitrogen, NP0007) and NuPage lOx sample reducing agent (Invitrogen NP0009), and incubated at 70°C, 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1.5 mm NuPage 4- 12% Bis-Tris Novex pre-cast gels (Invitrogen, NP0335PK2) with NuPage MES SOS running buffer under denaturing conditions. 10 pl aliquots of SeeBlue Plus 2 pre-stained molecular weight standards (Invitrogen, LC5925) and a control IgG4 antibody at 1 mg/ml were included on the gel. 1 pl of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISBOlL) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imagine System (UVP).

6.2.8 Endotoxin Analysis

[ 00233 ] Endotoxin levels purified protein from the larger scale (2.5 L) production was measured at 2.54 mg/ml using the Endosafe- PTS instrument, a cartridge based method based on the LAL assay (Charles River).

6.3 Results and Discussion

[ 00234 ] The transfectant culture from the initial expression at a volume of 200 ml was harvested on Day 6 post-transfection and clarified by centrifugation and sterile filtration. The clarified cell culture supernatant was purified using one-step Protein A

chromatography. Quantification was by absorbance at A280nm. Production quality analysis in the form of SE-HPLC and SDS-PAGE showed a high level of purity was achieved post- purification.

[ 00235 ] For scaling up the culture volume up to 2.5 litres, as before, Day 6 harvested, clarified cell culture supernatant was purified using one-step Protein A chromatography. Product quality analysis in the form of SE-HPLC, SDS-PAGE and endotoxin detection was carried out using purified material at a concentration of 1 mg/ml, alongside an in- house human IgG4 antibody as a control sample. High level of purity was observed from the purified ichorcumab S30A with a small trace of high molecular weight impurity (1.8%) and an endotoxin level below the detectable scale of <0.02 EU/mg.

[ 00236 ] Thereafter, analysis of the IgG S30A variant produced as above was performed using standard techniques to check in vitro and in vivo activity compared with the anti-exosite IgG antibody.

[ 00237 ] Figure 23 shows that IgG S30A has equivalent or higher binding affinity to thrombin than the IgG antibody, as determined by a standard ELISA binding assay.

[ 00238 ] Using a standard ex vivo activated partial thromboplastin time (APTT) coagulation assay, IgG S30A was found to be equivalent or more potent than IgG.

[ 00239 ] Table 5 shows IgG and IgG S30A binding affinities to thrombin using Biacore™ surface binding analysis (GE Healthcare, Little Chalfont, Buckinghamshire, UK). IgG S30A has equivalent or higher affinity to thrombin compared to IgG. Affinities were not affected for either IgG S30A or IgG by storage for one month at 4° C or by exposure to light (PO).

[ 00240 ] Table 6 shows that both IgG S30A and IgG have equivalent solubility and both are soluble to >100 mg/ml concentration, with little reduction in solubility (and no aggregate formation) on storage.

[ 00241 ] Figure 24 shows the potency of IgG and IgG S30A in an ex vivo activated partial thromboplastin time (APTT) coagulation assay. IgG S30A is equivalent or more potent than IgG.

[ 00242 ] Figure 25 shows that both IgG S30A and IgG are equivalent in the rat tail clip bleeding model (see experimental section 3 above), with both showing no difference to vehicle control in time to stop bleeding for 30 seconds.

[ 00243 ] Figure 26 shows that both IgG S30A and IgG are equivalent in the rat tail clip bleeding model, with both showing no difference to vehicle control in total bleeding time. [ 00244 ] Figure 27 shows that both IgG S30A and IgG are equivalent in the rat tail clip bleeding model, with both showing no difference to vehicle control in total haemoglobin lost.

[ 00245 ] Figure 28 shows that both IgG S30A and IgG are equivalent in the rat venous thrombosis model using ferric chloride (FeCl³; see experimental section 2 above) at 2.5% concentration, with both IgG S30A and IgG causing total prevention of thrombus formation.

[ 00246 ] Figure 29 shows that both IgG S30A and IgG are equivalent in the rat venous thrombosis model using ferric chloride (FeCl³) at 5% concentration, with both IgG S30A and IgG causing similar reduction of thrombus formation.

[ 00247 ] The results showed that the removal of the S30 glycosylation site in the IgG antibody to form the IgG S30A variant did not negatively impact on the binding or other beneficial characteristics of the antibody. The IgG S30A variant thus may be preferable from a manufacturing and production perspective for reasons described above.

[ 00248 ] Specific anti-exosite 1 antibody molecules disclosed herein include the following:

1) a wild-type anti-exosite 1 IgA antibody;

2) a synthetic anti-exosite 1 IgG antibody (also referred to herein as "IgG"), re-formatted from the wild-type IgA antibody; and

3) a synthetic anti -exosite 1 IgG S30A variant antibody (also referred to herein as "IgG S30A"), which compared with the IgG antibody above has an S30A substitution.

[ 00249 ] The IgG antibody has the wild-type sequence of IgA in the VH and VL domains. The IgG S30A antibody has the wild type sequence of IgA and IgG in the VH and VL domains, except that a glycosylation site in VL domain of SEQ ID NO 6 has been mutated out by introducing a substitution (alanine for serine) at S30.

[ 00250 ] In the specific examples, the synthetic monoclonal antibodies IgG and IgG S30A are also referred to by the name "ichorcumab". 7. Large-scale production of IgG S30A variant antibody

7.1 Introduction

[ 00251 ] In experimental section 6 above, the IgG S30A variant was expressed transiently using standard techniques for the purposes of analysing the variant. Here, we show that large scale production of IgG S30A following stable cell transfection using standard techniques is also possible.

7.2 Materials and Methods

[ 00252 ] In outline, double gene vector (DGV) was constructed using previously established single gene vectors (see experimental section 6 above) in Lonza's GS Xceed vectors (pXC IgG4pro DK for the heavy chain constant domain encoding region and pXC Kappa for light chain constant domain encoding region). The DGV was amplified and stably transfected into CHOK1SV GS-KO cells and analysed.

[ 00253 ] The methods used will be described below. Where manufacturer' s instructions were followed, this will be indicated.

7.2.1 Vector Construction

[ 00254 ] Single gene vectors (SGVs) established in Lonza' s GS Xceed vectors from the previous transient production of ichorcumab S30A (see experimental section 6 above) were used to generate a double gene vector (DGV). The DGV was constructed by restriction digest of the established SGVs using Pvul (Roche, 10650129001) and Notl (Roche, 11014714001) in a total reaction volume of 20 mΐ and incubated at 37°C for 2 hours. 4.0 mΐ of 6x DNA loading buffer was added to the digested samples and electrophoresed at 120 V for 40 min on a 1% w/v agarose gel stained with ethidium bromide. 10 mΐ Lonza Simply Load Tandem DNA ladder was used as a reference ladder. The agarose gel was imaged using BioSpectrum Imaging System (IVP).

[ 00255 ] The relevant fragments were gel-extracted using a QIAquick gel extraction kit (QIAGEN, 28704) according to manufacturer' s instructions. Ligations were set-up using Roche' s quick ligation kit (Roche, 11635379001) with a 1:3 ratio of vector backbone to insert DNA, 1 mΐ T4 quick ligase, 10 mΐ of 2x T4 quick ligation buffer, 2 mΐ of lOx DNA dilution buffer, reaction volume adjusted to 21 mΐ with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 10 minutes. 10 mΐ aliquots of the ligation reactions were used to transform One Shot Top 10 Chemically Competent Escherichia coli cells (Invitrogen, C404003) using the heat-shock method according to manufacturer' s instructions.

[ 00256 ] Cells were spread onto ampicillin-containing (50 pg/ml) APS Media (APS LB Broth base, BO 292438) agar plates and incubated overnight at 37°C until bacterial colonies were evident. Positive clones were screened by PCT amplification and verified by restriction digest (using a Hindlll/EcoRI double restriction digest) and nucleotide sequencing of the coding regions through a 3^rd party provider.

7.2.2 DNA Amplification

[ 00257 ] For DNA amplification, 5 ml of the growth cultures produced during the colony screening were used to inoculate 1 L APS medium (APS LB Broth base, BD 292438) containing 50 pg/ml ampicillin, incubated at 37°C overnight and shaking at 220 rpm. Vector DNA was isolated using the QIAGEN Plasmid Plus Gigaprep system (QIAGEN, 12991) and quantified using a Nanodrop 1000 spectrophotometer (Thermo- Scientific).

7.2.3 Routine Culture of CHOK1SV GS-KO Cells

[ 00258 ] CHOK1SV GS-KO cells were cultured in CD-CHO media (Invitrogen, 10743- 029) supplemented with 6 mM L-glutamine (Invitrogen, 25030-123). Cells were incubated in a shaking incubator at 36.5°C, 5% CO², 85% humidity, 140 rpm. Cells were routinely sub-cultured every 3-4 days, seeding at 2 x 10⁵ cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20

7.2.4 Stable Pooled Transfection of CHOK1SV GS-KO Cells

[ 00259 ] Double gene vector DNA plasmids were prepared for transfection by linearizing with Pvul followed by ethanol precipitation and resuspension in EB buffer to a final concentration of 400 pg/ml. Transfections were carried out via electroporation using either the Gene Pulse XCell (Bio-Rad). For each transfection, viable cells were resuspended in a pre-warmed CD-CHO media to l .43x 10⁷ cells/ml. 100 pl linearized DNA at a concentration of 400 pg/ml was aliquoted into a 0.4 cm gap electroporation cuvette and 700 pl cell suspension added. Three cuvettes of cells and DNA were electroporated at 300 V, 900 pF and immediately recovered to 30 ml pre-warmed CD- CHO supplemented with 10 ml/L SP4 (Lonza, BESP1076E) to generate a stable pool. The transfectants were incubated in a shaking incubator at 36.5°C, 5% CO², 85% humidity, 140 rpm.

[ 00260 ] A total of 5 stable pool transfectants were established. 24 h post-transfection the cultures were centrifuged and resuspended into pre-warmed CD-CHO supplemented with 50 mM MSX (L- Methionine Sulfoximine, Sigma-Aldrich, M5379) and 10 ml/L SP4. Cell growth and viability were periodically checked post-transfection.

[ 00261 ] When the viable cell density reached >0.6x 10⁵ cells/ml, the transfectant cultures were suitable to process. Cells were seeded at 0.2x 10⁶ cells/ml in a final volume of 100 ml in CD-CHO medium supplemented with 50 pM MSX/ lOml/L SP4, in a 500ml vented Erlenmeyer flask (Fisher Scientific (Coming), 10352742) and incubated in a shaking incubator at 36.5°C, 5% CO², 85% humidity, 140 rpm. Cell cultures were monitored and expanded once cultures had adapted to exponential growth. Cultures were then expanded to the appropriate production volume.

7.2.5 Protein A HPLC

[ 00262 ] Duplicate samples of clarified cell culture supernatant were analysed by Protein A HPLC on an Agilent 1200 series HPLC system, using a POROS Protein A cartridge (Applied Biosystems, 2-1001-00). 100 pl aliquots of supernatant samples, 0.22 pm filtered, were injected and run in 50 mM glycine, 150 mM sodium chloride, pH 8.0 at 2 ml/min for 5 minutes eluting with 50 mM glycine, 150 mM sodium chloride, pH 2.5. An 8-point standard curve was generated with 2-fold dilutions of a 1 mg/ml IgG4 in- house standard. All sample chromatograms were analysed using Chemstation software.

7.2.6 Cryopreservation of Cells

[ 00263 ] Five (5) vials each of the top two producing stable pools, as screened by Protein A HPLC during the suspension adaptation phase, were cryopreserved. Each vial contains 1.5 ml cell culture at lx 10⁷ cells/ml, passage number 3, with viability in excess of 98% prior to cryopreservation. Cells were centrifuged at 900 rpm for 5 minutes, the supernatant discarded and the cell pellet resuspended in ambient CD-CHO supplemented with 7.5% v/v DMSO. The vials were transferred into a Mr. Frosty™ (ThermoFisher) to minus 80°C before the frozen vials were transferred into vapour phase nitrogen storage. 7.2.7 Abridged Fed-Batch Overgrow Study

[ 00264 ] Cells were propagated to production volume by seeding the appropriate culture at 0.2x 106 cells/ml in Lonza's CM42 base media supplemented with 4 ml/L SPE using the established stable pools. The production volume was established in 5 L shake flasks (Generon, 931116). Shake flask cultures were incubated in a shaking incubator at 36.5°C, 5% CO², 85% humidity, and 140 rpm. Two batches of culture were initiated with a preliminary 1 L culture to deduce production titre followed by a 40 L production initiated one week later. Cell count and viability were monitored on day 4, before feeding was initiated, and periodically until the culture was harvested on day 12. The bolus feeds were administered on day 4 and 8 consisting of a mixture of Lonza's proprietary feeds.

7.2.8 Harvesting and Concentrating of Production Culture

[ 00265 ] 2.9L of the 40 L production culture was harvested by centrifugation at 6000 rpm prior to depth filtration using a KLEENPAK nova cartridge (PALL, NT6UBP1G), followed by filter sterilisation using a KLEENPAK 0.22 pm filter cartridge (PALL, KA2EKVP1G). The remaining supernatant was centrifuged as above and subject to clarification using pilot scale systems. The supernatant was frozen and stored at -20°C.

7.2.9 Protein A Affinity Chromatography

[ 00266 ] Clarified supernatant was purified using a 100 ml HiTrap MabSelect SuRE column (GE Healthcare, 17-5438-02) on an AKTA purifier (20 ml/min). The column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0

(equilibration buffer) for 5 column volumes (CVs) After sample loading, the column was washed with 2 CVs of equilibration buffer followed by 3 CVs of 50 mM sodium phosphate, 1 M sodium chloride pH 7.0 and a repeat wash of 2 CVs of equilibration buffer. The product was then eluted with 10 mM sodium formate, pH 3.5 over 5 CVs. Protein containing, eluted fractions were immediately pH adjusted to pH 7.2 and filtered through a 0.2 pm filter.

7.2.10 SE-HPLC Analysis

[ 00267 ] Duplicate samples were analysed by SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 pm 9.4 mm ID x 250 mm column (Agilent). Aliquots of sample at a concentration of 1 mg/ml were filtered through a 0.2 pm filter prior to injection. 80 mΐ aliquots were injected respectively and run at 1 ml/min for 15 minutes. Soluble aggregate levels were analysed using Chemstation (Agilent) software.

7.2.11 SDS-PAGE Analysis

[ 00268 ] Reduced samples were prepared for analysis by mixing with NuPage 4x LDS sample buffer (Invitrogen, NP0007) and NuPage lOx sample reducing agent (Invitrogen, NP0009), and incubated at 70°C, 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1.5 mm NuPage 4- 12% Bis-Tris Novex pre-cast gels (Invitrogen, NP0335PK2) with NuPage MES SOS running buffer under denaturing conditions. 10 mΐ aliquots of SeeBlue Plus 2 pre-stained molecular weight standards (Invitrogen, LC5925) and a control IgG4 antibody at 1 mg/ml were included on the gel. 1 mΐ of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISBOlL) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imaging System (UVP).

7.2.12 Endotoxin Analysis

[ 00269 ] Endotoxin levels of the purified product were tested once concentrating to 20 mg/ml was completed. The product was tested at 1 mg/ml using the Endosafe-PTS instrument, a cartridge based method based on the LAL assay (Charles River).

7.3 Results and Discussion

[ 00270 ] Initially, 5 stable pools of transfectant cultures were produced. The transfectant cultures were screened by Protein A HPLC to identify the top 2 expressing pools. A 1 L preliminary culture followed by a 40 L production culture were initiated and subjected to an abridged fed-batch overgrow study including the administration of bolus feeds on days 4 and 8. Cultures were harvested on Day 12 and supernatant titre determined prior to harvest. A volume of the sample culture was clarified by

centrifugation followed by depth and sterile filtration. The clarified cell culture supernatant was purified using one-step Protein A chromatography.

[ 00271 ] Product quality analysis in the form of SE-HPLC, SOS-PAGE and endotoxin detection showed a high level of purity was achieved post-purification. The remaining supernatant was clarified using a pilot scale filtration system due to high viscosity and large amount of product present within the supernatant. Sequences

[ 00272 ] Amino acid sequence of human preprothrombin (GeneID:2l47;

NP_000497. l; GI:4503635; exosite 1 residues underlined): (SEQ ID NO: l)

1 MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQR VRRANTFLEE VRKGNLEREC

61 VEETCSYEEA FEALESSTAT DVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHV

121 NITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNP DSSTTGPWCY TTDPTVRRQE

181 CSI PVCGQDQ VTVAMTPRSE GSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASA

241 QAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKP GDFGYCDLNY CEEAVEEETG

301 DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYI

361 DGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRW VLTAAHCLLY PPWDKNFTEN

421 DLLVRIGKHS_ RTRYERNIEK I SMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHP

481 VCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQP SVLQWNLPI VERPVCKDST

541 RIRITDNMFC AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY

601 GFYTHVFRLK KWIQKVIDQF GE

[ 00273 ] Amino acid sequence of anti-exosite 1 IgA and IgG VH domain with Kabat Numbering (CDRs underlined): (SEQ ID NO: 2).

QVQLIQSGSAVKKPGASVRVSCKVSGYTLTEAAIHWVRQAPGKGLEWMG

10 20 30 40 50

LDPQDGETVYAQQFKGRVTMTEDRSTDTAYMEVNNLRSEDTATYYCTTGD

52a 60 70 8082abc 90

FSEFEPFSMDYFHFWGQGTWTVAS

lOOabcdefgh 110

[ 00274 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDR1: (SEQ ID NO:3).

GYTLTEAAIH

[ 00275 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDR2: (SEQ ID NO:4).

GLDPQDGETVYAQQFKG

[ 00276 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDR3: (SEQ ID NO:5).

GDFSEFEPFSMDYFHF

[ 00277 ] Amino acid sequence of anti-exosite 1 IgA and IgG VL domain with Kabat Numbering (CDRs underlined): (SEQ ID NO: 6).

EIVLTQSPATLSLSPGERATLSCRASQNVSSFLAWYQHKPGQAPRLLIYD

10 20 30 40 50

ASSRATDI PIRFSGSGSGTDFTLTI SGLEPEDFAVYYCQQRRSWPPLTFG 60 70 80 90 95a

GGTKVEIKR

100 108

[ 00278 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR1: (SEQ ID NO:7).

RASQNVSSFLA

[ 00279 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR2: (SEQ ID NO:8).

DASSRAT

[ 00280 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR3: (SEQ ID NO:9).

QQRRSWPPLT

[ 00281 ] Amino acid sequence of anti-exosite 1 IgG4 (JNJ-64179375) heavy chain with CDRs underlined: (SEQ ID NO: 14). SEQ ID NO: 14 includes S228P substitution (numbered according to the EU numbering system) to stabilize hinge region and the C- terminal lysine of the HC was removed to eliminate heterogeneity.

1 QVQLIQSGSA VKKPGASVRV SCKVSGYTLT EAAIHWVRQA PGKGLEWMGG LDPQDGETVY

61 AQQFKGRVTM TEDRSTDTAY MEVNNLRSED TATYYCTTGD FSEFEPFSMD YFHFWGQGTV

121 VTVASASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA

181 VLQSSGLYSL SSWTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL

241 GGPSVFLFPP KPKDTLMI SR TPEVTCVWD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ

301 FNSTYRWSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT I SKAKGQPRE PQVYTLPPSQ

361 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS

421 RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G

[ 00282 ] Amino acid sequence of anti-exosite 1 IgG4 (JNJ-64179375) light chain with CDRs underlined (SEQ ID NO: 15). SEQ ID NO: 15 includes S30A substitution to remove glycosylation site.

1 EIVLTQSPAT LSLSPGERAT LSCRASQNVA SFLAWYQHKP GQAPRLLIYD ASSRATDIPI 61 RFSGSGSGTD FTLTI SGLEP EDFAVYYCQQ RRSWPPLTFG GGTKVEIKRT VAAPSVFIFP 121 PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 181 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

[ 00283 ] Amino acid sequence of anti-exosite 1 IgG4 (JNJ-64179375) VL domain with S30A substitution to remove the glycosylation site (CDRs underlined): (SEQ ID NO: 16).

1 EIVLTQSPAT LSLSPGERAT LSCRASQNVA SFLAWYQHKP GQAPRLLIYD ASSRATDIPI

61 RFSGSGSGTD FTLTI SGLEP EDFAVYYCQQ RRSWPPLTFG GGTKVEIKR [ 00284 ] Amino acid sequence of anti-exosite 1 IgG4 (JNJ-64179375) LCDR1: (SEQ ID NO: 17). SEQ ID NO: 17 includes the alanine (underlined) for serine substitution that corresponds the S30A substitution in SEQ ID NO:6.

RASQNVAS FLA

Table 1: Coagulation Screening results

Caatroi/Nosmalised

Test Result Ratio (NR)

Frothrom bis Tim 43 see. NR = 11-13 see.

50: (S 35 see,

correction

Act. part 157 see, NR - 22-23 see,

Thro mbopiasti n

50:50 105 sec,

correction

Thrombln Time >150 sec. - 10- 13 sec

Reptilxse Time 16 sec. Control - 15 sec,

Fibrinogen Cl suss 0.7 g/J NR - 1 5-4.5 g/1

Antigenic 5.0 g?

Table 2: Effect of anti-exosite 1 IgA on thrombin hydrolysis of procoagulant substrates

Ttorabm substrate Activity Antibody Effect

Fibrinogen Formation of fibrin No detectable

clot cleavage

Platelet receptor Activation of 15-foM decrease

PAS-1 peptide platelets in hydrolysis

FVIII Feedback activation 7-fold decrease

of thrombin via in hydrolysis

X&se complex

Table 3: Effect of saturating concentration of anti-exosite 1 IgA (Fab) on thrombin inhibition by antithrombin (AT) in the absence and presence of 1 nM heparin (Hep).

Rate of InMbftioaiM ¹ s ⁴) Heparin effect

AT 4.8 * 0.2 x K 2.4'· fold

AT f Hep 11.8 ± 0.3 x Ufo

AT + Fab 1.7 ± 0.1 x V? 3.3 -fold

AT 't Hep a- Fab 5.6 ± 0.3 10³

Table 4: Binding constants of anti-exosite 1 IgA (n=l under this precise condition), IgG (n=3) antibodies, and IgG-derived FAB to S195A thrombin (active site free, recombinant thrombin).

IgA 1.8 3.3 x 10^s 3.7 x !CT⁴ 1.2

IK 1.5 * 0.3 13 ¾ 0.5 x 10⁵ 6,8 * 1.1 x lO ⁴ 2.1 s 0.3

IgG FAB NO 5,0 x JO⁵ 2.7 x ίίT³ 5.3

igG FAB⁺ 3.3 * 0.3 4.3 x 10^s 2.1 x ID"³ 4.9

TE d detenmsjcd from sicady-statc analysis of response vs. concentration,

&Kd calculated from rales..

Dstetni5tie<l naia immobilised FAB, Table 5: Binding affinities of IgG and IgG S30A to thrombin using Biacore™ surface binding analysis. Binding at ambient condition ("Control") was compared with binding (1) after storage for one month at 4°C or (2) after exposure to light ("PO").

Ligand Kd (riM)

Day 0 128 * 0 129 ± 0

Day 6 12 * 0 122 ± 0

Day 28 113 ± 1 123 ± 2

8. Rat AY shunt model

[ 00285 ] A widely used rat model of thrombosis, the arterial-venous (AV) shunt model, was established and characterized with reference agents in-house. Reference agents included Apixaban, Dabigatran, Bivalirudin, and Heparin. Evaluations in the AV shunt model included Thrombin Time (TT), activated Partial Thrombin Time (aPTT),

Prothrombin Time (PT), Ecarin Clotting Time (ECT), and blood clot weight. Results with the reference agents were compared to JNJ-64179375.

Methods

[ 00286 ] The rat AV-shunt model is done under anesthesia. The left jugular vein and right carotid artery are cannulated with 8cm long PE- 100 tubing. A baseline blood sample (lml) is collected and then compounds are administered either as an intravenous bolus or l5min infusion. After administering a compound, the shunt is assembled by connecting the jugular vein and carotid artery cannulae with a 6cm long tubing containing a 6cm 2-0 silk surgical thread. The 6cm long connection is the shunt and the silk thread in the shunt acts as a foreign body to activate the intrinsic cascade to cause a blood clot (thrombus). Blood is allowed to flow through the shunt for 15 min. At the end of 15 min, the external tubing with the thread and blood clot are removed and the blood clot is weighed. After removing the thread, additional blood samples (2x4.5ml) are collected for subsequent testing and the animal is euthanized.

Results

[ 00287 ] JNJ-64179375, dabigatran, and apixaban all maximally inhibited thrombus formation in dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, IV, 0.1 mg/kg, IV and 1 mg/kg, IV with JNJ-64179375, dabigatran, and apixaban, respectively (Figure 30). Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-64179375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT) (Figure 31). In addition, there were dose dependent increases in drug exposure with JNJ-64179375 (Table 7) and with Apixaban, Dabigatran, and Bivalirudin (Figure 32).

Table 7: Pharmacokinetic (PK) results of JNJ-64179375 (JNJ-9375) in AV shunt model

9. Translational Model for Gastrointestinal (GI) Bleeding

Introduction

[ 00288 ] Despite advances in anticoagulation with direct acting oral anticoagulants (DOAC’s), bleeding, including gastrointestinal (GI) bleeding remains a significant concern. This is especially true for patients with risk factors for GI bleeding, e.g., older aged patients, patients previously or currently treated with other anticoagulants, patients currently treated with antiplatelet agents, patients with GI disorders, patients with a family history of GI disorders, patients with prior GI bleeding, cancer patients, patients who had a prior stroke, patients with reduced eGFR, patients with renal impairment, patients with severe liver disease, patients with anemia, and patients with lower bodyweight. This patient population with risk factors for GI bleeding includes patients that may be advised not to take other anticoagulants such as direct acting oral anticoagulants (DOACs), e.g., factor Xa (FXa) inhibitors (e.g., apixaban), thrombin inhibitors (e.g., dabigatran), or factor XIa (FXIa) inhibitors. The animal models used to assess bleeding risk are typically acute, provoked injury models that do not mimic spontaneous bleeds or the extent of bleeding in patients. The aim of this study was to use Apc^mn/+ mice to determine the potential for spontaneous GI bleeding with JNJ-64179375 compared to Eliquis (apixaban) and Pradaxa (dabigatran).

Background

[ 00289 ] Coagulation cascade activation leading to fibrin formation is central to thrombosis. Anticoagulant agents are of proven benefit in many thrombotic and/or embolic disorders but despite improvements, treatment-related bleeding continues to be a major concern and event rates remain unacceptably high [Lozano et al. Lancet

2012;380:2095-128.; Chai-Adisaksopha et al. JThromb Haemost 2015;13:2012-20.; Chai-Adisaksopha et al. Blood 2014;124:2450-8.]. All the currently available agents act to either prevent thrombin generation (e.g. vitamin K antagonists, factor Xa inhibitors, low molecular weight heparin) or block the catalytic site of the protease directly (e.g. dabigatran, bivalirudin). Consequently, they provide broad inhibition of all thrombin activity, which, given thrombin has key roles in both thrombosis and haemostasis, invariably predisposes to a narrow therapeutic window. Novel anticoagulant strategies are required to provide equivalent (or superior) efficacy with an acceptable safety profile, e.g., a favorable risk:benefit ratio with a relatively low or reduced frequency and/or low or reduced severity of adverse events, including reduced adverse gastrointestinal (GI) bleeding events, compared to the standard of care or to another comparator.

[ 00290 ] JNJ-64179375 is a first-in-class, recombinant, fully human immunoglobin (Ig)

G4 monoclonal antibody that binds reversibly with high affinity and specificity to the exosite-l region of thrombin. JNJ-64179375 was engineered to mimic the pharmacologic effects of an IgA antibody that was found in a patient with markedly abnormal clotting times but with a lack of spontaneous bleeding episodes over a prolonged follow-up period, representing the profile of an anticoagulant that might have a beneficial therapeutic index in terms of anticoagulation efficacy with low bleeding risk [Baglin et al. J Thromb Haemost. 2015;14: 137-42] JNJ-64179375 comprises a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15; a variable heavy chain (VH) domain amino acid sequence of SEQ ID NO:2 and a variable light chain (VL) domain amino acid sequence of SEQ ID NO: 16; heavy chain CDR amino acid sequences of SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), and SEQ ID NO:5 (HCDR3); and the light chain CDR amino acid sequences of SEQ ID NO: 17 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3).

[ 00291 ] The JNJ-64179375 sequences include an S30A substitution in the LC to remove a glycosylation site, a serine 228 to proline substitution (S228P, as numbered according to the EU numbering system) in the HC to stabilize the hinge region [Labrijn et al. Nature Biotech. 27, 767 - 771 (2009).; Silva et al. JBiol Chem. 2015 Feb

27;290(9):5462-9.] and the C-terminal lysine was removed from the HC to eliminate heterogeneity. Subsequent characterisation of the antibody found it specifically bound to thrombin exosite 1. Exosite 1, together with exosite 2, regulate thrombin enzymatic activity by providing initial binding sites for substrates, inhibitors, or co-factors, and by allosteric modification or steric hindrance of the active site [Becker et al. J Biol Chem 1999;274:6226-33.; Lane. Blood 2005;106:2605-12.; Bock et al. J Thromh Haemost. 2007;5:81-94.]. Exosite 1 is predominantly the fibrinogen Aa recognition site, while exosite 2 binds to heparin and glycoprotein (GP) Iba. JNJ-64179375 therefore acts as an anticoagulant by preventing binding of fibrinogen Aa whilst leaving the catalytic activity of the protease intact. This mechanism of action is distinct from the current anticoagulants and by avoiding inhibiting all thrombin activity has the potential for a wider therapeutic window in terms of antithrombotic efficacy and haemorrhagic risk. For example, the mechanism of action is distinct from currently available direct thrombin inhibitors that block the active site only (eg, dabigatran, argatroban) or that block both the active site and exosite 1 (eg, bivalirudin, hirudin) (Figure 33). The mechanism of action is also distinct from other mechanisms that inhibit thrombin generation (eg, Factor Xa [FXa] inhibitors such as apixaban). This distinct mechanism of action for JNJ-64179375 may provide benefits, for example:

• JNJ-64179375 may expand therapeutic index compared to other thrombin

inhibitors by selectively inhibiting fibrinogen interaction at exosite 1 region of thrombin without affecting catalytic activity at thrombin active site

• Specific blockade at exosite 1 by JNJ-64179375 may preferentially inhibit fibrin- rich venous thrombosis with relative preservation of platelet-mediated hemostasis

• Thrombin can also activate platelets through PAR-4/GPIb interactions via exosite 2 to generate a hemostatic platelet plug Active site blockade (e.g., hirudin, bivalirudin, dabigatran) can prevent both fibrin-rich and platelet-rich clot formation

[ 00292 ] The most common complication of the treatment with an antithrombotic agent is bleeding, e.g., gastrointestinal (GI) bleeding, intracranial haemorrhage and trauma or surgical related bleeding. Major bleeding can cause long-term debilitating effects and can even be life-threatening. In three large studies, OASIS (Organisation to Assess Ischaemic Syndromes), OASIS-2 and CURE (the Clopidogrel in Unstable Angina to Prevent Recurrent Events), major bleeding associated with the antithrombotic agents correlated with a five-fold increase in the risk of death during the first 30 days and a 1.5 -fold higher rate of mortality between 30 days and six months [Eikelboom et al. Circulation 2006;

114: 774-782.]. The newly developed anticoagulant drugs apixaban, rivaroxaban and dabigatran were shown to exhibit significantly lower frequency of major bleeding

[Connolly et al. New Engl JMed 2009; 361: 1139-1151.; Mantha and Ansell. Thromb Haemost 2012; 108: 476-484.], however, patients treated with these new oral anticoagulants had an increased risk of GI bleeding compared to those who received the standard of care [Holster et al. Gastroenterology 2013 Jul;l45(l): 105-112.].

[ 00293 ] GI bleeding is the predominant form of major bleeding in patients using antithrombotic agents. For example, in the Randomized Evaluation of Long-Term Anti coagulation Therapy (RE-LY) trial, GI bleeding accounted for 48.5% of all major bleeding observed in patients taking 150 mg dabigatran twice daily [Connolly et al. New Engl JMed 2009; 361: 1139-1151.]. In clinic trials, forearm template or Ivy bleeding time is the most frequently used method for predicting the bleeding liability of antithrombotic drugs. Similarly, many preclinical animal models, such as tail bleeding [Morowski et al. Blood 2013; 121: 4938-4947.], cuticle bleeding [Littlewood et al.

Thromb Haemost 1996; 76: 743-748.], ear bleeding [Weitz. J Vase Interv Radiol 1995; 6 (6 Pt 2 Suppl): 19S-23S.] and tongue bleeding [Hong et al . J Cardiovasc Pharmacol 2005; 46: 526-533.], have been developed for predicting bleeding liability. These models are usually performed in healthy animals with some form of skin or cuticle injury.

However, GI bleeding and intracranial haemorrhage are typically spontaneous in nature. Even in surgery-related bleeding, the predictive value of bleeding time measurements performed in humans or in animal models is questionable [Lind. Blood 1991; 77: 2547- 2552.; Lind. Am JMed 1984; 77: 305-312.; Rodgers and Levin. Thromb Haemost 1990;16: 1-20.]. Hydrochloric acid/ethanol- or indomethacin induced GI damage in rodents has been used to study GI bleeding [Horisawa et al. Thromb Haemost 1999; 82: 1743-1748.; Melarange et al. Alimentary Pharmacol Therap 1992; 6: 67-77.], but indomethacin has antiplatelet effects and thus may interfere with the testing of antithrombotic agents. In addition, GI bleeding was induced within 24 hours (h) in these studies, which is neither chronic, nor spontaneous.

[ 00294 ] The Apc^mm/+ mouse is a model for Familial Adenomatous Polyposis (FAP) and harbours a dominant mutation at the Ape (Adenomatous polyposis coli) gene, the mouse homolog of the human Ape gene, resulting in a truncated product at amino acid 850 [Moser et al. Science 1990; 247: 322-324.]. Ape contributes to the degradation of cytosolic b-catenin. Inactivation of Ape leads to a constitutive accumulation of [3-catenin, which triggers transcription activation of b-catenin/Tcf target genes, such as Myc and Cyclin Dl [Korinek et al. Science 1997; 275: 1784-1787.; Morin et al. Science 1997; 275: 1787-1790.]. Apc^min/+mice develop multiple adenomas through the entire intestinal tract at an early age. The development of adenomas leads to chronic blood loss, resulting in progressive anemia in Apc^mm/+rnice [Moser et al. Science 1990; 247: 322-324.]. The spontaneous and chronic bleeding in Apc^min/+ mice make them ideal candidates for studying the bleeding risk of antithrombotic agents. In this study, we characterised GI bleeding and coagulation parameters of Apc^mn/+ mice, and assessed the spontaneous bleeding risk for dabigatran, apixaban and JNJ-64179375.

Methods

Overview

[ 00295 ] Apc^mn/+ mice (7 weeks old) were administered vehicle (buffer) or JNJ- 64179375 at 0.5, 1.5 and 4.5 mg/kg, s.c. every 4 days for 16 days. Robust antithrombotic efficacy was demonstrated with JNJ-64179375 at a dose of 0.3 mg/kg, i.v. in a rat Arterial- Venous (AV) shunt model, e.g., there was a dose dependent reduction in thrombus formation with a 50% reduction in thrombus weight at 0.3 mg/kg (Example 8, supra). Plasma level of JNJ-64179375 at 0.5 mg/kg, s.c. dosing was in line with the level associated with robust antithrombotic efficacy in rats, while the 1.5 and 4.5 mg/kg, s.c doses represent approximately 3X and 10X multiples of the dose for efficacy in rats based on results in the rat AV shunt model. Apixaban (0.6, 1.2 and 3.6 mg/gm) and dabigatran (2 and 5 mg/gm) were formulated in the diet 5001 and fed to wild type (WT) and Apc^mm/+ mice. [Wei et al. Thromb Haemost. 2014 Jun; l l l(6): l l2l-32.] The doses of apixaban and dabigatran were selected based on the published studies in which the clinical relevant exposures of the compounds were achieved. Controls were WT and Apc^mn/+ mice fed diet 5001 without drug. [Lei et al. Clin Pharmacol Ther. 2010 Sep;88(3):375-82.; Clemens et al. CurrMed Res Opin. 2012 Feb;28(2): 195-201.; van Ryn et al. Thromb Haemost. 2010 Jun;l03(6): 1116-27.] A small (50 ul) blood sample was obtained on day 0 and at the end of the study to assess levels for hemoglobin (Hgb), hematocrit (Hct), and red blood cells (RBC). A terminal blood sample was also collected to assess drug levels and to measure thrombin time (TT).

Animals

[ 00296 ] Male APC^min/+ mice (heterozygotes for a mutation in Ape gene in C57BL/6J background) and age-matched male wild type C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The mice were 4-6 weeks of age at arrival and were housed in a temperature-controlled room with 12-hour light/dark cycle. They were allowed ad libitum access to water and maintained on a regular diet (diet 5001). Experiments were initiated when the mice were 7 weeks old and the animals were then housed at 1 mouse per cage so that food and/or water consumption could be accurately measured during the study period. Throughout the treatment period, animals were observed for any signs of toxicity. Body weight, food and/or water intake were recorded biweekly. Animal experimentation was carried out in accordance with the National Institute of Health’s Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act in a program accredited by the American Association for Accreditation of Laboratory Animal Care.

Blood sample collection

[ 00297 ] A small tail snip (~2mm) was made on restrained animals and about 50 ul of blood was collected into tubes containing EDTA. Samples were analysed using a Hemavet® (analyser) (Drew Scientific, Dallas, TX, USA) to measure several parameters including, e.g., hemoglobin (Hgb), hematocrit (Hct), and red blood cells (RBC). At the end of study, animals were anesthetized under ketamine/xylazine anesthesia and terminal blood samples were collected into syringes containing 3.2% sodium citrate from the abdominal aorta or from cardiac puncture. Samples of twenty microliters of whole blood samples were mixed with 60 ul of 1 uM sodium citrate to assess drug levels by mass spectrometry. Mouse plasma was prepared by centrifuging mouse blood at 2,400 x g for 15 minutes (min) at 4°C.

Thrombin Time (TT)

[ 00298 ] Evaluation of thrombin time (TT) was performed as described previously (17). Briefly, for the TT assay, a IOOmI prewarmed plasma sample was incubated with IOOmI of reagents from Diagnostica Stago, S.A.S (Parsippany, NJ, USA) to initiate clot formation. The time of clot formation was detected and measured with Coag-A-Mate (Diagnostica Stago, Inc).

Statistical analysis

[ 00299 ] Data are expressed as the mean with Standard Error (SE) or Standard Deviation (SD) reported, and statistical analysis was done by ANOVA and Dunnett’s Multiple Comparison Test. A p-value <0.05 was considered significant.

Compounds and Treatment Groups

JNJ-64179375

[ 00300 ] JNJ-64179375 was administered to APC^min/+ mice by s.c. injection every 4 days (q4d) for 16 days, at 0.5, 1.5, and 4.5 mg/kg. The control was vehicle (buffer) administered to APC^mn+ mice by s.c. q4d

Table 8: Treatment Groups for JNJ-64179375

Procedure

• The study was done for 16-days • Day 1 (Monday): Basal levels for Hgb, Hct, RBC, Plt, and body weight (BW) were measured. Mice were then allocated into groups based on their basal blood measurement and BW

• Day 1 (Monday): mice receiving JNJ-64179375 or buffer were injected s.c. as indicated in Table 8

• Body weight (BW) and food intake (FI) were measured twice per week (every Tuesday and Friday)

• Weekly (Mondays) and at the end of the study, ~50 ul of blood from each mouse was collected into tubes containing EDTA via a small tail snip for the

measurements of Hgb, Hct, RBC, and Plt

• Fecal occult blood was measured weekly (every Monday) via fecal blood drop test kit

• Mice were monitored daily, including weekends

• At the end of the study (day 16), mice were anesthetized and sacrificed for

subsequent sampling, including:

blood samples collected for 8 mice per group from cardiac puncture bleeding into syringes containing 3.2% sodium citrate for compound exposure levels in plasma and for an in vitro clotting assay, thrombin time (TT)

GI systems removed from 4 mice in each group and evaluated by CT- scanning.

Dabigatran

[ 00301 ] Dabigatran (PharmaBlock Sciences, Cat# PBN20120440) was formulated in diet 5001 at 2 and 5 mg/gram using a blending machine.

Table 9: Treatment Groups for Dabigatran

Procedure

• The study was done for 16-days

• Day 1 (Monday): Basal levels for Hgb, Hct, RBC, Plt, and body weights (BW) were measured. Mice were then allocated into groups based on their basal blood measurement and BW

• Day 1 (Monday): pre-weighed diet 5001 or dabigatran formulated diets were provided to mice in different treatment groups as indicated in Table 9

measurements of Hgb, Hct, RBC, and Plt

• Mice were monitored daily, including weekends

• At the end of the study (day 16), mice were anesthetized and sacrificed for

subsequent sampling, including:

blood samples collected for 8 mice per group from abdominal aorta into syringes containing 3.2% sodium citrate for compound exposure levels in plasma and for an in vitro clotting assay, thrombin time (TT) small intestines were opened at necropsy to observe tumor growth in APC^mn+ mice Apixaban

[ 00302 ] Apixaban (AChemBlock, Cat# 10525) was formulated in diet 5001 at 0.6, 1.2, and 3.6 mg/gram using a blending machine.

Table 10: Treatment Groups for Apixaban

Procedure

• The study was done for 16-days

• Day 1 (Monday): pre-weighed diet 5001 or apixaban formulated diets were

provided to mice in different treatment groups as indicated in Table 10

• Body weight (BW) and food intake (FI) were measured twice per week (every Tuesday and Friday) • Weekly (Mondays) and at the end of the study, ~50 ul of blood from each mouse was collected into tubes containing EDTA via a small tail snip for the

measurements of Hgb, Hct, RBC, and Pit

• Mice were monitored daily, including weekends

• At the end of the study (day 16), mice were anesthetized and sacrificed for

subsequent sampling, including:

blood samples collected for 8 mice per group from abdominal aorta into syringes containing 3.2% sodium citrate for compound exposure levels in plasma and for an in vitro clotting assay, thrombin time (TT) small intestines were opened at necropsy to observe tumor growth in APC^mn+ mice

Results

Summary

[ 00303 ] Compared to vehicle controls, mean levels of hemoglobin (Hgb), hematocrit (Hct), and red blood cells (RBC) decreased significantly with apixaban treatment of APC^mm/+ mice at all three doses tested (Table 17 and Table 21). Furthermore, the change in Hgb levels after apixaban treatment indicated major bleeding as defined by the ISTH bleeding scale, e.g., bleeding causing a fall in hemoglobin level of 2 g/dF or more. Mean Hgb, Hct, and RBC levels also decreased significantly with dabigatran treatment in APC"""^/~ mice at both doses tested (Table 14 and Table 20). In contrast, Hgb, Hct, and RBC levels were not significantly different with JNJ-64179375 treatment of APC^min/+ mice at any of the 3 doses tested (Table 11 and Table 19). Plasma levels for JNJ- 64179375 were IX, 3X and 10X multiples of the antithrombotic dose. Plasma levels of apixaban and dabigatran measured at the end of the study were in line with the levels seen in patients. Mean plasma concentration of dabigatran in the APC min mice dosed at 2 and 5 mg/g in the diet were 172hM and 399 nM, respectively. These levels are in line with those drug levels reported in the literature with dabigatran at 150 mg BID dose in patients (191-390 nM). Drug levels of apixaban in the APC min mice were 192, 227 and 620 nM at the 0.6, 1.2 and 3.6 mg/g in diet, respectively. These levels of apixaban are comparable to the drug levels reported with apixaban at 5 mg, BID dose in patients (233-326 nM). See, for example, Table 18 and Table 15, respectively, and published reports [Lei et al. Clin Pharmacol Ther. 2010 Sep;88(3):375-82.; Clemens et al. Curr Med Res Opin. 2012 Feb;28(2): l95-20l .; van Ryn et al. Thromh Haemost. 2010 Jun;l03(6): 1116-27.].

Table 11: Blood sample results and starting body weight (BW) for J J-64179375 administered to APC^mn/+ mice by s.c. injection every 4 days (q4d) for 16 days, at 0.5,

1.5, and 4.5 mg/kg. The control was buffer s.c. q4d. (*: no terminal blood samples collected for TT)

Table 12: Exposure results for J J-64179375 administered to APC^mn/+ mice by s.c. injection every 4 days (q4d) for 16 days, at 0.5, 1.5, and 4.5 mg/kg.

Table 13: Blood sample results and starting body weight (BW) for dabigatran administered to wild type (WT) mice at 2 and 5 mg/gram. The control was WT mice fed diet 5001 without drug. (* : sampling error)

Table 14: Blood sample results and starting body weight (BW) for dabigatran administered to APC^mn/+ mice at 2 and 5 mg/gram. The control was APC^mm/+ mice fed diet 5001 without drug. (NS: no sample taken, * : sampling error)

Table 15: Exposure results for dabigatran administered to WT and APC^mm+ mice at 2 and

5 mg/gram. (NS: no sample taken)

Table 16: Blood sample results and starting body weight (BW) for apixaban administered to wild type (WT) mice at 0.6, 1.2, and 3.6 mg/gram. The control was WT mice fed diet

5001 without drug. (NS: no sample taken)

Table 17: Blood sample results for apixaban administered to APC^min/+ mice at 0.6, 1.2, and 3.6 mg/gram. The control was APC^mn/+ mice fed diet 5001 without drug. (NS: no sample taken)

Table 18: Exposure results for apixaban administered to WT and APC^mn/+ mice at 0.6,

1.2, and 3.6 mg/gram. (NS: no sample taken)

Statistical analysis

Table 19. Statistical analysis of JNJ-64179375 in APC^mm+ mice

Table 20. Statistical analysis of Dabigatran in APC^min+ mice

Table 21. Statistical analysis of Apixaban in APC^mm+ mice

Conclusion

[ 00304 ] In a clinically relevant translational GI bleeding model using Apc^mn/+ mice, experimental results showed significant decreases in Hgb, Hct, and RBC levels that reflected significant increases in spontaneous, chronic and major bleeding with apixaban and dabigatran treatment at drug exposures that were comparable to exposure levels seen in the clinic. In contrast, JNJ-64179375 did not significantly increase GI bleeding in the model even at 3X and 10X multiples of the antithrombotic dose.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing treatment-related adverse gastrointestinal (GI) bleeding events in a patient at risk for gastrointestinal (GI) bleeding and in need of a treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to said patient an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15.

2. The method of claim 1, wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: older aged patient, patient previously or currently treated with an anticoagulant, patient currently treated with an antiplatelet agent, patient with GI disorders, patient with a family history of GI disorders, patient with prior GI bleeding, a cancer patient, patient who had a prior stroke, patient with reduced estimated glomerular filtration rate (eGFR), patient with renal impairment, patient with severe liver disease, patient with anemia, and patient with lower bodyweight.

3. The method of claims 1 or 2, wherein treatment with said anti -thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

4. The method of claim 3, wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

5. The method of claim 3, wherein the DOAC is selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor.

6. The method of claim 3, wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

7. The method of claim 3, wherein the DOAC is the FXa inhibitor apixaban.

8. The method of claim 3, wherein the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.

9. A composition for use in inhibiting a thrombotic and/or embolic disorder in a patient at risk for GI bleeding, comprising an anti-thrombin antibody comprising a HC amino acid sequence of SEQ ID NO: 14 and a LC amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent, and wherein treatment with said composition comprising the anti-thrombin antibody causes significantly reduced adverse GI bleeding events.

10. The composition of claim 9, wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: older aged patient, patient previously or currently treated with an anticoagulant, patient currently treated with an antiplatelet agent, patient with GI disorders, patient with a family history of GI disorders, patient with prior GI bleeding, a cancer patient, patient who had a prior stroke, patient with reduced estimated glomerular filtration rate (eGFR), patient with renal impairment, patient with severe liver disease, patient with anemia, and patient with lower bodyweight.

11. The composition of claims 9 or 10, wherein the treatment with said anti -thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

12. The composition of claim 11, wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

13. The method of claim 11, wherein the DOAC is selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor.

14. The composition of claim 11, wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

15. The composition of claim 11, wherein the DOAC is the FXa inhibitor apixaban.

16. The composition of claim 11, wherein the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.

17. A method of reducing treatment-related adverse gastrointestinal (GI) bleeding in a patient at risk for gastrointestinal (GI) bleeding and in need of treatment for inhibiting a thrombotic and/or embolic disorder, comprising administering to said patient a composition comprising an anti-thrombin antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO: 14 and a light chain (LC) amino acid sequence of SEQ ID NO: 15, wherein the composition also comprises at least one pharmaceutically acceptable carrier or diluent.

18. The method of treatment of claim 17, wherein the patient at risk for GI bleeding is a patient selected from the group consisting of: older aged patient, patient previously or currently treated with an anticoagulant, patient currently treated with an antiplatelet agent, patient with GI disorders, patient with a family history of GI disorders, patient with prior GI bleeding, a cancer patient, patient who had a prior stroke, patient with reduced estimated glomerular filtration rate (eGFR), patient with renal impairment, patient with severe liver disease, patient with anemia, and patient with lower bodyweight.

19. The method of claims 17 or 18, wherein the treatment with said anti-thrombin antibody causes significantly reduced adverse GI bleeding events compared to treatment with a direct acting oral anticoagulant (DOAC).

20. The method of treatment of claim 19, wherein the significantly reduced adverse GI bleeding events is a 35-50% reduction compared to the DOAC.

21. The method of claim 19, wherein the DOAC is selected from the group consisting of: a factor Xa (FXa) inhibitor, a thrombin inhibitor, and a factor XIa (FXIa) inhibitor.

22. The method of treatment of claim 19, wherein the DOAC is selected from the group consisting of: the FXa inhibitor apixaban and the thrombin inhibitor dabigatran.

23. The method of treatment of claim 19, wherein the DOAC is the FXa inhibitor apixaban.

24. The method of treatment of claim 19, wherein the significantly reduced adverse GI bleeding events is significantly reduced major GI bleeding events.